Torulaspora delbrueckii, lactobacillus fermentum, coffee compound leavening agent, preparation method of lemongrass-flavored coffee and lemongrass-flavored coffee beans

Abstract

The invention discloses torulaspora delbrueckii, lactobacillus fermentum, a coffee compound leavening agent, a preparation method of lemongrass-flavored coffee and lemongrass-flavored coffee beans, and relates to the technical field of coffee fermentation. The preparation method comprises the following steps: preparing a coffee composite leavening agent liquid from torulaspora delbrueckii which is preserved in the China General Microbiological Culture Collection Center (CGMCC) and has a preservation number of CGMCC No.35270 and lactobacillus fermentum with a preservation number of CGMCC No.30567 according to a volume ratio of 1: 1, inoculating the coffee composite leavening agent liquid into a rehydrated and sterilized coffee bean fermentation tank, and fermenting for 1-2 hours at a temperature of 20-30 DEG C to obtain the coffee composite leavening agent liquid. After the primary fermentation, adding the treated lemongrass for secondary fermentation, and then washing, drying and baking to obtain the lemongrass-flavored coffee. The method comprises the following steps: rehydrating coffee beans in a bean-water ratio of 1: 2, inoculating a mixed bacterium suspension with the concentration of 8 g CFU / mL, fermenting for 24 hours under the conditions that the temperature is 32 + / -2 DEG C and the humidity is 85%, and adding lemongrass which is 5% of the mass of the green coffee beans in the second fermentation. By precisely regulating and controlling mixed fermentation and secondary fermentation of lemongrass, the coffee is endowed with unique lemongrass flavor, and the quality and diversity of the coffee are improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation and food deep processing, and particularly relates to a strain of Delbryo sp. and a strain of Lactobacillus fermentum and a coffee compound starter obtained by mixing preparation, and further application in a coffee fermentation process, and particularly relates to a fermentation method and application of the coffee in a lemongrass-flavored coffee. BACKGROUND

[0002] Catimor is one of the widely planted hybrid varieties in the coffee industry, which is a hybrid of Timor and Caturra, bred by the Portuguese Coffee Institute (CIFC) in the late 1950s. Timor is a naturally rust-resistant hybrid (a mix of Arabica and Robusta), contributing disease-resistant genes, and Caturra is a Bourbon variety in Brazil, which is dwarf and high-yielding, improving taste and yield. Therefore, Catimor combines the disease resistance of Robusta and the flavor of Arabica to cope with the threat of leaf rust, and is known for its improved disease resistance, adaptability and yield, playing an important role in coffee-producing regions in Asia and Latin America.

[0003] Due to the unique geographical environment and climate conditions in Yunnan, Yunnan Catimor has a unique flavor, but there are still some obvious defects in flavor and planting, which limit its performance in the specialty coffee market. Due to the influence of Robusta genes, Catimor coffee beans may have a bitter and astringent taste, especially when over-extracted, which is prone to wood, earthy or herbal flavors; low and monotonous acidity, lack of bright fruit acidity and layers of high-quality Arabica; low flavor complexity, insufficient aroma, weak floral and fruity notes, more nutty, cereal or spice base notes. Catimor is susceptible to planting conditions, and in areas with an altitude of less than 1,000 meters, the flavor is further flattened, and the bitterness is increased; if over-fertilized, the beans may have a "hollow" feeling; if sun or honey treatment is not properly handled, it is prone to over-fermentation, producing moldy or off-flavors. Therefore, the defects of Catimor are essentially the "cost of balancing disease resistance and flavor", suitable for commercial plantations that require stable output, but more investment is needed on the path of specialization, and for consumers, choosing high-altitude, finely processed Catimor may be a pleasant surprise.

[0004] Therefore, adding a fermenting agent (such as yeast, lactic acid bacteria or other microbial preparations) as an emerging process during coffee fermentation aims to improve coffee flavor, shorten fermentation time or enhance fermentation stability by directional regulation of microbial activity. Secondary fermentation is a further fermentation using exogenous microorganisms on the basis of preliminary fermentation. During the fermentation process, specific microorganisms will secrete hydrolytic enzymes to decompose macromolecular pectin into monosaccharides and short-chain fatty acids. After the mucilage layer is degraded, the fermentation efficiency is accelerated, and the risk of over-fermentation is reduced. Microorganisms produce hundreds of volatile substances through metabolic activity, which directly affect the aroma of coffee, such as esters ethyl acetate and ethyl hexanoate, which have a fruity aroma, aldehydes such as benzaldehyde, which have an almond and caramel aroma, ketones such as furanone, which have a caramel sweetness, and terpenes such as linalool, which have a citrus and floral aroma. At the same time, the addition of dominant bacterial species inhibits the growth of harmful microorganisms such as Escherichia coli and molds through rapid proliferation, acid production (reducing pH to 4.0-5.0) or releasing bacteriocin, reducing the production of undesirable flavors and ensuring fermentation safety and controllability.

