Lactobacillus rhamnosus AIV-37 and application thereof in preparation of Thelephora ganbajun flavor coffee beans
By using the co-fermentation technology of Lactobacillus rhamnosus AIV-37 and Gynostemma pentaphyllum, the problems of uncontrollable flavor and unstable quality during the fermentation process of Catimor coffee beans have been solved, achieving efficient and stable production of Gynostemma pentaphyllum flavored coffee, reducing the risk of contamination by other microorganisms, and ensuring the safety and flavor consistency of the product.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-03
- Publication Date
- 2026-03-13
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
In existing technologies, the fermentation process of Catimor coffee beans results in uncontrollable flavor, poor quality uniformity and stability, and the fermentation process is subject to contamination by miscellaneous bacteria and safety risks, making it difficult to achieve efficient and stable production of coffee with specific flavors.
Lactobacillus rhamnosus AIV-37 and Gynostemma pentaphyllum were co-fermented. By controlling the fermentation conditions and inoculum concentration, combined with pasteurization, Gynostemma pentaphyllum flavored coffee beans were prepared to ensure flavor consistency and safety.
This achieves stability and sustainability of the Ganba mushroom-flavored coffee beans, balances flavor, reduces the risk of contamination by other microorganisms, and improves the controllability of the fermentation process and the consistency of product quality.
Smart Images
Figure CN121653014A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial fermentation and deep food processing technology, specifically relating to a strain of Lactobacillus rhamnosus ( Lactobacillus rhamnosus AIV-37 and its application in the preparation of Ganbazin-flavored coffee beans. Background Technology
[0002] Catimor coffee is Arabica coffee. Coffea arabica ) and Robusta ( Coffea canephora Catimor is a hybrid variety developed by the Portuguese Coffee Research Institute (CIFC) in 1959 through crossbreeding Timor (a naturally disease-resistant variety) and Caturra (a dwarf, high-yielding Arabica). This variety was created to combine the flavor advantages of Arabica with the disease resistance of Robusta, especially against leaf rust, a major threat to coffee cultivation. Catimor inherited Timor's leaf rust resistance genes, exhibiting outstanding resistance in high-humidity growing regions such as Latin America and Southeast Asia, significantly reducing pesticide costs. Compared to traditional Arabica, Catimor has a shorter fruiting period (producing within 2-3 years of planting) and a 20%-30% increase in yield per unit area, making it suitable for growers seeking efficiency. It can grow at altitudes of 800-1600 meters, but optimal quality is typically found above 1000 meters. It is drought-tolerant and suitable for mountainous regions with variable climates.
[0003] Catimor coffee beans have traditionally been processed primarily using the wet washed method, which involves removing the skins from fresh coffee cherries, fermentation, washing, and finally slow air-drying. This method maximizes the preservation of its unique flavor and highlights its cleanliness. Honey treatment can also be used to enhance the sweetness of Catimor, but this method requires strict control of the fermentation time; otherwise, it can easily cause off-flavors in the coffee beans.
[0004] Catimor flavor assessments are polarized. High-altitude regions like Colombia and Yunnan are considered its superior growing areas. Refined Catimor from these regions can exhibit nutty and caramel aromas, medium acidity, and good body. Typically, Catimor has a relatively soft acidity, prominent caramel or brown sugar notes, and a noticeable oiliness. However, some cuppers believe it has a woody or bitter aftertaste reminiscent of Robusta, with a pronounced straw flavor, especially noticeable in low-altitude or over-fertilized growing conditions.
