Leuconostoc mesenteroides, saccharomyces cerevisiae, fermentation baking method of dried orange peel flavor coffee beans, coffee beans and application

Abstract

The invention is suitable for the technical field of microbial fermentation and food deep processing, and provides leuconostoc mesenteroides LB01, saccharomyces cerevisiae PB01, a microbial starter containing two strains, a fermentation method and a baking method of dried orange peel flavored coffee beans, a product and application. The bacterial strain is derived from Ktill coffee fresh fruits and is obtained through separation, purification and identification. The fermentation method comprises the following steps: mixing and sterilizing the green coffee beans and the pericarpium citri reticulatae powder, and performing ultraviolet sterilization on the orange shred powder; the preparation method comprises the following steps: activating and centrifuging a bacterial strain, preparing a compound bacterial solution, mixing the compound bacterial solution with raw materials, fermenting for 20-28 hours at 30-35 DEG C and 65-75% humidity, drying, and performing a sectional baking method to obtain a coffee bean product with the flavor of dried orange peel. The product has compound flavors such as pericarpium citri reticulatae fragrance and citrus fragrance, the bitterness is obviously reduced, tartaric acid is soft, and the aftertaste is sweet for a long time. The method is controllable in fermentation, high in efficiency, simple and convenient in process, stable in product quality, suitable for industrial production and capable of meeting the requirements of consumers for novel and healthy compound-flavor coffee.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and food deep processing technology, and in particular relates to a fermentation and roasting method for preparing tangerine peel flavored coffee beans based on Leuconostoc mesenteroides and Saccharomyces cerevisiae, as well as the coffee beans themselves. Background Technology

[0002] As one of the world's most consumed beverage crops, coffee's flavor quality is a core factor determining its product value. The formation of coffee flavor is influenced by multiple factors, including variety, origin, and processing technology. Among these, fermentation, as a key step in the initial processing of coffee, directly regulates the generation and transformation of flavor compounds and plays a decisive role in the sensory characteristics of the final product.

[0003] Traditional coffee fermentation primarily relies on natural fermentation methods such as sun-drying, washing, and honey processing, which depend heavily on the natural fermentation process using wild microbial communities in the environment. However, natural fermentation has significant drawbacks: the fermentation process is easily affected by external factors such as climate and environmental hygiene; the microbial community structure is unstable, leading to large differences in flavor between batches of coffee beans, inconsistent flavor across batches, and unstable quality. Furthermore, under-fermentation or over-fermentation can easily produce undesirable flavors such as sourness and off-flavors, severely restricting the stable mass production of high-quality coffee. In addition, while single-strain fermentation technology can partially improve flavor, its metabolic products are limited, making it difficult to achieve a rich flavor profile and a smooth mouthfeel.

[0004] To overcome the drawbacks of natural fermentation, targeted inoculation fermentation technology, which involves adding specific functional microorganisms to regulate the fermentation process, has gradually become a research hotspot in recent years. Existing technologies include methods that use yeast strains alone to ferment coffee, aiming to enhance the fruity and floral aromas of coffee by producing ethanol and esters through yeast metabolism. However, the metabolic pathways of a single strain are limited, making it impossible to simultaneously achieve multiple goals such as aroma enhancement, mouthfeel thickening, and improved flavor harmony, thus failing to create a rich and complex flavor profile. Other technologies disclose the staged fermentation of lactic acid bacteria and yeast to prepare tangerine peel-flavored coffee. For example, patent document CN115868566A discloses a method for preparing tangerine peel-flavored coffee using staged fermentation of lactic acid bacteria and yeast, attempting to integrate hesperidin from tangerine peel into coffee beans through biotransformation. However, the method used in this patent leads to the loss of a large amount of bioactive substances in the coffee. Furthermore, the fermentation process reduces the coffee's body and results in insufficient flavor harmony. In addition, this patent does not address the subsequent roasting method for green coffee beans. In summary, there are still many areas for improvement in fermented tangerine peel-flavored coffee.

[0005] Meanwhile, as consumers' demand for health and flavor diversity increases, the combination of tangerine peel (Chenpi) and other food-medicine homologous ingredients with coffee is becoming a new direction. Tangerine peel is rich in functional components such as limonene and hesperidin, possessing both a unique citrus aroma and physiological activities such as anti-oxidation and regulation of intestinal flora. However, existing tangerine peel coffees mostly use physical mixing methods (such as blending and grinding, co-infusion), which have shortcomings such as insufficient flavor integration, low bioavailability of functional components, and poor flavor stability, failing to achieve a deep synergy between the base flavor of coffee and the flavor of tangerine peel.

[0006] Therefore, there is an urgent need for a new technology that can integrate specific microbial synergistic fermentation with natural raw material bioprocessing to achieve a deep fusion of tangerine peel and coffee flavor, thereby enhancing the flavor quality and functional value of coffee. This is of great significance for the quality upgrade of the coffee industry. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a strain of Leuconostoc mesenteroides with bitterness-reducing and sweetness-enhancing effects. Leuconostoc dextranicum )LB01 and a brewing yeast with flavor-enhancing effects ( Saccharomyces cerevisiae )PB01, and the fermentation and roasting method of tangerine peel flavored coffee beans using the above strain, the coffee bean products obtained therefrom, and their applications.

[0008] This invention first provides Leuconostoc mesenchyme ( Leuconostoc dextranicum )LB01 was deposited at the China Center for Type Culture Collection on August 4, 2025, with accession number CCTCC No: M20251759.

[0009] It also provides brewing yeast ( Saccharomyces cerevisiae PB01 was deposited at the China Center for Type Culture Collection on August 4, 2025, with accession number CCTCC No: M20251760.

[0010] The present invention also provides a microbial fermentation agent comprising Leuconostoc mesenteroides (Leuconostoc mesenteroides). Leuconostoc dextranicum )LB01 and brewer's yeast ( Saccharomyces cerevisiae )PB01.

[0011] Another object of the present invention is to provide a fermentation and roasting method for tangerine peel flavored coffee beans, the fermentation and roasting method comprising the following steps: S1. Pre-processing stage: Mix green Catimor coffee beans with dried tangerine peel powder ground to 80-100 mesh, sterilize and cool to 20-30℃; sterilize orange peel powder ground to 80-100 mesh by ultraviolet irradiation and set aside. S2, Bacterial Liquid Preparation Stage: Leuconostoc des extranicum LB01, which has undergone three generations of activation, and Saccharomyces cerevisiae (…) are prepared. Saccharomyces cerevisiae After centrifugation, the bacterial concentration of PB01 was adjusted to 5 × 10⁻⁶ cells with sterile physiological saline. 7 CFU / mL - 1×10 8 CFU / mL, take 0.5ml of Leuconostoc mesenteroides LB01 bacterial solution and 1mL of Saccharomyces cerevisiae PB01 bacterial solution respectively, and add them to 200mL of sterile water to prepare fermentation solution; S3. Fermentation stage: Mix the green Catimor coffee beans, dried tangerine peel powder, orange peel powder and fermentation liquid evenly, and ferment at 30-35℃ and 65-75% humidity for 20-28 hours, shaking once every 6 hours during the process. S4. Post-processing stage: Separate the fermented coffee beans from the powder, dry the coffee beans at 50-55℃ to a moisture content of 10%-12%, and then roast them in stages to obtain tangerine peel flavored coffee beans.

