Rhodotorula sp. And moschus coffee bionic fermentation process

Abstract

The invention discloses a biomimetic fermentation process of rhodotorula daliensis and coffee, which is characterized in that the collection number of the rhodotorula daliensis YD202501 is CCTCC NO: M 20251796, and the purified and identified rhodotorula daliensis is inoculated into a PDB seed culture medium for high-density multiplication culture; filtering and collecting thallus precipitate, and adding a composite protective agent in proportion; preparing fermentation liquor; sterilizing the Ktill coffee mung beans at high temperature and high pressure; sealing and cooling to room temperature; inoculating a leavening agent and an enzyme preparation; performing sealed fermentation on a shaking table; washing away fermentation liquid with clear water; and drying until the water content is 10-12% to obtain the decaisne fargesii flavored fermented beans. According to the method, an in-vitro simulation fermentation method and a time sequence enzyme method are combined, so that the quality of the simulation fermentation cat excrement coffee reaches or even possibly exceeds that of traditional cat excrement coffee, and meanwhile, the problems of yield, sanitation, ethics and the like are solved.

Description

Technical Field

[0001] This invention relates to the field of coffee fermentation technology, and in particular to a red yeast and civet coffee biomimetic fermentation process that enhances the body and fruity aroma of coffee beans. Background Technology

[0002] Kopi Luwak (also known as civet coffee), one of the world's most expensive coffees, has always attracted much attention for its unique flavor formation mechanism. Traditional Kopi Luwak relies on wild civets naturally feeding on coffee cherries, which undergo a series of complex microbial fermentation processes in their digestive tract, ultimately resulting in coffee beans excreted with their feces. This biological digestive process endows the coffee beans with unique flavor characteristics, including reduced bitterness, enhanced body, and distinctive chocolate, caramel, and cooling sensations.

[0003] The existing production of civet coffee is extremely low and unstable, relying entirely on the foraging behavior of wild civets. Furthermore, hygiene conditions are difficult to control, posing a risk of pathogenic microorganism contamination. At the same time, animal ethics issues are receiving increasing attention, resulting in high market prices that are unaffordable for ordinary consumers. Summary of the Invention

[0004] The purpose of this invention is to provide a biomimetic fermentation process for red yeast and civet coffee. This process involves scientifically screening strains from the intestinal microorganisms of civets that have specific performance advantages (high temperature resistance, fast growth, and acid resistance) and can produce excellent flavor. By combining these two core technologies of simulated biological digestion and fermentation, the quality of simulated fermented civet coffee not only meets or may even exceed that of traditional civet coffee, but also solves problems related to production volume, hygiene, and ethics.

[0005] The above-mentioned technical objective of the present invention is achieved through the following technical solution: In one aspect, this invention provides a red yeast (Rhodotorula dairenensis) YD202501, with accession number CCTCC NO: M 20251796. YD202501 was deposited on August 7, 2025, at the China Center for Type Culture Collection, and classified as (Rhodotorula dairenensis) YD202501.

[0006] The red yeast (Rhodotorula dairenensis) YD202501 described in this invention was obtained through isolation and purification.

[0007] Specifically: Sample source: The intestinal microbiota of civets was collected from Xiangshu Village, Lujiang Town, Longyang District, Baoshan City, Yunnan Province. It is located on the eastern foot of Gaoligong Mountain, at the southern end of the Nujiang Grand Canyon, at an altitude of 900m. The annual precipitation exceeds 1000mm, and the climate is mild, making it a golden area for coffee planting and animal breeding.