[0005] The key to controlling coffee bean fermentation using specific starter cultures lies in selecting the right starter culture and incorporating secondary fermentation. Starter cultures used in coffee refer to microorganisms (such as yeast and bacteria) or enzyme preparations used to regulate the coffee fermentation process. Their core functions are to break down pectin and sugars, accelerating the fermentation process; producing flavor precursors to enhance the coffee's acidity, sweetness, body, and aroma complexity; and inhibiting contamination by other microorganisms, thus improving fermentation stability and safety. Secondary fermentation technology involves artificially controlling the fermentation environment (such as anaerobic / aerobic conditions, adding microorganisms or fruits) to further process coffee beans that have already undergone primary processing. Its core advantage lies in significantly enhancing flavor complexity and market competitiveness. By precisely controlling temperature (18-22℃), time (24-72 hours), and microbial strains (such as brewer's yeast), this technology can impart prominent wine aromas, berry or tropical fruit notes to coffee (such as the wine-like fermentation notes of sun-dried Yunnan red wine), while simultaneously compensating for the deficiencies of insufficient primary fermentation. Despite being criticized by traditionalists for "over-intervention," secondary fermentation remains a key technology for specialty coffee regions to overcome flavor homogenization due to its high reproducibility and adaptability to high-sugar beans (such as Geisha). By incorporating dominant microorganisms and other flavor compounds into the coffee beans through secondary fermentation, undesirable flavors such as straw, astringency, and woodiness in Catimor coffee beans can be mitigated. Summary of the Invention
[0006] To address the problems existing in the prior art, this invention provides a Lactobacillus rhamnosus ( Lactobacillus rhamnosus AIV-37 was deposited on May 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34562. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Lactobacillus rhamnosus AIV-37 and Bacillus thuringiensis ( ) Thelephora ganbajun This method is applied to the fermentation of Catimor coffee beans to produce high-quality coffee beans with a dabba flavor. After professional cupping, the coffee beans produced have a distinct edible mushroom aroma, a mellow acidity with a honey-like sweetness, and a hint of bitterness in the nutty and chocolate notes. The aftertaste is sweet, with a long finish, a smooth and delicate mouthfeel, and a good balance. The overall flavor is pleasant, and the sensory evaluation score is 84.25 points.
[0007] The objective of this invention is achieved through the following technical solution: 1. Lactobacillus rhamnosus ( Lactobacillus rhamnosus AIV-37 acquisition and separation Milk cake obtained from Dali Prefecture, Yunnan Province, China was pulverized and placed in sterile physiological saline. After shaking and mixing, it was serially diluted 10-fold. The diluted solutions were spread on MRS agar medium and incubated at 37°C for 24-48 hours until single colonies grew. The dominant colonies were selected by observing the characteristics of colony morphology, size, color, gloss, texture, surface and edge conditions. The strain AIV-37 was obtained by streaking on MRS solid plate medium. 2. Identification of strain AIV-37 (1) Morphological characteristics of strain AIV-37: On MRS solid medium, the colonies are round, white, raised, smooth and moist, with neat edges; the bacteria are Gram-positive. (2) Molecular identification Genomic DNA was extracted from strain AIV-37 using a bacterial genomic DNA extraction kit. Using the extracted genome as a template, PCR amplification was performed using universal primers 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'. After sequencing the PCR products, the sequencing results were compared with sequences on NCBI. Combined with morphological characteristics and molecular identification results, the strain was ultimately identified as *Lactobacillus rhamnosus*. Lactobacillus rhamnosus ); 3. Strain AIV-37 was used to prepare Ganba mushroom-flavored coffee beans. (1) Preparation of AIV-37 bacterial suspension Remove the frozen *Lactobacillus rhamnosus* glycerol tubes from the -80℃ freezer and thaw at room temperature. Under aseptic conditions, inoculate the thawed *Lactobacillus rhamnosus* into MRS liquid medium. Incubate at 37±2℃ for 24-36 hours in a constant-temperature shaking incubator to obtain an activated strain. Incubate the activated strain at 37℃ in a constant-temperature shaking incubator until the logarithmic growth phase. Inoculate the logarithmic growth phase culture medium into fresh MRS liquid medium and incubate at 37±2℃ in a constant-temperature shaking incubator until the logarithmic growth phase is reached, completing the first subculture. Inoculate the first-generation culture medium into fresh MRS liquid medium and incubate at 37±2℃ in a constant-temperature shaking incubator until the logarithmic growth phase is reached, completing the second subculture. Centrifuge the second-generation culture medium at 4000-6000 rpm at 4℃ for 10-15 minutes, collect the bacterial sludge, and resuspend the sludge in sterile physiological saline to obtain a concentration of 10. 8 CFU / mL bacterial suspension.