[0012] A further technical solution of the present invention is as follows: In step S4, the segmented roasting uses an HB-M6-SE direct-fire semi-hot air roaster. After each segment of roasting, the coffee beans are removed from the roaster and cooled to 20-30°C before proceeding to the next segment. This process is repeated until the coffee beans are removed from the roaster. The segmented roasting includes the following steps: S41. Place the fermented coffee beans in the roaster, adjust the heat to 1.5-1.8kW, roast the beans to 149.5℃, and then remove them from the roaster to cool. S42. Place the cooled coffee beans in a roaster with the heat set to 0 and roast to 149.5°C, then remove from the roaster and let cool. S43. After roasting, place the cooled coffee beans in a roasting oven with the heat set to 0 and the bean surface temperature set to 150℃, then remove from the oven and cool. S44. Place the cooled coffee beans in a roaster with the heat adjusted to 2.8-3.0 kW until the coffee beans enter the first crack. Immediately adjust the damper from level 4 to level 9-10, keep the heat unchanged, and roast for another 25-30 seconds after the first crack to bring the coffee bean temperature to 195±10℃. Remove the beans from the roaster to obtain roasted tangerine peel flavored coffee beans.

[0013] A further technical solution of the present invention is: the sterilization in step S1 is high-pressure steam sterilization at 110°C for 10 minutes; The culture medium for activating the strains in step S1 is as follows: MRS medium is used for Leuconostoc mesenteroides LB01, and PDB medium is used for Saccharomyces cerevisiae PB01.

[0014] A further technical solution of the present invention is as follows: the MRS medium (1L) consists of: 20.0g glucose, 10.0g peptone, 8.0g beef extract, 4.0g yeast extract, 2.0g dipotassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 5.0g sodium acetate, 1mL Tween, 0.2g magnesium sulfate, 0.04g manganese sulfate, distilled water added to 1L, pH 7.0, and 20.0g agar added to form a solid medium; The PDB medium (1L) is made by adding 12g of potato extract powder, 20g of glucose, distilled water to 1L, and 20.0g of agar to form a solid medium.

[0015] Another object of the present invention is to provide a tangerine peel flavored coffee bean, prepared by the aforementioned fermentation and roasting method.

[0016] A further technical solution of the present invention is that it has at least one of the following flavors: tangerine peel aroma, citrus aroma, fruit aroma, wood aroma, nut aroma and chocolate aroma, and the bitterness is significantly reduced, while having both fruit acidity and sweet aftertaste.

[0017] This invention provides Leuconostoc mesenteroides ( Leuconostoc dextranicum )LB01 and brewer's yeast ( Saccharomyces cerevisiae The combined application of PB01 in the fermentation of green coffee beans; or Leuconostoc mesenteroides ( Leuconostoc dextranicum )LB01 and brewer's yeast ( Saccharomyces cerevisiae The combined application of PB01 in improving coffee flavor and reducing coffee bitterness.

[0018] The beneficial effects of this invention are as follows: By synergistically combining the sweet-producing characteristics of *Leuconostoc mesenteroides* LB01 with the aroma-enhancing function of *Saccharomyces cerevisiae* PB01, and integrating the flavor compounds of dried tangerine peel and orange peel, coffee beans acquire a complex flavor profile including dried tangerine peel aroma, citrus aroma, and nutty aroma. Bitterness is significantly reduced, fruit acidity is mellow, and the aftertaste is long-lasting. The flavor profile far surpasses that of single-strain fermentation or physically mixed products. Using a directional inoculation compound fermenting agent instead of natural or single-strain fermentation ensures clear and controllable fermentation parameters, stable microbial community structure, minimal flavor differences between batches of coffee beans, and high reproducibility. This solves the core problem of unstable quality in traditional fermentation, making the fermentation process controllable and stable. The fermentation cycle is only 20-28 days. The fermentation time is significantly shorter than some traditional fermentation processes, and the pre-treatment, fermentation, and roasting steps are simple, requiring no complex equipment. This makes it suitable for standardized and industrialized production, reducing production costs and technical barriers, improving fermentation efficiency, simplifying the fermentation process, and innovating roasting methods. Through microbial co-fermentation, the flavor substances of tangerine peel and the base flavor of coffee are biotransformed and deeply integrated, avoiding flavor separation problems caused by physical mixing. The flavor remains harmonious and unified from high to medium and low temperatures, improving the degree of flavor integration. At the same time, the segmented roasting method, through the "roasting-cooling" cyclic heat treatment, achieves the staged control and layer-by-layer integration of chemical reactions such as dehydration, heat penetration, caramelization, and flavor development within the coffee beans. Attached Figure Description

[0019] Figure 1 This is a flowchart of the fermentation method for tangerine peel flavored coffee beans provided in an embodiment of the present invention.

[0020] Figure 2 The fresh fruit sample provided in this embodiment of the invention is diluted 10 times. 4 Illustration of colony count on plate.

[0021] Figure 3 This is a schematic diagram showing the droplet OD value measured by MISS cell in MRS culture medium for sorting fresh fruit samples according to an embodiment of the present invention.

[0022] Figure 4 This is a heatmap of OD values ​​from MRS fresh fruit sample output plates 1-4 provided in this embodiment of the invention.

[0023] Figure 5 This is a schematic diagram showing the droplet OD value measured by the MISS cell in the PDB culture medium for sorting fresh fruit samples according to an embodiment of the present invention.

[0024] Figure 6 This is a heat map of 10-15 OD values ​​from the PDB fresh fruit sample output plate provided in this embodiment of the invention.

[0025] Figure 7This is the filtering effect provided by the embodiments of the present invention. Figure 1 .

[0026] Figure 8 This is the filtering effect provided by the embodiments of the present invention. Figure 2 .

[0027] Figure 9 This is the filtering effect provided by the embodiments of the present invention. Figure 3 .

[0028] Figure 10 This is a schematic diagram of the three stages of fermentation preparation (pretreatment), fermentation, and post-fermentation provided in the embodiments of the present invention.

