A Kluyveromyces marxianus yeast, a method for fermenting rose-flavored coffee beans, and coffee beans

Abstract

The present invention discloses a Kluyveromyces marxianus yeast strain, a method for fermenting rose-flavored coffee beans and the coffee beans. The Kluyveromyces marxianus yeast strain is selected as the fermentation strain. By obtaining and preserving microorganisms from sour meat, the green coffee beans are co-fermented with Damascus rose flowers, thereby endowing the coffee beans with a unique rose flavor. The green coffee beans are subjected to high-temperature sterilization and the rose petals are subjected to ultraviolet sterilization treatment; then inoculation is carried out using a fermentation strain with a specific concentration; then multiple fermentations are carried out in a constant temperature and humidity environment to fully integrate the rose aroma and coffee flavor. Through selective fermentation and precise control of the inoculation concentration, it is ensured that the flavor compounds generated during the fermentation process are consistent, achieving a stable flavor performance. The finally obtained rose-flavored coffee has a unique flavor, moderate acidity, good body, balanced sweetness, appropriate roasting degree and uniform particle size. Its moisture content and trace element content are precisely controlled, and the tasting score reaches 81.3 points.

Description

Technical Field

[0001] The present invention belongs to the technical field of food engineering, and particularly relates to a Kluyveromyces marxianus, a method for fermenting rose-flavored coffee beans, and coffee beans. Background Art

[0002] Catimor coffee beans were first produced in the Catimor region of southern Karnataka, India. Catimor coffee beans belong to the Arabica variety, and mainly have two variants, Ginger and Cillón, among which the quality of the Cillón variety is slightly better than that of Ginger. The Arabica species to which Catimor coffee beans belong is famous for its delicate fragrance, moderate acidity, and long aftertaste, and presents soft cocoa, nut, and dried fruit aromas. After brewing, the soup is smooth and clear. All along, Catimor coffee beans have mainly been processed by the wet method, that is, the fresh coffee fruits are subjected to processes such as peeling, fermentation, and washing, and finally slowly dried in the shade, so as to retain its unique flavor to the greatest extent.

[0003] There has always been a traditional skill of fermenting coffee in the Catimor region. The locals in the Catimor region adopt a special way of ancient brine fermentation, that is, during the fermentation process, an appropriate amount of salt is added, and the antibacterial effect of the salt is used to prevent the fermentation process from getting out of control. This has become a unique process of using salt to assist in controlling fermentation and is also a traditional feature of Catimor coffee beans. However, the salt remains in the coffee beans, affecting the final taste of the coffee, increasing the saltiness, and reducing the purity of the flavor. This makes the market acceptance of Catimor coffee beans processed by the traditional brine fermentation process not high.

[0004] Using a specific fermenting agent to control the fermentation of coffee beans is a new coffee flavor adjustment technology. By scientifically regulating the fermentation process of coffee beans, the flavor of coffee can be improved and enhanced. The key points of using a specific fermenting agent to control the fermentation of coffee beans are to select a specific fermenting agent and conduct secondary fermentation. The fermenting agents used for coffee beans are usually specific microorganisms, such as lactic acid bacteria, yeasts, etc. These microorganisms can produce specific metabolites during the fermentation process. The metabolites can significantly affect the flavor of coffee, increasing acidity, sweetness, or imparting specific aromas. Secondary fermentation is based on the primary fermentation and further uses exogenous microorganisms for fermentation treatment. During the fermentation process, exogenous microorganisms can produce specific enzymes and metabolites, further improving the chemical composition of the coffee beans. These changes can enhance the flavor quality of coffee, such as increasing floral and fruity aromas, and enhancing acidity and sweetness. This can promote the richness of coffee flavor and make the coffee taste more mellow and unique. It can be seen that using scientific fermenting agents for control can reduce the generation of bad flavors and achieve the high-quality rate of coffee.

[0005] In Yunnan, most of the coffee plants are Catimor, which has a distinctive flavor due to the unique geographical environment and climate conditions in Yunnan. In terms of the sensory evaluation of coffee beans, commercially washed Catimor beans usually have a moderate acidity, a full body, and often carry unique fruit aromas such as citrus and berries, as well as floral and nutty aromas. In terms of the quality of coffee beans, since the main production areas of commercially washed Catimor beans in Yunnan include Pu'er City, Lincang City, Baoshan City, Dehong Dai and Jingpo Autonomous Prefecture, and Xishuangbanna Dai Autonomous Prefecture, the production management in these areas is relatively standardized, and the planting technology and processing technology are also relatively mature, ensuring the stable quality of commercially washed Catimor beans. In addition, Yunnan has rich flower resources and a long history of edible flowers. By adding flower elements during the coffee fermentation process, the aromatic substances in the flowers can penetrate into the coffee beans during the fermentation process, enhancing the flavor quality of the original coffee beans and having the potential to develop innovative flavored coffee products.

[0006] CF Silva et al. proposed in the article "Succession of bacterial and fungal communities during natural coffee (Coffea arabica) fermentation" (2008, Food Microbiology) that during the natural coffee fermentation process, the bacterial and fungal communities show a complex succession pattern, and different microorganisms play different roles at different stages of fermentation. And yeasts such as Pichia anomala and Pichia guilliermondii have an important impact on the flavor and quality of coffee during the fermentation process.

[0007] Similarly, Wafa Masoud et al. proposed in the article "Yeast involved in fermentation of East African coffee genotyped and determined by direct denaturing gradient gel electrophoresis" (2004, Yeast) that during the coffee fermentation process, different locations and processing methods may lead to differences in microbial communities. In particular, the processing sites in Tanzania may have a higher content of Pichia kluyveri yeast due to the microbial residues from early production. The diversity and dynamic changes of microorganisms during the coffee fermentation process in different geographical locations such as East Africa indicate the necessity to improve the existing coffee bean fermentation process.

[0008] Chinese Invention (CN 114365784A) discloses a method for improving coffee flavor by microbial fermentation. Different composite bacteria are used for co-fermentation to obtain coffee with different flavors. Adding granulated sugar or brown sugar during fermentation will significantly increase the sugar content in the fermentation substrate. This will cause the rapid reproduction of fermenting bacteria (such as yeast), consuming sugars to produce ethanol and carbon dioxide. Excessive sugar is likely to lead to too fast fermentation speed, generating excessive ethanol, resulting in an obvious alcohol taste in coffee beans and affecting their original flavor characteristics. Too much sugar is prone to promoting the overgrowth of certain strains (such as yeast) and inhibiting the growth of other beneficial strains (such as lactic acid bacteria), leading to microbial ecological imbalance during fermentation. Microbial ecological imbalance will cause uneven fermentation and produce bad flavor compounds, such as bad sourness or bitterness. Even more, the high-sugar environment may change the texture of the fermented product, making the coffee beans become too sticky or difficult to dry.

[0009] Chinese Invention (CN 115868566A) discloses a method for preparing directionally flavored fermented coffee. Gas chromatography-time of flight mass spectrometry technology is used to determine the volatile components of coffee roasted beans, obtaining chemical components related to sensory evaluation, and selecting natural edible materials containing the chemical components to obtain the flavor substrate; then Lactococcus lactis is added for closed culture to obtain directionally flavored fermented coffee beans. The defect of this method is that after the flavor substrate is mixed with green coffee beans and sterilized at 100 °C for 30 min, after long-term sterilization at 100 °C, the flavor substrate will change or volatilize and dissipate. The high-temperature environment will cause the degradation of some heat-sensitive flavor compounds, especially esters and alcohols, which will directly affect the flavor quality. Further, the loss of moisture and heat treatment are prone to making the texture of the substrate and coffee beans harden, affecting the taste and quality of coffee. Even more, high-temperature sterilization will promote the Maillard reaction (the reaction between amino acids and reducing sugars), resulting in a darker color and generating bitterness and bad flavors.

[0010] Chinese Invention (CN 111543534A) discloses a fermentation method capable of enhancing the aromatic substances of coffee beans. Aerobic fermentation and anaerobic fermentation are carried out under pressurized and constant temperature and humidity conditions, and soluble fructose and / or glucose are added to obtain coffee beans. The defect of this method is that adding fructose or glucose will directly increase the sweetness of coffee, even masking the original complex flavor of coffee beans and making the coffee flavor unnatural. The increase in sugar content may affect the acidity balance of coffee. The fermentation process under a high-sugar environment is prone to generating more acidic substances, such as acetic acid, resulting in too high acidity and affecting the overall taste of coffee. At the same time, the increase in sugar content leads to an increase in the viscosity of the fermentation broth, affecting gas exchange and heat conduction during fermentation and being unfavorable for uniform fermentation. Summary of the Invention

[0011] In order to solve the problems and deficiencies existing in the above-mentioned prior art, the present invention aims at a Kluyveromyces marxianus, a method for fermenting rose-flavored coffee beans, and coffee beans. By using the fermentation method of the present invention, a rose-flavored coffee can be prepared. This coffee has obvious rose flower fragrance, a sweet taste like brown sugar, a gentle acidity with a sweet aftertaste, and tones of almond, chocolate, and nut. It is a fermented coffee that can feel the rose flower fragrance from high temperature to low temperature, and its tasting score is 81.25 points.

[0012] Specifically, the first object of the present invention is to deposit the Kluyveromyces marxianus strain, and attach the acquisition method and identification results of the strain. The second object of the present invention is to carry out directional flavor fermentation on coffee beans with Kluyveromyces marxianus, achieve coffee flavor control, exclude harmful microorganism contamination, reduce the safety risk during the fermentation process, and improve the safety of coffee products. The third object of the present invention is to add edible roses during the fermentation process. The addition of edible roses requires pretreatment, and then under the action of yeast, the rose flavor is integrated into the coffee to prepare a rose-flavored coffee with stable quality. The fourth object of the present invention is to obtain a rose-flavored coffee through the fermentation method described above, which has obvious rose flower fragrance, a sweet taste like brown sugar, a gentle acidity with a sweet aftertaste, and tones of almond, chocolate, and nut, and can feel the characteristics of rose flower fragrance from high temperature to low temperature.

[0013] The first object of the present invention is to deposit the Kluyveromyces marxianus strain, and attach the acquisition method and identification results of the strain.

[0014] First, the Kluyveromyces marxianus CI-06 is deposited in the China General Microbiological Culture Collection Center. CI-06 is named Kluyveromyces marxianus, and the deposit number is CGMCC No: 30564. The deposit date is May 9, 2024, and the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0015] Second, the acquisition method of the Kluyveromyces marxianus is divided into strain screening, strain isolation, purification and preservation, and strain identification.

[0016] (1) Screening of Kluyveromyces marxianus strain: The Kluyveromyces marxianus strain sample was collected from traditional fermented food sour meat in Lincang City, Yunnan Province, China. After pasting the label, it was stored in the refrigerator for cold storage and transported back to the laboratory on the same day and stored at ultra-low temperature in a -80°C refrigerator.

[0017] Specifically, 5 g of the sour meat sample was weighed into 15 mL of sterile physiological saline, and after mixing, it was made into 10 -4 ~10 -910-fold serial dilutions. Respectively, 0.5 mL of the dilution solutions with different concentration gradients were pipetted onto the solidified YPD agar plates. Three replicates were made for each different dilution, and a spreader was used for spreading to make the dilution solutions evenly mixed within the plates. After mixing evenly, the plates were inverted and cultured in an incubator at 28 °C for 24 - 48 h.

[0018] (2) Isolation, purification, and preservation of Kluyveromyces marxianus strains: Observe the colony morphology on the plates, pick single colonies with different sizes, morphologies, and colors that grow well, and use the streak plate method to streak and culture on YPD agar plates, and culture in an incubator at 28 °C for 1 - 2 d. After three generations of streak purification of the strains, transfer them to numbered test tube slants and culture for 2 d. Store the slant test tubes in a 4 °C refrigerator for later experimental needs, and preserve the strains in glycerol tubes at -80 °C. Finally, select the isolates with consistent morphology and send them for identification.

[0019] (3) Identification of Kluyveromyces marxianus strains: After treating the above-mentioned isolated bacteria with sodium hydroxide solution, extract 16S rRNA from them for amplification. The amplification system is as shown in Table 1 below:

[0020]

[0021] Furthermore, perform PCR product sequencing: After the PCR products are detected to be qualified, cut the target bands for purification and recovery, and use the recovered products for Sanger sequencing.

[0022] DNA extraction: Use the TSINGKE Plant DNA Extraction Kit (Universal, product number: TSP101).

[0023] PCR amplification: Amplify using universal primers for strain identification, and analyze whether the PCR product bands are consistent with the target size, whether they are single, and whether there is smearing through agarose gel electrophoresis of the amplification products.

[0024] As Figure 1 shown, for the comparative analysis of the PCR product test results, the Sanger sequencing results are spliced using the software ContigExpress, and the inaccurate parts at both ends are removed. Furthermore, batch blastn (latest version v2.13) alignment of the spliced sequences is performed against the nucleic acid database. Among them, the nucleic acid database selects the latest version of the nt library. By performing blastn alignment with the nt library, the Accession Number of the homologous sequence, species identification, and annotation can be obtained.

[0025] The second object of the present invention is to provide a fermentation method for rose-flavored coffee, which is realized by three stages: preparation before fermentation (pretreatment), fermentation, and subsequent treatment:

[0026] (1) Pretreatment stage: Select coffee beans, rehydrate the coffee beans, sterilize, and cool.

[0027] (2) Fermentation stage: Prepare the bacterial suspension (including activating the strain, centrifugal separation, and formulating the bacterial suspension), inoculate (or seed), ferment, add rose flowers, and secondary fermentation.

[0028] (3) Post-treatment stage: Separate the bean curd and dry.

[0029] Specifically, as Figure 2 shown, the present invention aims at a fermentation method for rose-flavored coffee, which is realized by the following technical solutions:

[0030] (1) Pretreatment stage: Select coffee beans, rehydrate the coffee beans, sterilize and cool.

[0031] S1. Coffee bean selection: Pick out defective beans such as broken beans, moldy beans, worm-eaten beans, spotted beans, shell beans, shriveled beans, etc. and other impurities from the raw materials (Yunnan small bean coffee - Catimor washed - commercial beans), and reserve them for later use.

[0032] S2. Coffee bean rehydration: Use water at room temperature, put it into the fermentation tank at a ratio of bean to water of 1:3, and the volume after adding water accounts for about half of the total volume of the tank. Soak until the coffee beans are grayish-white and the appearance color is uniform.

[0033] S3. Sterilization (disinfection): Seal with a sterile sealing film, sterilize at a temperature of 105 - 115 °C for 5 - 10 min. After sterilization, conduct natural cooling until the temperature drops to 25 - 35 °C.

[0034] (2) Fermentation stage: Preparation of the bacterial suspension, inoculation (or seeding), fermentation, addition of rose flowers, and secondary fermentation.

[0035] S4. Preparation of the bacterial suspension: Centrifuge the activated fermentation strain to discard the culture medium, and then add sterile normal saline to adjust to an appropriate concentration; preferably, the concentration is adjusted to 8 lgCFU / mL.

[0036] S5. Inoculation: Sprinkle the prepared bacterial suspension on the surface of sterile coffee beans, and the microbial concentration reaches 6 - 7 lgCFU / mL after inoculation.

[0037] S6. Fermentation: After inoculating the coffee beans, transfer them to a constant temperature room, keep the temperature at 28 ± 2 °C, and the relative humidity above 85%. Ferment for 24 h.

[0038] S7. Addition of rose flowers: Add the rose petals sterilized by ultraviolet light into the fermentation tank in layers at 10 - 15% of the mass of the green coffee beans.

[0039] S8. Secondary fermentation: Transfer the fermenter after adding flowers to a constant temperature room, keep the temperature at 28 ± 2 °C, and the relative humidity above 85%. Ferment for 48 - 60 h.

[0040] S9. Sealing: From sterilization to the end of fermentation, the fermenter is sealed with a sealing film for microbial culture; the pore size of this sealing film prevents microorganisms from passing through, and gas can pass through freely.

[0041] (3) Subsequent treatment stage: Separating soybeans and drying.

[0042] S10. Separating soybeans: Pick out the roses from the coffee beans after fermentation and only keep the coffee beans.

[0043] S11. Drying: Dry the coffee beans at 40 - 45 °C until the moisture content is less than 12%, then a fermented rose - flavored coffee is made.

[0044] S12. Roasting: Take 300 g of fermented rose coffee beans and put them into a drum - type coffee roaster. The bean - inlet temperature is 180 °C, keep the firepower at 60%, the roasting time is 8 - 12 min, and the best out - of - pot temperature is 205 - 218 °C, then medium - roasted coffee beans (recommended medium - light) are obtained.

[0045] Supplementally, to meet the fermentation conditions, the preparation of the bacterial suspension described in the present invention is an important step before fermentation starts, specifically including three steps: activating the Kluyveromyces marxianus strain, centrifugal separation, and adding sterile physiological saline.

[0046] S41. Step of activating the fermentation strain: Select the Kluyveromyces marxianus strain as the fermentation strain and carry out activation culture in a suitable medium to ensure that the strain is in the best growth state.

[0047] S42. Centrifugal separation step: Centrifugally separate the culture solution of the activated Kluyveromyces marxianus strain. The centrifugation conditions (such as rotation speed and time) need to be set according to the characteristics of the strain and the experimental equipment, generally centrifuging at 5000 r / min for 10 min. After centrifugation, carefully discard the supernatant and retain the precipitated bacteria.

[0048] S43. Step of resuspending with sterile physiological saline: Resuspend the centrifuged precipitated bacteria with sterile physiological saline (usually 0.85% NaCl solution). Aseptic operation is very important to avoid contamination. Adjust the concentration of the bacterial suspension to reach a suitable concentration range, usually 6 - 7 lgCFU / mL. The adjustment of the concentration can be carried out by measuring the optical density of the bacterial solution or the direct counting method.

[0049] Furthermore, the step of activating the fermentation strain is to activate the Kluyveromyces marxianus strain and obtain the culture medium.

[0050] Specifically, the activated Kluyveromyces marxianus strain uses YPD medium (yeast extract, peptone, glucose medium). The specific formula of the YPD medium is 10 g / L of yeast extract, 20 g / L of peptone, and 20 g / L of glucose. Dissolve the above three components in 1 L of distilled water and perform autoclaving (121 °C, 15 min).

[0051] Specifically, after the medium is prepared, inoculation and activation culture are required. The inoculation is to inoculate the freeze-dried or preserved Kluyveromyces marxianus strain into a small amount of YPD liquid medium under sterile conditions. Usually, aseptic operation techniques are used to ensure that the inoculation process is not contaminated by miscellaneous bacteria. The activation culture is carried out on a constant temperature shaker at 25 °C to 30 °C, with a shaking speed of about 150 rpm and a culture time of 24 h. The activation culture helps to activate yeast cells and make them enter an active growth state from a dormant state.

[0052] Specifically, after inoculation and activation culture are completed, microscopic examination is required, and sometimes subculture is needed. For the microscopic examination, take a small amount of the culture solution and observe it under a microscope to check the activity and purity of the yeast cells. The subculture is carried out when more culture solution is needed. In this case, the activated culture solution is inoculated into more YPD medium and continuously cultured under the same conditions until the required amount is obtained.

[0053] Furthermore, the centrifugation separation step is to perform centrifugation separation on the culture solution of the activated Kluyveromyces marxianus strain.

[0054] Specifically, the equipment used in the centrifugation separation step is a high-speed refrigerated centrifuge, centrifuge tubes, and a sterile operating table; the material for centrifugation separation is the culture solution of the activated Kluyveromyces marxianus strain.

[0055] Specifically, the method of centrifugation separation is as follows: First, prepare and load the tubes. Divide the culture solution into centrifuge tubes according to the amount, with equal liquid volume in each centrifuge tube. Then, seal the centrifuge tubes and place them on the rotor of the centrifuge, ensuring symmetric placement. Next, set the centrifugation conditions of the high-speed refrigerated centrifuge, specifically, the centrifuge speed is 5000 r / min and the centrifugation time is 10 min. Then, perform centrifugation operation, that is, start the centrifuge and carry out 10 min of centrifugation separation; after centrifugation is completed, slowly turn off the centrifuge and take out the centrifuge tubes. Finally, remove the supernatant and collect the cells. Carefully pour out the supernatant, avoiding disturbing the precipitated cells, and reserve the precipitated part (Kluyveromyces marxianus cells) for use.