[0005] And, since Yunnan has rich plant resources, Yunnan citronella grass has strong local characteristics, therefore, in order to produce coffee with Yunnan characteristics and enrich the diversity of coffee flavors, it is planned to produce a coffee bean with citronella grass flavor to enrich the variety of coffee flavors. SUMMARY

[0006] To achieve the above-mentioned purpose, the application adds citronella grass in the secondary fermentation of coffee, which fully integrates coffee beans and citronella grass under the action of microorganisms, thereby imparting coffee with fascinating citronella grass flavor and increasing the diversity of coffee aroma and flavor. Through scientific control of the process of coffee secondary fermentation, the addition of exogenous substances under mixed fermentation can unlock the potential flavor of coffee, realize the leap from "natural random fermentation" to "precise flavor design", increase the diversity of coffee flavor, improve the quality of Yunnan coffee, and broaden the path and choice for the future development of coffee industry. The specific technical scheme is as follows:

[0007] The first object of the application is to screen and obtain Torulaspora delbrueckii and Lactobacillus fermentum strains, and to preserve the strains,

[0008] The Torulaspora delbrueckii CII-19 strain was preserved in the China General Microbiological Culture Collection Center on July 17, 2025, with the preservation number being CGMCC No. 35270 and the preservation address being No. 3, Beichen West Road, Chaoyang District, Beijing;

[0009] The Lactobacillus fermentum AI-08 strain is preserved in the China General Microbiological Culture Collection Center on May 9, 2024, with a preservation number of CGMCC No. 30567, and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing.

[0010] A second object of the present application is to prepare a mixed seed bacteria suspension as a composite ferment for coffee fermentation by using the two strains obtained in the present application (Torulaspora delbrueckii and Lactobacillus fermentum), and the volume ratio of the Torulaspora delbrueckii CII-19 strain and the Lactobacillus fermentum AI-08 in the composite ferment is 1:1, and the prepared mixed seed bacteria suspension can also be made into a mixed freeze-dried bacteria powder.

[0011] A third object of the present application is to provide a method for preparing a citronella-flavored coffee by mixed fermentation of Torulaspora delbrueckii No. XX and Lactobacillus fermentum AI-08, which comprises the following steps:

[0012] 1) Sorting coffee beans: sorting high-quality beans from cleaned coffee bean raw materials, and removing defective beans and other impurities for standby;

[0013] 2) Rehydrating coffee beans: using sterile water, putting the beans into the fermentation tank at a bean-to-water ratio of 1:2, the water volume accounts for about 1 / 2 of the total volume of the tank after adding water, and the coffee beans are soaked to a grayish white color with uniform appearance and color;

[0014] 3) Sterilization: sealing with a sterile sealing film, sterilizing at a temperature of 110 ℃ for 5 min; after sterilization, natural cooling is performed, and the temperature is reduced to 25-35 ℃;

[0015] 4) Preparation of bacteria suspension: centrifuging the activated Torulaspora delbrueckii and Lactobacillus fermentum fermentation strains, discarding the culture medium, and then adding sterile physiological saline to adjust the concentration to 6-8 lg CFU / mL to prepare a bacteria suspension, and preparing a mixed seed bacteria suspension by mixing the prepared Torulaspora delbrueckii and Lactobacillus fermentum bacteria suspensions at a ratio of (1-2):(1-2);

[0016] 5) Inoculation: inoculate the mixed seed bacteria suspension prepared in step 4) to the surface of the sterile coffee beans, mix well, and the inoculated microorganisms have a concentration of 5-7 lg CFU / mL in the fermentation tank;

[0017] 6) Fermentation: move the inoculated fermentation tank into a constant temperature room, keep the temperature at 32±2 DEG C, the relative humidity is above 85%, and ferment for 12-36 h;

[0018] 7) Add lemongrass: grind the dried lemongrass into powder, sterilize by ultraviolet, and add 5% of the lemongrass powder to the fermentation tank according to the mass of the green coffee beans, and shake well;

[0019] 8) Secondary fermentation: move the lemongrass added fermentation tank into a constant temperature room, keep the temperature at 32±2 DEG C, the relative humidity is above 85%, and continue to ferment for 12-36 h;

[0020] 9) Washing and drying: wash the lemongrass in the fermented coffee beans and only keep the coffee beans, dry the coffee beans in a drying device at 40-45 DEG C to a moisture content of less than 11-12%, and obtain lemongrass flavored coffee beans;

[0021] 10) Roasting: take the lemongrass flavored coffee beans, put them into a pan with a temperature of 190-200 DEG C, keep the fire at 60%, roast for 7-10 min, and take out the pan with a temperature of 205-218 DEG C to obtain medium light roasted coffee beans.

[0022] Further, the ratio of Torulaspora delbrueckii and Lactobacillus fermentum in the mixed seed bacteria suspension in step 4) is 1:1 in volume ratio; wherein the concentration of the Torulaspora delbrueckii bacteria suspension is 8 lg CFU / mL; and the concentration of the Lactobacillus fermentum bacteria suspension is 8 lg CFU / mL.

[0023] Further, the fermentation time in steps 5) and 8) is 24 h.

[0024] The fourth object of the present application is to provide a lemongrass flavored coffee bean prepared according to the method as described above.

[0025] The fifth object of the present application is to provide an application of a coffee compound starter in the production and preparation of coffee.

[0026] Compared with the prior art, the beneficial effects of the present application are:

[0027] (1) The coffee beans after fermentation by the method of the present application have a pH value of 4.13, a soluble solid content of 8.7%, a tannin content of 14.57 mg / g, and a coffee cup evaluation score of 84, showing a flavor characteristic of citronella grass fragrance and acidity, and the sensory quality is significantly improved;

[0028] (2) By optimizing the fermentation process, a characteristic coffee with Yunnan characteristic citronella grass flavor is successfully developed, a new type of fermented product is developed, and a new path is opened up for the innovative development of the coffee industry and the high-value utilization of agricultural products. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is a process flowchart of the present application;

[0030] Figure 2 is a single colony map of a strain of Delbruekia sp. ;

[0031] Figure 3 is a single colony map of a strain of Lactobacillus fermentum;

[0032] Figure 4 is the sorted coffee beans, with high-quality beans on the left and defective beans on the right;

[0033] Figure 5 is a schematic diagram of the treated citronella grass;

[0034] Figure 6 is a schematic diagram of the rehydrated coffee. DETAILED DESCRIPTION

[0035] The present application will be further described in detail below with specific embodiments in conjunction with the accompanying drawings.