[0008] Strain strain isolation steps: Step 1: Sample separation and purification: Mix 1g of fresh feces with 100mL of sterile physiological saline and vortex for 3 minutes to prepare a sample solution; perform serial dilutions (10⁻¹-10⁻⁶) on MRS and PDA media respectively; MRS medium was cultured at 37°C under anaerobic conditions for 48 hours, and PDA medium was cultured at 28°C under aerobic conditions for 48 hours; single colonies were picked for purification culture to ensure that various possible candidate strains were isolated from the complex intestinal microbial community. Step 2: 425 pure strains were initially isolated and then subjected to multiple rounds of rigorous screening. The screening criteria mainly included three aspects: First, physiological characteristics screening, focusing on the strains' heat resistance (ability to grow at 45-50℃), acid resistance (high survival rate under pH 3.0-4.0 conditions), and rapid growth ability (short logarithmic growth phase). Second, the flavor-producing ability was screened. The flavor of the coffee after fermentation was evaluated through small-scale fermentation experiments. After analysis by GC-MS, cupping and sensory evaluation, the strain was finally identified as GP-2. Third, protein degradation and pectin degradation capabilities were selected as indicators for screening strains. Strains were cultured on screening medium (plates with added casein) and the presence of transparent hydrolysis zones was observed. Alternatively, pectin-containing medium (pectin plates) was used, and after staining with iodine solution, the presence of transparent hydrolysis zones was observed. Step 3, Identification and Preservation: The strain was identified by molecular biology through morphological, physiological and biochemical characteristics and ITS sequence analysis (ITS1-5.8S-ITS2). The results showed that the strain was Rhodotorula dairenensis. This invention also provides a biomimetic fermentation process for civet coffee, comprising the following steps: Step 1: Inoculate the purified and identified red yeast into PDB seed culture medium and culture the seed liquid under controlled temperature and pH conditions. After the cell concentration reaches the peak of the logarithmic growth phase, transfer it to a fermenter for high-density proliferation culture. Step 2: The high-density fermentation broth is continuously centrifuged or membrane filtered to collect the bacterial precipitate. A compound protective agent is added in proportion, and the mixture is thoroughly mixed to form a bacterial sludge. Pre-frozen at 40°C and then freeze-dried under vacuum to obtain an active dry powder yeast preparation (hereinafter referred to as fermentation agent). Step 3: Preparation of fermentation broth: After juicing the coffee fruit peels, adjust the pH to 4-5 to facilitate subsequent simulated digestion; Step 4: High-temperature and high-pressure sterilization of Catimor coffee beans: After mixing the fermentation liquid with coffee beans, sterilize by high-pressure steam for 20 minutes; Step 5: Cooling: Cool to room temperature in a sealed environment.

[0009] Step 6: Inoculate with starter culture and enzyme preparation: Under aseptic conditions, inoculate with starter culture at a dosage of 0.2%, and add protease and cellulase separately using the sequential enzymatic method and mix well; Step 7: Shaking fermentation: Ferment in a sealed environment at 38℃ for 24-36 hours; Step 8: Wash: Rinse with clean water to remove the fermented liquid; Step 9: Drying: Dry the coffee beans to a moisture content of 10%~12% to obtain civet coffee flavored fermented beans; Step 10, Roasting: Place the dried Kopi Luwak-flavored fermented beans into a roaster and roast them at a medium-dark setting. Observe the color change of the beans during roasting. Cool them immediately after roasting to stabilize the flavor compounds of the coffee beans.

[0010] Furthermore: In step one, the fermentation temperature is controlled at 38±1℃, the pH value is 5.0, and the fermentation time is 24-36 hours, at which point the cell concentration can reach 10. 10 CFU / mL or higher.

[0011] Furthermore: In step two, the composite protective agent is trehalose and glycerin.

[0012] Furthermore: In step four, the temperature for high-pressure steam sterilization is controlled at 121°C and the pressure is controlled at 1.5 MPa.

[0013] Further: In step ten, the baking parameters are as follows: heat at 80℃ for 30 minutes, bean temperature controlled at 190℃; when the temperature reaches about 140℃, increase the heat to 65% to promote the Maillard reaction, reduce to 55% heat for 5-6 minutes to develop sweetness, reduce the heat to 45% before the first crack, remove from the oven 1 minute 10-30 seconds after the first crack begins, development rate 15-20%, total time 9-10 minutes, color value 65-70.

[0014] In summary, the present invention has the following beneficial effects: Firstly, this invention scientifically screens strains from the intestinal microorganisms of civets that have specific performance advantages (high temperature resistance, fast growth, and acid resistance) and can produce excellent flavor; and by combining the two core technologies of simulated biological digestion and fermentation, the quality of simulated fermented civet coffee not only meets or may even exceed that of traditional civet coffee, while solving problems such as production volume, hygiene, and ethics. Secondly, the functional microorganisms isolated from the intestinal flora of civets in this invention can efficiently metabolize flavor precursor substances, and these microorganisms can precisely execute a series of complex synergistic biochemical reactions, ultimately forming the material basis for the characteristic rich nutty, caramel chocolate flavor and mellow taste of civet coffee through Maillard reaction during the roasting stage. Attached Figure Description

[0015] Figure 1 This is a process flow diagram of the present invention. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to the accompanying drawings.