[0056] Specifically, a high-speed refrigerated centrifuge is used in the centrifugation separation step. It is necessary to ensure that the temperature is set at 4°C to maintain the activity of yeast. Before centrifugation, it is necessary to check whether the centrifuge tubes are balanced to avoid failure caused by equipment damage. Throughout the centrifugation separation process, aseptic operation should be maintained to prevent external contamination.

[0057] Furthermore, the step of resuspending with sterile normal saline is to resuspend the precipitated Kluyveromyces marxianus cells with sterile normal saline; preferably, the concentration of the cell suspension is adjusted to 8lgCFU / mL. This can ensure the sterility and appropriate concentration of the cell suspension, providing a high-quality bacterial source for the subsequent fermentation process.

[0058] Specifically, the tools for the step of resuspending with sterile normal saline are sterile pipettes or pipettors, spectrophotometers or plate counting method equipment; the materials for the step of resuspending with sterile normal saline are sterile normal saline (0.85% NaCl) and the precipitated Kluyveromyces marxianus cells after centrifugation.

[0059] Specifically, the method of resuspending with sterile normal saline is as follows: First, prepare sterile normal saline and formulate a 0.85% NaCl solution, sterilize it and cool it to room temperature. Then resuspend the cells. First, carefully discard the supernatant in the centrifuge tube, and then use a sterile pipette to add an appropriate amount of sterile normal saline to the centrifuge tube and gently mix to completely resuspend the precipitated cells. Next, adjust the concentration. The optical density measurement method or the direct counting method can be used. The optical density measurement method is to transfer a part of the cell suspension to a spectrophotometer cuvette and measure the OD 600 value; according to the measured OD 600 value, dilute or concentrate the cell suspension with sterile normal saline to adjust to the target OD 600 value (such as 0.1 - 0.2). The direct counting method is to take an appropriate amount of cell suspension, perform serial dilution, spread it on an agar medium, and culture it at 28°C for 24h; then count the number of colonies, calculate the number of colonies in the original cell suspension according to the dilution factor; finally, adjust the concentration of the cell suspension according to the counting result. (In the scheme of the present invention, preferably, the concentration is adjusted to 8lgCFU / mL).

[0060] Specifically, the whole process of resuspending with sterile normal saline needs to be carried out under aseptic operation. During the resuspension process, mix well to prevent cell aggregation from affecting measurement and subsequent use. Mix well during the resuspension process to prevent cell aggregation from affecting the measurement results.

[0061] Supplementally, the third object of the present invention is to add edible roses during the fermentation process. Then, under the promotion of yeast, the rose flavor is integrated into the coffee to prepare a coffee with a stable quality and a rose flavor. The added edible roses need to be pretreated separately as follows:

[0062] Furthermore, in order to integrate the conditions of rich rose flavor in fermentation, the addition of roses in the present invention is an important step after the first fermentation, specifically requiring three steps: proportioning rose petals, ultraviolet sterilization, and layering rose petals.

[0063] S71. Proportioning rose petals: Select fresh, pest-free, and pollution-free rose petals ((the variety is Yunnan edible rose - Damascus rose dried flowers), gently wash and dry them to ensure that there is no soil or impurities on the surface; then calculate the mass ratio of coffee beans and rose petals, and prepare rose petals according to the ratio of 10 - 15% of the mass of green coffee beans.

[0064] S72. Ultraviolet sterilization: Lay the proportioned rose petals evenly on a clean, light-proof tray, ensuring that the petals do not overlap so that ultraviolet light can evenly irradiate the surface of each petal; place the tray under the ultraviolet sterilization lamp, start the ultraviolet sterilization lamp, and irradiate for 15 - 30 minutes; after irradiation, put the petals into a sterile container for standby.

[0065] S73. Layering rose petals: Evenly spread a layer of coffee beans at the bottom of the fermentation tank, accounting for about one-fourth to one-third of the total amount; then, evenly sprinkle a layer of rose petals on the first layer of coffee beans to cover the coffee beans; lay another layer of coffee beans (the second layer of coffee beans) to ensure that the previous layer of rose petals is covered; continue to evenly sprinkle another layer of rose petals on the second layer of coffee beans; finally, lay another layer of coffee beans (layer) to cover the rose petals above. Repeat the above steps until all the rose petals and coffee beans have been layered into the fermentation tank.

[0066] Supplemented, in order to detect the rose coffee prepared by the present invention using Kluyveromyces marxianus strain fermentation, the present invention conducted physical and chemical tests such as moisture determination, pH determination, and tannin content analysis.

[0067] (1) Moisture determination

[0068] Determine the moisture content of the coffee beans in Examples 2, 3, 4, and 5 after aerobic fermentation for 72 hours. The determination method is as follows: Use an HS153 halogen moisture analyzer to accurately weigh 2 g of coffee samples, put them into a constant-weight aluminum box, heat and dry at 105 °C until the weight no longer changes within 90 s, record the moisture content displayed by the instrument, and repeat each sample three times. The experimental results are shown in Table 2 below:

[0069] Table 2 Determination results of moisture content values in different examples

[0070]

[0071] (2) pH determination

[0072] The pH values of the coffee beans after aerobic fermentation for 72 h in Examples 2, 3, 4, and 5 were measured. The measurement method is as follows: Weigh 5.00 g of coffee sample, add 25 mL of distilled water, shake for 20 min, and directly measure using a pH meter. The pH measurement results of different examples are shown in Table 3 below:

[0073] Table 3 pH measurement results of different examples

[0074]

[0075] (3) Analysis of tannin content

[0076] The tannin contents of the coffee beans after aerobic fermentation for 72 h in Examples 2, 3, 4, and 5 were measured. The measurement method is as follows:

[0077] 1) Drawing of the standard curve: Accurately pipette 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1 mL of the standard tannic acid solution (200 mg / L) into 10-mL colorimetric tubes containing 6 mL of water respectively. Add 0.5 mL of Folin-Ciocalteu phenol, 1 mL of 7.5% sodium carbonate solution, and make up the volume to the scale line with distilled water. Shake well, let stand for 30 min for color development, and then, with the same operation of the reagent blank as the blank control, measure the absorbance of the standard solution at a wavelength of 760 nm. Draw the standard curve with the tannic acid concentration as the abscissa and the absorbance value as the ordinate.

[0078] 2) Measurement of coffee samples: Precisely pipette 0.2 mL of the coffee powder extract into a 10-mL colorimetric tube containing 6 mL of water. Add 0.5 mL of Folin-Ciocalteu phenol, 1 mL of 7.5% sodium carbonate solution, and make up the volume to the scale line with distilled water. Shake well. Let stand for 30 min for color development, and then, with the same operation of the reagent blank as the blank control, measure the absorbance of the sample solution at a wavelength of 760 nm. Calculate the concentration of tannic acid in the test solution according to the standard curve. The results are shown in Table 4.

[0079] Table 4 Tannin content concentration values of different examples

[0080]

[0081] Supplementary, in order to detect the rose coffee prepared by fermenting with Kluyveromyces marxianus strains in the present invention, the present invention conducted a sensory evaluation.

[0082] After aerobic fermentation for 72 h in Examples 2, 3, 4, and 5, the coffee beans were separated by bean curd, dried, baked, etc. until the moisture content reached 10 - 12%, and then baked and subjected to sensory evaluation. The specific evaluation process is as follows:

[0083] S1. Grinding: The roasted coffee beans pass through a sieve with a 20-mesh aperture, and the passing rate of the coffee powder is preferably 70-75%.

[0084] S2. Brewing: Pour 200 mL of hot water at 90-94 °C into 11 g of coffee powder. After 4 minutes, break the dregs and remove the dregs in sequence, and then conduct a sensory evaluation. The cup-tested samples are randomly coded, labeled, and presented.

[0085] S3. Five coffee tasters with Q-grader ratings score the coffee samples during the sniffing, sipping, and aftertaste procedures. The flavor characteristics are evaluated from ten comprehensive aspects: fragrance / aroma, clean cup, sweetness, acidity, body, flavor, aftertaste, balance, uniformity, and overall.

[0086] Sensory evaluation conclusion: Example 2 obtained a total score of 81.25 points, Example 3 obtained a total score of 80.25 points, Example 4 obtained a total score of 80.75 points, and Example 5 obtained a total score of 77.50 points. In addition, the panelists unanimously described the coffee beans fermented with Kluyveromyces marxianus by directed flavor fermentation and added with rose flavor enhancement technology as having an obvious rose floral fragrance, a sweet taste like brown sugar, a soft acidity with a sweet aftertaste, and tones of almond, chocolate, and nut. The fermented coffee with rose floral fragrance can be felt from high temperature to low temperature, while the intensity and flavor of the fermented coffee in the non-inoculated treatment group are weaker.

[0087] According to the SCA scoring requirements, coffee with a score above 80 is considered specialty coffee. Using Kluyveromyces marxianus for directed flavor fermentation and adding rose flavor enhancement technology has a relatively significant improvement in coffee flavor. The review group scored the coffee beans according to the SCA international cup-tasting standard, as shown in Table 5 below. This coffee received a high score of 81.25.

[0088] Table 5 Sensory scoring results of different examples

[0089]

[0090]

[0091] Furthermore, the fourth object of the present invention is to obtain a rose-flavored coffee through the described fermentation method. This is a fermented coffee with an obvious rose floral fragrance, a sweet taste like brown sugar, a soft acidity with a sweet aftertaste, and tones of almond, chocolate, and nut. The rose floral fragrance can be felt from high temperature to low temperature, and the sensory evaluation score is 81.25 points.

[0092] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention directly adds edible roses, and the green coffee beans and roses are co-fermented under the action of fermenting bacteria, endowing the green coffee beans with rose flavor, effectively solving the technical problems that the natural fermentation of existing coffee leads to uncontrollable coffee flavor, poor uniformity and stability of coffee quality, and the difficulty in preservation of coffee fruit fermentation, and the seasonality of coffee fruit restricts the industrialization of fermented products; using the dominant microorganisms in traditional Yunnan fermented foods as fermenting bacteria and green coffee beans as raw materials to produce coffee beans with specific flavors, which has sustainability and stability; the added flavor is integrated with the original coffee flavor through biotransformation without a sense of separation; reducing bitterness and astringency, with a balanced flavor and a soft taste. Based on this method, the dominant aroma-producing microorganisms in other fermented foods can be applied to coffee fermentation to make specific flavor coffee beans have sustainability and product stability.

[0093] Specifically, in the fermentation method of a rose flavor coffee of the present invention, rose petals sterilized by ultraviolet light are added during the fermentation process, so that the aromatic compounds of roses penetrate into the coffee beans. Through secondary fermentation, the rose aroma and coffee flavor are fully integrated to form a unique rose flavor coffee. By selective fermentation and controlling the inoculation concentration, it is ensured that the flavor compounds produced during the fermentation process are consistent, thereby achieving a stable flavor performance.

[0094] Specifically, in the fermentation method of a rose flavor coffee of the present invention, a specific bacterial suspension (inoculation concentration of 6 - 7 lgCFU / mL) and constant temperature and humidity fermentation conditions are used in the fermentation stage to ensure the controllability and repeatability of the fermentation process and improve the consistency of product quality. By setting a clear fermentation time (primary fermentation for 24 h, secondary fermentation for 48 - 60 h), it is ensured that each batch of coffee beans reaches the ideal flavor and quality within the optimal fermentation time.

[0095] Specifically, in the fermentation method of a rose flavor coffee of the present invention, rose petals are sterilized by high temperature (105 - 115 °C, 5 - 10 min) and ultraviolet light in the pretreatment stage, significantly reducing the risk of miscellaneous bacteria and pathogenic bacteria, and ensuring the safety of the fermentation process and the final product. Using a specific concentration of fermentation strains to exclude harmful microorganism contamination, reducing the risk of the growth of harmful bacteria during the fermentation process, and improving the safety and stability of the product.

[0096] Specifically, in the fermentation method of a rose flavor coffee of the present invention, an activated specific fermentation strain (such as Kluyveromyces marxianus) is used to optimize the fermentation process, improve the fermentation efficiency, shorten the fermentation time, and ensure production efficiency and economic benefits. Maintaining an appropriate temperature (28 ± 2 °C) and humidity (relative humidity above 85%) during the fermentation process provides the best growth environment for the fermenting bacteria and ensures an efficient fermentation process.

[0097] Specifically, for the fermentation method of the rose - flavored coffee of the present invention, from coffee bean selection, re - hydration, sterilization, inoculation, fermentation to subsequent processing (soybean curd separation, drying), the entire technological process is clear and systematic, which is easy for standardized operation and large - scale production application. The operating conditions of each step are clear, facilitating implementation and control in actual production, reducing process fluctuations and operation errors. Description of the Drawings

[0098] Figure 1 It is the molecular identification result of the microbial strain of the Kluyveromyces marxianus strain of the present invention;

[0099] Figure 2 It is the process flow chart of the fermentation method of the rose - flavored coffee of the present invention;

[0100] Figure 3 It is the trend chart of the influence of the inoculation concentration of the rose - flavored coffee of the present invention on the coffee sensory score

[0101] Figure 4 It is the cupping score and standard deviation chart of the rose - flavored coffee of the present invention under different inoculation concentrations (log10CFU / mL);

[0102] Figure 5 It is the trend chart of the influence of the fermentation temperature of the rose - flavored coffee of the present invention on the coffee sensory score;

[0103] Figure 6 It is the cupping score and standard deviation chart of the rose - flavored coffee of the present invention at different fermentation temperatures;

[0104] Figure 7 It is the trend chart of the influence of the fermentation time of the rose - flavored coffee of the present invention on the coffee sensory score;

[0105] Figure 8 It is the cupping score and standard deviation chart of the rose - flavored coffee of the present invention at different fermentation times;

[0106] Figure 9 It is the trend chart of the influence of the rose addition amount of the rose - flavored coffee of the present invention on the coffee sensory score;

[0107] Figure 10 It is the cupping score and standard deviation chart of the rose - flavored coffee of the present invention under different rose addition amounts;

[0108] Figure 11 It is the pH change trend chart during the fermentation of the rose - flavored coffee of the present invention;

[0109] Figure 12Data chart of the change in pH during the fermentation process of a rose - flavored coffee according to the present invention;

[0110] Figure 13 Microbial growth trend chart during the fermentation process of a rose - flavored coffee according to the present invention;

[0111] Figure 14 Data chart of the growth of strains during different fermentation times of a rose - flavored coffee according to the present invention;

[0112] Figure 15 Water content chart during different step - stages of a rose - flavored coffee according to the present invention;

[0113] Figure 16 Water content comparison chart of the re - hydration / sterilization methods of a rose - flavored coffee according to the present invention;

[0114] Figure 17 Physical and chemical comparison chart before and after fermentation of the obtained rose - flavored coffee beans of a rose - flavored coffee according to the present invention;

[0115] Figure 18 Flavor description chart of the obtained rose - flavored coffee beans of a rose - flavored coffee according to the present invention;

[0116] Figure 19 Change chart of the contents of tannins, polyphenols and flavonoids in the coffee beans fermented by different yeasts and unfermented coffee beans of a rose - flavored coffee according to the present invention;

[0117] Figure 20 Chart of the change in color (evaluated by a color difference meter) during the fermentation process of a rose - flavored coffee according to the present invention;

[0118] Figure 21 Physical comparison chart of the obtained rose - flavored coffee beans of a rose - flavored coffee and the raw material Catimor washed commercial beans according to the present invention;

[0119] Figure 22 Overall evaluation chart of the cupping results of the obtained rose - flavored coffee beans of a rose - flavored coffee according to the present invention;

[0120] Figure 23 Tasting score sheet of the obtained rose - flavored coffee beans of a rose - flavored coffee according to the present invention;

[0121] Figure 24 Process physical chart of the fermentation method of a rose - flavored coffee according to the present invention. Detailed implementation manners

[0122] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0123] The first object of the present invention is to deposit the Kluyveromyces marxianus strain, and attach the acquisition method and identification results of the strain. The second object of the present invention is to use Kluyveromyces marxianus to carry out directional flavor fermentation on coffee beans, achieve coffee flavor control, eliminate harmful microorganism contamination, reduce the safety risk during the fermentation process, and improve the safety of coffee products. The third object of the present invention is to add edible roses during the fermentation process. The addition of edible roses requires pretreatment, and then under the action of yeast, the rose flavor is fused into the coffee to prepare a coffee with a stable quality and rose flavor. The fourth object of the present invention is to obtain a rose-flavored coffee through the fermentation method described above, which has obvious rose flower fragrance, a sweet taste like brown sugar, a soft acidity with a sweet aftertaste, and tones of almond, chocolate, and nut, and the rose flower fragrance can be felt from high temperature to low temperature.

[0124] The fermentation strain used in the present invention is the Kluyveromyces marxianus strain. As described above, the Kluyveromyces marxianus strain is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. CI-06 is named Kluyveromyces marxianus, the deposit number is CGMCC No: 30564, the deposit date is May 9, 2024, and the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The edible rose variety used is Yunnan edible rose - dried Damascus rose.

[0125] The acquisition method of the Kluyveromyces marxianus used in the present invention is divided into strain screening, strain isolation, purification and preservation, and strain identification.

[0126] (1) Screening of Kluyveromyces marxianus strain: The Kluyveromyces marxianus strain sample was collected from traditional fermented food sour meat in Lincang City, Yunnan Province, China. After labeling, it was stored in a refrigerator for cold storage and transported back to the laboratory on the same day and stored at ultra-low temperature in a -80°C refrigerator.

[0127] Specifically, 5 g of the sour meat sample was weighed into 15 mL of sterile physiological saline, mixed evenly, and then a 10-fold gradient dilution solution of 10 -4 ~10 -9 was prepared. 0.5 mL of dilution solutions with different concentration gradients were respectively pipetted onto the solidified YPD agar plate. Three parallels were made for different dilution degrees, and a spreader was used for spreading to make the dilution solution evenly mixed in the plate. After mixing, the plate was inverted and cultured in an incubator at 28°C for 24 - 48 h.

[0128] (2) Isolation, purification and preservation of Kluyveromyces marxianus strains: Observe the colony morphology on the plate, pick single colonies with different sizes, morphologies and colors that grow well, and streak-culture them on a YPD agar plate using the streak plate method. Incubate in an incubator at 28 °C for 1 - 2 days. After three generations of streak purification of the strains, transfer them to a slant tube, number them and culture for 2 days. Store the slant tubes in a 4 °C refrigerator for later experimental needs, and preserve the strains in glycerol tubes at -80 °C. Finally, select the isolates with consistent morphology and send them for identification.

[0129] (3) Identification of Kluyveromyces marxianus strains: After treating the above-mentioned isolated bacteria with sodium hydroxide solution, extract 16S rRNA from them for amplification. The amplification system is shown in Table 1:

[0130] Furthermore, perform PCR product sequencing on Kluyveromyces marxianus strains: After the PCR products are detected to be qualified, cut the target bands for purification and recovery, and use the recovered products for Sanger sequencing.

[0131] DNA extraction: Use the TSINGKE Plant DNA Extraction Kit (universal type).

[0132] PCR amplification: Amplify using the universal primers for strain identification shown in Table 1, and analyze whether the PCR product bands match the target size, are single, and have no smear through agarose gel electrophoresis.

[0133] As Figure 1 shown, perform comparative analysis on the PCR product test results. The Sanger sequencing results are spliced using the software ContigExpress, and the inaccurate parts at both ends are removed. Furthermore, batch-align the spliced sequences with the nucleic acid database using blastn (latest version v2.13). Among them, the nucleic acid database selects the latest version of the nt library. By performing blastn alignment with the nt library, the Accession Number of the homologous sequence, species identification and annotation can be obtained.

[0134] Example 1 Fermentation method of a rose-flavored coffee according to the present invention

[0135] As Figure 2 shown, for the fermentation method of a rose-flavored coffee according to the present invention, the coffee bean variety used is Yunnan small bean coffee - Caturra washed - commercial bean; the fermentation strain used is Kluyveromyces marxianus strain, which has been microbially preserved as described above; the rose flower used is dried edible rose flower.

[0136] As Figure 2As shown in the figure, the present invention provides a fermentation method for rose - flavored coffee, which is realized through three stages: preparation before fermentation (pretreatment), fermentation, and subsequent treatment.

[0137] (1) Pretreatment stage: Select coffee beans, re - hydrate the coffee beans, sterilize, and cool.

[0138] (2) Fermentation stage: Prepare a bacterial suspension, inoculate (or inoculate with bacteria), ferment, add rose flowers, and conduct secondary fermentation.