[0008] (2) Pretreatment of coffee beans and ganba fungus S1. Coffee bean selection: Remove broken, moldy, insect-damaged, spotted, shelled, shriveled, and other defective beans and impurities from Yunnan small-bean coffee (Catim) washed coffee beans (green coffee beans) and set them aside; S2. Pretreatment of Ganba mushrooms: Fresh, disease-free, and pollution-free Yunnan native Ganba mushrooms are washed, crushed, and sterilized with ultraviolet light for 15-30 minutes for later use. S3. Sterilization: Boil purified water to 85-90℃. According to the bean-to-water ratio of 1:1.5-1.8 (g:mL), put the selected coffee beans and 3-5% of the pre-treated dried coffee bean bacteria into the 85-90℃ water for sterilization for 15-30 seconds. Then, put the sterilized mixture into a sterile fermentation tank. Seal the fermentation tank with a sealing film for microbial culture (the pore size of this sealing film is impenetrable to microorganisms, but allows gas to pass freely). Cool to a temperature of 25-35℃. (3) Fermentation S1. Inoculation: Spray the AIV-37 bacterial suspension into the cooled mixture of step (2) at a ratio of 0.05%-0.1% of the coffee bean weight, and mix well; S2. Fermentation: Transfer the inoculated mixture to a constant temperature room and ferment for 24-48 hours at 30-37℃ and relative humidity ≥85%. S3. Roasting: The coffee beans are separated from the ganba mushroom. The coffee beans are dried at 40-45℃ until the moisture content is 10%-12%. The dried coffee beans are placed in a drum roaster with an inlet temperature of 190-200℃ and a heat of 50%. The roasting time is 6-12 minutes, and the outlet temperature is 190-205℃, which yields medium-roasted ganba mushroom flavored coffee beans.
[0009] (4) Sensory evaluation S1. Grinding: The roasted coffee beans are ground using a grinder with a particle size of 65mm. S2. Brewing: Pour 200mL of hot water at 90-94℃ into 12.5g of coffee powder. After 4 minutes, break and remove the grounds one by one, and then conduct the evaluation. The cupping samples are randomly coded, labeled and sent. S3. Five baristas with Q-grader certification evaluate the coffee samples through smelling, sipping, and aftertaste procedures. Flavor characteristics are assessed based on ten comprehensive aspects: aroma, cleanliness, sweetness, acidity, body, flavor, aftertaste, balance, uniformity, and overall.
[0010] The mushroom-flavored coffee beans of this invention produce coffee beans with a distinct mushroom aroma, a mellow acidity with a honey-like sweetness, and a hint of bitterness in the nutty and chocolate notes. The aftertaste is sweet, with a long finish, a smooth and delicate mouthfeel, and a good balance. The overall flavor is pleasant, while the flavor of the non-mushroom-treated group of fermented coffee is weaker.
[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention employs the direct addition of edible *Lactobacillus rhamnosus*, allowing green coffee beans and *Lactobacillus rhamnosus* to co-ferment with each other under the action of *Lactobacillus rhamnosus*. This imparts a *Lactobacillus rhamnosus* flavor to the green coffee beans, effectively solving the technical problems of uncontrollable coffee flavor, poor uniformity and stability in coffee quality resulting from natural coffee fermentation, and the difficulty in preserving fresh coffee cherries and the seasonality of fresh coffee cherries hindering the industrialization of fermented products. Using dominant microorganisms from traditional fermented foods as fermentation bacteria and green coffee beans as raw materials, this invention produces coffee beans with a specific flavor, exhibiting sustainability and stability. The added flavor is integrated with the original coffee flavor through biotransformation, without any separation. It reduces the straw taste, bitterness, and astringency in Yunnan Catimor coffee beans, resulting in a balanced flavor and a smooth mouthfeel. Based on this method, dominant aroma-producing microorganisms from other fermented foods can be applied to coffee fermentation, ensuring the sustainability and product stability of coffee beans with specific flavors.
[0012] 2. This invention adds *Clerodendrum trichotomum* during the fermentation process, allowing its aromatic compounds to permeate the coffee beans. Through co-fermentation, the aroma of *Clerodendrum trichotomum* and the flavor of the coffee are fully integrated, creating a unique *Clerodendrum trichotomum* flavored coffee. By selectively fermenting and controlling the inoculum concentration, the consistency of flavor compounds produced during fermentation is ensured, thereby achieving a stable flavor profile.
[0013] 3. Specific bacterial suspensions and constant temperature and humidity fermentation conditions are used during the fermentation stage to ensure the controllability and repeatability of the fermentation process, thereby improving product quality consistency. By setting specific fermentation times, each batch of coffee beans is ensured to achieve ideal flavor and quality within the optimal fermentation period.