[0029] Figure 11 This is a functional schematic diagram of the HB-M6-SE direct-fire semi-hot air baking machine provided in an embodiment of the present invention.

[0030] Figure 12 This is a schematic diagram of the morphology of dried coffee beans after fermentation (before roasting in the first stage) provided in an embodiment of the present invention.

[0031] Figure 13 This is a schematic diagram of the morphology of roasted coffee beans provided in the first embodiment of the present invention.

[0032] Figure 14 This is a schematic diagram of the second segment of the coffee bean morphology after roasting, provided in an embodiment of the present invention.

[0033] Figure 15 This is a schematic diagram of the third segment of the coffee bean morphology after roasting, provided in an embodiment of the present invention.

[0034] Figure 16 This is a schematic diagram of the fourth segment of the coffee bean morphology (finished product morphology) provided in an embodiment of the present invention. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Those skilled in the art can make modifications or improvements without departing from the spirit and scope of the present invention.

[0036] Specifically, the present invention provides two functional microbial strains with synergistic effects, and a fermentation agent containing the strains, thereby providing a fermentation method for tangerine peel flavored coffee beans that is process-controllable and highly reproducible, ultimately obtaining tangerine peel flavored coffee beans with unique flavor, stable quality, and health benefits.

[0037] Two strains with clearly defined functions are provided: Leuconostoc mesenteroides LB01 and Saccharomyces cerevisiae PB01. Their synergistic effect can achieve multiple benefits such as enhancing aroma, reducing bitterness, increasing sweetness, and improving flavor harmony. A microbial fermentation agent based on the above-mentioned strain is provided to provide a stable and controllable functional microbial community for coffee fermentation.

[0038] A fermentation method for tangerine peel flavored coffee beans is provided to achieve a deep fusion of tangerine peel flavor and coffee base flavor, solving the flavor separation problem caused by physical mixing; We offer a tangerine peel-flavored coffee bean with a rich flavor, low bitterness, and a long-lasting sweet aftertaste, meeting consumers' demand for novel, healthy, and complex flavored beverages.

[0039] This study provides the combined application of Leuconostoc mesenteroides LB01 and Saccharomyces cerevisiae PB01 in the fermentation of green coffee beans, as well as their combined application in improving coffee flavor and reducing bitterness.

[0040] Strain identification and preservation 1. Leuconostoc mesentery LB01 Classification and nomenclature: Leuconostoc mesenteroides ( Leuconostoc dextranicum )LB01; Preservation information: Deposited on August 4, 2025 at the China Center for Type Culture Collection, accession number CCTCC No: M20251759; deposit address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Strains were obtained from fresh Catimor coffee cherries from Ziyuan Coffee Factory in Simao District, Pu'er City, through isolation, purification, and screening. Functional characteristics: It has the ability to produce sweetness, metabolize and produce aromatic compounds such as creamy and nutty flavors, gently lower the pH value of the fermentation system, inhibit the growth of miscellaneous bacteria, and improve the roundness of the coffee taste.

[0041] 2. Saccharomyces cerevisiae PB01 Classification and naming: Brewing yeast ( Saccharomyces cerevisiae )PB01 Preservation information: Deposited on August 4, 2025 at the China Center for Type Culture Collection, accession number CCTCCNo: M20251760, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0042] Strain source: Similar to Leuconostoc mesenteroides ( Leuconostoc dextranicum Both LB01 and LB01 are derived from fresh Catim coffee cherries from Ziyuan Coffee Factory in Simao District, Pu'er City, and were obtained through separation, purification, and screening.

[0043] Functional characteristics: It has a strong metabolic capacity, efficiently converting sugars into flavor compounds such as ethanol, esters, and higher alcohols. Its esters, such as ethyl acetate and ethyl hexanoate, impart pleasant aromas like fruit and wine to coffee. It also interacts with Leuconostoc mesenteroides (…). Leuconostoc dextranicum )LB01 synergistically enriches the flavor profile.

[0044] Microbial fermentation agent A microbial fermentation agent containing Leuconostoc mesenteroides ( Leuconostoc dextranicum )LB01 and brewer's yeast ( Saccharomyces cerevisiae )PB01. In this microbial fermentation agent, the ratio of the two strains is based on the principle of optimal fermentation effect, and Leuconostoc mesenteroides is preferred. Leuconostoc dextranicum )LB01 and brewer's yeast ( Saccharomyces cerevisiae The cell volume ratio of PB01 is 5:1, and its corresponding bacterial liquid volume ratio is 5:1.

[0045] The present invention provides a fermentation method for tangerine peel flavored coffee beans, which includes four stages: pretreatment, bacterial culture preparation, fermentation, and posttreatment, detailed below: Pre-processing stage: Mix green Catimor coffee beans with dried tangerine peel powder ground to 80-100 mesh, sterilize, and cool to 20-30℃; sterilize orange peel powder ground to 80-100 mesh by UV irradiation and set aside; select 500g of green Catimor coffee beans, 30g of dried tangerine peel from Dongjia, Xinhui, Guangzhou (aged 3-5 years), and 30g of orange peel from Linyi, Shandong; grind the dried tangerine peel to 80-100 mesh in a grinder, mix with the green Catimor coffee beans, put into a fermentation tank, seal with sealing film, and sterilize with high-pressure steam at 110℃ for 10 minutes. After sterilization, place in a clean bench and cool naturally to 20-30℃; grind the orange peel to 80-100 mesh, place in a clean bench and sterilize by UV irradiation for 30 minutes and set aside.

[0046] Bacterial culture preparation stage: Enterocytozoa that has undergone three generations of activation (… Leuconostoc dextranicum )LB01 and brewer's yeast ( Saccharomyces cerevisiae After centrifugation, the bacterial concentration of PB01 was adjusted to 5 × 10⁻⁶ cells with sterile physiological saline. 7 CFU / mL - 1×10 8 CFU / mL, 5ml of *Leuconostoc mesenteroides* LB01 bacterial suspension and 1ml of *Saccharomyces cerevisiae* PB01 bacterial suspension were respectively added to 200mL of sterile water to prepare fermentation broth; the preserved *Leuconostoc mesenteroides* ( Leuconostoc dextranicum LB01 was inoculated into MRS medium, and Saccharomyces cerevisiae ( Saccharomyces cerevisiae PB01 was inoculated into PDB medium and activated for three generations at 32℃ to obtain stable bacterial cultures. The activated cultures of both strains were centrifuged at 10,000 rpm for 10 minutes, and the supernatant was discarded. The cell concentration of both strains was adjusted to 5 × 10⁻⁶ cells / mL with sterile physiological saline. 7 CFU / mL - 1×10 8CFU / mL; Take 5mL of Leuconostoc mesenteroides LB01 bacterial solution and 1mL of Saccharomyces cerevisiae PB01 bacterial solution, add them to 200mL of sterile water, shake well, and prepare a compound fermentation solution.