[0139] (3) Subsequent treatment stage: Separate the bean curd and dry.

[0140] Specifically, as Figure 2 shown in the figure, the fermentation method for rose - flavored coffee of the present invention is realized by the following technical solutions:

[0141] (1) Pretreatment stage: Select coffee beans, re - hydrate the coffee beans, sterilize, and cool.

[0142] S1. Coffee bean selection: Pick out defective beans such as broken beans, moldy beans, worm - eaten beans, spotted beans, shell beans, shriveled beans, etc. and other impurities from the raw materials (Yunnan small - bean coffee - Catimor washed - commercial beans), and then set them aside for later use.

[0143] S2. Coffee bean re - hydration: Use water at room temperature, put it into the fermentation tank at a bean - to - water ratio of 1:3, and the volume of water added accounts for about half of the total volume of the tank. Soak until the coffee beans are gray - white and have a uniform appearance color.

[0144] S3. Sterilization (disinfection): Seal with a sterile sealing film, sterilize at a temperature of 105 - 115 °C for 5 - 10 min. After sterilization, conduct natural cooling until the temperature drops to 25 - 35 °C.

[0145] (2) Fermentation stage: Preparation of the bacterial suspension, inoculation (or inoculation with bacteria), fermentation, addition of rose flowers, and secondary fermentation.

[0146] S4. Preparation of the bacterial suspension: Centrifuge the activated fermentation strain to discard the culture medium, and then add sterile normal saline to adjust to an appropriate concentration; preferably, the concentration is adjusted to 1.5×10 8 CFU / mL.

[0147] S5. Inoculation: Spray the prepared bacterial suspension onto the surface of sterile coffee beans, and the inoculated microorganism concentration reaches 6 - 7 CFU / mL.

[0148] S6. Fermentation: After inoculation, transfer the coffee beans to a constant - temperature room, keep the temperature at 28 ± 2 °C, and the relative humidity above 85%. Ferment for 24 h.

[0149] S7. Addition of rose flowers: Layer - add the rose petals sterilized by ultraviolet light into the fermentation tank at 10 - 15% of the mass of the green coffee beans.

[0150] S8. Secondary fermentation: Move the fermenter after adding flowers into a constant temperature room, keep the temperature at 28 ± 2 °C, and the relative humidity above 85%. Ferment for 48 - 60 h.

[0151] S9. Sealing: From sterilization to the end of fermentation, the fermenter is sealed with a sealing film for microbial culture; the pore size of this sealing film cannot be passed by microorganisms, and gas can pass freely.

[0152] (3) Subsequent treatment stage: Tofu separation, drying.

[0153] S10. Tofu separation: Pick out the roses from the coffee beans after fermentation and only keep the coffee beans.

[0154] S11. Drying: Dry the coffee beans at 40 - 45 °C until the moisture content is lower than 12%, then a fermented rose - flavored coffee is made.

[0155] S12. Roasting: Take 300 g of fermented rose coffee beans and put them into a drum - type coffee roaster. The temperature when putting into the pot is 180 °C, keep the firepower at 60%, the roasting time is 8 - 12 min, and the best temperature when taking out of the pot is 205 - 218 °C, then medium - roasted coffee beans (recommended medium - light) are obtained.

[0156] More specifically, the present invention provides a fermentation method for rose - flavored coffee, which is realized by the specific operation steps of the foregoing S.1 - S.12. The following is the specific operation implementation method:

[0157] S1. Coffee bean selection

[0158] Through coffee bean selection, ensure the quality and hygiene standards of coffee beans, and provide high - quality raw materials for subsequent fermentation. Specifically, the tools prepared in the coffee bean selection can be manual selection tools (including trays, workbenches with sufficient light), or mechanical equipment in food engineering (including screening machines, photoelectric sorting machines, air - separation machines, etc.). Specifically, the manual selection is to evenly spread the coffee beans on the tray, and in an environment with sufficient light, carefully check and pick out broken beans, moldy beans, insect - eaten beans, spotted beans, shell beans, and shriveled beans; the mechanical screening is to pour the coffee beans into a screening machine, and through sieves with different pore sizes, screen out beans of different sizes and remove beans that are too large or too small; the photoelectric sorting is to use a photoelectric sorting machine to identify and remove defective beans according to color and shape; the air - separation is to separate light impurities such as wood chips and stones through wind power.

[0159] Specifically, the method for selecting coffee beans is as follows: First, perform manual selection. Evenly spread the coffee beans on a tray and carefully inspect and pick out broken beans, moldy beans, insect-infested beans, spotted beans, shell beans, and shriveled beans in an environment with sufficient light. Then, conduct mechanical screening. Pour the coffee beans into a screening machine and screen out beans of different sizes through sieves with different pore sizes to remove beans that are too large or too small. Next, perform optoelectronic sorting. Use an optoelectronic sorter to identify and remove defective beans based on color and shape. Finally, perform air separation to separate light impurities such as wood chips and stones by wind power.

[0160] S2. Rehydration of coffee beans

[0161] Rehydration of coffee beans is to make the dried coffee beans reabsorb moisture, become plump, promote the attachment and penetration of fermenting bacteria, and provide a good environment for subsequent fermentation.

[0162] Use water at room temperature and put it into a fermentation tank at a ratio of coffee beans to water of 1:3. After adding water, the volume accounts for about half of the total volume of the tank. Soak until the coffee beans are grayish-white and have a uniform appearance color. Specifically, the tools required for the rehydration of coffee beans are a fermentation tank, a stirring rod, and measuring instruments. The ratio of coffee beans to water in the rehydration of coffee beans is 1:3. For example, if 1 kg of coffee beans is used, 3 kg of water is required.

[0163] Specifically, the method for rehydrating coffee beans is to use water at room temperature and put the coffee beans into a fermentation tank at a ratio of 1:3 (coffee beans: water); then add water three times the weight of the coffee beans and stir; after adding water, ensure that the volume of water and beans accounts for about half of the total volume of the fermentation tank to fully soak the coffee beans and avoid water overflow; then, let the coffee beans soak in water until the beans are grayish-white and have a uniform appearance color. This process usually takes several hours, but the specific time depends on the drying degree and variety of the coffee beans. The preferred soaking time range is 8 - 12 h.

[0164] S3. Sterilization (disinfection)

[0165] Sterilization is a crucial step in the preparation process of fermented coffee beans, aiming to eliminate harmful microorganisms on the surface and inside the coffee beans and ensure the purity and controllability of the subsequent fermentation process. Seal with a sterile sealing film and sterilize at a temperature of 105 - 115 °C for 5 - 10 min. Specifically, the equipment for sterilization is an autoclave or a high-temperature oven and a high-temperature-resistant sterile container; the materials for sterilization are coffee beans after rehydration treatment and a sterile sealing film.

[0166] Specifically, the method for sterilizing coffee beans is as follows: First, seal the container. Put 1 kg of pre-treated coffee beans into a high-temperature resistant sterile container, and use a sterile sealing film to tightly seal the container to ensure sterility after sterilization. Then, perform sterilization. The first method of sterilization is to put the sealed container into an autoclave, set the temperature to 110 °C, and the sterilization time to 8 minutes. The second method of sterilization is to put the sealed container into a high-temperature oven, set the temperature to 115 °C, and the sterilization time to 5 minutes. Finally, after sterilization, naturally cool the container to room temperature (25 - 35 °C).

[0167] During the sterilization process, it is necessary to ensure that the coffee beans in the container are evenly distributed to achieve uniform heating and sterilization. At the same time, it is necessary to ensure that the sealing film is intact to prevent external contaminants from entering after sterilization.

[0168] S4. Prepare the bacterial suspension

[0169] Preparing the bacterial suspension is to adjust the activated fermentation strain to an appropriate concentration (1.5×10 8 CFU / mL) to ensure that a sufficient number of effective bacteria are provided during inoculation and improve the fermentation efficiency. Preparing the bacterial suspension is an important step before fermentation starts. Specifically, it requires three steps: activating the Kluyveromyces marxianus strain, centrifugal separation, and adding sterile physiological saline.

[0170] Activating the fermentation strain step: Select the Kluyveromyces marxianus strain as the fermentation strain and perform activation culture in an appropriate medium to ensure that the strain is in the best growth state.

[0171] Centrifugal separation step: Centrifuge the culture solution of the activated Kluyveromyces marxianus strain. The centrifugation conditions (such as rotation speed and time) need to be set according to the characteristics of the strain and the experimental equipment. Generally, centrifuge at 5000 r / min for 10 minutes. After centrifugation, carefully discard the supernatant and retain the precipitated cells.

[0172] Adding sterile physiological saline to resuspend step: Resuspend the centrifuged precipitated cells with sterile physiological saline (usually 0.85% NaCl solution). Aseptic operation is very important to avoid contamination. Adjust the concentration of the bacterial suspension to reach an appropriate concentration range, usually 6 - 7 lgCFU / mL. The adjustment of the concentration can be carried out by measuring the optical density of the bacterial solution or by direct counting methods.

[0173] Furthermore, the step of activating the fermentation strain is to activate the Kluyveromyces marxianus strain and obtain the culture medium.

[0174] Specifically, the activated Kluyveromyces marxianus strain uses YPD medium (yeast extract, peptone, glucose medium). The specific YPD medium formula is 10 g / L of yeast extract, 20 g / L of peptone, and 20 g / L of glucose. Dissolve the above three components in 1 L of distilled water and perform autoclaving (121 °C, 15 min).

[0175] Specifically, after the medium is prepared, inoculation and activation culture are required. The inoculation is to inoculate the freeze-dried or preserved Kluyveromyces marxianus strain into a small amount of YPD liquid medium under sterile conditions. Usually, aseptic operation techniques are used to ensure that the inoculation process is not contaminated by miscellaneous bacteria. The activation culture is carried out on a constant temperature shaker at 25 - 30 °C, with a shaking speed of about 150 rpm and a culture time of 24 h. Activation culture helps to activate yeast cells and make them enter an active growth state from a dormant state.

[0176] Specifically, after inoculation and activation culture are completed, microscopic examination is required, and sometimes expansion culture is needed. For the microscopic examination, take a small amount of the culture solution and observe it under a microscope to check the activity and purity of the yeast cells. The expansion culture is when more bacterial liquid is needed. In this case, the activated bacterial liquid needs to be inoculated into more YPD medium and continue to be cultured under the same conditions until the required amount is reached.

[0177] Further, the centrifugation separation step is to perform centrifugation separation on the culture solution of the activated Kluyveromyces marxianus strain.

[0178] Specifically, the equipment used in the centrifugation separation step is a high-speed refrigerated centrifuge, centrifuge tubes, and a sterile operating table; the material for centrifugation separation is the culture solution of the activated Kluyveromyces marxianus strain.

[0179] Specifically, the method of centrifugation separation is as follows: First, prepare and load the tubes. Dispense the culture solution into centrifuge tubes according to the amount, with equal liquid volume in each centrifuge tube. Then seal the centrifuge tubes and place them on the rotor of the centrifuge, ensuring symmetric placement. Next, set the centrifugation conditions of the high-speed refrigerated centrifuge, specifically, the centrifuge speed is 5000 r / min and the centrifugation time is 10 min. Then carry out centrifugation operation, that is, start the centrifuge and perform 10 min of centrifugation separation; after centrifugation ends, slowly turn off the centrifuge and take out the centrifuge tubes. Finally, remove the supernatant and collect the bacterial cells. Carefully pour out the supernatant, avoiding disturbing the precipitated bacterial cells, and reserve the precipitated part (yeast cells) for use.

[0180] Specifically, a high-speed refrigerated centrifuge is used in the centrifugation separation step. It is necessary to ensure that the temperature is set at 4 °C to maintain the activity of the yeast. Before centrifugation, it is necessary to check whether the centrifuge tubes are balanced to avoid equipment damage and failure. Maintain aseptic operation throughout the centrifugation separation process to prevent external contamination.

[0181] Further, the step of resuspending with sterile normal saline is to resuspend the precipitated Kluyveromyces marxianus cells with sterile normal saline; preferably, the concentration of the cell suspension is adjusted to 1.5×10 8 CFU / mL. This can ensure the sterility and appropriate concentration of the cell suspension, providing a high-quality cell source for the subsequent fermentation process.

[0182] Specifically, the tools for the step of resuspending with sterile normal saline are sterile pipettes or pipettors, spectrophotometers or plate counting method devices; the materials for the step of resuspending with sterile normal saline are sterile normal saline (0.85% NaCl) and the precipitated Kluyveromyces marxianus cells after centrifugation.

[0183] Specifically, the method of resuspending with sterile normal saline is as follows: First, prepare sterile normal saline, formulate it into a 0.85% NaCl solution, sterilize it and cool it to room temperature. Then resuspend the cells. First, carefully discard the supernatant in the centrifuge tube, and then use a sterile pipette to add an appropriate amount of sterile normal saline to the centrifuge tube, and gently mix it to completely resuspend the precipitated cells. Next, adjust the concentration. The optical density measurement method or the direct counting method can be used. The optical density measurement method is to transfer a part of the cell suspension to a spectrophotometer cuvette and measure the OD 600 value; according to the measured OD value, dilute or concentrate the cell suspension with sterile normal saline to adjust it to the target OD value (for example, 0.1 - 0.2). The direct counting method is to take an appropriate amount of cell suspension, perform serial dilution, spread it on an agar medium, and culture it at 28°C for 24 h; then count the number of colonies, calculate the number of colonies in the original cell suspension according to the dilution factor; finally, adjust the concentration of the cell suspension according to the counting result. (In the scheme described in the present invention, preferably, the concentration is adjusted to 1.5×10 8 CFU / mL).

[0184] Specifically, the whole process of resuspending with sterile normal saline needs to be carried out under aseptic conditions, and mix well during the resuspension process to prevent cell aggregation from affecting measurement and subsequent use. Mix well during the resuspension process to prevent cell aggregation from affecting the measurement result.

[0185] S5. Inoculation

[0186] Inoculation is to evenly spray the prepared cell suspension onto the surface of sterile coffee beans, so that the surface of the coffee beans is covered with a certain concentration of fermenting cells, ensuring the smooth progress of the subsequent fermentation process.

[0187] The equipment used for inoculation is a sterile sprayer or atomizer, a sterile tray or fermentation container, and a sterile operating table; the materials for inoculation are the prepared cell suspension (the optimal concentration is 1.5×10 8(CFU / mL) and the aforementioned aseptic coffee beans (variety: Yunnan Arabica - Caturra Washed - Commercial Beans).

[0188] The method of inoculation is as follows: First, prepare a sterile tray or fermentation container in a sterile operation table or sterile room, and lay the sterilized coffee beans flat. Then, spray the bacterial suspension. Pour the prepared bacterial suspension into a sterile sprayer, gently shake the bacterial suspension to ensure uniform distribution; starting from one end of the tray or container, evenly spray the bacterial suspension onto the surface of the coffee beans, and gradually move to the other end to ensure that all coffee beans are evenly covered. Next, it is necessary to confirm the inoculation concentration. Use the plate counting method to confirm that the concentration of bacteria on the surface of the inoculated coffee beans is 6 - 7 lg CFU / mL, that is, the sample inspection is completed.

[0189] During the inoculation, it is necessary to ensure that the bacterial suspension evenly covers all coffee beans, avoiding excessive or insufficient amounts in local areas, which may affect the fermentation effect; the entire inoculation process must be aseptic operation, and the sprayer and container used need to be sterilized in advance to ensure sterility.

[0190] S6. Fermentation

[0191] Fermentation is a process of biological transformation of the inoculated coffee beans in a controlled environment. By controlling the temperature and humidity, it promotes the growth and metabolism of the fermenting bacteria, generating unique flavor compounds.

[0192] The equipment for fermentation is a sterile tray or fermentation container and a constant temperature room (with temperature and humidity control functions); the material for fermentation is the inoculated coffee beans.

[0193] The method of fermentation is as follows: First, prepare a constant temperature room, set the temperature to 28 ± 2°C, and the relative humidity to over 85%; then place the coffee beans. Evenly spread the inoculated coffee beans in a sterile tray or fermentation container, maintaining an appropriate gap; and move the tray or container into the constant temperature room and place it in a well - ventilated position. Next, carry out fermentation. The fermentation time is 24 - 48 h, and during this period, regularly check the temperature and humidity to ensure the stability of the environmental conditions.

[0194] During the fermentation, it is necessary to monitor the fermentation environment, regularly check the temperature and humidity of the constant temperature room, and adjust if necessary to ensure that the fermentation conditions are always within the set range; in large - scale coffee production, different batches of fermentation should be treated separately to avoid cross - contamination affecting the fermentation effect and quality.

[0195] S7. Add rose flowers

[0196] In the middle stage of coffee bean fermentation, rose petals sterilized by ultraviolet light are added in layers at 10-15% of the mass of green coffee beans to further enrich the flavor of coffee and give it a unique rose aroma. The tool for adding rose flowers is a sterile fermentation tank; the materials used for adding rose flowers are coffee beans after inoculation and 24-hour fermentation and rose petals after ultraviolet sterilization.

[0197] The method of adding rose petals is as follows: First, prepare rose petals and weigh the rose petals at a ratio of 10-15% (for example, 1 kg of green coffee beans requires 100-150 g of rose petals); then, in a sterile environment, put the rose petals into an ultraviolet sterilization device and sterilize them for 15-30 minutes. Then, add the rose petals after ultraviolet sterilization in layers. First, evenly spread a part of the coffee beans on the bottom of the fermentation tank, and then evenly sprinkle a layer of rose petals; keep repeating the above steps until all the coffee beans and rose petals are added in layers. Finally, it is necessary to check and ensure that the rose petals in each layer are evenly distributed so that each coffee bean can fully contact the rose petals and absorb their aroma.

[0198] The whole process of adding rose petals must be kept under sterile operation to prevent external contamination; at the same time, it is necessary to ensure that the rose petals are evenly distributed in each layer to achieve the best aroma penetration effect.

[0199] Furthermore, when adding rose petals during the coffee fermentation process, it is necessary to ensure that the rose petals are free of miscellaneous bacteria and pathogens. Therefore, ultraviolet sterilization of the rose petals before addition is a necessary step.

[0200] The tools for ultraviolet sterilization are ultraviolet sterilization lamps, clean trays, and sterile containers; the materials for ultraviolet sterilization are 100-150 g of rose petals (taking 1 kg of green coffee beans as an example).

[0201] The method of ultraviolet sterilization is as follows: First, select fresh, pest-free, and pollution-free rose petals, gently wash and dry them to ensure that there is no soil or impurities on the surface. Then, evenly spread the washed and dried rose petals on a clean, light-proof tray; it is necessary to ensure that the petals do not overlap so that ultraviolet light can evenly irradiate the surface of each petal. Then, prepare an ultraviolet sterilization lamp (UV-C lamp), place the tray within the irradiation range of the ultraviolet sterilization device, and ensure that each petal can be irradiated by ultraviolet light. During the ultraviolet sterilization process, turn on the ultraviolet sterilization lamp and irradiate the rose petals. Usually, the irradiation time is 15-30 minutes to ensure that the ultraviolet intensity and irradiation time are sufficient throughout the process to effectively kill bacteria, molds, and other microorganisms on the surface of the petals. Finally, after the sterilization treatment, place the rose petals in a sterile container or packaging bag to avoid secondary contamination. If the rose petals cannot be used immediately, it is best to store them under sterile conditions or use them in the coffee fermentation process as soon as possible.

[0202] S8. Secondary fermentation

[0203] In the secondary fermentation stage, the coffee beans added with roses are further fermented to ensure that the aroma of roses is fully incorporated into the coffee beans, while further developing and optimizing the flavor of the coffee beans.

[0204] The equipment used for each direct shot in the secondary fermentation is a sterile tray or fermentation container and a constant temperature room (with temperature and humidity control functions); the material in the secondary fermentation is the coffee beans after adding roses.

[0205] The method of the secondary fermentation is as follows: First, prepare a constant temperature room, set the temperature to 28±2°C, and the relative humidity to more than 85%. Then carry out the secondary fermentation. Evenly spread the coffee beans with added flowers in layers on a sterile tray or fermentation container, keeping an appropriate gap; then move the tray or container into the constant temperature room and place it in a well-ventilated position. The temperature in the constant temperature room needs to be maintained at 28±2°C and the humidity at more than 85% throughout the fermentation process. The fermentation time is 48 - 60h. Check the temperature and humidity regularly during the fermentation period to ensure the stability of the environmental conditions.