[0014] 4. Pasteurizing coffee beans and guarana during the pretreatment stage significantly reduces the risk of contaminating and pathogenic bacteria, ensuring the safety of the fermentation process and the final product, while preventing flavor loss from both the coffee beans and guarana. Using specific concentrations of fermentation strains eliminates harmful microbial contamination, reducing the risk of harmful bacteria growth during fermentation and improving product safety and stability.
[0015] 5. Utilize activated specific fermentation strains (Lactobacillus rhamnosus) to optimize the fermentation process, improve fermentation efficiency, shorten fermentation time, and ensure production efficiency and economic benefits. Maintain suitable temperature and humidity during fermentation to provide the optimal growth environment for the fermenting cells, ensuring a highly efficient fermentation process.
[0016] 6. From coffee bean selection, sterilization, inoculation, fermentation to subsequent processing (bean-microbead separation, drying), the entire process is clear and systematic, facilitating standardized operation and large-scale production. The operating conditions for each step are clearly defined, making it easy to execute and control in actual production, reducing process fluctuations and operational errors. Attached Figure Description
[0017] Figure 1 This is a colony morphology diagram of Lactobacillus rhamnosus AIV-37; Figure 2 Phylogenetic tree of Lactobacillus rhamnosus AIV-37; Figure 3 This is a diagram showing the state of the bacteria after the fermentation process is completed and the dried bacteria have been washed away, as described in this invention. Figure 4 Image of dried Ganbazha flavored coffee beans; Figure 5 Image of finished product: Dried mushroom flavored coffee beans; Figure 6 This is a diagram showing the state of coffee bean fermentation failure in control group 4. Detailed Implementation
[0018] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the scope of protection of the present invention is not limited to the contents described. Unless otherwise specified, the reagents and methods used in the embodiments are all conventional reagents and conventional methods. The MRS solid culture medium formulation in the examples is as follows: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, Tween-80 1 mL / L, K2HPO4•7H2O 2 g / L, sodium acetate•3H2O 5 g / L, triammonium citrate 2 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, agar 15 g / L, pH 6.2±0.2; the MRS liquid culture medium formulation in the examples is as follows: casein digest 10 g / L, beef extract 10 g / L, yeast extract 4 g / L, triammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, dipotassium hydrogen phosphate 2 g / L, glucose 20 g / L, Tween-80 1.08 g / L, pH 5.7±0.2.
[0019] Example 1: Lactobacillus rhamnosus ( Lactobacillus rhamnosus Isolation and identification of AIV-37 1. Isolation of strain AIV-37 One g of milk cake obtained from Dali Prefecture, Yunnan Province, China, was pulverized and placed in 9 mL of sterile physiological saline. After shaking and mixing, it was serially diluted 10-fold to prepare 10... -4 ~10-9 The dilutions were prepared by taking 0.2 mL of each of the different concentration gradients and spreading them onto solidified MRS agar medium. The culture was carried out at 37°C until single colonies grew. The dominant colonies were selected by observing the morphology, size, color, gloss, texture, surface and edge characteristics of the colonies. The strain AIV-37 was obtained by streaking the MRS solid plate medium for purification. 2. Identification of strain AIV-37 (1) Morphological characteristics of strain AIV-37: On MRS solid medium, the colonies are white, raised, round, with a smooth and moist surface and neat edges; the bacteria are Gram-positive. Figure 1 ); (2) Molecular identification Genomic DNA was extracted from strain AIV-37 using a bacterial genomic DNA extraction kit. Using the extracted genome as a template, PCR amplification was performed using universal primers 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'. After sequencing the PCR products, the sequencing results were compared with sequences on NCBI. Combined with morphological characteristics and molecular identification results, the strain was ultimately identified as *Lactobacillus rhamnosus*. Lactobacillus rhamnosus ); see phylogenetic tree Figure 2 .