[0047] Fermentation stage: Mix Catimor green coffee beans, dried tangerine peel powder, orange peel powder, and fermentation starter culture evenly, and ferment at 30-35℃ and 65-75% humidity for 20-28 hours, shaking once every 6 hours during the process; add the pre-treated orange peel powder to the fermentation tank containing coffee beans and dried tangerine peel powder, then pour in the prepared fermentation starter culture, and shake thoroughly to ensure even contact between the coffee beans, dried tangerine peel powder, orange peel powder, and fermentation starter culture; place the fermentation tank in a climate simulation incubator and ferment at 30-35℃ and 65-75% humidity for 20-28 hours, shaking once every 6 hours during the fermentation process, for a total of 4 times, to ensure uniform fermentation; fermentation temperature 32℃, humidity 70%, fermentation time 24 hours.

[0048] Post-processing stage: Separate the fermented coffee beans from the powder. Dry the coffee beans at 50-55℃ until the moisture content is 10%-12%, then roast them to obtain tangerine peel flavored coffee beans. After fermentation, separate the coffee beans from the tangerine peel powder and orange peel powder, and collect the fermented coffee beans. Spread the separated coffee beans on a roasting rack and place them in a 50-55℃ oven to dry until the moisture content of the coffee beans drops to 10%-12%. Use an HB-M6-SE type direct-fire semi-hot air roaster to carry out a segmented roasting process of "roasting-cooling-re-roasting". After each roasting stage, the coffee beans need to be removed from the oven and actively cooled to room temperature of 20-30℃ before being put back in according to the parameters of the next stage. First stage: Roast at an initial heat of 1.5-1.8 kW until the surface temperature of the beans reaches 149.5℃, then remove them from the oven and cool them to 20-30℃. At this time, the moisture content of the beans is about 9.3%. The second stage: Before adding the beans, adjust the heat to 0 kW and use residual heat to roast them to a surface temperature of 149.5°C. Remove them from the oven and cool them to 20-30°C, at which point the moisture content will be reduced to approximately 8.3%. The third stage: Continue roasting at 0 kW, raising the surface temperature to 150°C before removing them from the oven. After cooling, the moisture content will be approximately 7.8%. The fourth stage: Use a stronger heat of 2.8-3.0 kW for roasting. When the coffee beans experience the first crack, immediately adjust the air damper from level 4 to level 9-10, while maintaining the same heat. After the first crack, continue roasting for 25-30 seconds, until the surface temperature of the beans reaches approximately 195°C, at which point they are finally removed from the oven, completing the roasting process. The core characteristic of this roasting method is that after each stage, the beans must be removed from the oven and cooled to 20-30°C before the conditions are reset for the next stage.

[0049] A type of tangerine peel flavored coffee bean is prepared by the above-mentioned fermentation and roasting method. The tangerine peel flavored coffee bean has at least one or more complex flavors among tangerine peel aroma, citrus aroma, fruit aroma, woody aroma, nutty aroma and chocolate aroma. The inherent bitterness of coffee is significantly reduced, and it has a mellow fruit acidity and a long-lasting sweetness. The flavor is rich, harmonious and unified, and the tangerine peel flavor and citrus notes can be perceived from high temperature to medium and low temperature drinking.

[0050] Application of strains Leuconostoc mesenteroides ( Leuconostoc dextranicum )LB01 and brewer's yeast ( Saccharomyces cerevisiae )The combined application of PB01 in the fermentation of green coffee beans; or the combined application in improving coffee flavor, reducing coffee bitterness, and enhancing coffee aftertaste.

[0051] Experimental culture medium and reagent formulations MRS medium (1L): 20.0g glucose, 10.0g peptone, 8.0g beef extract, 4.0g yeast extract, 2.0g dipotassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 5.0g sodium acetate, 1mL Tween, 0.2g magnesium sulfate, 0.04g manganese sulfate, add distilled water to 1L, adjust pH to 7.0; add 20.0g agar to make a solid medium.

[0052] PDB medium (1L): 12g potato extract powder, 20g glucose, add distilled water to 1L for mold culture; add 20.0g agar for solid culture medium.

[0053] Experimental equipment and related instruments Microbial screening equipment: MISS cell high-throughput single-cell microbial screening equipment; Fermentation equipment: YTQP-300B artificial climate chamber; Roasting equipment: HB-M6-SE direct-fire semi-hot air roaster.

[0054] Isolation, purification and identification of strains Strains Isolation and Purification The present invention contains Leuconostoc mesenteroides (Leuconostoc mesenteroides). Leuconostoc dextranicum )LB01 and brewer's yeast ( Saccharomyces cerevisiae Screening of PB01: The sample was obtained by isolation and purification from fresh Catimor coffee cherries collected from Ziyuan Coffee Factory in Simao District, Pu'er City, Yunnan Province, China, as detailed below: Sample pretreatment: Mix the sample bag thoroughly, weigh out 8g of fresh coffee fruit sample and put it into a 50mL centrifuge tube to form a sample tube, add 40mL of sterile 1×PBS, crush the pulp and vortex for 5 minutes, shake at 28℃ and 180rpm for 15 minutes, and then sonicate for 10 minutes to prepare a bacterial suspension; transfer the 50mL sample tube to a clean bench and let the bacterial suspension stand at room temperature for 10 minutes to settle, take the supernatant and filter it through a 40μm sterile cell filter, and collect the filtered bacterial suspension.

[0055] Plate counting: The bacterial suspension obtained after sample pretreatment and filtration was serially diluted 10⁻¹⁰ with sterile 1×PBS. 1 10², 10³, 10 4 150 μL of each sample was spread onto MRS and PDB plates for yeast isolation. MRS plates were incubated at 36°C for 48 hours, and PDB plates at 28°C for 48 hours. Colony growth was recorded, and the bacterial concentration of the suspension was calculated based on the colony count on the plates. The bacterial concentration for fresh fruit samples was 8.36–8.66 × 10⁻⁶. 6 cfu / mL, such as Figure 2 As shown.