[0206] In the secondary fermentation, the coffee beans need to be evenly spread in the fermentation container to avoid excessive stacking, which may affect air circulation and fermentation uniformity; at the same time, regularly check the temperature and humidity in the constant temperature room and adjust if necessary to ensure that the fermentation conditions are always within the set range. In the scenario of large-scale coffee bean preparation, the fermentation of different batches should be treated separately to avoid cross-contamination affecting the fermentation effect and quality.

[0207] S9. Sealing

[0208] Sealing is to ensure that the contents of the fermentation tank are not invaded by external pollutants (especially microorganisms) after the secondary fermentation is completed, while allowing the gas generated during the fermentation process to be discharged smoothly. Use special equipment to fix the sealing film at the mouth of the fermentation tank to ensure a tight fit and no leakage. In this way, both the aseptic environment inside the fermentation tank is maintained and the smooth gas exchange is ensured.

[0209] The equipment used for sealing is a sealing machine, a sealing film for microbial culture made of polytetrafluoroethylene (PTFE) and polypropylene (PP) materials with pores, and a sealed fermentation tank; the material for sealing is the rose coffee after secondary fermentation.

[0210] The method of sealing is as follows: First, prepare the sealing film, select a sealing film of appropriate size, and ensure its cleanliness and no damage. Then place the sealing film at the mouth of the fermentation tank and fix it with a sealing machine; to ensure firm fixation, clamps and compression rings are needed to ensure that the sealing film fits tightly against the mouth of the fermentation tank. Finally, check and confirm that the sealing film is firmly fixed and there is no air leakage; check by detecting pressure changes or using a gas leak detector.

[0211] S10. Separation of bean curd flowers

[0212] The separation of bean curd flowers is to pick out the rose petals from the coffee beans after fermentation and only retain the fermented coffee beans. This step is to ensure that the final product (coffee beans with rose flavor) does not contain rose petals, but retains the aroma and flavor of roses to the greatest extent.

[0213] The equipment used for the separation of bean curd flowers is sterile tweezers, a sterile sieve, a sterile container, and a sterile workbench; the materials for the separation of bean curd flowers are coffee beans (containing rose petals) that have been fermented twice.

[0214] The method of separating bean curd flowers is as follows: First, lay a sterile mat on the sterile workbench and prepare sterile tweezers, a sieve, and a container; then carry out manual selection and screening. Pour the coffee beans and rose petals in the fermentation tank onto the sterile workbench, and use sterile tweezers to carefully pick out the rose petals and put them into a sterile container; then use a sieve for preliminary separation, gently shake the sieve to separate the coffee beans and petals. Finally, carefully check the coffee beans on the sieve and the workbench to ensure that all petals have been separated.

[0215] S11. Drying

[0216] Drying is a crucial step in reducing the moisture content of the fermented coffee beans to less than 12%. Appropriate drying can not only preserve the flavor of the coffee beans but also extend their shelf life and prevent the growth of mold and other microorganisms.

[0217] The equipment used in the drying process is drying equipment (a dryer or an oven), a sterile tray, and a moisture meter or a precision electronic scale; the materials in the drying process are rose-flavored coffee beans that have been fermented twice and separated from bean curd flowers.

[0218] The method of drying is as follows: First, prepare the drying environment, set the temperature of the drying equipment to 40 - 45 °C, and prepare a sterile tray. Spread the coffee beans evenly on the tray. Then conduct moisture measurement, take samples every 2 hours to measure the moisture content of the coffee beans until the moisture content is less than 12%. Finally, carry out cooling and storage. After drying is completed, take out the coffee beans and let them cool naturally to room temperature in a sterile environment; then put the cooled coffee beans into a sealed container for storage.

[0219] During the drying process, the coffee beans need to be turned regularly to ensure even heating and water evaporation. The drying temperature is strictly controlled at 40-45°C to prevent the impact of high temperature on the quality of coffee beans.

[0220] S12. Roasting

[0221] By strictly controlling the roasting temperature and time, the fermented rose coffee beans are roasted to 205-218°C, and medium-light roasted coffee beans can be obtained. Roasting can maximize the retention of the flavor and aroma of coffee beans, giving them rich flavor layers and a pleasant drinking experience.

[0222] The equipment used in the roasting is a drum coffee roaster; the material for roasting is the fermented rose coffee beans obtained from the above fermentation steps.

[0223] The roasting method is as follows: First, preheat the roaster, set the inlet temperature of the roaster to 180°C, and preheat the roaster to the set temperature. Then, put the fermented rose coffee beans into the preheated drum roaster. Furthermore, adjust the firepower to 60%, maintain a stable heat input to ensure that the coffee beans

[0224] are evenly heated. At the same time, set the roasting time to 8-12 minutes, and pay attention to observing the color change and aroma release of the coffee beans during this period. Gradually increase the temperature during the roasting process, and the optimal outlet temperature is 205-218°C. Pay attention to observing the color change of the coffee beans during this period. Preferably, it is medium-light roasted rose coffee beans. Finally, after reaching the target temperature, quickly take out the rose coffee beans from the roaster and perform rapid cooling to complete the preparation of this rose coffee bean.

[0225] Furthermore, through the above fermentation method, a rose-flavored coffee is prepared. This is a fermented coffee with obvious rose flower fragrance, having a sweet taste like brown sugar, a soft acidity with a sweet aftertaste, and tones of almond, chocolate, and nut. The rose flower fragrance can be felt from high temperature to low temperature, and the tasting score is 81.25 points.

[0226] Specific example of using Damascus rose flowers (dry flower petals) as an additive in Example 2

[0227] As Figure 2 shown, this example is a fermentation method for Damascus rose flower flavor coffee, which is specifically as follows:

[0228] First, in the pretreatment stage, select Yunnan small - bean coffee - Catimor washed - commercial beans as raw materials, remove various defective beans and impurities, then re - hydrate the coffee beans. Soak them in a fermentation tank with water at room temperature in a bean - to - water ratio of 1:3 until they turn gray - white and have a uniform color. Then sterilize them. After sealing with a sealing film, sterilize at 105 - 115 °C for 5 - 10 min, and then cool naturally. Enter the fermentation stage. First, prepare a bacterial suspension. Centrifuge the activated Kluyveromyces marxianus yeast strain and adjust the concentration to 1.5×10 8 CFU / mL with sterile physiological saline, and then inoculate to make the inoculation concentration reach 6 - 7 lgCFU / mL. After inoculation, transfer the coffee beans to a constant - temperature room and ferment at 28 ± 2 °C and humidity above 85% for 24 h. Then add Yunnan Damascus rose petals that have been sterilized by ultraviolet light and account for 10 - 15% of the mass of the green coffee beans for secondary fermentation for 48 - 60 h. During the sterilized fermentation stage, seal with a specific sealing film, then separate the bean curd, pick out the rose petals and keep the coffee beans. Then dry at 40 - 45 °C until the moisture content is less than 12%. Finally, perform roasting. Put 300 g of fermented rose coffee beans into a drum - type coffee roaster, with the bean - inlet temperature of 180 °C, the firepower of 60%, roast for 8 - 12 min, and the best out - of - pot temperature of 205 - 218 °C, thus obtaining medium - light roasted rose - flavored coffee beans.

[0229] Test results: The cup - tested rose fragrance has rich layers, strong flavor, and mellow rose fragrance.

[0230] Cup - testing score: 81.25 points. Flavor description: Strong rose fragrance, almond flavor, chocolate, nuts, slightly sour, slightly bitter, sweet aftertaste, long aftertaste, and rose fragrance can be felt from high temperature to low temperature.

[0231] Conclusion: Damascus rose flowers (dry flower petals) have the best effect on preparing this rose coffee and are the preferred production plan for high - end rose coffee products.

[0232] Example 3: Specific example using Rosa rugosa Thunb. (dry flower petals) as an additive (aroma - enhancing additive control example)

[0233] As Figure 2 shown, this example is a fermentation method for Rosa rugosa Thunb. - flavored coffee, specifically as follows:

[0234] First, in the pretreatment stage, select Yunnan small - bean coffee - Catimor washed - commercial beans as raw materials, remove various defective beans and impurities. Then, rehydrate the coffee beans. Soak them in a fermentation tank with water at room temperature at a bean - to - water ratio of 1:3 until they turn gray - white and have a uniform color. Then, sterilize them. After sealing with a sterile sealing film, sterilize at 105 - 115 °C for 5 - 10 minutes, and then cool naturally. Enter the fermentation stage. First, prepare the bacterial suspension. After centrifuging the activated Kluyveromyces marxianus yeast strain, add sterile physiological saline to adjust the concentration to 1.5×10^8 CFU / mL, and then inoculate to make the inoculation concentration reach 6 - 7 lgCFU / mL. After inoculation, transfer the coffee beans to a constant - temperature room and ferment at 28 ± 2 °C and a humidity of more than 85% for 24 hours. Then, add Carthamus tinctorius petals that have been sterilized by ultraviolet light and account for 10 - 15% of the mass of the green coffee beans for a secondary fermentation for 48 - 60 hours. During the sterilized fermentation stage, seal with a specific sealing film, then separate the coffee pulp to pick out the rose petals and retain the coffee beans. Then, dry at 40 - 45 °C until the moisture content is less than 12%. Finally, roast. Put 300 g of fermented rose coffee beans into a drum - type coffee roaster. The temperature when putting into the pot is 180 °C, the firepower is 60%, roast for 8 - 12 minutes, and the best temperature when taking out of the pot is 205 - 218 °C, thus obtaining medium - light roasted rose - flavored coffee beans.

[0235] Test results: The rose fragrance in the cup - tasting is plain, and the layering of the rose fragrance is not prominent.

[0236] Cup - tasting score: 80.25 points. Flavor description: Slight rose flower fragrance, caramel, nuts, slightly sour, slightly bitter, sweet aftertaste, short aftertaste, and no rose flower fragrance in the later stage.

[0237] Conclusion: Carthamus tinctorius (dry flower petals) has a general effect on preparing this rose coffee and is used as an ordinary alternative product.

[0238] Example 4: Specific example using Dianhong rose (dry flower petals) as an additive (aroma - enhancing additive control example)

[0239] As Figure 2 shown, the fermentation method of this example for Dianhong rose - flavored coffee is as follows:

[0240] First, in the pretreatment stage, select Yunnan small - bean coffee - Catimor washed - commercial beans as raw materials, remove various defective beans and impurities. Then, rehydrate the coffee beans. Soak them in a fermentation tank with water at room temperature at a bean - to - water ratio of 1:3 until they turn gray - white and have a uniform color. Then, sterilize them. After sealing with a sterile sealing film, sterilize at 105 - 115 °C for 5 - 10 minutes, and then cool naturally. Enter the fermentation stage. First, prepare the bacterial suspension. After centrifuging the activated Kluyveromyces marxianus yeast strain, add sterile physiological saline to adjust the concentration to 1.5×10 8CFU / mL, and then inoculate to make the inoculation concentration reach 6 - 7 lgCFU / mL. After inoculation, transfer the coffee beans to a constant temperature room and ferment at 28 ± 2°C and humidity above 85% for 24 h. Then add Dianhong rose petals that have been sterilized by ultraviolet light and account for 10 - 15% of the mass of the raw coffee beans for secondary fermentation for 48 - 60 h. During the sterilized fermentation stage, seal with a specific sealing film, then separate the bean curd, pick out the rose petals and retain the coffee beans. Then dry at 40 - 45°C until the moisture content is less than 12%. Finally, perform roasting. Put 300 g of fermented rose coffee beans into a drum coffee roaster, with the bean inlet temperature of 180°C and the fire power of 60%, roast for 8 - 12 min, and the optimal out-of-pot temperature is 205 - 218°C, thus obtaining medium-light roasted rose-flavored coffee beans.

[0241] Test results: The cup aroma of roses is strong, but the aroma layer of roses is single.

[0242] Cup score: 80.75 points. Flavor description: Obvious rose flower fragrance, almond flavor, caramel, nuts, slightly sour, slightly bitter, sweet aftertaste, the aftertaste is flat, and there is a weak rose flower fragrance in the latter stage.

[0243] Conclusion: Dianhong rose (dry flower petals) has a general effect on the preparation of this rose coffee and is used as an ordinary alternative product.

[0244] Example 5: An example using Phnom Penh rose (dry flower petals) as an additive (aroma-enhancing additive control example)

[0245] As Figure 2 shown, this example is a fermentation method for Phnom Penh rose-flavored coffee, specifically as follows:

[0246] First, in the pretreatment stage, select Yunnan small bean coffee - Caturra washed - commercial beans as raw materials, remove various defective beans and impurities, then rehydrate the coffee beans, soak them in a fermentation tank at a bean-to-water ratio of 1:3 with water at room temperature until they are grayish-white and have a uniform color, and then sterilize. After sealing with a sterile sealing film, sterilize at 105 - 115°C for 5 - 10 min, and then cool naturally. Enter the fermentation stage. First, prepare a bacterial suspension. After centrifuging the activated Kluyveromyces marxianus strain, add sterile physiological saline to adjust the concentration to 1.5×10 8CFU / mL, and then inoculate to make the inoculation concentration reach 6 - 7 lgCFU / mL. After inoculation, the coffee beans are transferred to a constant temperature room and fermented at 28 ± 2°C and a humidity of over 85% for 24 h. Then, add sterilized golden-edge roses accounting for 10 - 15% of the mass of the green coffee beans for secondary fermentation for 48 - 60 h. During the sterilized fermentation stage, seal with a specific sealing film, then separate the bean curd to pick out the rose petals and retain the coffee beans. Then dry at 40 - 45°C until the moisture content is less than 12%. Finally, perform roasting. Put 300 g of fermented rose coffee beans into a drum coffee roaster, with the inlet temperature of 180°C and the firepower of 60%, roast for 8 - 12 min, and the optimal outlet temperature is 205 - 218°C, thus obtaining medium-light roasted rose-flavored coffee beans.

[0247] Test results: The rose fragrance in the cup test is weak, and there is almost no rose fragrance. The comprehensive evaluation of the test fails.

[0248] Cup test score: 77.5 points. Flavor description: Light floral fragrance, caramel, slightly sour, slightly bitter, astringency, slightly sweet aftertaste, no aftertaste.

[0249] Conclusion: Golden-edge roses (dry flower petals) are not effective for preparing this rose coffee and are not suitable for mass production.

[0250] Example 6 Example using Pichia kudriavzevii as the fermentation bacterium (fermentation strain control example)

[0251] In this example, Pichia kudriavzevii is used as the fermentation bacterium, and the other steps and processes are the same as those in the fermentation method of the aforementioned Kluyveromyces marxianus strain.

[0252] As Figure 2 shown, the fermentation method of rose-flavored coffee using Pichia kudriavzevii as the fermentation bacterium in this example is as follows: First, in the pretreatment stage, select Yunnan small bean coffee - Caturra washed - commercial beans as raw materials, remove various defective beans and impurities, then rehydrate the coffee beans, soak them in a fermentation tank at a bean-to-water ratio of 1:3 with water at room temperature until they are grayish-white and have a uniform color, and then sterilize them. Seal with a sealing film and sterilize at 105 - 115°C for 5 - 10 min, and then cool naturally. Enter the fermentation stage. First, prepare a bacterial suspension. Centrifuge the activated Pichia kudriavzevii and adjust the concentration to 1.5×10 8CFU / mL, and then inoculate to make the inoculation concentration reach 6-7 lgCFU / mL. After inoculation, the coffee beans are transferred to a constant temperature room and fermented at 28±2°C and a humidity of more than 85% for 24 h. Then, add 10-15% of the mass of the raw coffee beans of Yunnan Damask rose petals sterilized by ultraviolet light for secondary fermentation for 48-60 h. During the sterilized fermentation stage, all are sealed with a specific sealing film, and then the rose petals are separated by bean curd to pick out the rose petals and retain the coffee beans. Then, it is dried at 40-45°C until the moisture content is lower than 12%. Finally, roasting is carried out. Take 300 g of fermented rose coffee beans and put them into a drum-type coffee roaster. The bean inlet temperature is 180°C, the firepower is 60%, and roasting is carried out for 8-12 min. The best out-of-pot temperature is 205-218°C, so as to obtain rose-flavored coffee beans with a medium-light roast.

[0253] Test results: Cup testing of rose coffee fermented with Pichia kudriavzevii. The quality of this coffee bean is stable, but the overall flavor is on the sour side.

[0254] Cup testing score: 80.5 points. Flavor description: obvious rose flower fragrance, almond flavor, chocolate, nuts, citrus acid, slightly bitter, sweet aftertaste, long aftertaste, and the fragrance decays in the later stage.

[0255] Conclusion: Using Pichia kudriavzevii to prepare this rose coffee has a stable effect, but the sour taste is too heavy. The degree of retention and integration of the rose flower fragrance is not as good as that of Kluyveromyces marxianus. The total score for the coffee with a sour taste in the tasting is 80.5, which is not the optimal production plan.

[0256] Example 7 Example using the raw material's own bacteria as the fermenting bacteria (fermentation strain control example)

[0257] In this example, the raw material's own bacteria are used as the fermenting bacteria, and the other steps and processes are the same as those in the fermentation method of the aforementioned Kluyveromyces marxianus strain.

[0258] The details are as follows: First, in the pretreatment stage, select Yunnan small - bean coffee - washed - Catimor - commercial beans as raw materials, remove various defective beans and impurities. Then, rehydrate the coffee beans. Soak them in a fermentation tank with water at room temperature in a bean - to - water ratio of 1:3 until they turn grayish - white and have a uniform color. Transfer the coffee beans to a constant - temperature room and ferment them at 28 ± 2 °C and a humidity of over 85% for 24 hours. After that, add 10 - 15% of Yunnan Damascus rose petals that have been sterilized by ultraviolet light based on the mass of the raw coffee beans and conduct a secondary fermentation for 48 - 60 hours. During the sterilized fermentation stage, seal them with a specific sealing film. Then, separate the bean curd and pick out the rose petals, retaining the coffee beans. Next, dry them at 40 - 45 °C until the moisture content is lower than 12%. Finally, conduct roasting. Put 300 g of fermented rose coffee beans into a drum - type coffee roaster, with the bean - inlet temperature of 180 °C, a firing power of 60%, roast for 8 - 12 minutes, and the optimal discharging temperature is 205 - 218 °C, thus obtaining light - medium roasted rose - flavored coffee beans. (Except for killing the microorganisms in the raw materials and inoculating fermentation bacteria, the other operations are the same as the patent method)

[0259] Test results: There was a foul smell during the fermentation process, a large amount of foam on the surface. At the end of fermentation, it was found that some of the coffee beans turned green, with musty, pickled - like and other abnormal smells. The test failed and no cup - testing score was carried out.

[0260] Conclusion: The raw materials carry various microorganisms, using their own bacteria as fermentation bacteria is not only difficult to control the fermentation, but also poses food - safety risks. It is not recommended to use and is not suitable for production.

[0261] Example 8: Example using Lactobacillus plantarum as the fermentation bacteria (fermentation strain control example)

[0262] In this example, Lactobacillus plantarum is used as the fermentation bacteria, and the other steps and processes are the same as those in the fermentation method of the aforementioned Kluyveromyces marxianus yeast strain.

[0263] As Figure 2 shown, the fermentation method of rose - flavored coffee using Lactobacillus plantarum as the fermentation bacteria in this example is as follows: First, in the pretreatment stage, select Yunnan small - bean coffee - washed - Catimor - commercial beans as raw materials, remove various defective beans and impurities. Then, rehydrate the coffee beans. Soak them in a fermentation tank with water at room temperature in a bean - to - water ratio of 1:3 until they turn grayish - white and have a uniform color. Then, sterilize them, seal them with a sealing film and sterilize at 105 - 115 °C for 5 - 10 minutes, and then cool naturally. Enter the fermentation stage. First, prepare a bacterial suspension. Centrifuge the activated Lactobacillus plantarum bacteria and adjust the concentration to 1.5×10 8CFU / mL, and then inoculate to make the inoculation concentration reach 6-7 lgCFU / mL. After inoculation, the coffee beans are transferred to a constant temperature room and fermented at 28±2°C and a humidity of more than 85% for 24 hours. Then, add 10-15% of the mass of the raw coffee beans of Yunnan Damask rose petals sterilized by ultraviolet light for secondary fermentation for 48-60 hours. During the sterilized fermentation stage, seal with a specific sealing film, then separate the bean curd, pick out the rose petals and retain the coffee beans, then dry at 40-45°C until the moisture content is less than 12%, and finally roast. Put 300 g of fermented rose coffee beans into a drum coffee roaster, with the bean inlet temperature of 180°C, the firepower of 60%, roast for 8-12 minutes, and the best out-of-pot temperature of 205-218°C, so as to obtain medium-light roasted rose-flavored coffee beans.