[0020] Example 2: Preparation of Dried Mushroom Flavored Coffee 1. The coffee bean variety used is Yunnan small-bean coffee, Catimor washed commercial bean; the fermentation strain is Lactobacillus rhamnosus AIV-37, and the Ganba fungus is a local edible Ganba fungus from Yunnan. 2. Preparation of Lactobacillus rhamnosus AIV-37 bacterial suspension (1) Take out the frozen Lactobacillus rhamnosus glycerol tubes from the -80℃ freezer and thaw them at room temperature; under aseptic conditions, inoculate Lactobacillus rhamnosus AIV-37 into MRS liquid medium and culture it in a constant temperature shaking incubator at 37℃ and 160rpm for 24h to obtain the activated strain; (2) Primary culture: The activated strain was cultured in a constant temperature shaking shaker at 37°C and 160 rpm for 24 h. The turbidity and growth of the culture medium were monitored and observed in real time to ensure that the lactic acid bacteria reached the logarithmic growth phase. (3) First subculture: Take 1% of the initial culture medium and inoculate it into a new MRS liquid medium. Shake and culture it in a constant temperature shaking shaker at 37°C and 160 rpm for 24 h. Repeat the same method, monitor and observe the turbidity and growth of the culture medium in real time to ensure that the lactic acid bacteria reach the logarithmic growth phase. (4) Second subculture: Take 1% of the first generation culture solution and inoculate it into a new MRS liquid culture medium. Shake and culture in a constant temperature shaking shaker at 37°C and 160 rpm for 24 h. Repeat the same method, monitor and observe the turbidity and growth of the culture solution in real time to ensure that the lactic acid bacteria reach the logarithmic growth phase. (5) Pour the second-generation culture medium into centrifuge tubes, centrifuge at 5000 rpm and 4℃ for 12 minutes, collect the bacterial sludge, resuspend the bacterial sludge in sterile physiological saline, and obtain 10 8 AIV-37 bacterial suspension at CFU / mL; 3. Pretreatment of coffee beans and dried mushrooms S1. Coffee bean selection: Remove broken, moldy, insect-damaged, spotted, shelled, shriveled, and other defective beans and impurities from Yunnan small-bean coffee (Catim) washed coffee beans (green coffee beans) and set them aside; S2. Pretreatment of Ganba mushrooms: Fresh, disease-free, and pollution-free Yunnan native Ganba mushrooms are washed, crushed, and sterilized under ultraviolet light for 30 minutes for later use. S3. Sterilization: Boil purified water to 85℃, and sterilize the selected coffee beans and 3% of the pre-treated dried bacon by weight together in water at a bean-to-water ratio of 1:1.5 (g:mL) for 30 seconds. Place the sterilized mixture into a sterile fermentation tank and seal the fermentation tank with a sealing film for microbial culture (the pore size of this sealing film is impenetrable to microorganisms, but allows gas to pass freely). Cool to 30℃. 4. Fermentation S1. Inoculation: Spray the Lactobacillus rhamnosus AIV-37 bacterial suspension from step 2 onto sterile coffee beans at 0.05% of the coffee bean weight and mix well; S2. Fermentation: The inoculated mixture was transferred to a constant temperature chamber and fermented for 48 hours at 37°C and 85% relative humidity. The coffee beans were then separated from the kiwifruit powder. Figure 3 Coffee beans are dried at 45°C to a moisture content of 11%. Figure 4 ); S3. Roasting: Place the dried coffee beans in a drum roaster. The bean temperature is 200℃, the heat is maintained at 50%, the roasting time is 8 minutes, and the output temperature is 200℃ to obtain medium-roasted Brussels flavored coffee beans. Figure 5 (Experimental group).
[0021] A control group (control group 1) was set up, consisting of untreated Catim roasted coffee beans. Control group 2: The fermentation strain was Lactobacillus plantarum ( Lactobacillus plantarum The remaining steps are the same as above; Control group 3: The preparation method is the same as that of the experimental group, except that Lactobacillus rhamnosus AIV-37 bacterial suspension is not inoculated. The rest of the operation is the same as above. Control group 4: Yunnan small-bean coffee (Catim) green coffee beans were placed in a fermentation tank at a bean-to-water ratio of 1:1.5 g:mL. The fermentation tank was sealed with a microbial culture sealing film. After fermentation at 37℃ and 85% relative humidity for 48 hours, the coffee beans were dried at 45℃ to a moisture content of 11%. The dried coffee beans were then placed in a drum roaster with an input temperature of 200℃, a heat of 50%, a roasting time of 8 minutes, and an output temperature of 200℃, resulting in medium-to-light roasted Ganbajun-flavored coffee beans.