[0036] Example 1: Strain acquisition

[0037] 1. Delbruekia sp. strain acquisition method:

[0038] (1) Screening of Delbruekia sp. strain: Delbruekia sp. strain samples were collected from soy sauce in Tuojiang County, Chuxiong Yi Autonomous Prefecture, Yunnan Province, China, and were stored in a refrigerator after labeling. The samples were transported back to the laboratory on the same day and stored in a -80℃ refrigerator. Specifically, 5 mL of soy sauce sample was weighed into 15 mL of sterile physiological saline, mixed, and then 1 mL of sample solution was diluted to 10 -6 , 10 -7 , 10 -8 to obtain the diluent. 0.2 mL of diluent of different concentration gradients was taken and spread on the solidified YPD agar medium using a spreader to mix the diluent evenly on the plate. After mixing, the plate was inverted and cultured in a 28℃ incubator for 24-48 h, with three parallel samples for each dilution.

[0039] (2) Isolation and preservation of Torulaspora delbrueckii strain: observe the colony morphology on the plate, adopt plate streaking method, pick single colonies with different size, state, color and good growth, streak culture on YPD agar medium, and culture in 28°C incubator for 1-2 days. After three generations of strain streaking and purification, transfer to test tube slant, number and culture for 2 days. After good growth, select the isolated strain CII-19 with consistent morphological characteristics and send to Shanghai Jizhen Biotechnology Company for identification. The CII-19 strain is identified as Torulaspora delbrueckii, and the colony morphology is round or oval, regular edge, smooth surface, intermediate convex, buttery texture, and milk white.

[0040] 2. Lactobacillus strain acquisition method:

[0041] (1) Lactobacillus strain screening: Lactobacillus strain samples are collected from traditional fermented food pickles in Fuyuan County, Qujing City, Yunnan Province, China, labeled and stored in a refrigerator. On the same day, take 5 g of pickle sample in 15 mL of sterile normal saline, mix well, take 1 mL of sample solution, dilute to 10-6, 10-7, 10-8 by 10-fold gradient dilution method to obtain dilution solution. Take 0.2 mL of dilution solution with different concentration gradient on the solidified YPD agar medium, use a coating rod to coat the dilution solution evenly on the plate, mix well, invert the plate, and culture in a 28°C incubator for 24-48 h. Three parallel plates are prepared for different dilutions.

[0042] (2) Isolation and preservation of Lactobacillus strain: observe the colony morphology on the plate, adopt plate streaking method, pick single colonies with different size, state, color and good growth, streak culture on YPD agar medium, and culture in 28°C incubator for 1-2 days. After three generations of strain streaking and purification, transfer to test tube slant, number and culture for 2 days. After good growth, select the isolated strain A I-08 with consistent morphological characteristics and send to Shanghai Jizhen Biotechnology Company for identification. The A I-08 strain is identified as Lactobacillus fermentum, and the colony morphology is round, regular or slightly irregular edge, smooth surface, opaque, milk white or light yellow.

[0043] Example 2: Specific steps of a method for preparing citronella grass flavored coffee by mixed fermentation of Torulaspora delbrueckii CII-19 strain and Lactobacillus fermentum A I-08 strain:

[0044] 1. Sorting coffee beans: Sort out high-quality beans from coffee bean raw materials, and remove defective beans and other impurities for use;

[0045] 2. Coffee bean rehydration: Use sterile water to put into the fermentation tank at a bean-to-water ratio of 1:2, the volume after adding water accounts for about 1 / 2 of the total volume of the tank, soak the coffee beans to grayish white, and the appearance color is uniform;

[0046] 3. Sterilization (bactericidal): Use sterile sealing film to seal, sterilize at a temperature of 110 ℃ for 5 min; after sterilization, natural cooling is performed, and the temperature is reduced to 25-35 ℃;

[0047] 4. Preparation of bacterial suspension: Centrifuge the activated two strains of fermentation bacteria and discard the culture medium, then add sterile physiological saline to adjust the concentration to the appropriate concentration; preferably, the concentration is adjusted to 8 lg CFU / mL;

[0048] 5. Inoculation of bacterial strains: Delbruei asporogenous round yeast and Lactobacillus fermentum are inoculated onto the surface of sterile coffee beans at a ratio of 1:1, mixed thoroughly, and the concentration of microorganisms reaches 5-7 lg CFU / mL;

[0049] 6. Fermentation: After inoculation, the coffee beans are moved into a constant temperature room, the temperature is kept at 30±2 ℃, the relative humidity is above 85%, and the fermentation is carried out for 24 h;

[0050] 7. Add lemongrass: Dry lemongrass is ground into powder, sterilized by ultraviolet light, and added to the fermentation tank at a ratio of 5% of the mass of the green coffee beans, and shaken evenly;

[0051] 8. Second fermentation: The fermentation tank with added lemongrass is moved into a constant temperature room, the temperature is kept at 30±2 ℃, the relative humidity is above 85%, and the fermentation is carried out for 24 h;

[0052] 9. Washing and drying: The lemongrass in the fermented coffee beans is washed away, only the coffee beans are left, and the coffee beans are dried at 40-45 ℃ to a moisture content of less than 11%-12%, thereby producing a kind of fermented lemongrass flavored coffee beans.