[0264] Test results: The rose coffee fermented with Lactobacillus plantarum has a strong acid taste in the cup test. The fusion effect of the rose flower fragrance and the coffee fragrance is not as good as that of yeast. Since lactic acid bacteria grow rapidly, it is very easy to cause over-fermentation; and negative flavors are produced.

[0265] Cup test score: 78.25 points. Flavor description: Weak rose flower fragrance, nuts, sharp acid, slightly bitter, slightly aftertaste, short aftertaste, and no rose flower fragrance at the end.

[0266] Conclusion: The rose coffee prepared with Lactobacillus plantarum has a relatively strong sour taste, the fusion effect of the rose flower fragrance is not as good as that of yeast, and the total score of the coffee bean acidity tasting is 78.25. It is very easy to cause over-fermentation and is not suitable for production.

[0267] Furthermore, the specific mechanism of the fermentation method of a rose-flavored coffee of the present invention is as follows: Specifically, the fermentation method of a rose-flavored coffee includes a pretreatment stage, a fermentation stage, and a subsequent treatment stage.

[0268] Furthermore, the pretreatment stage is a crucial step in the fermentation process. The present invention creates an ideal initial environment for the subsequent fermentation through scientific treatment methods, ensuring the quality and safety of coffee beans before the start of fermentation.

[0269] Specifically, in the selection of coffee beans, selecting coffee beans without defects can ensure the quality and consistency of the final product. Defective beans may carry bad flavors or toxins, affecting the taste and safety of coffee. In particular, removing defective beans can reduce the introduction of impurities and bad microorganisms, reduce the competition and pollution risks of miscellaneous bacteria during the fermentation process, and improve the growth advantage of the target strain (Kluyveromyces marxianus strain).

[0270] Specifically, during the rehydration treatment of coffee beans, the coffee beans absorb moisture during the rehydration process, enabling the moisture content inside the beans to reach a uniform and appropriate level. Subsequently, the beans with sufficient moisture are more readily utilized by microorganisms, facilitating the growth and metabolism of microorganisms during the fermentation process. The entire rehydration treatment renders the external color and luster of the coffee beans uniform, such that each coffee bean can undergo fermentation evenly during the fermentation process, thereby avoiding the situation of over-fermentation or under-fermentation of some beans.

[0271] Specifically, during the sterilization process, high-temperature sterilization treatment can effectively kill harmful microorganisms on the surface and inside the coffee beans, preventing contamination by miscellaneous bacteria during the fermentation process. This step ensures the sterility of the fermentation environment, providing good conditions for the inoculation and growth of the target strain (Kluyveromyces marxianus strain). Meanwhile, through heat-sensitive control (controlling the sterilization time (5 - 10 min) and temperature (105 - 115 °C)), it is possible to avoid the generation of off-flavors or texture changes in the coffee beans due to excessive high-temperature treatment time.

[0272] Specifically, during the cooling process, cooling to a temperature range of 25 - 35 °C can not only ensure that the growth of the inoculated Kluyveromyces marxianus strain is not inhibited by high temperature but also provide a suitable temperature environment for the Kluyveromyces marxianus strain to rapidly adapt and reproduce.

[0273] Furthermore, the process principle of the fermentation stage mainly involves the growth, metabolism of the strain, the generation of flavor substances, and the penetration of aroma components. The purpose of the entire fermentation stage is to achieve the best effects in the metabolic activities of the strain and the release of the aroma of roses through scientific control of fermentation conditions and technological steps, thereby producing coffee with a unique rose flavor.

[0274] Specifically, during the preparation of the bacterial suspension, centrifuging to remove the culture medium can reduce the impact of impurities and residues in the culture medium on subsequent fermentation, ensuring the purity of the bacterial suspension. Adjusting the concentration using sterile physiological saline can ensure that the bacterial suspension is not contaminated by other microorganisms, maintaining the activity and purity of the strain. Adjusting the concentration of sterile physiological saline to 1.5×10 8 CFU / mL can ensure that the strain has sufficient quantity and activity during inoculation, facilitating the rapid initiation and stable progress of fermentation.

[0275] Specifically, during the inoculation process, spraying the bacterial suspension can spray the prepared bacterial suspension onto the surface of sterile coffee beans, enabling the concentration of the inoculated microorganisms to reach an ideal optimal value (1.5×10 8 CFU / mL); furthermore, the Kluyveromyces marxianus strain with the optimal inoculation concentration can ensure the rapid proliferation of the strain in the initial stage of fermentation, inhibit the growth of miscellaneous bacteria, and promote fermentation growth.

[0276] Specifically, during the fermentation process, maintaining a constant temperature of 28 ± 2°C is the optimal growth temperature for Kluyveromyces marxianus, which can promote the metabolic activities of the strain and generate flavor substances. A high-humidity environment with a humidity above 85% helps to keep the surface of the coffee beans moist, promoting the growth and fermentation metabolism of the strain. Furthermore, according to the control of the fermentation environment, Kluyveromyces marxianus generates various flavor compounds through metabolic activities, such as organic acids, alcohols, esters, etc., which endow the coffee beans with a unique flavor.

[0277] Specifically, during the fermentation, dried petals of Rosa damascena are added in layers. The rose petals contain rich volatile aroma components. Through the pretreatment of ultraviolet sterilization, the sterility of the petals can be ensured, avoiding the introduction of miscellaneous bacteria. The method of adding rose petals in layers can make the aroma components evenly distributed in the fermenter, facilitating the release and penetration of the aroma.

[0278] Specifically, during the secondary fermentation, the secondary fermentation continues in a constant-temperature and high-humidity environment, which helps the aroma components of the roses to fully penetrate into the coffee beans. At the same time, the strain continues to metabolize, further enriching the flavor of the coffee. Furthermore, the aroma components of the roses and the flavor substances of the coffee beans are mutually fused during the re-fermentation process, forming a unique rose-flavored coffee (i.e., the product obtained according to the fermentation method of the present invention).

[0279] Specifically, from sterilization to the end of fermentation, the fermenter is sealed with a sealing film for microbial culture. Using a sealing film for microbial culture can ensure aerobic fermentation. Because during the fermentation process of coffee beans and roses, the presence of oxygen is essential; the air permeability of the sealing film allows oxygen to enter the fermenter, promoting aerobic fermentation; and because the pore size of the sealing film is small enough, it can prevent external microorganisms from entering the fermenter, maintaining the sterility of the fermentation environment and ensuring the purity and activity of the fermentation strain. Specifically, the sealing film itself allows gases to pass freely, enabling oxygen to enter the fermenter, and carbon dioxide and other gases to exchange and escape, maintaining the aerobic environment required during the fermentation process.

[0280] Furthermore, from the overall fermentation process, the fermentation conditions of constant temperature (28 ± 2°C) and high humidity (above 85%) can simulate the optimal growth environment of the strain, promote its metabolic activities, and generate the required flavor substances. The primary fermentation and the re-fermentation after adding roses help to control the fermentation process in stages, enabling different flavor substances to be optimally generated and released at different stages.

[0281] Furthermore, in terms of fermentation kinetics and metabolic kinetics, under the fermentation conditions of constant temperature (28±2°C) and high humidity (above 85%), the inoculated strains can grow and reproduce rapidly, metabolize to generate a series of complex flavor compounds such as organic acids, alcohols, esters, etc. These compounds together endow coffee beans with a unique rose flavor. Especially during the fermentation process, there may be interactions between different microorganisms, such as co-metabolism and mutualism. Microbial interaction helps to generate richer flavor components.

[0282] Furthermore, the process principle of the subsequent treatment stage involves the separation of coffee beans and rose petals and the drying process, ensuring the integrity, pure flavor, and long-term storage stability of the fermented coffee beans. The ultimate goal is to ensure the quality and stability of the rose coffee described in the present invention.

[0283] Specifically, the separation of the flowers can achieve the integrity and pure flavor of rose coffee through physical separation and flavor protection; picking out the rose petals and sorting them from the coffee beans can ensure that each coffee bean is completely retained and prevent breakage; the aroma and flavor released by the rose petals during the fermentation process have penetrated into the coffee beans. After separating the petals, the coffee beans can maintain their unique rose flavor and avoid the petals from releasing other flavor-affecting components.

[0284] Specifically, during the drying process, reducing the moisture content of the coffee beans to less than 12% can effectively prevent the growth of molds and other microorganisms, ensuring the long-term storage and stability of the coffee beans; furthermore, the low moisture content can reduce the chance of hydrolysis and oxidation reactions occurring during the storage of the coffee beans, extending their shelf life.

[0285] Specifically, a drying temperature of 40-45°C is selected to avoid damage to the aroma components and flavor substances in the coffee beans caused by too high a temperature, and at the same time prevent the deterioration or texture change of the coffee beans caused by high temperature; furthermore, it ensures the uniform evaporation of moisture inside and on the surface of the coffee beans, avoiding the phenomenon of dry outside and wet inside.

[0286] Furthermore, for the fermentation method of a rose-flavored coffee described in the present invention, in order to achieve the fermentation conditions, the preparation of the bacterial suspension described in the present invention is an important link before the start of fermentation. Specifically, it requires three steps: activating the Kluyveromyces marxianus strain, centrifugal separation, and adding sterile physiological saline. The following is the specific principle:

[0287] Specifically, in the step of activating the fermentation strain, the Kluyveromyces marxianus strain is selected because of its excellent fermentation performance and environmental adaptability and is chosen as the fermentation strain. These strains can efficiently convert the sugars in the culture medium into the target fermentation products.

[0288] Specifically, using YPD medium (yeast extract, peptone, glucose medium) provides rich nutrients for yeast, ensuring its rapid growth and reproduction; and the autoclaving process (121 °C, 15 min) is used to sterilize the medium, ensuring a sterile environment and preventing contamination by miscellaneous bacteria. Further, in the inoculation and activation culture, inoculating the strain into a small amount of medium under sterile conditions can ensure that the medium is not contaminated by external miscellaneous bacteria; and then culturing it on a shaker at 25-30 °C and 150 rpm for 24 h to activate the yeast cells to enter the active growth state from the dormant state. Inoculation and activation culture can increase the number and activity of the bacterial cells and improve the subsequent fermentation efficiency.

[0289] Specifically, the mechanism of the centrifugal separation is to extract high-concentration yeast cells from the culture solution. The centrifugal force (RCF) generated by high-speed centrifugation can quickly sediment the yeast cells suspended in the culture solution to the bottom of the tube, forming a precipitate. After the centrifugal separation is completed, the supernatant mainly contains the medium and metabolic wastes (which need to be removed), and the precipitate part is high-concentration yeast cells (for standby).

[0290] Specifically, in the centrifugal separation step, the centrifuge uses centrifugal force to separate the yeast cells with a larger density from the culture solution; and a rotation speed of 5000 revolutions per minute and a centrifugation time of 10 minutes are sufficient to effectively precipitate the yeast cells, and the low temperature of 4 °C ensures that the activity of the yeast cells is not damaged by high temperature. Further, the whole process of centrifugal separation is carried out under sterile conditions, which can prevent the influence of any external contamination on the yeast cells, and the supernatant after centrifugation is carefully poured out, and the remaining precipitate is high-concentration yeast cells.

[0291] Specifically, in the step of resuspending with sterile normal saline, the centrifuged yeast cells are resuspended with normal saline (0.85% NaCl solution) to simulate physiological conditions, which helps to maintain the activity and stability of the yeast cells. Specifically, the osmotic pressure of the 0.85% NaCl solution is equivalent to the osmotic pressure inside the yeast cells, providing an isotonic environment. This can avoid cell lysis (in a hypotonic environment) or dehydration (in a hypertonic environment) caused by osmotic pressure differences; and then maintaining an isotonic environment can protect the integrity of the yeast cells, prevent the cell membrane from rupturing or being damaged, and then maintain the activity of the cells. Specifically, + Na - and Cl

[0292] Specifically, the NaCl solution in the physiological saline provides a microenvironment similar to the natural physiological environment of yeast cells, which is necessary for the normal physiological functions and metabolic activities of the cells. Yeast cells need to maintain basic metabolic activities during the resuspension process to maintain their viability. The stable environment provided by the physiological saline helps the smooth progress of cell metabolism.

[0293] Specifically, the 0.85% NaCl solution is relatively chemically inert and will not undergo adverse chemical reactions with the components in yeast cells, thus protecting the stability of the cells. At the same time, the physiological saline can be sterilized to provide a sterile environment and prevent external microbial contamination of yeast cells.

[0294] Furthermore, the optical density measurement method (OD6 00 value) and the direct counting method are used to determine and adjust the concentration of the bacterial suspension to 1.5×10 8 CFU / mL, which can not only quickly estimate the bacterial concentration but also provide an accurate and reliable number of bacteria. This double-verification method ensures an appropriate concentration of viable bacteria at the start of fermentation, thus guaranteeing the efficiency, stability, and ultimately the quality of the rose coffee described in the present invention during the fermentation process.

[0295] Preferably, adjusting the concentration of the bacterial suspension to the optimal value of 1.5×10 8 CFU / mL can ensure that there are sufficient viable bacteria at the start of fermentation, thus quickly entering the fermentation state and contributing to a stable and controllable fermentation process; furthermore, this precise concentration value of 1.5×10 8 CFU / mL can ensure the repeatability and controllability of each fermentation process, which is particularly important for industrial food production.

[0296] Furthermore, for the fermentation method of a rose-flavored coffee described in the present invention, the specific mechanisms and characteristics of the raw material coffee beans, the added dried rose flower petals, and the fermentation strain are as follows:

[0297] In terms of the selection of coffee bean raw materials, for the fermentation method of a rose-flavored coffee described in the present invention, the coffee bean variety used is Yunnan small bean - Caturra washed - commercial bean, and the specific characteristics are as follows:

[0298] Specifically, the washed commercial Caturra Timor coffee bean is a coffee variety obtained by crossing the Caturra variety and the Timor variety, with high yield and disease resistance. The washed commercial Caturra Timor coffee bean is a high-yield fermentation raw material, avoiding the seasonality of coffee fresh fruits and providing an adequate raw material basis for microbial fermentation. Using this as the raw material can ensure fermentation production throughout the year, facilitating the monitoring of microbial dynamics under different fermentation conditions, and thus ensuring a stable output of coffee quality. The flavor characteristics of the washed commercial Caturra Timor coffee bean are closer to those of Arabica, enabling it to produce richer and more complex flavor substances during the fermentation process. Specifically, the original flavor of the washed commercial Caturra Timor coffee bean has a strong sour taste and a weak aroma; after fermentation, the sour taste of the washed commercial Caturra Timor coffee bean weakens, the sweetness increases, and it has a rose aroma. Fourthly, the washed commercial Caturra Timor coffee bean is low in cost, can solve inventory backlogs, and effectively handle aged coffee beans.

[0299] Furthermore, the mechanism of coffee bean selection is to pick out defective beans such as broken beans, moldy beans, worm-eaten beans, spotted beans, shell beans, shriveled beans, and other impurities in the raw material (Yunnan small bean coffee - washed Caturra Timor - commercial bean) for later use. Broken beans are suitable for manual selection or mechanical screening because broken beans are damaged during processing or transportation, with an increased surface area, making them prone to contamination and oxidation, which affects flavor and quality; moldy beans are suitable for visually inspecting the surface of coffee beans, and beans with obvious mold spots or strange odors need to be picked out because moldy beans contain mycotoxins, which are harmful to the human body, and mold also affects the flavor of coffee; worm-eaten beans are suitable for manual selection or optoelectronic sorting technology to remove beans with wormholes because worm-eaten beans are eroded by insects and may contain insect eggs or feces inside, seriously affecting hygiene and flavor; spotted beans are suitable for visual inspection to pick out beans with abnormal spots on the surface because spotted beans are usually caused by diseases or poor environments, and beans with abnormal color spots may have quality problems; shell beans are suitable for removal after manual selection or machine screening because shell beans are beans that did not develop into complete coffee beans normally during growth, with a shape similar to a shell, usually having poor internal quality; shriveled beans are suitable for screening by size screening equipment to remove beans with too small a volume because shriveled beans are coffee beans with poor growth and development, significantly smaller in volume than normal beans, and may have differences in taste; other impurities in coffee beans are suitable for removal by physical methods such as magnetic separation and air separation because these impurities are stones, wood chips, etc. that may be mixed in during processing and transportation, which will affect the operation of processing equipment and the quality of the final product.

[0300] Furthermore, the mechanism of rehydrating the coffee beans is to allow moisture to penetrate into the coffee beans and restore the cell structure of the coffee beans, thereby achieving an ideal state of hydration. Rehydrating the coffee beans is to enable the dry coffee beans to reabsorb moisture and restore the water content close to its fresh state. Moisture penetrates through the epidermis of the coffee beans and gradually enters the internal tissues of the beans. The coffee beans are in a dry state during the previous storage and will lose a large amount of moisture. Therefore, the cell walls and cell membranes of the coffee beans will shrink. Rehydration can cause the cell structure to expand again, restoring its elasticity and softness, which is beneficial to the action of microorganisms during the subsequent fermentation process. A uniform rehydration process can ensure that each coffee bean can absorb moisture equally and reach an ideal state of hydration, thus making the subsequent fermentation process more uniform and controllable.

[0301] Furthermore, the sterilization of the coffee beans is a key step in the pretreatment stage of fermenting the coffee beans. The purpose is to eliminate harmful microorganisms on the surface and inside of the coffee beans to ensure the purity and controllability of the subsequent fermentation process. Sealing with the sterile sealing film means putting the pretreated coffee beans into a high-temperature resistant sterile container and using the sterile sealing film to seal the container to ensure that external contaminants cannot enter during the sterilization process. Therefore, the sterile sealing film can effectively prevent miscellaneous bacteria and particles in the air from entering the container during the sterilization process and maintain the sterile state inside the container. The high-temperature sterilization is to put the sealed container into a high-temperature sterilization device (such as an autoclave or an oven), set the temperature at 105 - 115 °C, and the sterilization time is 5 - 10 min. This conforms to the principle of thermal sterilization, that is, high temperature can destroy the cell membranes and proteins of microorganisms, causing them to lose their activity and reproductive ability; and the set temperature range of 105 - 115 °C is sufficient to kill most bacteria, yeasts, and molds without causing excessive damage to the coffee beans themselves. The sterilization time of 5 - 10 min can ensure sufficient heat transfer to the inside of the coffee beans to completely kill potential pathogenic microorganisms, while avoiding the negative impact of long-term high temperature on the quality of the coffee beans.

[0302] In terms of the selection of additives, for the fermentation method of a rose-flavored coffee of the present invention, the variety of dried rose flowers added during the fermentation process is Yunnan edible rose - Damascus rose (preferred), and the specific mechanism is as follows:

[0303] Specifically, Damascus rose is famous for its strong and complex aroma. Its main aroma components include aromatic alcohols and aromatic esters. During the fermentation process, aromatic alcohols (such as geraniol and nerol) have a sweet floral fragrance and can form a complement with the alcohol and ester flavor substances in the coffee; aromatic esters (such as ethyl phenylacetate and methyl methacrylate) can provide complex aroma levels, combine with the acids and alcohols in the coffee, and enhance the overall flavor of the coffee.

[0304] Specifically, during the fermentation process, Kluyveromyces marxianus can promote the release and transformation of aroma components in Rosa damascena. Kluyveromyces marxianus contains a variety of microbial enzymes that can degrade the rose flower cell wall and release aromatic compounds; Kluyveromyces marxianus can combine the aromatic alcohols in roses with the organic acids in coffee to generate new aromatic ester compounds, enhancing flavor complexity. Further, as described in the preparation method of the present invention, the control of fermentation conditions can optimize the release and transformation of rose aroma components. A lower temperature is beneficial for retaining and releasing floral fragrance components and avoiding aroma loss caused by over-fermentation; appropriately extending the fermentation time (including secondary fermentation) can ensure the full release of aroma components and their integration with coffee flavor.

[0305] Specifically, the synergistic effect between the aroma components of Rosa damascena and the original flavor components of coffee. The synergistic effect between the aroma components of Rosa damascena and the original flavor components of coffee; at the same time, the sweet fragrance of roses can neutralize the bitterness and acidity in coffee, making the coffee taste more balanced and mellow.