[0022] Control group 5: The preparation method is the same as that of the experimental group above, except that the selected coffee beans and 3% of the bean weight of pretreated Ganba fungus are put into room temperature water at a ratio of 1:1.5 g:mL. After mixing, the mixture is sterilized at 110℃ for 10 min. The rest of the operation is the same as above. Control Group 6: The selected coffee beans and purified water were placed in a fermentation tank at a bean-to-water ratio of 1:1.5 (g:mL). The coffee beans were soaked until they turned grayish-white and had a uniform color. The fermentation tank was then sealed with sterile sealing film and sterilized at 110℃ for 10 minutes. After cooling, 0.05% of the weight of the coffee beans in Lactobacillus rhamnosus suspension was added and fermented at 37℃ and 85% relative humidity for 24 hours. Then, UV-sterilized Dried Bacillus powder was added to the tank containing the fermented coffee beans and fermented for another 24 hours under the same conditions. The remaining drying and roasting steps were the same as those in the experimental group. Example 3: Performance evaluation of coffee beans obtained from the experimental and control groups 1-6 in Example 2 1. Sensory evaluation S1. Grind the roasted coffee beans using a grinder with a particle size of 65mm; S2. Pour 200mL of 92℃ hot water into 12.5g of coffee powder. After 4 minutes, break up and remove the coffee grounds one by one. Then conduct the tasting. The cupping samples are randomly coded, labeled and submitted. S3. Five baristas with Q-grader certification evaluate the coffee samples through smelling, sipping, and aftertaste procedures. Flavor characteristics are assessed based on ten comprehensive aspects: aroma, cleanliness, sweetness, acidity, body, flavor, aftertaste, balance, uniformity, and overall.
[0023] The results showed that the coffee beans prepared using the experimental method had a rich and complex aroma of dried mushrooms, with a well-integrated flavor profile of dried mushrooms and coffee, and a long finish. Cupping score: 84.25 points. Flavor description: A rich aroma of dried mushrooms, nuts, and chocolate, with bright acidity and a slight bitterness, followed by a sweet aftertaste, a long finish, a smooth and delicate mouthfeel, good balance, and an overall pleasant flavor. The results indicate that the coffee beans prepared using the method of this invention have the best effect, possessing excellent flavor, body, and balance.
[0024] The control group 1 coffee beans had only a bitter and slightly acidic taste, a strong smoky flavor, a straw-like taste, and a slightly astringent taste. The cupping score was 78.25.
[0025] Control group 2 used *Lactobacillus plantarum* as the fermentation agent to ferment Catimor coffee beans. The resulting coffee beans had a distinct *Lactobacillus plantarum* aroma and stable quality, but the overall flavor was relatively weak. Cupping score: 81.75 points. Flavor description: distinct *Lactobacillus plantarum* aroma, nutty, chocolate, slightly acidic, slightly bitter, slightly sweet aftertaste, short finish. The results indicate that the *Lactobacillus plantarum*-fermented *Lactobacillus plantarum*-fermented *Lactobacillus plantarum* flavored coffee beans were relatively stable, but the overall flavor was weak, and the *Lactobacillus plantarum* aroma was not well integrated with the coffee beans. The control group consisted of three Catimorum coffee beans fermented with *Lactobacillus rhamnosus*. The overall fermentation process was normal, but the roasted beans exhibited a limited range of flavor profiles and inconsistent quality. Cupping score: 81.00 points. Flavor description: Distinct *Lactobacillus rhamnosus* aroma, nutty notes, chocolate notes, and slight bitterness, but the earthy taste of the *Lactobacillus rhamnosus* was quite pronounced. The results indicate that the *Lactobacillus rhamnosus* flavor in the Catimorum coffee beans, without inoculation but with added *Lactobacillus rhamnosus* to adjust the flavor, did not blend well with the coffee, resulting in a limited range of flavor profiles and a bitter taste. Overall, the flavor was inferior to the *Lactobacillus rhamnosus*-infused *Lactobacillus rhamnosus* flavored coffee.
[0026] In control group 4, some coffee beans turned green during fermentation, accompanied by musty, sour, and other off-odors. The liquid became cloudy and yellowish (see...). Figure 6 If the experiment fails, no cupping score will be given.