[0053] 10. Roasting: 300 g of fermented lemongrass coffee beans are put into a drum-type coffee roasting machine, the pan temperature is 190-200 ℃, the fire power is kept at 60%, the roasting time is 7-10 min, and the optimal pan temperature is 205-218 ℃, thereby obtaining medium roasted coffee beans (recommended medium light).

[0054] The coffee beans with the flavor of citronella grass obtained by the above method have good thermal stability, the flavors of citronella grass and coffee are well fused together after high-temperature roasting, and the fascinating citronella grass flavor can be felt from high temperature to low temperature brewing, and the taste is soft and delicate, clean and refreshing.

[0055] Example 3: Optimization experiment of mixed bacteria fermentation

[0056] In order to illustrate the advantages of mixed bacteria fermentation, a control experiment was carried out, and four groups were set: natural fermentation of coffee with the flavor of citronella grass (without adding T. d and L. f fermentation), single bacteria fermentation of coffee with the flavor of citronella grass (adding T. d fermentation, adding L. f fermentation), mixed bacteria fermentation of coffee with the flavor of citronella grass (adding T. d and L. f fermentation), and comparing and analyzing the pH value, soluble solids content after 36 h of fermentation and sensory evaluation of coffee beans in different groups.

[0057] 1. pH determination

[0058] 5.00 g of coffee sample was weighed, 25 mL of distilled water was added, and oscillation was carried out for 20 min, and pH meter was used for direct determination. The pH determination results are shown in Table 1.

[0059] Table 1 pH value of natural fermentation, single bacteria fermentation and mixed bacteria fermentation of coffee with the flavor of citronella grass

[0060] Group Group pH First group Coffee bean + Cymbopogon citratus 4.35 Second group Coffee bean + Cymbopogon citratus + T. d 5.17 Third group Coffee bean + Cymbopogon citratus + L. f 4.87 Fourth group Coffee bean + Cymbopogon citratus + T. d + L. f 4.67

[0061] Analysis:

[0062] The first group (coffee beans + citronella grass), the pH value is 4.35: the group without adding microorganisms has the lowest pH value, which indicates that natural fermentation produces more acidic substances, which will make the sour taste particularly prominent and the flavor unbalanced.

[0063] The second group (coffee beans + citronella grass + T. d), the pH value is 4.87: the pH value is the highest after adding T. d fermentation, which indicates that this treatment method can effectively control the acidic substances, so that the sour taste is not so prominent.

[0064] The third group (coffee beans + citronella grass + L. f), the pH value is 4.15: the pH value is lower after adding L. f fermentation, which may be related to the fact that lactic acid bacteria are dominant strains in the fermentation system, which will cause the pH to slowly decrease.

[0065] The fourth group (coffee beans + citronella grass + T. d + L. f), the pH value is 4.13: the pH value is lower after adding T. d and L. f, which indicates that the co-fermentation of the two strains can make the sour taste more prominent.

[0066] 2. Soluble solids content determination

[0067] A 5.00 g coffee sample was weighed, ground, and added to 25 mL distilled water. The sample was filtered with gauze, and an appropriate amount of sample liquid was taken and measured directly using a saccharimeter. The soluble solids content determination results are shown in Table 2.

[0068] Table 2 Soluble solids content of different groups

[0069] Group Group Soluble solids content First group Coffee bean + Cymbopogon citratus 11.2% Second group Coffee bean + Cymbopogon citratus + T. d 10.6% Third group Coffee bean + Cymbopogon citratus + L. f 9.3% Fourth group Coffee bean + Cymbopogon citratus + T. d + L. f 8.7%

[0070] Analysis:

[0071] The first group (coffee beans + citronella grass) had a soluble solids content of 11.2%, and the microbial activity was slow under natural fermentation, and the carbohydrate consumption was slow.

[0072] The second group (coffee beans + citronella grass + T.d) had a soluble solids content of 10.6%, and the Delbrueckia spore yeast was the dominant bacteria, which grew rapidly at a suitable temperature and fermented quickly.

[0073] The third group (coffee beans + citronella grass + L.f) had a soluble solids content of 9.3%, and the Lactobacillus fermentum grew at a relatively fast rate and utilized sugar very quickly.

[0074] The fourth group (coffee beans + citronella grass + T.d + L.f) had a soluble solids content of 8.7%, and the Delbrueckia spore yeast and Lactobacillus fermentum cooperated to ferment, which improved the fermentation efficiency and shortened the fermentation time.

[0075] 3. Tannin content determination

[0076] Preparation of standard curve: Accurately pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of tannic acid standard solution (200 mg / L) into 10 mL colorimetric tubes containing 6 mL water, add 0.5 mL of Folin phenol, 1 mL of 7.5% sodium carbonate solution, and distilled water to the mark, shake well, stand for 30 min for color development, and then measure the absorbance of the standard solution at a wavelength of 760 nm. Take the reagent blank as the blank control. Plot the standard curve with the tannic acid concentration as the abscissa and the absorbance value as the ordinate.