[0306] Preferably, Yunnan edible roses are selected as raw materials for fermentation. As for the additive 0 of flavored coffee, when comparing the coffee beans fermented with Dianhong roses, Carmine roses and Rosa damascena, the rose flavor in the coffee fermented with Rosa damascena is obvious and integrates well with the original coffee flavor. Therefore, Rosa damascena (dry flower petals) is the most preferred additive variety.

[0307] Comparatively, compared with rose hydrosol, rose liquid and rose essence, the edible rose - Rosa damascena dry flowers added in the present invention have a better integration of flower fragrance and coffee flavor. The integration degree of additives such as essence and coffee flavor is low, showing a quality of strong brewing color and light taste, without aftertaste.

[0308] In comparison, rose hydrosol, rose liquid and rose essence are made by extracting or synthesizing rose aroma components. These components can be chemically extracted from ornamental roses or obtained by chemical synthesis. Although rose hydrosol, rose liquid and rose essence can be directly added to coffee, there is controversy. Specifically, the naturally extracted rose aroma is more in line with the natural and healthy expectations of high-end boutique coffee, but the cost is high, the ingredients are complex and the stability is poor; while the chemically synthesized rose aroma components are low in cost, simple in ingredients, and have low acceptance in high-end and boutique coffees, and are usually not added. Specifically, natural rose extracts are usually more complex and delicate, but there may be large differences between different batches, affecting the consistency of coffee products; while synthetic flavors can provide a stable aroma, but lack the layering and complexity of natural aromas. Specifically, natural extracts are generally considered safer, but may also contain certain allergens or impurities; and although synthetic flavors have undergone rigorous safety assessments, the long-term health assessment results are unstable. Specifically, whether natural or synthetic, the addition of flavors in coffee is controversial, especially in the high-end and boutique coffee markets, where the tasting trend is more inclined to not add any flavors to maintain the pure flavor of coffee.

[0309] Furthermore, the mechanism of adding rose petals in layers is to ensure that the coffee beans fully absorb the aroma of roses, and to generate new aromatic compounds through enzymatic reactions, making the coffee flavor more complex and rich. Adding rose petals in layers effectively utilizes the aromatic components of roses and enhances the overall flavor performance of coffee.

[0310] Specifically, adding rose petals in layers can ensure that the aroma of roses is evenly permeated into each coffee bean, thereby increasing the contact surface between coffee beans and rose petals, and facilitating better transmission and absorption of aroma components. During the aforementioned fermentation process, aromatic compounds (such as aromatic alcohols and aromatic esters) in rose petals are released under the action of microbial enzymes and evenly diffused into coffee beans, allowing the coffee beans to absorb the aroma of roses.

[0311] Specifically, from the perspective of microbial enzyme action, the enzymes secreted by the fermentation strain can promote the decomposition of the cell walls of rose petals and release the aromatic compounds therein; under the enzymatic reaction, the aromatic compounds diffuse into the coffee beans through the fermentation medium. Under the action of microbial enzymes, the aromatic alcohols of roses combine with the organic acids in coffee to achieve aroma transformation and form new aromatic ester compounds, further enriching the flavor of coffee.

[0312] Specifically, the rose petals are added in layers so that each layer of coffee beans can fully contact the rose petals, ensuring that every part of the coffee beans can evenly absorb the rose aroma. During the fermentation process, the rose petals at different layers gradually release the aroma, ensuring the continuous penetration and stable release of the aroma throughout the fermentation process.

[0313] In terms of the selection of fermentation strains, for the method of fermenting a rose-flavored coffee according to the present invention, the method for obtaining Kluyveromyces marxianus strains and the specific mechanism are as follows:

[0314] The method for obtaining Kluyveromyces marxianus according to the present invention mainly goes through three steps: strain screening, isolation, purification and preservation of the strain, and identification of the strain.

[0315] Further, the strain screening process goes through three links: sample collection, sample treatment, and plate coating. Specifically, in the sample collection link, the Kluyveromyces marxianus strain sample is collected from traditional fermented food, sour meat, in Lincang City, Yunnan Province, China, and stored refrigerated; specifically, in the sample treatment link, after weighing the sample, it is put into sterile physiological saline and mixed evenly to make dilution solutions with different gradients; specifically, in the plate coating link, the dilution solutions with different concentration gradients are coated on YPD agar plates and cultured at 28°C for 24-48 h.

[0316] Further, the specific mechanism of the raw material for obtaining the strain (traditional fermented food, sour meat, in Lincang City, Yunnan Province, China) is as follows: The traditional fermented food, sour meat, is used as a sample. Sour meat contains a rich microbial community. These microorganisms have adapted to the specific environment for a long time and have diversity and uniqueness; collecting samples from this environment can obtain strains with special fermentation properties. Refrigerated storage of the sour meat sample can inhibit the growth and metabolic activities of microorganisms, prevent changes in the sample during transportation and storage, maintain the original state of the sample, and contribute to accurate subsequent separation and identification.

[0317] Further, the specific mechanism of the sour meat sample treatment is as follows: First, the sour meat sample is put into sterile physiological saline and mixed evenly, which can ensure the uniform distribution of microorganisms in the sample, so that individual colonies can be more evenly separated during subsequent dilution and culture processes. Further, the gradient dilution method is used for dilution with different gradients, which can effectively reduce the interference of miscellaneous bacteria in the sample, making it easier to separate and purify the target strain. In particular, high-dilution gradients can reduce the density of colonies, making it easier to identify and select individual colonies. Furthermore, the gradient dilution method can disperse the microorganisms in the sample through dilution, increasing the chance of contact between the target strain and the culture medium, thereby improving the separation efficiency.

[0318] Further, the mechanism included in the strain screening is as follows: The screening of the target strain is through gradient dilution and plate coating with different dilution degrees to ensure that individual colonies can be separated, which can increase the probability of screening the target strain. The culture environment is set at a culture temperature of 28°C and the YPD agar plate provides the nutrients and environment required for the growth of Kluyveromyces marxianus.

[0319] Furthermore, the isolation, purification, and preservation of the strain went through three steps: colony selection, streak plating, and slant culture. Specifically, in the colony selection step, single colonies with good growth were picked based on the morphology, size, and color of the colonies. Specifically, in the streak plating step, the strain was further purified by the streak plating method. Specifically, in the slant culture, the purified strain was transferred to a test tube slant for culture and stored at low temperature.

[0320] Furthermore, the mechanism involved in the isolation, purification, and preservation of the strain is as follows: In the streak plating step, through repeated streak culture, the purity of the strain is ensured and contamination by miscellaneous bacteria is avoided. For slant culture, low-temperature preservation is adopted. Preservation at 4°C and in glycerol tubes at -80°C can maintain the activity and stability of the strain, facilitating subsequent experimental use.

[0321] Furthermore, the strain identification went through three steps: nucleic acid extraction, PCR amplification, and sequence analysis. Specifically, in nucleic acid extraction, the isolated bacteria were treated with sodium hydroxide solution to extract 16S rRNA for amplification. Specifically, in PCR amplification, specific primers were used for PCR amplification and Sanger sequencing was performed. Specifically, in sequence analysis, accurate sequence information was obtained through alignment analysis and software splicing, and blastn was used to compare the nucleic acid database to obtain the Accession Number of homologous sequences and species identification.

[0322] Furthermore, the mechanism involved in the strain identification is: As Figure 1 shown, the 16S rRNA gene is the standard for bacterial molecular identification. The strain can be accurately identified through PCR amplification and sequencing. By using the latest version of the nucleic acid database for comparison, precise species identification and annotation information can be obtained.

[0323] In terms of the application of the fermenting strain, for the fermentation method of a rose-flavored coffee described in the present invention, the fermenting strain used is the Kluyveromyces marxianus strain as the dominant strain. The specific mechanism is as follows:

[0324] Firstly, Kluyveromyces marxianus contains various microbial enzymes, and these enzymes play important roles in the metabolic process of fermentation. The first category is carbohydrate-metabolizing enzymes, specifically including: α-glucosidase, which is used to break down starch and glycogen to generate glucose; alcohol dehydrogenase, which is involved in the production and metabolism of ethanol; hexokinase, which converts glucose to glucose-6-phosphate during glycolysis. Specifically, the following is a chemical reaction showing that alcohol dehydrogenase (ADH) can oxidize ethanol to acetaldehyde:

[0325] CH3CH2OH + NAD + →CH3CHO + NADH + H +

[0326] Specifically, the following chemical reaction shows that α-glucosidase can hydrolyze starch or glycogen into glucose:

[0327] (C6H 1O O5) n +H2O→(C6H 12 O6)

[0328] The second category is organic acid metabolic enzymes, specifically including: citrate synthase, which participates in the tricarboxylic acid cycle and combines acetyl-CoA with oxaloacetic acid to generate citric acid; lactate dehydrogenase, which can reduce pyruvate to lactate and is particularly active under anaerobic conditions. Specifically, the following chemical reaction shows that citrate synthase can generate citric acid in the tricarboxylic acid cycle:

[0329] CH3CO-SCoA + C4H4O4 → C6H8O7 + CoA-SH

[0330] The third category is lipid metabolic enzymes, specifically including: lipase, which can decompose fat to generate fatty acids and glycerol; acetyl-CoA carboxylase, which can carboxylate acetyl-CoA to malonyl-CoA in the initial step of fatty acid synthesis. The fourth category is amino acid metabolic enzymes, specifically including: glutamate dehydrogenase, which can convert glutamate to α-ketoglutarate in amino acid metabolism; enzymes related to the Ehrlich pathway, including enzymes that convert amino acids to higher alcohols (such as isopentanol). The fifth category is esterifying enzymes, specifically including: alcohol acetyltransferase, which can combine ethanol and acetyl-CoA to generate esters such as ethyl acetate.

[0331] Second, Kluyveromyces marxianus can regulate the red-ox metabolism. Under aerobic fermentation conditions, Kluyveromyces marxianus can still carry out the red-ox metabolism and continue to use NAD + / NADH and NADP + / NADPH as key cofactors to support energy metabolism and biosynthesis processes.

[0332] Specifically, under aerobic conditions, NAD + / NADH and NADP + / NADPH continue to maintain the redox balance in the cell. NADH is generated in glycolysis and the tricarboxylic acid cycle (TCA cycle), and electrons are transferred to oxygen through the electron transport chain (ETC) to generate water and ATP, and this process is called oxidative phosphorylation.

[0333] The following key chemical reactions show NADH generation and oxidative phosphorylation:

[0334] (1) Glycolysis: C6H2O6 + 2NAD + + 2ADP + 2Pi → 2C3H4O3 + 2NADH + 2ATP + 2H2O

[0335] During glycolysis, glucose is broken down into pyruvate, while generating NADH and ATP.

[0336] (2) Tricarboxylic acid cycle (TCA cycle): C3H4O3 + CoA + NAD + → C2H3OCoA + CO2 + NADH

[0337] Pyruvate is converted to acetyl-CoA and NADH is produced.

[0338] 1) Acetyl-CoA + Oxaloacetate → Citrate

[0339] 2) Citrate → Isocitrate → a-Ketoglutarate → Succinyl-CoA → Succinate

[0340] → Fumarate → Malate → Oxaloacetate

[0341] In the TCA cycle, acetyl-CoA is further oxidized to generate more NADH and FADH2.

[0342] The following chemical reactions show the electron transport chain (ETC) and oxidative phosphorylation: (3) NADH + H + + 1 / 2O2 → NAD+ + H2O NADH is oxidized in the electron transport chain, and the released electrons pass through a series of protein complexes and finally transfer to oxygen to generate water. At the same time, ATP synthesis is driven by the proton gradient.

[0343] The following chemical reactions show the generation and use of NADPH: (4) Pentose phosphate pathway (PPP): Glucose-6-phosphate + NADP + → 6-Phosphoglucono-o-lactone + NADP + → 6-Phosphoglucono-δ-lactone + NADPH + H +

[0344] NADPH is mainly generated in the pentose phosphate pathway and is used for biosynthesis and antioxidant defense.

[0345] Specifically, aerobic conditions contribute to the generation of more volatile organic compounds (such as esters and aldehydes), which play an important role in the aroma of coffee. In particular, higher alcohols and esters can form unique floral and fruity aromas under specific conditions. Under aerobic conditions, the metabolic pathway of yeast tends to produce secondary metabolites, such as phenylethyl alcohol (a higher alcohol with a rose aroma), which is very important for the presentation of rose flavor.

[0346] The following chemical reactions show the formation of phenylethyl alcohol:

[0347] (1) Phenylalanine metabolism: Phenylalanine → Phenylpyruvate → Phenylacetaldehyde → Phenylethanol

[0348] Phenylalanine forms phenylethyl alcohol through a series of reactions, which is a higher alcohol with a rose aroma.

[0349] The following key chemical reaction formula shows the formation of phenylethyl acetate:

[0350] (2) Esterification reaction: Phenylethanol + Acetyl-CoA → Phenylethyl acetate + CoA

[0351] Phenylethanol reacts with acetyl coenzyme A to form phenylethyl acetate, giving coffee a unique floral aroma.

[0352] Thirdly, Kluyveromyces marxianus plays a key role in the preparation of rose-flavored coffee through its efficient fermentation ability of hexoses (such as glucose). The volatile organic compounds such as higher alcohols and esters generated by its metabolic activities bring unique floral aromas and tastes to coffee. As mentioned above, by optimizing the fermentation conditions in the use of Kluyveromyces marxianus, the generation of these aroma components can be further improved, thus producing high-quality rose-flavored coffee. Therefore, Kluyveromyces marxianus becomes an ideal fermentation strain for the preparation of flavored coffee. The following chemical reactions show the formation of these aroma components:

[0353] (1) Glycolysis reaction of glucose: C6H 12 O6 → 2C3H4O3 + 2ATP + 2NADH

[0354] Glucose (C6H 12 06) is decomposed into two molecules of pyruvate (C3H4O3) in the glycolysis pathway, while generating 2 ATP and 2 NADH molecules.

[0355] (2) Formation of phenylethyl alcohol (rose aroma component): C9H 10 O ← Phenylalanine

[0356] Phenethyl alcohol (C9H 10 O) is produced by the conversion of phenylalanine in the yeast metabolic pathway. This process involves multiple steps, including deamination and reduction reactions.

[0357] (3) Formation of phenethyl acetate (floral fragrance component): C9H1O2 + C2H4O2 → C6H5CH2OCOCH3 + H2O

[0358] Phenethyl acetate (C6H5CH2OCOCH3) is produced by the reaction of phenethyl alcohol and acetic acid (C2H4O2) in an esterification reaction.

[0359] Thirdly, by selecting specific Kluyveromyces marxianus, the chemical reactions occurring during fermentation can be targeted controlled, thereby achieving selective fermentation.

[0360] Specifically, Kluyveromyces marxianus can direct the formation of flavor compounds through the Ehrlich pathway and the acetate pathway:

[0361] (1) Ehrlich pathway: Amino acids are converted into higher alcohols under the action of yeast and then further converted into flavor compounds such as esters. Specific chemical formula: Amino Acid → Fusel Alcohol → Ester

[0362] Leucine contained in Kluyveromyces marxianus is converted into isopentyl alcohol through the Ehrlich pathway and then further converted into isopentyl acetate.

[0363] (2) Acetate pathway: Yeast combines ethanol with acetyl-CoA to produce acetate esters such as ethyl acetate. Specific chemical formula: CH3CO-SCoA + C2H5OH → CH3COOC2H5 + CoA.

[0364] Second, Kluyveromyces marxianus can exclude the interference of harmful microorganisms. Through selective fermentation, Kluyveromyces marxianus can ferment under strictly controlled aseptic conditions, excluding the interference of miscellaneous bacteria and harmful microorganisms that may exist in the environment. The method of directed fermentation ensures that only specific yeasts dominate the fermentation process, reducing the risk of competition and contamination by miscellaneous bacteria. Specifically, Kluyveromyces marxianus can grow rapidly and metabolize efficiently. As mentioned above, Kluyveromyces marxianus can grow rapidly and efficiently metabolize sugars and other organic substances, producing specific flavor compounds such as alcohols, esters, and acids. This efficient metabolic ability ensures the rapid progress of the fermentation process and a stable flavor output. Specifically, Kluyveromyces marxianus has strong environmental adaptability and can remain active within a wide range of temperatures and pH values, enabling it to perform well under different fermentation conditions. Therefore, Kluyveromyces marxianus can ensure stable operation in the coffee bean project (industry) and ensure the consistency of fermentation. Specifically, Kluyveromyces marxianus has the ability to quickly occupy the fermentation environment, inhibit the growth of other microorganisms, thereby reducing the risk of contamination by harmful microorganisms and ensuring the purity and safety during the fermentation process.

[0365] Furthermore, for the fermentation method of a rose-flavored coffee described in the present invention, determinations were made for various fermentation indicators (the determination methods were disclosed in the foregoing invention content), and the specific determination results are as follows:

[0366] Determination 1: Influence of different inoculation concentrations on the sensory score of coffee

[0367] According to Figure 3 (The trend chart of the influence of inoculation concentration on the sensory score of coffee) and Figure 4 (The cupping score and standard deviation chart at different inoculation concentrations (log10CFU / mL)), which shows the influence of inoculation concentration on the sensory score of coffee and includes the standard deviation (error bar) for each score.

[0368] Specifically, from the trend of the cupping score, the score gradually increases when the inoculation concentration is 4 - 6 lgCFU / mL. After reaching the peak at 6 lgCFU / mL, the score starts to decline as the inoculation concentration continues to increase. Especially when the inoculation concentration is 6 lgCFU / mL, the cupping score reaches the highest, which is 79.75 points.

[0369] Specifically, from the standard deviation, when the inoculation concentration is 6 lgCFU / mL, the standard deviation of the score is the smallest, only 1.94. This indicates that at this concentration, the sensory scores of the coffee are more consistent and stable; while when the inoculation concentration is 7 lgCFU / mL, the standard deviation is the largest, 4.29, indicating that at this concentration, the fluctuations of the sensory scores are larger and the consistency of the scores is poor.

[0370] Furthermore, at low inoculum concentrations (4 lgCFU / mL and 5 lgCFU / mL), the score for 4 lgCFU / mL was 72.25 points and the score for 5 lgCFU / mL was 75.25 points. This indicates that at low inoculum concentrations, the number of strains is small and the fermentation process is relatively slow, resulting in insufficient fermentation. Furthermore, due to incomplete fermentation, the generation of flavor substances (such as organic acids, esters, etc.) in coffee beans is insufficient, and the flavor of coffee is not rich and complex enough, and the taste is relatively plain.

[0371] Furthermore, at a medium inoculum concentration (6 lgCFU / mL), the score was 79.75 points, which was the optimal inoculum concentration. This indicates that the medium inoculum concentration provides sufficient active strains, enabling the fermentation process to reach the optimal state and the flavor substances to be fully generated. And at the medium inoculum concentration, the flavor performance of coffee is the most rich and balanced, with a moderate acidity and a long aftertaste, reflecting the best taste of coffee.

[0372] Furthermore, at high inoculum concentrations (7 lgCFU / mL and 8 lgCFU / mL), the score for 7 lgCFU / mL was 77.0 points and the score for 8 lgCFU / mL was 71.5 points. This indicates that at high inoculum concentrations, the number of strains is too large, and the fermentation process is too fast or excessive, resulting in the accumulation of adverse metabolites. Excessive fermentation produces too much sourness or other off-flavors, leading to an unbalanced taste of coffee, and the flavor is too sharp or complex, affecting the overall sensory experience.

[0373] Conclusion: It can be seen from the measured data that when the inoculum concentration is 6 lgCFU / mL, the sensory score of coffee is the highest and the stability is the best, indicating that at this concentration, the fermentation process of coffee is the most ideal and the flavor is the best. This concentration can not only fully ferment the flavor substances in coffee beans, but also avoid the adverse effects brought by excessive fermentation.

[0374] Determination 2: Effect of fermentation temperature on the sensory score of coffee

[0375] According to Figure 5 (Trend chart of the effect of fermentation temperature on the sensory score of coffee) and Figure 6 (Cupping score and standard deviation chart at different fermentation temperatures), which shows the effect of fermentation temperature on the sensory score of coffee, with the standard deviation (error bar) of each score attached.