[0017] In the description of this invention, it should be understood that the terms "upper", "lower", "left", "right", "front", "rear", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0018] A red yeast strain, Rhodotorula dairenensis YD202501, with accession number CCTCCNO: M 20251796.

[0019] A biomimetic fermentation process for civet coffee includes the following steps: Step 1: Inoculate the purified and identified red yeast into PDB seed culture medium and culture the seed liquid under controlled temperature and pH conditions. After the cell concentration reaches the peak of the logarithmic growth phase, transfer it to a fermenter for high-density proliferation culture. Control the fermentation temperature at 38±1℃ and the pH value at 5.0. Ferment for 24-36 hours. The cell concentration can reach more than 10¹⁰ CFU / mL. Step 2: The high-density fermentation broth is continuously centrifuged or membrane filtered to collect the bacterial precipitate. A compound protective agent made of trehalose and glycerol is added in proportion, and the mixture is thoroughly mixed to form a bacterial sludge. Pre-frozen at 40°C and then freeze-dried under vacuum to obtain an active dry powder yeast preparation (hereinafter referred to as fermentation agent). Step 3: Preparation of fermentation broth: After juicing the coffee fruit peels, adjust the pH to 4-5 to facilitate subsequent simulated digestion; Step 4: High-temperature and high-pressure sterilization of Catimor coffee beans: After mixing the fermentation liquid with coffee beans, sterilize by high-pressure steam for 20 minutes, and control the temperature at 121℃ and the pressure at 1.5Mpa. Step 5: Cooling: Seal and cool to room temperature; Step 6: Inoculate with starter culture and enzyme preparation: Under aseptic conditions, inoculate with starter culture at a dosage of 0.2%, and add protease and cellulase separately using the sequential enzymatic method and mix well; Step 7: Shaking fermentation: Ferment in a sealed environment at 38℃ for 24-36 hours; Step 8: Wash: Rinse with clean water to remove the fermented liquid; Step 9: Drying: Dry the coffee beans to a moisture content of 10%~12% to obtain civet coffee flavored fermented beans; Step 10, Roasting: The roasting parameters are as follows: heat 80℃ for 30 minutes, bean temperature controlled at 190℃; when the temperature reaches about 140℃, increase the heat to 65% to promote the Maillard reaction, reduce to 55% heat for 5-6 minutes to develop sweetness, reduce the heat to 45% before the first crack, remove from the oven 1 minute 10-30 seconds after the first crack begins, development rate 15-20%, total time 9-10 minutes, color value 65-70, to obtain Kopi Luwak flavored fermented beans.

[0020] Testing process 1. Component analysis: The contents of chlorogenic acid and caffeine were determined by high performance liquid chromatography (HPLC) (referencing the chlorogenic acid detection method in "Food Science" 2021). 2. Flavor substance detection: Volatile components were analyzed by gas chromatography-mass spectrometry (GC-MS). Detection conditions: HP-5MS capillary column, programmed temperature rise to 50℃ and hold for 2 min, then increased to 250℃ at 5℃ / min and hold for 10 min. 3. Sensory evaluation: Referring to the SCAA coffee cupping standards, 5 senior tasters scored bitterness, body, flavor characteristics (chocolate / caramel aroma), and overall acceptability (out of 10), and the average score was taken.

[0021] Example 1: Catimor Coffee with Green Bean Roast Flavor The same batch of Catimor coffee green beans were selected, washed, and then directly dried to control the final moisture content at 10%. The green beans were then added to a 3D Coffee R500 roaster with the following roasting parameters set: damper 4, heat 45%, bean temperature 200℃, air temperature 210℃, and speed 70%. The total roasting time was 13 minutes. The resulting coffee beans had a color value between 65 and 68 and served as a non-fermented and non-digested control group.

[0022] Example 2: Refer to Figure 1 Catim coffee green beans inoculated with GP-2 simulated digestion, fermentation, and roasting flavor Sterilized coffee beans were inoculated with GP-2 strain and Pichia kudrica, followed by the addition of protease and cellulase. The mixture was then anaerobically fermented in a shaker at 38°C for 36 hours. After fermentation, the coffee beans were washed and dried to a moisture content of 10%. Subsequently, they were roasted according to similar roasting parameters to obtain simulated digestion and fermentation roasted coffee beans.