[0077] Determination of coffee sample: precisely take 0.2 mL coffee powder extract, put it in a 10 mL colorimetric tube containing 6 mL water, add 0.5 mL of Folin phenol, add 1 mL of 7.5% sodium carbonate solution, and distill water to the calibration line, shake well. After color development for 30 min, the reagent blank is operated as a blank control, and the absorbance of the sample solution is measured at a wavelength of 760 nm. The concentration of tannic acid in the test solution is calculated according to the standard curve. The tannin content of each example is calculated from the tannic acid standard curve, and the results are shown in Table 3.

[0078] Table 3 Tannin content of coffee in different groups

[0079] Group Group Tannin content First group Coffee bean + Cymbopogon citratus 13.62 mg / g Second group Coffee bean + Cymbopogon citratus + T. d 12.21 mg / g Third group Coffee bean + Cymbopogon citratus + L. f 12.39 mg / g Fourth group Coffee bean + Cymbopogon citratus + T. d + L. f 14.57 mg / g

[0080] Analysis:

[0081] The first group (coffee beans + citronella grass) had a tannin content of 13.62 mg / g, indicating that adding microorganisms could also produce tannin, but the effect was not as good as mixed fermentation.

[0082] The second group (coffee beans + citronella grass + T.d) had a tannin content of 12.21 mg / g, which indicated that the tannin content was lower after adding Delbruekia spore yeast treatment. This treatment method made the tannin release effect worse.

[0083] The third group (coffee beans + citronella grass + L.f) had a tannin content of 12.29 mg / g, which indicated that the tannin content after adding Lactobacillus fermentum treatment was higher than that of the second group (coffee beans + citronella grass + T.d), indicating that this treatment method had a certain effect on releasing tannin.

[0084] The fourth group (coffee beans + citronella grass + T.d + L.f) had a tannin content of 14.57 mg / g. During the complex fermentation of Delbruekia spore yeast and Lactobacillus fermentum, due to the action of microorganisms and their enzyme systems, some substances in coffee can be degraded and converted into useful compounds, and bioactive substances are released, improving antioxidant activity.

[0085] 4. Sensory evaluation

[0086] In order to determine the actual sensory differences between different fermentation groups, 10 professional coffee sensory evaluators evaluated the roasted beans of the four fermentation systems according to the SCAA standard. The flavor characteristics were evaluated from fragrance (Fragrance / Aroma), cleanliness (Clean cup), sweetness (Sweetness), acidity (Acidity), body (Body), flavor (Flavor), aftertaste (Aftertaste), balance (Balance), uniformity (Uniformity) and overall (Overall) ten comprehensive aspects. Different fermentation methods (Table 4), different mixed bacteria ratios (Table 5), different inoculation concentrations (Table 6), different bacteria addition amounts (Table 7), different fermentation times (Table 8), different fermentation temperatures (Table 9) and different citronella grass addition amounts (Table 10) were evaluated respectively.

[0087] Table 4 Sensory evaluation of coffee in different fermentation method groups

[0088] Group Group Sensory evaluation First group Coffee bean + Cymbopogon citratus 77 points Second group Coffee bean + Cymbopogon citratus + T. d 81 points Third group Coffee bean + Cymbopogon citratus + L. f 80 points Fourth group Coffee bean + Cymbopogon citratus + T. d + L. f 84 points

[0089] Analysis:

[0090] The first group (coffee beans + citronella grass) scored 77 points: this combination also blended the flavor of citronella grass in the fermented coffee, but the acidity was prominent, the bitterness was slightly strong, and the mouthfeel was unbalanced.

[0091] The second group (coffee beans + citronella grass + T.d) scored 81 points: this combination had a strong citronella flavor in the fermented coffee, but the citronella flavor was too strong, causing discomfort, the bitterness was slightly strong, and the acidity was very weak.

[0092] The third group (coffee beans + citronella grass + L.f) scored 80 points: this combination scored higher, the growth rate of Lactobacillus fermentum was fast, consuming a large amount of nutrients in the coffee beans, resulting in a strong acidity, the coffee was mixed with grass and butter flavors, and the citronella flavor was not prominent.

[0093] The fourth group (coffee beans + citronella grass + T.d + L.f) scored 84 points: this combination scored the highest, the fermentation of Torulaspora delbrueckii and Lactobacillus fermentum made the coffee blend evenly with the aroma and flavor of citronella, the mouthfeel was soft, the bitterness and acidity were moderate, the fragrance was mild, and the coffee was mixed with the fragrance of citronella.

[0094] Four, mixed bacteria ratio

[0095] In order to determine the best ratio of Torulaspora delbrueckii and Lactobacillus fermentum, according to the ratio of Torulaspora delbrueckii (T.d) and Lactobacillus fermentum (L.f) as 1:1, 1:2, 2:1, add them with coffee beans and citronella grass for fermentation experiment, take sensory evaluation as evaluation index, find out the best mixed bacteria ratio.

[0096] Table 5 Comparison of sensory evaluation of coffee with different proportions of mixed bacteria

[0097] Group Group Sensory evaluation First group T. d : L. f = 1 : 1 82.5 points Second group T. d : L. f = 1 : 2 79 points Third group T. d : L. f = 2 : 1 81 points

[0098] Analysis:

[0099] The first group (T.d:L.f =1:1) scored 82.5: this combination had a distinct lemongrass flavor, moderate bitterness, and a short-lasting sour taste with a slight aftertaste, and had a nutty, vanilla cake flavor.