[0027] The control group of coffee beans underwent high-temperature sterilization, resulting in beans with a pronounced *Gnaphalium affine* flavor, slightly bitter and slightly acidic, but with a bland overall flavor. Cupping score: 79.50. Flavor description: *Gnaphalium affine* aroma, nutty, chocolate, slightly acidic, slightly bitter. The results indicate that the *Gnaphalium affine* flavored coffee beans treated with high-temperature sterilization had a weak, one-dimensional flavor profile and an overall bland taste.
[0028] The control group consisted of coffee beans that underwent high-temperature sterilization followed by sequential fermentation (with Lactobacillus rhamnosus AIV-37 and *Gynostemma pentaphyllum* added sequentially). The resulting coffee beans exhibited a weak *Gynostemma pentaphyllum* flavor, a weak coffee taste, and a disjointed flavor profile with a short finish. Cupping score: 79.25. Flavor description: *Gynostemma pentaphyllum* aroma, nutty notes, chocolate, earthy notes, and noticeable acidity. The results indicate that sequential fermentation of coffee beans with *Gynostemma pentaphyllum* resulted in insufficient integration of the *Gynostemma pentaphyllum* flavor with the coffee beans, a short finish, and a lack of flavor complexity.
[0029] 2. Determination of pH content, caffeine content, total polyphenol content, and lactic acid content. The pH content, caffeine content, total polyphenol content, and lactic acid content of coffee beans obtained from the experimental group and control group 1-6 were determined.
[0030] pH measurement: Weigh 5.00g of coffee sample, add 25mL of distilled water, shake for 20min, and measure directly using a pH meter. The results are shown in Table 1.
[0031] Caffeine content determination (HPLC method): 1) Sample preparation: Weigh 1g (accurate to 0.001g) of homogeneous sample pulverized to less than 30 mesh into a 250mL Erlenmeyer flask, add about 200mL of water, heat in a boiling water bath for 30min, shaking occasionally, remove and cool under running water for 1min, add 5g of magnesium oxide, shake, then heat in a boiling water bath for 20min, remove the Erlenmeyer flask, cool to room temperature, transfer to a 250mL volumetric flask, add water to make up to the mark (so that the caffeine content in the sample solution is within the range of the standard curve), shake well, let stand, take the supernatant and filter through a microporous membrane for later use.
[0032] 2) Preparation of standard curve: Inject the standard series working solutions into the liquid chromatograph and measure the corresponding peak areas. Plot the standard curve with the concentration of the standard working solution as the abscissa and the peak area as the ordinate.
[0033] 3) Determination of sample solution: Inject the sample solution into the liquid chromatograph and determine the qualitative analysis by retention time. Record the peak area at the same time. Obtain the concentration of caffeine in the test solution according to the standard curve. The number of parallel determinations shall not be less than two. Total polyphenol content determination: The total polyphenol content was determined using the Folin-Ciocalteu method. 1.00 g of green coffee bean powder was weighed into a beaker, and 20 mL of 50% ethanol solution was added. The mixture was ultrasonically extracted at 60℃ for 40 min, then diluted to volume and filtered. 1.0 mL of the coffee extract was placed in a 10 mL colorimetric tube, and 0.2 mL of Folin-Ciocalteu was added. After 3–8 min, 2 mL of 75 g / L Na₂CO₃ solution was added, and the mixture was diluted to volume with pure water. The mixture was allowed to stand in the dark for 1 h, and the absorbance was measured at 765 nm. The results are shown in Table 1.
[0034] Lactic acid content determination: The lactic acid content of coffee beans in the experimental group and the control group was determined according to the method in GB12456-2021 "National Food Safety Standard - Determination of Total Acid in Food". The results are shown in Table 1.
[0035] As shown in Table 1, compared with control group 1, the experimental group method had the least impact on the caffeine content of coffee beans and could increase the total polyphenol and lactic acid content, indicating that pasteurization and co-fermentation are beneficial to the preservation of caffeine; control groups 5 and 6 used high-temperature sterilization, which easily caused the loss of polyphenols and lactic acid; control group 3 did not have inoculation, and the lactic acid content was similar to that of control group 1, indicating that inoculation can increase the lactic acid content in coffee; Table 1. Indicators of Ganbajun-flavored coffee beans .