[0056] MISS cell isolation and culture: Fresh fruit samples were diluted 10 times by machine based on plate results. 4 times (i.e., sample A) and 10 5 The bacterial suspension (i.e., sample B) was prepared into MRS injection bottles, generating a consumable cartridge containing 3750 droplets and a control of 1250 MRS empty droplets. After offline incubation at 36°C for 22 hours, OD was performed. 600 Droplets were sorted, with 48 droplets of sample A having an OD value greater than 2.1 and 243 droplets of sample B having an OD value greater than 0.5, for a total of 291 droplets. These droplets were then inoculated into 96-well microplates and 100 μL of MRS medium was added for incubation at 36°C. The 10-well microplates were then diluted... 4 times (sample A) and 10 5 A bacterial suspension of sample B was prepared into PDB injection bottles, each containing 2500 droplets. After offline incubation at 28°C for 26 hours, OD was performed. 600 Droplets were sorted and collected: 384 droplets with an OD value of 0.8 or higher from sample A and 119 droplets with an OD value of 1.2 or higher from sample B, for a total of 503 droplets. These droplets were then inoculated into 96-well microplates and 100 μL of PDB medium was added for incubation at 28°C.

[0057] Purification and preservation: The strains after sorting and expansion were inoculated into MRS solid medium and PDA solid medium by streak method. After cultivation, single colonies were picked and purified three times to obtain pure strains. The strains were preserved in a mixture of glycerol (3): water (1) in an ultra-low temperature freezer at -80℃.

[0058] Strain identification Bacterial molecular identification (Leuconostoc mesenteroides LB01): Using the strain's genomic DNA as a template, 16S rDNA gene amplification was performed using primers 27F (5′-AGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR reaction conditions were: initial denaturation at 98℃ for 2 min, followed by 35 amplification cycles, including denaturation at 98℃ for 10 s, annealing at 56℃ for 10 s, extension at 72℃ for 10 s, and final extension at 72℃ for 5 min. The amplified products were detected by agarose gel electrophoresis and then sequenced. The sequenced sequence was compared with a nucleic acid sequence database using BLAST, and the strain was identified as Leuconostoc mesenteroides. Leuconostoc dextranicum ), named LB01.

[0059] Molecular identification of fungi (Saccharomyces cerevisiae PB01): DNA was extracted from the strain using a novel plant genomic DNA extraction kit. PCR amplification was performed using primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The PCR reaction system consisted of 45 μL Taq PCR Master Mix, 2.0 μL total DNA template, 2.0 μL ITS1, 2.0 μL ITS4, and 4.0 μL ddH2O, for a total volume of 25 μL. The PCR conditions were: 98℃ pre-denaturation for 2 min, 35 cycles (including denaturation at 94℃ for 10 s, annealing at 54℃ for 10 s, extension at 72℃ for 10 s, and final extension at 72℃ for 5 min). The amplified products were detected by 1% agarose gel electrophoresis and then sequenced. The sequencing results were compared with the BLAST sequence in the NCBI database, and the strain was identified as Saccharomyces cerevisiae. Saccharomyces cerevisiae It was named PB01.

[0060] Leuconostoc mesenteroides ( Leuconostoc dextranicum LB01 and brewer's yeast ( Saccharomyces cerevisiae ) Screening of strains of PB01 1. Sorting fresh fruit samples using MRS culture medium Sample A diluted 10 4 Droplet generation disk 1, MRS blank control droplet generation disk 2, Sample B diluted 10 5 Droplet generating trays 3 and 4 were incubated at 36℃ for 22 hours. Afterwards, the droplet trays were sorted. For sample A, 48 droplets with an OD value greater than 2.1 were collected. No droplets were collected in the MRS control group. For sample B, 243 droplets with an OD value greater than 0.5 were collected. The OD values ​​of the droplets were measured using a MISS cell. Figure 3 As shown.

[0061] A total of 291 droplets were sorted from the MRS fresh fruit sample, and the output plate OD value heatmap was generated, as shown below. Figure 4 As shown.

[0062] 2. Sorting fresh fruit samples using PDB culture medium Sample A diluted 10 4 Consumable Kit 1 - Droplet Pan 2, Sample B Diluted 10 5 The droplet collection kit (1-4) was incubated at 28°C for 26 hours and then sorted. For sample A, 384 droplets with an OD value greater than 0.8 were collected; for sample B, 119 droplets with an OD value greater than 1.2 were collected. The OD values ​​of the droplets were measured using a MISS cell, as shown below. Figure 5 As shown.

[0063] A total of 503 droplets were sorted from the PDB fresh fruit sample, and the output plate OD heatmap was obtained, as shown below. Figure 6 As shown. The filtered result images are as follows. Figure 7 , 8 As shown in Figures 9 and 9.

[0064] The selected single-cell strains were streaked onto MRS and PDA solid media, respectively, in duplicate. One copy was used for PCR amplification, and the product was detected by agarose gel electrophoresis and then sequenced. The sequencing results were then compared with the BLAST sequence in the NCBI database. The other copy was used for preservation of the strain in a 3:1 mixture of glycerol and water at -80°C.

[0065] Sequence retrieval and comparison revealed a total of 59 microorganisms, including 10 bacteria, 48 yeasts, and 1 strain with a bimodal deletion that could not be matched.

[0066] After searching for strains LB01 and PB01, strain LB01 was found to be *Leuconostoc mesenteroides*. Leuconostoc dextranicum ), strain PB01 is a brewer's yeast ( Saccharomyces cerevisiae ).

[0067] The table below shows the strain of Leuconostoc mesenteroides (Leuconostoc mesenteroides). Leuconostoc dextranicum LB01 and brewer's yeast ( Saccharomyces cerevisiae The relevant sequence of PB01.

[0068] Core parameters of the compound fermentation process for tangerine peel flavored coffee A fermentation method for a tangerine peel flavored coffee, using Yunnan small-bean coffee beans. Katim Wash Commercial beans; the fermentation strains used were Leuconostum dextranicum LB01 and Saccharomyces cerevisiae PB01. As mentioned above, the Leuconostum dextranicum LB01 and Saccharomyces cerevisiae PB01 strains were preserved microbially (glycerol preservation at -80℃); the edible tangerine peel used were Dongjia tangerine peel from Xinhui, Guangzhou (3-5 years old) and tangerine peel from Linyi, Shandong.

[0069] like Figure 10 As shown, to achieve the above objectives, this invention provides a method for preparing tangerine peel flavored coffee beans based on the co-fermentation of Leuconostoc mesenteroides and Saccharomyces cerevisiae, characterized by the following steps: This invention provides a fermentation method for tangerine peel flavored coffee, which is achieved through three stages: pre-fermentation preparation (pretreatment), fermentation, and post-fermentation. Pretreatment stage: Pure strains of Leuconostoc dextranicum PB01 and Saccharomyces cerevisiae PB01, which were stored at -80℃, were placed in MRS medium and PDB medium respectively and activated for three generations at 32℃.

[0070] Choose 500g of green Catimor coffee beans, 30g of dried tangerine peel (3-5 years old) from Dongjia, Xinhui, Guangzhou, and 30g of dried tangerine peel from Linyi, Shandong.