[0023] Example 3: Catimor coffee mung beans inoculated with other yeasts to simulate digestion, fermentation, and roasting flavor. Sterilized coffee beans were inoculated with Angel brewer's yeast strain and Pichia pastoris, followed by the addition of protease and cellulase. The mixture was then anaerobically fermented in a shaker at 38°C for 36 hours. After fermentation, the coffee beans were washed and dried to a moisture content of 10%. They were then roasted according to similar roasting parameters to obtain simulated digestion and fermentation roasted coffee beans.

[0024] Results Analysis Technical effect Flavor matching: The content of characteristic flavor substances and sensory scores of Example 2 are close to those of traditional Kopi Luwak, and the degradation rate of chlorogenic acid reaches 68.5%, which solves the problem of "impurity of simulated flavor".

[0025] Strain advantages: Compared with ordinary brewing yeast, GP-2 strain (red yeast) has a 192% higher ability to generate flavor substances, confirming the effectiveness of its "superior flavor strain" screening criteria.

[0026] Process reliability: The synergistic effect of anaerobic fermentation and enzymatic hydrolysis reduced the bitterness of coffee beans by 57.3% and increased the body by 102%, verifying the scientific validity of the simulated intestinal digestion process.

[0027] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make inventive modifications to this embodiment as needed, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A red yeast, characterized in that: The red yeast (Rhodotorula dairenensis) YD202501 has the accession number CCTCC NO: M 20251796.

2. A biomimetic fermentation process for civet coffee, characterized in that, Includes the following steps: Step 1: Inoculate the purified and identified red yeast into PDB seed culture medium and culture the seed liquid under controlled temperature and pH conditions. After the cell concentration reaches the peak of the logarithmic growth phase, transfer it to a fermenter for high-density proliferation culture. Step 2: The high-density fermentation broth is continuously centrifuged or membrane filtered to collect the bacterial precipitate. A compound protective agent is added in proportion, and the mixture is thoroughly mixed to form a bacterial sludge. Pre-frozen at 40°C and then freeze-dried under vacuum to obtain an active dry powder yeast preparation (hereinafter referred to as fermentation agent). Step 3: Preparation of fermentation broth: After juicing the coffee fruit peels, adjust the pH to 4-5 to facilitate subsequent simulated digestion; Step 4: High-temperature and high-pressure sterilization of Catimor coffee beans: After mixing the fermentation liquid with coffee beans, sterilize by high-pressure steam for 20 minutes; Step 5: Cooling: Seal and cool to room temperature; Step 6: Inoculate with starter culture and enzyme preparation: Under aseptic conditions, inoculate with starter culture at a dosage of 0.2%, and add protease and cellulase separately using the sequential enzymatic method and mix well; Step 7: Shaking fermentation: Ferment in a sealed environment at 38℃ for 24-36 hours; Step 8: Wash: Rinse with clean water to remove the fermented liquid; Step 9: Drying: Dry the coffee beans to a moisture content of 10%~12% to obtain civet coffee flavored fermented beans; Step 10, Roasting: Place the dried Kopi Luwak-flavored fermented beans into a roaster and roast them at a medium-dark setting. Observe the color change of the beans during roasting. Cool them immediately after roasting to stabilize the flavor compounds of the coffee beans.

3. The biomimetic fermentation process for civet coffee according to claim 2, characterized in that: In step one, the fermentation temperature is controlled at 38±1℃, the pH value is 5.0, and the fermentation time is 24-36 hours, at which point the cell concentration can reach 10. 10 CFU / mL or higher.

4. The biomimetic fermentation process for red yeast and civet coffee according to claim 2, characterized in that: In step two, the composite protective agent is trehalose and glycerin.

5. The biomimetic fermentation process for civet coffee according to claim 2, characterized in that: In step four, the temperature for high-pressure steam sterilization is controlled at 121°C and the pressure is controlled at 1.5 MPa.

6. The biomimetic fermentation process for civet coffee according to claim 2, characterized in that: In step ten, the baking parameters are as follows: heat at 80℃ for 30 minutes, bean temperature controlled at 190℃; when the temperature reaches about 140℃, increase the heat to 65% to promote the Maillard reaction, reduce to 55% heat for 5-6 minutes to develop sweetness, reduce the heat to 45% before the first crack, remove from the oven 1 minute 10-30 seconds after the first crack begins, development rate 15-20%, total time 9-10 minutes, color value 65-70.

Images