[0376] Specifically, from the trend of the cupping score, the score gradually increases when the fermentation temperature is 20°C to 28°C, reaches the peak at 28°C, and then starts to decline as the fermentation temperature continues to increase. Especially when the fermentation temperature is 28°C, the cupping score reaches the highest, which is 80.25 points.

[0377] Specifically, in terms of the standard deviation, when the fermentation temperature is 28°C, the standard deviation of the score is the smallest, which is 2.11. This indicates that at this temperature, the sensory scores of the coffee are more consistent and stable. And

[0378] when the fermentation temperature is 32°C, the standard deviation is the largest, which is 4.01, indicating that at this temperature, the fluctuations of the sensory scores are relatively large and the consistency of the scores is poor.

[0379] Furthermore, in the case of low-temperature fermentation (20°C and 24°C), it may lead to relatively low strain activity, a slower fermentation process, less flavor substances produced, and the coffee taste may be relatively plain.

[0380] Furthermore, under the optimal temperature fermentation (28°C), the strain activity is the best, the fermentation process is moderate, rich flavor substances are generated, the flavor of the coffee is complex and balanced, and the taste is the best.

[0381] Furthermore, under high-temperature fermentation (32°C and 36°C), it is easy to cause too fast fermentation, generating bad flavor substances, such as excessive sourness or bitterness, which affects the overall taste of the coffee. At the same time, the consistency of fermentation at high temperature is poor and the score fluctuations are relatively large.

[0382] Conclusion: Considering the scores and standard deviations comprehensively, when the fermentation temperature is 28°C, the coffee has the highest sensory score and the best stability, which is the optimal fermentation temperature. This temperature can not only fully stimulate the strain activity and generate rich flavor substances, but also avoid the out-of-control fermentation and flavor deterioration caused by high temperature.

[0383] Measurement 3: Influence of fermentation time on the sensory score of coffee

[0384] According to Figure 7 (Trend chart of the influence of fermentation time on the sensory score of coffee) and Figure 8 (Cup test score and standard deviation chart at different fermentation times), which shows the influence of fermentation time on the sensory score of coffee, with the standard deviation (error bar) of each score attached.

[0385] Specifically, in terms of the trend of cup test scores, the scores gradually increase from 36h to 72h of fermentation time, reach the peak at 72h, and then start to decline as the fermentation time continues to increase. Especially when the fermentation time is 72h, the cup test score reaches the highest, which is 81.50 points.

[0386] Specifically, in terms of the standard deviation, when the fermentation time is 36h and 60h, the standard deviations of the scores are relatively small, which are 2.17 and 2.21 respectively. This indicates that at these two time points, the sensory scores of the coffee are more consistent and stable. However, when the fermentation time is 48h, the standard deviation is the largest, which is 2.94, indicating that at this time point, the fluctuations of the sensory scores are relatively large and the consistency of the scores is poor.

[0387] Furthermore, in the case of short-time fermentation (36h and 48h), the score at 36h is 73.5 points and at 48h is 77.75 points. It can be seen that short-time fermentation easily leads to insufficient strain activity, a shorter fermentation process, incomplete generation of flavor substances, and the coffee taste may be relatively bland.

[0388] Furthermore, in the case of optimal-time fermentation (60h and 72h), the score at 60 hours is 79.00 points and at 72 hours is 81.50 points. It can be seen that within the optimal-time fermentation (60h and 72h), the strain activity is the best, the fermentation process is moderate, the generated flavor substances are rich, the flavor of the coffee is complex and balanced, and the taste is the best.

[0389] Furthermore, in the case of long-time fermentation (above 84h), the score is 76.25 points. It can be seen that long-time fermentation may lead to over-fermentation, generating adverse flavor substances, such as excessive sourness or bitterness, which affects the overall taste of the coffee. At the same time, the consistency of long-time fermentation is poor, and the score fluctuates greatly.

[0390] Conclusion: Considering the score and standard deviation comprehensively, when the fermentation time is 72h, the sensory score of the coffee is the highest and the stability is better, which is the optimal fermentation time. This time can not only fully stimulate the strain activity and generate rich flavor substances, but also avoid the flavor deterioration caused by long-time fermentation.

[0391] Measurement 4: Influence of rose addition amount on the sensory score of coffee

[0392] According to Figure 9 (Trend chart of the influence of rose addition amount on the sensory score of coffee) and Figure 10 (Cup test score and standard deviation chart under different rose addition amounts), which shows the influence of rose addition amount on the sensory score of coffee, with the standard deviation (error bar) of each score attached.

[0393] Specifically, from the trend of the cup test score, the score gradually rises when the rose addition amount is 5% - 10%, reaches the peak at 10%, and then starts to decline as the rose addition amount continues to increase. Especially when the rose addition amount is 10%, the cup test score reaches the highest, with a value of 80.00 points.

[0394] Specifically, from the standard deviation, when the rose addition amount is 5%, the standard deviation is the largest, with a value of 4.153, indicating that at this addition amount, the fluctuation of the sensory score is large and the consistency of the score is poor. However, when the rose addition amount is 15%, the standard deviation is the smallest, with a value of 2.410, indicating that at this addition amount, the fluctuation of the sensory score is small and the consistency of the score is good.

[0395] Furthermore, at low addition levels (5% and 8%), the scores were 73.50 for 5% and 74.25 for 8%. It can be seen that at low addition levels, the rose flavor is relatively weak and does not significantly enhance the overall flavor of the coffee.

[0396] Furthermore, at the optimal addition level (10%), the score was 80.00. It can be seen that at the optimal addition level (10%), the aroma and flavor of the rose are perfectly integrated with the coffee, with a complex and balanced flavor and the best taste.

[0397] Furthermore, at high addition levels (12% and 15%), the scores were 75.00 for 12% and 72.75 for 15%. It can be seen that at high addition levels, the rose flavor may be too strong, masking the original flavor of the coffee, causing the overall taste of the coffee to be unbalanced and reducing the sensory score instead.

[0398] Conclusion: Considering the score and standard deviation comprehensively, when the rose addition level is 10%, the coffee has the highest sensory score and good stability, which is the optimal rose addition level. This can perfectly integrate the aroma and flavor of the rose with the coffee, enhancing the overall sensory experience of the coffee. This addition level can fully bring out the aroma of the rose without masking the original flavor of the coffee.

[0399] Measurement 5: pH change during fermentation

[0400] According to Figure 11 (pH change trend graph during fermentation) and Figure 12 (data chart of pH change during fermentation), which show the pH change during fermentation, and the pH values and their standard deviations of the fermentation group and the control group at different time points are marked respectively.

[0401] Specifically, in terms of the pH change trend, at the initial stage (0 h), the pH values of the fermentation group and the control group were close, 5.90 and 5.87 respectively; after 24 h, the pH value of the fermentation group dropped to 5.10, and the control group dropped to 4.94; after 48 h, the pH value of the fermentation group slightly rebounded to 5.19, and the control group continued to drop to 4.71; after 72 h, the pH value of the fermentation group further rebounded to 5.43, and the control group slightly rebounded to 4.73.

[0402] Specifically, in terms of the standard deviation, at different time points, the standard deviation of the pH value of the fermentation group remained small, with a range of 0.01 to 0.02, indicating good consistency in the pH value measurement of the fermentation group. However, the standard deviation of the pH value of the control group also remained small, with a range of 0.01 to 0.02, indicating good consistency in the pH value measurement of the control group.

[0403] Furthermore, the pH change in the fermentation group is as follows: In the initial stage of fermentation, microorganisms are active and produce a large amount of organic acids (such as lactic acid, acetic acid, etc.), resulting in a rapid decrease in pH value; in the middle stage of fermentation, the production and consumption of acidic substances reach equilibrium, and the pH value remains relatively stable; in the late stage of fermentation, some microorganisms may start to utilize organic acids or produce some alkaline metabolites (such as ammonia), resulting in a rise in pH value. It can be seen that during the fermentation process, the pH value of the fermentation group decreased significantly in the first 24 hours because microorganisms produce organic acids during fermentation, leading to a decrease in pH value. However, as the fermentation time extends, the pH value rises slightly, which may be due to a reduction in certain metabolic activities or the production of certain alkaline metabolites.

[0404] Furthermore, in the control group, no specific fermenting microorganisms were inoculated, but natural microorganisms in the environment still metabolized to produce acidic substances, resulting in a decrease in pH value. In the later stage, the pH value of the control group rose slightly, which may be due to a reduction in the metabolic activities of some natural microorganisms or some chemical reactions resulting in the generation of alkaline substances. It can be seen that the pH value of the control group continued to decrease in the first 48 hours, indicating that even under non-fermentation conditions, certain chemical or microbial activities in the sample were still ongoing, leading to the production of acidic substances. After 72 hours, the pH value of the control group rose slightly but was still lower than that of the fermentation group.

[0405] Conclusion: In actual production, the pH change during the fermentation process should be closely monitored, and by adjusting factors such as fermentation time, temperature, and inoculum size, the stability of the fermentation process and product quality can be ensured. Regularly measuring the pH value during the fermentation process can promptly detect and correct abnormal situations that occur during fermentation, ensuring the smooth progress of fermentation.

[0406] Determination 6: Growth of microorganisms during fermentation

[0407] According to Figure 13 (Microbial growth trend chart during fermentation) and Figure 14 (Data chart of strain growth at different fermentation times), which show the growth of microorganisms during the fermentation process, and the number changes of fermenting bacteria and total bacteria at different time points are respectively marked.

[0408] Specifically, in terms of the change trend of the number of bacteria, in the initial stage (0 h), the numbers of fermenting bacteria and total bacteria were close, being 6.290 and 6.371 lgCFU / mL respectively; after 24 h, the number of fermenting bacteria increased to 7.648 lgCFU / mL, and the total bacteria increased to 7.885 lgCFU / mL; after 48 h, the number of fermenting bacteria increased significantly to 9.255 lgCFU / mL, and the total bacteria increased to 9.423 lgCFU / mL; after 72 h, the number of fermenting bacteria further increased to 9.710 lgCFU / mL, and the total bacteria increased to 9.758 lgCFU / mL.

[0409] Specifically, in terms of the growth rate, in the first 24 hours, the number of microorganisms increased significantly, indicating that the microorganisms were in the rapid growth phase at the initial stage of fermentation; between 24 and 48 hours, the number of microorganisms continued to increase, but the growth rate slowed down, entering the steady-state growth phase; between 48 and 72 hours, the number of microorganisms approached stability, and the growth rate further slowed down, indicating that it was approaching the growth saturation phase.

[0410] Furthermore, at the initial stage (0 h), when fermentation began, the number of fermenting bacteria was close to that of the total bacteria, indicating that the initial inoculation amount was relatively balanced, and the microorganisms began to adapt to the fermentation environment, preparing for subsequent rapid growth.

[0411] Furthermore, in the rapid growth phase (0 - 24 h), the number of fermenting bacteria and the total bacteria increased significantly, indicating that the microorganisms were metabolically active and reproduced rapidly; at this time, the microorganisms metabolized to produce a large amount of metabolic products such as organic acids, affecting the pH value of the fermentation environment (as shown in the previous pH value change graph).

[0412] Furthermore, in the steady-state growth phase (24 - 48 h), the number of microorganisms continued to increase, but the growth rate slowed down, indicating that the consumption of nutrients and the accumulation of metabolic products began to affect the growth rate of the microorganisms; furthermore, the microorganisms gradually adapted to the new environmental conditions and entered the stable growth stage.

[0413] Furthermore, in the growth saturation phase (48 - 72 h), the number of microorganisms approached stability, and the growth rate further slowed down, indicating that the nutrients in the environment were gradually exhausted, and the metabolic products had accumulated to a certain extent; furthermore, the competition among the microorganisms and the inhibitory effect of the metabolic products became apparent, and the growth entered the plateau phase.

[0414] Conclusion: During the fermentation process, the number of fermenting bacteria and the total bacteria both showed a typical growth curve, experiencing the rapid growth phase, the steady-state growth phase, and the growth saturation phase. The change trend of the number of fermenting bacteria was similar to that of the total bacteria, indicating that the fermenting bacteria played a dominant role throughout the fermentation process and made a significant contribution to the total number of bacteria. During the fermentation production process, the fermentation time and the growth of microorganisms should be closely monitored, and an appropriate fermentation time should be selected to ensure that the microorganisms ferment under the optimal growth state to optimize the product quality. At the same time, by combining the monitoring of other environmental parameters such as the pH value, the fermentation process can be better regulated to improve the fermentation efficiency and the flavor of rose coffee.

[0415] Measurement 7: Moisture content chart in different step stages

[0416] According to Figure 15 (Moisture content chart in different step stages), it shows that appropriate moisture content helps the growth of fermenting bacteria and the formation of flavor substances.

[0417] Specifically, in terms of the changing trend of water content, the water content of coffee beans before rehydration was 9.3%; immediately after rehydration, fermentation was carried out, and the water content increased significantly to 50.94% at 0 h of fermentation; as the fermentation time increased, the water content of coffee beans gradually increased and reached the highest 53.32% at 72 h of fermentation; after drying, the water content dropped back to 9.4%.

[0418] Specifically, in terms of the change in water content during the fermentation process, in the initial stage of fermentation (0 - 24 h), the water content increased relatively fast, from 50.94% to 51.76%; in the middle stage of fermentation (24 - 48 h), the water content continued to increase to 52.68%; in the late stage of fermentation (48 - 72 h), the water content further increased to 53.32%.

[0419] Conclusion: The water content gradually increases during the fermentation process. Appropriate water content is conducive to the growth of fermenting bacteria and the formation of flavor substances; the water content of coffee beans is the highest after 72 h of fermentation, but finally it needs to be reduced to an appropriate level (about 9.4%) after drying to ensure the storage stability of coffee beans.

[0420] Measurement 8: Comparison chart of water content of rehydration / sterilization methods

[0421] According to Figure 16 (Comparison chart of water content of rehydration / sterilization methods), it shows that different rehydration and sterilization methods have a slight impact on the final water content of coffee beans, and the key lies in the sterilization conditions.

[0422] Specifically, in terms of the water content comparison, different rehydration and sterilization methods have an impact on the final water content of coffee beans, and the water content ranges from 50.83% to 52.8%.

[0423] Preferably, the highest water content is "soaked at 4°C for 24 h, sterilized at 121°C for 15 min", which is 52.8%; the lowest water content is "sterilized at 110°C for 5 min", which is 50.83%.

[0424] Specifically, in terms of the sensory cupping evaluation, the sterilization method at a relatively high temperature and for a short time (such as sterilized at 110°C for 5 min) can obtain a relatively high sensory score.

[0425] Preferably, the highest sensory cupping score is "sterilized at 110°C for 5 min", which is 78.50 points; the lowest score is "sterilized at 95°C for 30 min", which is 71.50 points.

[0426] Specifically, in terms of the effects of rehydration and sterilization, the rehydration method (soaking at 4°C for 24 hours vs. soaking at room temperature for 24 hours) has a certain impact on the water content and sensory score, but the impact is not significant. The key lies in the sterilization conditions. However, long-term sterilization at a lower temperature (sterilization at 95°C for 30 minutes) results in the lowest sensory score because long-term low-temperature sterilization is insufficient to inhibit the production of off-flavor substances.

[0427] Conclusion: According to the sensory cupping results, select appropriate sterilization conditions (such as sterilization at 110°C for 5 minutes) to obtain the best flavor and quality.

[0428] Determination 9: Physicochemical experiment comparison of the rose-flavored coffee prepared by the present invention before and after fermentation

[0429] According to Figure 17 It is a physicochemical comparison chart of rose-flavored coffee before and after fermentation, showing the content changes of tannins, polyphenols, and flavonoids (unit: mg / g) under two conditions of fermenting bacteria and fermenting bacteria + rose flowers before and after fermentation.

[0430] Specifically, in terms of the tannin content analysis, the tannin content before fermentation is 9.314 mg / g; the tannin content after fermentation (fermenting bacteria) increases to 10.117 mg / g; after fermentation (fermenting bacteria + rose flowers): the tannin content further increases to 10.833 mg / g.

[0431] Conclusion on tannin content: The fermentation process increases the tannin content, especially in the case of adding rose flowers, the tannin content increases significantly. This may be due to the combined action of the fermentation process and certain components in the rose flowers, promoting the production or release of tannins.

[0432] Specifically, in terms of the polyphenol content analysis, the polyphenol content before fermentation is 2.965 mg / g; the polyphenol content after fermentation (fermenting bacteria) slightly decreases to 2.827 mg / g; the polyphenol content after fermentation (fermenting bacteria + rose flowers) increases to 3.024 mg / g.

[0433] Conclusion on polyphenol content: The polyphenol content slightly decreases after single fermentation, while in the case of adding rose flowers, the polyphenol content increases. Rose flowers may contain rich polyphenol substances, increasing the overall polyphenol content.

[0434] Specifically, in terms of the flavonoid content analysis, the flavonoid content before fermentation is 27.949 mg / g; the flavonoid content after fermentation (fermenting bacteria) significantly decreases to 22.857 mg / g; the flavonoid content after fermentation (fermenting bacteria + rose flowers) increases to 28.649 mg / g.

[0435] Conclusion on flavonoid content: After single fermentation, the flavonoid content decreased significantly, which might be due to partial degradation or transformation of flavonoids during the fermentation process. However, after adding rose flowers, the flavonoid content increased significantly, exceeding the level before fermentation, indicating that rose flowers are rich in flavonoids and can supplement and increase the overall flavonoid content.

[0436] Overall conclusion: First, regarding the impact of fermentation on components, the fermentation process generally leads to a slight reduction in certain components (such as polyphenols and flavonoids), probably because some components are consumed or transformed by microbial metabolism. Second, regarding the impact of rose flowers, adding rose flowers significantly increased the contents of tannins, polyphenols, and flavonoids, indicating that rose flowers are rich in these beneficial components and can make up for the loss of certain components during the fermentation process and even increase the total amount of these components. During the coffee fermentation process, adding an appropriate amount of rose flowers can significantly increase the contents of tannins, polyphenols, and flavonoids in coffee, thereby enhancing the antioxidant properties and health benefits of coffee. In addition, monitoring the changes in these components during the fermentation process can optimize the fermentation conditions and further improve the quality and flavor of coffee.

[0437] Determination 10: Effects of three different treatment methods, namely non-fermented, fermented only with fermentation bacteria, and co-fermented with fermentation bacteria and rose flowers, on the cupping score and flavor description of coffee

[0438] According to Figure 18 (Flavor description diagram of rose-flavored coffee), which shows the cupping scores and flavor descriptions of coffee under different treatment methods.

[0439] Specifically, in terms of cupping scores, the cupping score of non-fermented coffee was 78.00; the cupping score after fermentation (with fermentation bacteria) slightly increased to 79.75; the cupping score after fermentation (with fermentation bacteria + rose flowers) significantly increased to 81.25. It can be seen that the fermentation treatment, especially the fermentation treatment with the addition of rose flowers, can significantly improve the cupping score of coffee. This indicates that the fermentation process and the addition of rose flowers have a positive effect on improving the quality of coffee.

[0440] Specifically, in terms of flavor description, when unfermented, the flavor of coffee beans is light floral fragrance, bright acidity, strong sourness, bitterness, astringency, and sweet aftertaste. The characteristics of coffee beans are relatively single flavor, strong sourness, and overall taste biased towards sour and bitter, with a sweet aftertaste; after fermentation (adding fermentation bacteria), the flavor of coffee beans is nuts, spices, caramel, weak acidity, slight bitterness, and sweet aftertaste. The characteristics of coffee beans are that the flavor is more abundant, adding the flavors of nuts, spices, and caramel, the sourness is weakened, the bitterness is slightly alleviated, the overall flavor is more balanced, and there is a sweet aftertaste; after fermentation (adding fermentation bacteria + rose), the flavor of coffee beans is rose floral fragrance, almond flavor, chocolate, nuts, slight acidity, slight bitterness, and sweet aftertaste. The characteristics of coffee beans are that the flavor is the most complex, adding the flavors of rose floral fragrance, almond flavor, and chocolate, the nut flavor is more prominent, the sourness and bitterness are further weakened, the sweet aftertaste is obvious, and the overall flavor is more harmonious and unique.

[0441] Overall conclusion: First, in terms of the improvement of flavor by fermentation, the fermentation process can significantly enhance the flavor complexity and overall score of coffee. Fermentation alone makes coffee add the flavors of nuts, spices, and caramel, weakens the sourness, and makes the flavor more balanced. Second, in terms of the influence of roses, after adding roses, the flavor of coffee is more abundant and unique, adding floral fragrance, almond flavor, and chocolate flavor, making the overall flavor more complex and harmonious, and at the same time significantly improving the cupping score. Therefore, in the process of coffee production and processing, using fermentation treatment and adding roses can not only improve the sensory score of coffee, but also enrich its flavor level. This treatment method is suitable for coffee production and can meet the needs of consumers for complex flavors and high-quality coffee.