[0071] Grind 30g of Guangzhou Xinhui Dongjia ring-branched tangerine peel (3-5 years old) in a grinder to 80-100 mesh, then put it into a fermentation tank containing 500g of Catimor coffee beans. Seal the tank with sealing film, then sterilize it in a high-pressure steam sterilizer at 110℃ for 10 minutes. After sterilization, place it in a clean bench to cool naturally to 20-30℃.

[0072] Grind 30g of Shandong Linyi orange shreds in a grinder to 80-100 mesh, then place them in a clean bench and irradiate with ultraviolet light for 30 minutes.

[0073] Fermentation stage: Activated third-generation *Leuconostoc dextranicum* LB01 (*L. dextranicum* PB01) and *Saccharomyces cerevisiae* PB01 (*S. cerevisiae* PB01) were centrifuged at 10,000 rpm for 10 min. After discarding the culture medium, sterile physiological saline was added to adjust the concentration to 5 × 10⁸ CFU / mL–1 × 10⁸ CFU / mL. After centrifugation, 0.5 mL of *Leuconostoc dextranicum* LB01 and 1 mL of *Saccharomyces cerevisiae* PB01 were each added to 200 mL of cooled sterile water and thoroughly mixed to prepare the fermentation broth for later use.

[0074] Pour 30g of Shandong Linyi orange peel powder from the pre-processing stage into the sterilized fermentation tank and mix thoroughly to ensure that the orange peel powder, orange peel powder and sterilized Catimor coffee beans are evenly integrated.

[0075] Pour 200mL of fermentation liquid into the fermentation tank, shake well, and ensure that the fermentation strain, green Catimor coffee beans, dried tangerine peel powder, and orange peel powder are evenly distributed in the fermentation tank.

[0076] The fermentation tank was then placed in a climate simulation incubator for fermentation. The fermentation temperature was set at 32℃, the humidity at 70%, and the fermentation time at 24 hours. The tank was shaken thoroughly every 6 hours for a total of 4 times.

[0077] Subsequent processing: After the fermentation time is over, separate the beans, tangerine peel powder and orange peel powder, leaving only the fermented tangerine peel coffee-flavored beans.

[0078] Spread the separated tangerine peel coffee beans on a roasting rack and place them in an oven at 50-55℃ to dry until the moisture content of the coffee beans is maintained between 10% and 12%.

[0079] Roasting: Use an HB direct-fire semi-hot air roaster. The specific process is as follows: First stage: Use 1.5-1.8kW power to roast the beans to approximately 149.5℃, then remove and cool (moisture content approximately 9.3%). Second stage: Before adding the beans, reduce the power to 0 and roast to 149.5℃, then remove and cool (moisture content approximately 8.3%). Third stage: Maintain the power at 0 and set the target surface temperature of the beans to 150℃, then remove and cool (moisture content approximately 7.8%). Fourth stage: Use 2.8-3.0kW high power for roasting. When the coffee beans enter the first crack, immediately increase the airflow from 4 to 9-10, keeping the power constant. After the first crack develops for another 25-30 seconds, remove the beans when the temperature reaches approximately 195±10℃. The roaster should be as follows: Figure 11 As shown.

[0080] Example 1: Isolation, purification, and identification of strains Sample collection: Ripe Catimor coffee cherries were collected from Ziyuan Coffee Factory in Simao District, Pu'er City. Rotten and damaged cherries were removed and the samples were set aside.

[0081] Sample pretreatment: Weigh 8g of fresh coffee cherries into a sterile 50mL centrifuge tube, add 40mL of sterile 1×PBS buffer, and crush the pulp with a sterile grinder; place the centrifuge tube on a vortex mixer and vortex for 5 minutes, then place it on a shaker and shake at 28℃ and 180rpm for 15 minutes; remove it and place it in an ultrasonicator and sonicate for 10 minutes to prepare the original bacterial suspension; let the original bacterial suspension stand at room temperature for 10 minutes to allow the fruit residue and other particles to settle, take the supernatant and filter it through a 40μm sterile cell filter, and collect the filtered bacterial suspension.

[0082] Serial dilution and plate plating: The filtered bacterial suspension was plated with sterile 1×PBS for 10 minutes. 1 10², 10³, 10 4 Serial dilutions were performed. 150 μL of bacterial suspension from each dilution was evenly spread on MRS plates (for lactic acid bacteria isolation) and PDB plates (for yeast isolation), with three replicates for each dilution. MRS plates were incubated at 36°C for 48 hours, and PDB plates were incubated at 28°C for 48 hours.

[0083] Plate counting: After incubation, select plates with colony counts between 30 and 300 for counting. Calculate the concentration of the original bacterial suspension as 8.36–8.66 × 10⁻⁶. 6 cfu / mL.

[0084] MISS cell high-throughput separation: MRS culture medium sorting: Take 10% diluted... 4 times (sample A) and 10 5 The bacterial suspension (sample B) was divided into MRS injection bottles and a consumable cartridge was generated using the MISS cell high-throughput single-cell screening device. A total of 3750 droplets were generated for samples A and B, with 1250 empty MRS droplets used as a control. After offline incubation at 36°C for 22 hours, the consumable cartridges were analyzed by OD... 600 The samples were sorted and 48 droplets with an AOD value ≥ 2.1 and 243 droplets with a BOD value ≥ 0.5 were collected. The collected droplets were inoculated into a 96-well microplate, and 100 μL of MRS medium was added to each well. The plates were then incubated at 36°C for 24 hours.

[0085] PDB culture medium sorting: Take 10% diluted PDB medium. 4 times (sample A) and 10 5 The bacterial suspension (sample B) was divided into PDB injection bottles and a droplet generating plate was created using a MISS cell device, generating 2500 droplets for each concentration. After offline incubation at 28°C for 26 hours, the droplet was analyzed by OD... 600 The samples were sorted and 384 droplets with an AOD value ≥ 0.8 and 119 droplets with a BOD value ≥ 1.2 were collected and inoculated into 96-well microplates. 100 μL of PDB medium was added to each well and the plates were incubated at 28°C for 24 hours.

[0086] Strain purification: Select wells with high OD values ​​from the expanded 96-well microplates and inoculate them into MRS solid medium and PDA solid medium, respectively. Purify the culture by streak plating and repeat the purification process three times to obtain morphologically uniform single colonies.

[0087] Strain identification: 16S rDNA identification (bacteria): Single colonies were picked from MRS medium, genomic DNA was extracted, and PCR amplification was performed using primers 27F and 1492R. The amplified products were sequenced and compared with the NCBI database to identify one strain as *Leuconostoc mesenteroides*. Leuconostoc dextranicum ), named LB01.