[0100] The second group (T.d:L.f =1:2) scored 79: this combination had a less distinct lemongrass flavor, which was masked by the fermented lactic acid bacteria flavor, and had less aroma and taste than the first group (K.m:L.P=1:1).

[0101] The third group (T.d:L.f =2:1) scored 81: this combination also had a distinct lemongrass flavor, but the aroma and flavor were unbalanced, and the sourness was too strong (the sour taste lasted a long time in the mouth).

[0102] V. Fermentation condition determination

[0103] 1. Inoculum concentration

[0104] 200 μL of CII-19 and AI-08 bacterial liquid was taken from the glycerol tube and inoculated into 10 mL of YPD liquid medium and MRS liquid medium, respectively. CII-19 was cultured in a 28°C incubator for 48 h, and AI-08 was cultured in a 37°C incubator for 36 h. The two strains were activated for two generations in this way. 1 mL of each activated strain was centrifuged at 6000 r / min for 8 min, the supernatant was removed, and 1 mL of sterile saline was added for washing twice. Finally, 1 mL of bacterial suspension was obtained. 1 mL of bacterial suspension was added to a sterile physiological saline test tube containing 9 mL of sterile physiological saline, and 1.0 x 10 8 CFU / mL McFarland turbidity tube was used for comparison, and the bacterial liquid concentration was adjusted until the turbidity of the bacterial liquid was consistent with that of the McFarland turbidity tube, i.e., 8 Lg CFU / mL of bacterial liquid was obtained. 1 mL of 8 Lg CFU / mL bacterial liquid was taken to 9 mL of sterile physiological saline to obtain 7 Lg CFU / mL bacterial liquid, and 1 mL of 7 Lg CFU / mL bacterial liquid was taken to 9 mL of sterile physiological saline to obtain 6 Lg CFU / mL. In this way, the desired bacterial liquid concentration was obtained by gradient dilution. Under the condition that other conditions remained unchanged, the fermentation comparison experiment of different inoculum concentrations was carried out according to the strain addition amount of 3%, the fermentation time of 24 h, the fermentation temperature of 32°C, and the lemongrass addition amount of 5%. The comparison results of coffee cup evaluation scores are shown in Table 6.

[0105] Table 6 Comparison of coffee cup evaluation scores at different inoculation concentrations

[0106] Group Group Sensory evaluation First group 3 Lg CFU / mL 75 points Second group 4 Lg CFU / mL 78 points Third group 5 Lg CFU / mL 79 points Fourth group 6 Lg CFU / mL 82 points Fifth group 7 Lg CFU / mL 77 points

[0107] According to the results shown in Table 6, different inoculation concentrations have different effects on the flavor of coffee. Too low inoculation concentration can cause insufficient fermentation of coffee beans, incomplete utilization of nutrients in coffee beans, insufficient generation of flavor substances (organic acids, esters, etc.), and the generation of undesirable flavors such as wood or herbal flavors. Too high inoculation concentration can lead to over-fermentation, breaking the balance of coffee taste, and producing undesirable flavors such as excessive acidity or soy sauce. When the inoculation concentration is 6 Lg CFU / mL, the flavor of coffee is rich and balanced, with moderate acidity and bitterness, and the taste is soft and smooth. At this time, the sensory score is the highest, reaching 82 points. Therefore, the optimal inoculation concentration is 6 Lg CFU / mL.

[0108] 2. Strain addition amount

[0109] The strain addition amount is a key control parameter in coffee fermentation, directly affecting the direction and quality of flavor. When the addition amount is too low, the fermentation starts slowly, and wild bacteria are easy to dominate, which may lead to off-flavors (such as vinegar taste, putrefactive taste) and weak target flavor. When the addition amount is moderate, the target bacteria become dominant, the fermentation process is stable and pure, and the specific flavor (such as wine aroma, tropical fruit flavor) can be clearly expressed, and it is well balanced with the background flavor of coffee, forming a sense of hierarchy. When the addition amount is too high, the fermentation is too intense, which may produce excessive fermentation taste, sharp acidity or "artificial" fruit flavor, covering the regional characteristics of coffee itself and causing flavor imbalance. Therefore, precise control of inoculation amount is the premise of achieving flavor design and obtaining high-quality processed coffee. Under the condition that other conditions remain unchanged, the fermentation comparison experiment of different strain addition amounts was carried out at an inoculation concentration of 6 Lg CFU / mL, a fermentation time of 24 h, a fermentation temperature of 32℃, and a citronella grass addition amount of 5%. The comparison results of coffee cup evaluation scores are shown in Table 7.

[0110] Table 7 Comparison of coffee cup evaluation scores at different strain addition amounts

[0111] Group Group Sensory evaluation First group 1% 73 points Second group 2% 80 points Third group 3% 83.5 points Fourth group 4% 79 points Fifth group 5% 76 points

[0112] According to the results shown in Table 7, different inoculation concentrations have different effects on the flavor of coffee. Too low inoculation concentration can cause insufficient fermentation of coffee beans, incomplete utilization of nutrients in coffee beans, insufficient generation of flavor substances (organic acids, esters, etc.), and the generation of undesirable flavors such as wood or herbal flavors. Too high inoculation concentration can lead to over-fermentation, breaking the balance of coffee taste, and producing undesirable flavors such as excessive acidity or soy sauce. When the inoculation concentration is 6 Lg CFU / mL, the flavor of coffee is rich and balanced, with moderate acidity and bitterness, and the taste is soft and smooth. At this time, the sensory score is the highest, reaching 82 points. Therefore, the optimal inoculation concentration is 6 Lg CFU / mL.