[0442] Determination 11: Compare the bioactive components of rose coffee fermented by Kluyveromyces marxianus (denoted as KF) and Pichia kudriavzevii (denoted as PF) with the raw material (denoted as YL) coffee beans

[0443] After fermentation according to the coffee bean process described in the present invention, compare the bioactive components of rose coffee fermented by Kluyveromyces marxianus (denoted as KF) and Pichia kudriavzevii (denoted as PF) with the raw material (denoted as YL) coffee beans. The changes in the contents of tannins, polyphenols, and flavonoids in coffee before and after fermentation are shown in the figure.

[0444] According to Figure 19 (The left figure in the graph of the changes in the contents of tannins, polyphenols, and flavonoids in coffee beans and unfermented coffee beans) shows the influence of different treatments during fermentation on the contents of tannins and polyphenols in coffee beans. There are three sets of data in the graph, representing unfermented coffee beans (YL), coffee beans fermented with Kluyveromyces marxianus (KF), and coffee beans fermented with Pichia kudriavzevii (PF). The experimental results are shown in Table 6 below:

[0445] Table 6 Values of tannin content and polyphenol content in different groups

[0446] Yeast used / not used Tannin content (left Y-axis, mg / g) Polyphenol content (right Y-axis, mg / g) YL (raw material) 9.5 mg / g 2.0 mg / g KF (Kluyveromyces marxianus) 9.9 mg / g 3.0 mg / g PF (Pichia kudriavzevii) 9.6 mg / g 2.5 mg / g

[0447] Determination verification 1: After fermentation, the tannin content in the KF group was the highest, followed by the PF group, and the YL group was the lowest. This indicates that the fermentation process, especially using Kluyveromyces marxianus, significantly increased the tannin content.

[0448] Determination verification 2: After fermentation, the polyphenol content in the KF group was the highest, followed by the PF group, and the YL group was the lowest. This indicates that the fermentation process, especially using Kluyveromyces marxianus, significantly increased the polyphenol content.

[0449] Conclusion: First, fermentation with strains significantly increased the tannin and polyphenol content in coffee beans. Second, the coffee beans fermented with Kluyveromyces marxianus (KF) reached the highest values in both tannin and polyphenol content. Third, Pichia kudriavzevii (PF) came second, but was still significantly higher than the unfermented raw coffee beans (YL).

[0450] According to Figure 19 (The right figure in the graph of the changes in the contents of tannin, polyphenol and flavonoid in coffee beans and unfermented coffee beans) shows the effect of different treatments during fermentation on the flavonoid content in coffee beans. There are three sets of data in the graph, representing unfermented coffee beans (YL), coffee beans fermented with Kluyveromyces marxianus (KF), and coffee beans fermented with Pichia kudriavzevii (PF). The experimental results are as follows:

[0451] Table 7 Flavonoid content values in different groups

[0452]

[0453]

[0454] Determination verification 3: In terms of the increase in flavonoid content, fermentation significantly increased the flavonoid content in coffee beans. Specifically, the coffee beans fermented with Kluyveromyces marxianus (KF) had the highest flavonoid content, reaching 26.5 mg / g; the coffee beans fermented with Pichia kudriavzevii (PF) had the second highest flavonoid content, which was 25.0 mg / g; and the unfermented raw coffee beans (YL) had the lowest flavonoid content, which was 23.5 mg / g.

[0455] Determination verification 4: Statistically, the differences in flavonoid content between groups were statistically significant (P < 0.05). The flavonoid content in the KF group and the PF group was significantly higher than that in the YL group. And the flavonoid content in the KF group was significantly higher than that in the PF group.

[0456] Conclusion: The changes in the contents of tannin, polyphenol and flavonoid in coffee before and after fermentation are as Figure 19。Inoculation with yeast fermentation had an impact on the contents of bioactive components such as tannins, polyphenols and flavonoids in rose coffee. There was a significant difference in the tannin content between the rose coffee inoculated with yeast and the raw coffee (P < 0.05). Due to the combined action of the growth of strains and certain components in rose flowers during the fermentation process, the release of tannins was promoted. The flavonoid content increased significantly after fermentation. Rose flowers are rich in flavonoids, and the flavonoid content can be increased after co-fermentation with yeast.

[0457] Determination 12: Compare the color changes of rose coffee fermented with Kluyveromyces marxianus (denoted as KF) and Pichia kudriavzevii (denoted as PF) after different fermentation treatments

[0458] According to Figure 20 (Color changes of rose coffee after different fermentation treatments), this chart shows the color changes of rose coffee after different fermentation treatments, and the brightness, red-green chromaticity and yellow-blue chromaticity of coffee beans are evaluated by L value, a value and b* value. The experimental determination results are shown in Table 8 below:

[0459] Table 8 Color changes of rose coffee after different fermentation treatments

[0460]

[0461] Determination verification 1: After fermentation, the brightness of rose coffee beans decreased significantly, especially the coffee beans fermented with Pichia kudriavzevii (PF) had the lowest brightness.

[0462] Determination verification 2: After fermentation, the red chromaticity of rose coffee beans increased significantly, especially the coffee beans fermented with Pichia kudriavzevii (PF) were the most reddish.

[0463] Determination verification 3: After fermentation, the yellow chromaticity of coffee beans increased. The coffee beans fermented with Pichia kudriavzevii (PF) had the highest yellow chromaticity, but generally the change in b value was not as significant as that of L value and a* value.

[0464] Conclusion: The impact of rose coffee fermentation on color is visible to the naked eye. The color change can be evaluated by a color difference meter. Usually, the L value represents brightness, the a value represents red-green chromaticity, and the b value represents yellow-blue chromaticity. The larger the L value, the brighter the coffee bean; the larger the a value, the redder the coffee bean; and the larger the b value, the yellower the coffee bean. From Figure 20 it can be seen that after co-fermentation with yeast and rose flowers, the L value decreased, the brightness of coffee beans decreased, the a value increased, and the coffee beans were reddish, showing a rose red color. This is because the pigments in rose flowers were decomposed and released during the fermentation process and migrated to the coffee beans, while there was no significant difference in the b* value.

[0465] Determination 13: Compare the cupping scores of rose coffee beans fermented with Kluyveromyces marxianus (denoted as KF) and Pichia kudriavzevii (denoted as PF)

[0466] According to Figure 22 (Overall Evaluation Chart of Cup Testing Results of Rose-Flavored Coffee Beans), it shows the scoring of raw coffee beans (YL) and rose coffee beans fermented with two kinds of yeast (KF and PF) on different sensory attributes. These sensory attributes include aroma, flavor, aftertaste, sweetness, consistency, acidity, balance, body, cleanliness and overall evaluation. The tasting results are shown in Table 9 below:

[0467] Table 9 Overall Evaluation of Cup Testing Results of Rose-Flavored Coffee Beans

[0468]

[0469] Tasting and determination results:

[0470] The rose coffee fermented with KF (Kluyveromyces marxianus) is superior in aroma, flavor, aftertaste, sweetness and consistency, and obtains the highest overall evaluation and total score. The cup testing flavor description of this coffee is: rose flower fragrance, almond flavor, chocolate, nuts, slightly sour, slightly bitter, sweet aftertaste.

[0471] The rose coffee fermented with PF (Pichia kudriavzevii) also scores relatively high on most sensory attributes, second only to KF. Due to the traditional washing processing method, YL (raw coffee beans) has a higher cleanliness, but the acidity is relatively sharp, and the aroma and flavor are relatively plain, resulting in a lower overall score.

[0472] Determination 14: Compare the cup testing scores of rose coffee beans fermented with Kluyveromyces marxianus (denoted as KF) and Pichia kudriavzevii (denoted as PF)

[0473] According to Figure 23 (Tasting Score Table of Rose-Flavored Coffee Beans), the table shows the scoring of raw coffee beans (YL), coffee beans fermented with Kluyveromyces marxianus (KF) and coffee beans fermented with Pichia kudriavzevii (PF) on different sensory attributes. The following is the specific analysis

[0474] Table 10 Tasting Score Table of Rose-Flavored Coffee Beans

[0475]

[0476]

[0477] According to the tasting results in Table 10: the fermentation process, especially the use of Kluyveromyces marxianus (KF), significantly improved the aroma, flavor, consistency, aftertaste, sweetness and overall evaluation of coffee. Pichia kudrida (PF) was second, while the unfermented raw coffee beans (YL) performed better in cleanliness and balance, but scored lower in other sensory attributes. Overall, fermentation significantly improved the quality of coffee.

[0478] In addition, Figure 21 This is a physical comparison of rose-flavored coffee beans and raw material Catim washed commercial beans; it compares the difference between coffee beans after special processing and coffee beans in their original state. Specifically, the left side of the figure is rose coffee (KF), which represents the rose-flavored coffee beans prepared by the present invention. This is a processed coffee bean that has gone through the steps of fermentation and drying. The beans appear purple-red because roses are added (the roses in this figure are preferably Damascus roses). Specifically, the right side of the figure is raw material (YL), which is untreated green coffee beans, with a lighter color and no obvious processing marks on the surface.

[0479] In addition, Figure 24 The process diagram of the fermentation method of rose flavored coffee visually records each key stage of this traditional food processing method, showing the transformation process from raw materials (Catim commercial washed beans) to the final dried product.

[0480] a. Post-sterilization: The coffee beans have been sterilized by boiling or steaming to eliminate any unwanted bacteria or pathogens.

[0481] b. Add roses: Mix sterilized coffee beans with roses.

[0482] c. Fermenting: The mixture of coffee beans and rose flowers is placed in tanks for fermentation; these tanks are sealed and stored under controlled conditions for fermentation.

[0483] d. End of fermentation: The fermentation process has been completed and the appearance of the coffee beans has changed. The color and texture are also different due to the effects of fermentation.

[0484] e. Drying: The fermented coffee beans are dehydrated and dried.

[0485] 15. Special instructions for the Kluyveromyces marxianus strain (CI-06) of the present invention

[0486] The source of Kluyveromyces marxianus (CI-06) in the present invention is sour meat. Herein, the defined "sour meat" is a specific food, namely a traditional characteristic meat product in Gengma Dai and Va Autonomous County, Lincang City, Yunnan Province. It is made by mixing fresh lean pork, cooked pigskin strips, glutinous rice, salt, and chili, wrapping them with phrynium leaves and fermenting for 2 - 6 days. It is usually eaten raw or can also be eaten after heating. Therefore, as long as the defined "sour meat" is used, microbial isolation and purification can be carried out therefrom to obtain the CI-06 named Kluyveromyces marxianus in the present invention.

[0487] To determine that the source of Kluyveromyces marxianus (CI-06) in the present invention is sour meat, the following are the specific characteristics of the "sour meat" defined in the present invention. It should be particularly noted that as long as under the same limiting conditions, "sour meat" with the same source as the Kluyveromyces marxianus strain (CI-06) in the present invention can be found; specifically as shown in Table 11 below.

[0488] Table 11 Main limiting conditions of the "sour meat" which is the source of the Kluyveromyces marxianus strain (CI-06) in the present invention

[0489]

[0490] (2) For the "sour meat" samples which are the source of the Kluyveromyces marxianus strain (CI-06) in the present invention, sample collection and physical and chemical index detection were carried out: Samples were taken on the 0th, 2nd, 4th, and 6th days of fermentation to determine the physical and chemical indexes, as shown in Table 12 below:

[0491] Table 12 Detection results of physical and chemical indexes in the "sour meat" samples

[0492]

[0493]

[0494] The above data prove that microbial isolation and purification can be carried out from "sour meat" at different time periods to obtain the CI-06 named Kluyveromyces marxianus in the present invention.

[0495] Furthermore, in the above experiments, microbial isolation, purification, and preservation were carried out on the collected samples. One of the strains was identified as Kluyveromyces marxianus, and the Kluyveromyces marxianus strain (CI-06) was isolated from all the samples.

[0496] Furthermore, the above-mentioned 1 strain of bacteria was identified as Kluyveromyces marxianus, that is, the Kluyveromyces marxianus described in the present invention was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. CI-06 was named Kluyveromyces marxianus, with the deposit number of CGMCC No: 30564, the deposit date of May 9, 2024, and the deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0497] (3) Differences between the Kluyveromyces marxianus (CI-06) described in the present invention and other Kluyveromyces marxianus strains

[0498] Specifically, the Kluyveromyces marxianus (CI-06) described in the present invention is not the Kluyveromyces marxianus obtained from other source matrices. Therefore, the Kluyveromyces marxianus (CI-06) successfully used in the preparation method described in the present invention is the Kluyveromyces marxianus with the deposit number of CGMCC No: 30564 recognized by the General Microbiology Center of the China Committee for Culture Collection of Microorganisms; its deposit date is May 9, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0499] In order to further illustrate the differences between the Kluyveromyces marxianus (CI-06) described in the present invention and other Kluyveromyces marxianus strains, and that other Kluyveromyces marxianus strains are not applicable to the preparation method of coffee beans described in the present invention, multiple actual examples are used for demonstration; specifically as shown in the following table:

[0500] Table 13 Differences between the Kluyveromyces marxianus (CI-06) described in the present invention and other Kluyveromyces marxianus strains

[0501]

[0502]

[0503] Furthermore, other subspecies of Kluyveromyces marxianus strains can be isolated from relevant matrices and applied, and none of them are the Kluyveromyces marxianus strain (CI-06) described in the present invention for preparing coffee beans. Application examples of other subspecies of Kluyveromyces marxianus strains are as follows:

[0504] 1. The combination of Kluyveromyces marxianus B3 with proteolytic activity and esterolytic activity and Lactiplantibacillus plantarum Z43 is used for fermenting sausage, which can effectively improve the quality of fermented sausage;

[0505] 2. Kluyveromyces marxianus Y245 was isolated and purified from the fermented grains in the middle stage of the fermentation of light-flavor Chinese liquor and applied in the brewing of Chinese liquor to reduce the content of n-propanol;

[0506] 3. Kluyveromyces marxianus was compounded with Pichia pastoris and Saccharomyces cerevisiae for the fermentation of cider, wolfberry wine, yellow rice wine, wine, etc.;

[0507] 4. The compound fermentation of Kluyveromyces marxianus and Bacillus natto can improve the fermentation flavor of natto.

[0508] The above embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A Kluyveromyces marxianus Kluyveromyces marxianus CI-06, which was deposited at the China General Microbiological Culture Collection Center on May 9, 2024. The strain deposit number is CGMCC No: 30564, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A fermentation method for preparing rose-flavored coffee using Kluyveromyces marxianus according to claim 1, characterized in that, including the following steps: S1. Coffee bean selection: Defective beans and debris in the coffee bean raw materials are screened out and reserved; S2. Coffee bean rehydration: Use water at room temperature and put it into the fermentation tank at a ratio of coffee beans to water of 1:

3. After adding water, the volume accounts for half of the total volume of the tank, and soak until the coffee beans are grayish-white and the appearance color is uniform; S3. Sterilization: Seal with a sterile sealing film, sterilize at a temperature of 105 °C to 115 °C for 5 min to 10 min; after sterilization, perform natural cooling until the temperature drops to 25 - 35 °C; S4. Preparation of bacterial suspension: After centrifuging the activated Kluyveromyces marxianus fermentation strain to discard the culture medium, add sterile normal saline to adjust the concentration to 7 - 9 lgCFU / mL; S5. Inoculation: Spray the prepared bacterial suspension onto the surface of sterile coffee beans, and the inoculated microorganism concentration reaches 6 - 7 lgCFU / mL; S6. Fermentation: After inoculation, the coffee beans are transferred to a constant temperature room, the temperature is maintained at 28 ± 2 °C, the relative humidity is 85 ± 5%, and ferment for 24 hours; S7. Addition of rose petals: Add rose petals sterilized by ultraviolet light into the fermentation tank in layers according to 10 - 15% of the mass of green coffee beans; S8. Secondary fermentation: Transfer the fermentation tank with added flowers to a constant temperature room, the temperature is maintained at 28 ± 2 °C, the relative humidity is 85 ± 5%, and ferment for 48 - 60 h; S9. Sealing: From sterilization to the end of fermentation, the fermentation tank is sealed with a sealing film for microbial culture; the pore size of this sealing film cannot pass through microorganisms, and gas can pass through freely; S10. Separation of coffee flowers: Pick out the rose petals in the coffee beans after fermentation and only keep the coffee beans; S11. Drying: Dry the coffee beans at 40 - 45 °C until the moisture content is less than 12%; S12. Roasting: Take 300 g of fermented rose coffee beans and put them into a drum coffee roaster. The temperature when putting into the pot is 180 °C, keep the firepower at 60%, roast for 8 - 12 min, and the temperature when taking out of the pot is 205 - 218 °C.

3. The fermentation method according to claim 2, wherein: In the preparation of the bacterial suspension in S4, the concentration is adjusted to 8 lgCFU / mL by adding sterile normal saline.

4. The fermentation method according to claim 2, characterized in that: The preparation of the bacterial suspension in S4 also includes pretreatment: S41. Activation of fermentation strain link: Select Kluyveromyces marxianus strain as the fermentation strain and perform activation culture in YPD medium to ensure that the Kluyveromyces marxianus strain is in the best growth state; S42. Centrifugal separation link: Centrifuge the activated Kluyveromyces marxianus strain culture solution; the centrifugation conditions are 5000 r / min for 10 min, and the centrifugation temperature is 4 °C; after centrifugation, discard the supernatant and retain the precipitated bacteria; S43. Resuspension with sterile normal saline link: Resuspend the centrifuged precipitated bacteria with sterile normal saline; the sterile normal saline is a solution with a concentration of 0.85%, and adjust the concentration of the bacterial suspension to 8 lgCFU / mL by measuring the optical density of the bacterial solution or by direct counting method.

5. The fermentation method according to claim 4, characterized in that: The formula of the YPD medium is 10 g / L of yeast extract, 20 g / L of peptone, 20 g / L of glucose, which are dissolved in 1 liter of distilled water and autoclaved. The autoclaving temperature is 121 °C and the time is 15 min.

6. The fermentation method according to claim 2, characterized in that: The addition of rose flowers in S7 also includes the following rose pre-treatment methods: S71. Proportioning rose petals: Select fresh, pest-free and pollution-free rose petals, wash and dry them to ensure that there is no soil or impurities on the surface; then calculate the mass ratio of coffee beans and rose petals, and prepare rose petals at a ratio of 10-15% of the mass of green coffee beans. S72. Ultraviolet sterilization: Spread the proportioned rose petals evenly on a clean and light-proof tray, ensuring that the petals do not overlap so that the ultraviolet light can evenly irradiate the surface of each petal; place the tray under the ultraviolet sterilization lamp, turn on the ultraviolet sterilization lamp, and irradiate for 15-30 min; after irradiation, put the petals into a sterile container for standby. S73. Laminated addition of rose petals: Evenly spread a layer of coffee beans at the bottom of the fermentation tank, accounting for one-fourth to one-third of the total amount; then, evenly sprinkle a layer of rose petals on the first layer of coffee beans to cover the coffee beans; lay another layer of coffee beans as the second layer of coffee beans to ensure that the previous layer of rose petals is covered; continue to evenly sprinkle a layer of rose petals on the second layer of coffee beans; finally, lay another layer of coffee beans to cover the rose petals above; repeat the above steps until all the rose petals and coffee beans have been added to the fermentation tank in layers.

7. The fermentation method according to claim 6, characterized in that: The variety of rose flowers added is Yunnan edible rose - Damascus rose dried flowers, and the addition amount is at a ratio of 10% of the mass of green coffee beans.

8. The fermentation method according to claim 2, characterized in that: The fermentation strain is Kluyveromyces marxianus, the total fermentation duration is 72 h, the secondary fermentation is 48 h, and the microbial concentration in the fermentation system after inoculation is 6 lgCFU / mL.

9. The fermentation method according to claim 2, characterized in that: The variety of coffee bean raw materials selected in S1 is Yunnan small coffee - Caturra washed - commercial beans.

10. A rose-flavored coffee prepared by the fermentation method of a rose-flavored coffee according to any one of claims 2-9, characterized in that, The rose-flavored coffee has the fragrance of rose flowers.

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