[0088] ITS sequence identification (fungi): Single colonies were picked from PDA medium, genomic DNA was extracted, and PCR amplification was performed using primers ITS1 and ITS4. The amplified products were sequenced and compared with the NCBI database to identify one strain as *Saccharomyces cerevisiae*. Saccharomyces cerevisiae It was named PB01.

[0089] Strain preservation: The identified Leuconostoc mesenteroides LB01 and Saccharomyces cerevisiae PB01 were inoculated into their respective liquid culture media and cultured to the logarithmic developmental stage. They were then mixed with 30% sterile glycerol at a 1:1 volume ratio and stored in an ultra-low temperature freezer at -80℃. At the same time, the two strains were sent to the China Center for Type Culture Collection for preservation, with accession numbers CCTCC No: M20251759 (Leuconostoc mesenteroides LB01) and CCTCC No: M20251760 (Saccharomyces cerevisiae PB01), respectively, and the preservation date was August 4, 2025.

[0090] Example 2: Preparation of Tangerine Peel Flavored Coffee Beans Preprocessing: Select 500g of green Catimor coffee beans and remove impurities and damaged beans; select 30g of aged tangerine peel from Dongjia, Xinhui, Guangzhou, which has been stored for 3 years, grind it to 80 mesh, mix it with the green coffee beans, put it into a 5L fermentation tank, seal it with sealing film, and sterilize it with high-pressure steam at 110℃ for 10 minutes. After sterilization, place it on a clean bench to cool to 25℃.

[0091] Select 30g of shredded oranges from Linyi, Shandong, grind them to 80 mesh, and sterilize them by irradiating them with ultraviolet light for 30 minutes in a clean bench.

[0092] Preparation of bacterial culture: Strain activation: Leuconostoc mesenteroides LB01 and Saccharomyces cerevisiae PB01 were taken from the strains stored at -80℃ and inoculated into MRS liquid medium and PDB liquid medium, respectively. They were cultured at 32℃ with shaking for 24 hours to complete the first generation activation. The strains were then transferred to fresh medium at a 1% inoculum and the activation was repeated for two more generations to obtain the third generation activated strains.

[0093] Centrifugation and concentration adjustment: The cultures of the third-generation activated strain were placed in centrifuge tubes and centrifuged at 10,000 rpm for 10 minutes, discarding the supernatant. The bacterial cells were resuspended in sterile physiological saline, and the concentration of *Leuconostoc mesenteroides* LB01 was adjusted to 8 × 10⁻⁶. 7CFU / mL, adjust the concentration of Saccharomyces cerevisiae PB01 to 8×10⁻⁶. 7 CFU / mL.

[0094] Preparation of fermentation broth: Take 5 mL of Leuconostoc mesenteroides LB01 broth and 1 mL of Saccharomyces cerevisiae PB01 broth, add them to 200 mL of sterile water, shake well to prepare a compound fermentation broth.

[0095] Fermentation: Add the sterilized orange peel powder to the fermentation tank, pour in the compound fermentation liquid, tighten the lid and shake well; place the fermentation tank in a climate simulation incubator, set the temperature to 32℃ and humidity to 70%, and ferment for 24 hours. During this period, take out the fermentation tank and shake it well at 6h, 12h, 18h and 24h of fermentation, and shake it well each time for 1 minute.

[0096] Post-processing: Separation: After fermentation, the coffee beans are separated from the dried tangerine peel powder and orange peel powder through a sterile sieve, and the coffee beans are collected.

[0097] Drying: The coffee beans were evenly spread on the roasting rack and placed in a 52°C oven to dry for 12 hours. The moisture content of the coffee beans was measured to be 11%.

[0098] Roasting: Uses the HB-M6-SE type direct-fire semi-hot air roaster, such as... Figure 11 As shown, segmented baking is performed, and the detailed process and key technical points are shown in the table below; The first stage (initial dehydration and preheating): Starting with a medium-low heat of 1.5-1.8 kW, and maintaining a drum temperature of 210-215℃ and a hot air temperature of 210-215℃, steadily raise the surface temperature of the coffee beans to 180-185℃. The core objective of this stage is to ensure the bean core fully absorbs water, preheats evenly, and removes a large amount of residual moisture. When the bean surface temperature reaches 149.5℃ (±0.5℃), immediately remove the beans from the heat source and cool them to 20-30℃. At this point, the internal moisture content of the beans is approximately 9.3%, and the bean structure has opened up due to thermal expansion and contraction, preparing for the next stage of heat penetration.

[0099] The second stage (heat penetration and Maynard reaction preparation): The key operation is to pre-adjust the heat to 0 before adding the beans, that is, to use the residual heat from the drum (230-235℃) and hot air (220-230℃) for "zero-heat roasting". This avoids scorching the bean surface and promotes the slow and deep transfer of heat to the bean core. When the bean surface temperature rises again to 149.5℃, the beans are removed from the oven and cooled to a moisture content of about 8.3%. At this stage, the beans are further dried, and flavor precursors begin to form.

[0100] The third stage (flavor compound accumulation and early caramelization): Continuing with the zero-heat mode, the target surface temperature of the beans is explicitly set at 150℃, while the ambient temperature is slightly lowered (drum 210-215℃, hot air 205-210℃) to create a mild thermal environment. At this stage, the chemical reactions within the beans become more complex, and sugars begin to transform. After cooling, the moisture content is approximately 7.8%, the bean color is significantly darker, and the structure is more brittle, indicating the potential for flavor development.

[0101] The fourth stage (first crack and development stage): Switch to high heat (2.8-3.0kW) for a final burst, with the ambient temperature reaching its maximum (235-240℃ in the drum). The most critical operational point occurs at the onset of the first crack: the air damper must be immediately increased from 4 to 9-10. This is to quickly remove smoke and residual moisture, preventing astringency and clearly capturing the sound of the first crack. The heat must remain stable during this period to ensure sufficient heat supply. After the first crack, continue roasting for 25-30 seconds to allow the flavors to fully develop. Finally, remove the beans from the roaster when the surface temperature reaches approximately 195℃, completing the entire roasting process.

[0102] This process achieves precise "segmented cooling" control, enabling layered control over dehydration rate, heat penetration depth, and flavor development rhythm, making it particularly suitable for roasting coffee beans that demand high-sensory flavor and high cleanliness.

[0103] The roasting time is approximately 25-35 minutes, until the coffee beans turn a dark brown color. Figures 12 - 16 As shown, it has a distinct tangerine peel aroma. Stop roasting and cool to room temperature to obtain tangerine peel flavored coffee beans.