[0113] 3. Fermentation time

[0114] Fermentation time is the key to shaping coffee flavor. If the time is too short, the fermentation is insufficient, the flavor development is insufficient, it may have a bitter grassy taste and unpleasant sourness, and the sweetness is weak. If the time is moderate (usually 24-72 hours), the microbial metabolism is sufficient, and the rich flavor precursors such as alcohol and ester are produced, which can bring lively fruit acid, clear sweetness and complex tropical fruit, wine aroma and other levels, and it is more round. If the time is too long, it is easy to over-ferment, produce rotten smell, vinegar smell, alcohol smell or heavy mold smell, cover up the characteristics of coffee itself, and the taste becomes dull and unclean. Therefore, precise control of the length of time is crucial to the cleanliness and complexity of the flavor. Under the condition that other conditions remain unchanged, according to the inoculation concentration of 6 Lg CFU / mL, the strain addition amount of 3%, the fermentation temperature of 32℃, and the addition amount of lemongrass of 5%, the fermentation comparison experiment of different time is carried out, and the comparison results of coffee cup evaluation score are shown in Table 8.

[0115] Table 8 Comparison of coffee cup evaluation scores of different fermentation times

[0116] Group Group Sensory evaluation First group 20 h 78 points Second group 22 h 80 points Third group 24 h 82 points Fourth group 26 h 78 points Fifth group 28 h 77 points

[0117] According to the results shown in Table 8, different fermentation times have different effects on coffee flavor. Insufficient fermentation time or too long fermentation time will produce undesirable flavors in coffee. In a short time, the strain activity is insufficient, the fermentation process is short, the flavor substances are not completely generated, and the taste is relatively light. Long time fermentation, rich nutrients are depleted, and a large amount of undesirable ingredients are accumulated, which will cause over-acidification and vinegar taste. When the fermentation time is 24 h, the coffee flavor is complex and balanced, smooth and soft, and the sensory score is the highest, which is 82. Therefore, the most suitable fermentation time is 24 h.

[0118] 3. Fermentation temperature

[0119] Fermentation temperature is the core switch to regulate the direction of coffee flavor. Low-temperature fermentation (such as 15-20℃) is slow, which is beneficial to yeast ester production, and can form clean, delicate and elegant flavor with floral and fruit acid. However, it takes a long time. The medium-temperature fermentation (such as 20-30℃) is a common safe interval, the microbial activity is balanced, which can develop alcohol thickness and sweetness, form balanced stone fruit and caramel flavor, and has high fault tolerance. High-temperature fermentation (such as 30-40℃) greatly accelerates the reaction, lactic acid bacteria are active, which can bring rich wine fermentation, tropical fruit and even sauce aroma, but the risk is very high, and it is easy to produce pungent sour taste, rotten smell or dry taste of over-fermentation. Therefore, precise temperature control is the key to realizing flavor design and avoiding defects. Under the condition that other conditions remain unchanged, according to the inoculation concentration of 6 Lg CFU / mL, the strain addition amount of 3%, the fermentation time of 24 h, and the addition amount of lemongrass of 5%, the comparison experiment of different fermentation temperatures is carried out, and the comparison results of coffee cup evaluation score are shown in Table 9.

[0120] Table 9 Comparison of coffee cup evaluation scores at different fermentation temperatures

[0121] Group Group Sensory evaluation First group 28℃ 75 points Second group 30℃ 77 points Third group 32℃ 80 points Fourth group 34℃ 76 points Fifth group 36℃ 74 points

[0122] According to the results shown in Table 9, different culture temperatures have different effects on coffee flavor. When the culture temperature is too low, Lactobacillus fermentum does not grow enough; when the culture temperature is too high, the growth of Torula sp. is inhibited. When the culture temperature is 32°C, the growth activity of both is balanced, the fermentation state is optimal, the generated flavor substances are rich, the sour and bitter taste is moderate and balanced, and the sensory score is the highest, 80 points. Therefore, the optimal culture temperature is 32°C.

[0123] 4. Addition amount of lemongrass

[0124] The addition amount of lemongrass is the key to determining the characteristic aroma of lemongrass-flavored coffee. A small amount of addition can elegantly enhance the brightness of coffee, impart fresh lemon, citrus aroma and a touch of herbal sweetness, and harmonize with the flavor of coffee itself, playing a role of highlighting the main point. When the addition amount is moderate, the characteristic flavor of lemongrass will be more prominent, presenting a strong lemon sugar, lemon grass tea-like dominant flavor, which may suppress the regional characteristics of coffee itself, forming a unique but balanced special flavor style. Once the addition amount is excessive, the strong herbal and lemon aldehyde taste will become extremely aggressive, producing a chemical and artificial flavor similar to dishwashing liquid and essence, completely covering the original flavor of coffee, and the taste is dry and harsh, which is regarded as a processing defect. Under the condition that other conditions remain unchanged, the fermentation comparison experiment of different addition amounts of lemongrass was carried out according to the inoculum concentration of 6 Lg CFU / mL, the strain addition amount of 3%, the fermentation time of 24 h, and the lemongrass addition amount of 5%. The comparison results of coffee cup evaluation scores are shown in Table 10.