[0036] Example 4: Effects of different inoculum sizes, fermentation temperatures, and fermentation times of Lactobacillus rhamnosus AIV-37 on sensory scores The preparation method in this embodiment is the same as in Example 2, except that the inoculation amount of the bacterial suspension in step 4 is 0.01%, 0.05%, 0.1%, 0.15%, and 0.2%. The sensory evaluation method in Example 3 was used for scoring, and the results are shown in the table below:
[0037] The results showed that coffee beans produced with an inoculum concentration of 0.05-0.1% had higher sensory scores; coffee beans with the Ganba mushroom flavor had the best taste, moderate acidity, and the best balance when the inoculum concentration was 0.05%.
[0038] (2) Effect of fermentation temperature on coffee sensory scores The preparation method in this embodiment is the same as in Example 2, except that the fermentation temperature in step 4 is set to 28-40℃; the sensory evaluation method of Example 3 is used for scoring, and the results are shown in the table below:
[0039] The results showed that coffee beans fermented at 30-37℃ had higher sensory scores, and those fermented at 37℃ produced more flavor compounds and had the best coffee flavor and taste.
[0040] (3) The effect of fermentation time on coffee sensory scores The preparation method in this embodiment is the same as in Example 2, except that the fermentation temperature and time in step 4 are 24-72 hours; the sensory evaluation method of Example 3 is used for scoring, and the results are shown in the table below:
[0041] The optimal fermentation time for Ganba mushroom-flavored coffee beans is 36-48 hours, resulting in the best flavor and taste, optimal strain activity, more complete production of flavor compounds, and a balanced flavor profile. Fermentation of Ganba mushroom coffee beans for 24 hours is too short, leading to incomplete flavor compound formation and a bland taste. Fermentation times of 60 hours and 72 hours may result in over-fermentation, producing undesirable flavor compounds and causing the coffee to taste too acidic or bitter, leading to lower cupping scores.
[0042] (4) Effect of the amount of *Ganbaella* added on the sensory score of coffee The preparation method in this embodiment is the same as in Example 2, except that the amount of *Ganba* bacteria added in step 4 is 1-7%; the sensory evaluation method of Example 3 was used for scoring, and the results are shown in the table below:
[0043] The results showed that the scores were higher when the inoculation amount of *Gnaphalium affine* was 3% and 5%, at which point the flavor of *Gnaphalium affine* and coffee beans were moderately integrated, producing a pleasant flavor and a good taste. When the inoculation amount was below this range (1%), the coffee flavor was weak and the aftertaste was short. When the inoculation amount was above this range (7%), the flavor of *Gnaphalium affine* was too strong, masking the aroma of the coffee itself, resulting in an unbalanced taste and thus affecting the sensory score and lowering it.
Claims
1. A type of Lactobacillus rhamnosus ( Lactobacillus rhamnosus AIV-37, its accession number at the China General Microbiological Culture Collection Center is CGMCC No. 34562.
2. The use of Lactobacillus rhamnosus AIV-37 as described in claim 1 in the preparation of Ganbajun-flavored coffee beans.
3. The application according to claim 2, characterized in that: Selected dried green coffee beans and pre-treated dried bacon are rehydrated and sterilized, then inoculated with Lactobacillus rhamnosus AIV-37 bacterial solution and fermented together at 30-37℃ and relative humidity ≥85% for 24-48 hours. After removing the dried bacon, the beans are dried and roasted to obtain dried bacon flavored coffee beans.
4. The application according to claim 3, characterized in that: Pretreatment of Ganba mushrooms refers to the process of cleaning and crushing Ganba mushrooms and then sterilizing them with ultraviolet light for 15-30 minutes.
5. The application according to claim 4, characterized in that: The amount of pre-treated ganbazin added is 3-5% of the weight of the dried green coffee beans.
6. The application according to claim 3, characterized in that: The inoculum size of Lactobacillus rhamnosus AIV-37 was 0.05%-0.1% of the weight of dried green coffee beans.
7. The application according to claim 3, characterized in that: After removing the drier bacteria, the drying temperature is 40-45℃, and the moisture content of the dried coffee beans is 10%-12%.
8. The application according to claim 7, characterized in that: Roasting involves placing dried coffee beans in a drum roaster, with the bean temperature at 190-200℃, maintaining a heat of 50%, roasting for 6-12 minutes, and exiting at 190-205℃.