[0104] Example 3: Product Flavor Evaluation Ten professional coffee tasters were invited to conduct a sensory evaluation of the tangerine peel flavored coffee beans prepared in Example 2 according to the SCAA coffee flavor wheel standard. The evaluation results were compared with traditionally washed fermented Catimor coffee beans and Catimor coffee beans with physically mixed tangerine peel powder. The evaluation results are shown in the table below: Evaluation results show that the tangerine peel flavored coffee beans prepared by this invention are significantly superior to the comparative samples in terms of tangerine peel aroma, citrus aroma, bitterness control, aftertaste and flavor harmony, and have outstanding overall quality.

[0105] Industrial applicability The strain screening method of this invention is scientific and reliable, the fermentation process steps are clear and the parameters are controllable, and the required equipment are all commonly used equipment in the food processing field (such as high pressure steam sterilizers, climate simulation incubators, roasting machines, etc.), without the need for special customized equipment; the raw materials (Catim coffee beans, dried tangerine peel, orange peel) are widely available and the cost is controllable, making it suitable for large-scale industrial production.

[0106] The tangerine peel flavored coffee beans obtained by this invention have a unique flavor and stable quality. They are suitable for various brewing methods such as hand-drip and French press, and can be made into various end products such as coffee beans, coffee powder, and drip bags, meeting the demand of the coffee consumer market for novel, healthy, and complex flavor products, and have broad market application prospects.

[0107] This invention can also adjust the ratio of dried tangerine peel and orange peel, fermentation parameters, or baking degree according to different consumer needs, to develop a series of products with different flavor intensities and further expand application scenarios.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Leuconostoc mesenteroides ( Leuconostoc dextranicum )LB01, characterized in that, It was deposited at the China Center for Type Culture Collection on August 4, 2025, with accession number CCTCC No: M20251759.

2. Brewing yeast ( Saccharomyces cerevisiae PB01, characterized in that, It was deposited at the China Center for Type Culture Collection on August 4, 2025, with accession number CCTCC No: M20251760.

3. A microbial fermentation agent, characterized in that, Contains Leuconostoc mesenteroides as described in claim 1 ( Leuconostoc dextranicum )LB01 and the brewing yeast of claim 2 ( Saccharomyces cerevisiae )PB01.

4. A fermentation and roasting method for tangerine peel flavored coffee beans, characterized in that, The fermentation and baking method includes the following steps: S1. Pre-processing stage: Mix green Catimor coffee beans with dried tangerine peel powder ground to 80-100 mesh, sterilize and cool to 20-30℃; sterilize orange peel powder ground to 80-100 mesh by ultraviolet irradiation and set aside. S2, Bacterial Liquid Preparation Stage: Leuconostoc dextranicum LB01, which has undergone three generations of activation, and Saccharomyces cerevisiae (…) are prepared. Saccharomyces cerevisiae After centrifugation, the bacterial concentration of PB01 was adjusted to 5 × 10⁻⁶ cells with sterile physiological saline. 7 CFU / mL - 1×10 8 CFU / mL, take 0.5ml of Leuconostoc mesenteroides LB01 bacterial solution and 1mL of Saccharomyces cerevisiae PB01 bacterial solution respectively, and add them to 200mL of sterile water to prepare fermentation solution; S3. Fermentation stage: Mix the green Catimor coffee beans, dried tangerine peel powder, orange peel powder and fermentation liquid evenly, and ferment for 20-28 hours at a temperature of 30-35℃ and a humidity of 65-75%, shaking once every 6 hours during the process. S4. Post-processing stage: Separate the fermented coffee beans from the powder, dry the coffee beans at 50-55℃ to a moisture content of 10%-12%, and then roast them in stages to obtain tangerine peel flavored coffee beans.

5. The fermentation method according to claim 4, characterized in that, In step S4, the segmented roasting uses an HB-M6-SE direct-fire semi-hot air roaster. After each segment of roasting, the coffee beans are removed from the roaster and cooled to 20-30°C before proceeding to the next segment. This process is repeated until the coffee beans are removed from the roaster. The segmented roasting includes the following steps: S41. Place the fermented coffee beans in the roaster, adjust the heat to 1.5-1.8kW, roast the beans to 149.5℃, and then remove them from the roaster to cool. S42. Place the cooled coffee beans in a roaster with the heat set to 0 and roast to 149.5°C, then remove from the roaster and let cool. S43. After roasting, place the cooled coffee beans in a roasting oven with the heat set to 0 and the bean surface temperature set to 150℃, then remove from the oven and cool. S44. Place the cooled coffee beans in a roaster with the heat adjusted to 2.8-3.0 kW until the coffee beans enter the first crack. Immediately adjust the damper from level 4 to level 9-10, keep the heat unchanged, and roast for another 25-30 seconds after the first crack to bring the coffee bean temperature to 195±10℃. Remove the beans from the roaster to obtain roasted tangerine peel flavored coffee beans.

6. The fermentation method according to claim 4 or 5, characterized in that, The sterilization in step S1 is high-pressure steam sterilization at 110°C for 10 minutes. The culture medium for activating the strains in step S1 is as follows: MRS medium is used for Leuconostoc mesenteroides LB01, and PDB medium is used for Saccharomyces cerevisiae PB01.

7. The fermentation method according to claim 6, characterized in that, The MRS medium (1L) consisted of: 20.0g glucose, 10.0g peptone, 8.0g beef extract, 4.0g yeast extract, 2.0g dipotassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 5.0g sodium acetate, 1mL Tween, 0.2g magnesium sulfate, and 0.04g manganese sulfate, diluted with distilled water to 1L, pH 7.0, and 20.0g agar was added to form a solid medium. The PDB medium (1L) is made by adding 12g of potato extract powder, 20g of glucose, distilled water to 1L, and 20.0g of agar to form a solid medium.

8. A type of tangerine peel flavored coffee bean, characterized in that, It is prepared by the fermentation and baking method according to any one of claims 4-8.

9. The tangerine peel flavored coffee beans according to claim 9, characterized in that, It has at least one or more of the following flavors: tangerine peel, citrus, fruit, wood, nut, and chocolate. The bitterness is significantly reduced, and it has both fruit acidity and a sweet aftertaste.

10. The Enterobacter mesenchyme according to claim 1 ( Leuconostoc dextranicum )LB01 and the brewing yeast of claim 2 ( Saccharomyces cerevisiae The combined application of PB01 in the fermentation of green coffee beans; or The Enterobacterium leucocephala as described in claim 1 ( Leuconostoc dextranicum )LB01 and the brewing yeast of claim 2 ( Saccharomyces cerevisiae The combined application of PB01 in improving coffee flavor and reducing coffee bitterness.

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