[0125] Table 10 Comparison of coffee cup evaluation scores at different addition amounts of lemongrass

[0126] Group Group Sensory evaluation First group 3% 78 points Second group 4% 81 points Third group 5% 83 points Fourth group 6% 80 points Fifth group 7% 76 points

[0127] According to the results shown in Table 10, different addition amounts of lemongrass have different effects on coffee flavor. When the addition amount of lemongrass is too low, the lemongrass and microorganisms are not fully integrated, and the lemongrass flavor is too weak; when the addition amount of lemongrass is too high, the lemongrass flavor is too pungent and uncomfortable. When the addition amount of lemongrass is 5%, the lemongrass flavor is moderate and well integrated with coffee, which is soft and not conspicuous. At this time, the sensory score is the highest, 83 points. Therefore, the optimal addition amount of lemongrass is 5%.

[0128] In conclusion, by comparing the evaluation scores of the optimized experiments, it is known that in the method steps of the application, the optimal inoculum concentration of the compound starter is 6 Lg CFU / mL; the optimal addition amount of the compound starter is 3%; the optimal fermentation time is 24 h; the optimal fermentation temperature is 32 DEG C; and the optimal addition amount of the citronella grass is 5% of the coffee beans.

Claims

1. A strain of the yeast Torulaspora delbrueckii, characterized in that, The Torulaspora delbrueckii CII-19 strain is preserved in the China General Microbiological Culture Collection Center on July 17, 2025, with a preservation number of CGMCC No. 35270 and a preservation address of No. 3, Beichen West Road, Haidian District, Beijing.

2. A Lactobacillus fermentum strain, characterized in that, The Lactobacillus fermentum AI-08 strain is preserved in the China General Microbiological Culture Collection Center on May 9, 2024, with a preservation number of CGMCC No. 30567 and a preservation address of No. 3, Beichen West Road, Haidian District, Beijing.

3. A coffee composite starter culture, characterized in that, The composite starter culture comprises a mixed seed bacteria suspension or a fermentation bacteria powder prepared by mixing the activated Torulaspora delbrueckii CII-19 strain of claim 1 and the Lactobacillus fermentum AI-08 of claim 2 in a volume ratio of 1:

1.

4. A process for the preparation of a citronella-flavored coffee, characterized in that, The method comprises the following steps: 1) Sorting coffee beans: sorting high-quality beans from cleaned coffee bean raw materials, and reserving after removing defective beans and other impurities; 2) Rehydrating coffee beans: using sterile water, placing the beans in the fermentation tank at a bean-to-water ratio of 1:2, adding water to occupy about 1 / 2 of the total volume of the tank, and soaking the coffee beans until they are grayish white and have uniform color; 3) Sterilization: sealing with a sterile sealing film, sterilizing at a temperature of 110°C for 5 minutes, and naturally cooling after sterilization until the temperature drops to 25-35°C; 4) Preparing a bacteria suspension: centrifuging the activated Torulaspora delbrueckii CII-19 and Lactobacillus fermentum AI-08 fermentation strains, discarding the culture medium, and adding sterile physiological saline to adjust the concentration to 6-8 lg CFU / mL to prepare a bacteria suspension, and preparing a mixed seed bacteria suspension by mixing the prepared Torulaspora delbrueckii CII-19 and Lactobacillus fermentum AI-08 bacteria suspensions in a ratio of (1-2):(1-2); 5) Inoculation: inoculating the mixed seed bacteria suspension prepared in step 4 onto the surface of sterile coffee beans, mixing thoroughly, and inoculating the microorganisms to a concentration of 3-7 lg CFU / mL in the fermentation tank; 6) Fermentation: moving the inoculated fermentation tank into a constant-temperature room, maintaining the temperature at 32±2°C, maintaining the humidity at a relative humidity of 85% or higher, and continuing to ferment for 12-36 hours; 7) Adding citronella grass: grinding the washed and dried citronella grass into powder, sterilizing it with ultraviolet light, and adding it to the fermentation tank at a ratio of 3-7% of the mass of the green coffee beans, and shaking well; 8) Secondary fermentation: moving the fermentation tank with added citronella grass into a constant-temperature room, maintaining the temperature at 32±2°C, maintaining the humidity at a relative humidity of 85% or higher, and continuing to ferment for 12-36 hours; 9) Washing and drying: washing the citronella grass from the fermented coffee beans and retaining only the coffee beans, and drying the coffee beans in a drying device at 40-45°C until the moisture content is less than 12%, to obtain citronella-flavored coffee beans. 10) roasting: the above-mentioned citronella-flavored coffee beans are roasted at a pan temperature of 190-200 ℃, a fire power of 60%, a roasting time of 7-10 min, and a pan-out temperature of 205-218 ℃ to obtain medium-lightly roasted citronella-flavored coffee beans.

5. The method of claim 4, wherein, The ratio of the delbrueckii Torula CII-19 and the lactobacillus fermentum AI-08 in the mixed seed bacteria suspension in the step 4) is 1:1 in volume ratio; the concentration of the delbrueckii Torula CII-19 bacteria suspension is 8 lg CFU / mL; and the concentration of the lactobacillus fermentum AI-08 bacteria suspension is 8 lg CFU / mL.

6. The method of claim 4, wherein, The inoculation concentration in the fermenter of the step 5) is 6 lg CFU / mL.

7. The method of claim 4, wherein, The fermentation time in the steps 5) and 8) is 24 h.

8. The method of claim 4, wherein, The addition amount of the citronella grass in the step 7) is 5% of the coffee beans.

9. Citronella-flavored coffee beans prepared by the method of any one of claims 4-8.

10. Application of the coffee compound starter culture of claim 3 in the production of coffee.

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