Pichia pastoris, saccharomyces cerevisiae strain, screening method and application thereof

Abstract

Pichia pastoris, Saccharomyces cerevisiae strains, and screening methods and applications thereof. Among them, ester-scented Pichia kluyveri and floral-scented Saccharomyces cerevisiae strains are obtained from fresh coffee fruits of Katim, and are identified after isolation and purification. The fermentation method comprises the following steps: coffee green beans are mixed with ancient tea pollen and osmanthus pollen, and are sterilized; after centrifugation, a composite bacterial liquid is prepared, and is mixed with raw materials to ferment at 28-32 DEG C and 65-75% humidity for 20-28 hours; and after drying, the coffee bean product with the flavor of ancient tea flower is obtained by roasting. The product has the composite flavors of tea flower, osmanthus and tea, and has soft fruit acid, prominent floral scent and long-lasting tea flavor. The fermentation is controllable, efficient and simple in process, the product quality is stable, and the product is suitable for industrial production, and meets the needs of consumers for novel and healthy composite flavor coffee.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and food deep processing technology, and particularly relates to Pichia pastoris, Saccharomyces cerevisiae strains, their screening methods and applications. Background Technology

[0002] As a globally important economic crop and a widely popular beverage, coffee's flavor and quality are the core factors determining its market value. In the initial processing of green coffee beans, fermentation plays a crucial role. Fermentation not only degrades the coffee pectin layer but is also a key stage where microorganisms utilize bean substrates (such as sugars and proteins) to metabolize and generate flavor precursors such as organic acids, alcohols, and esters, directly establishing the sensory characteristics of the final roasted coffee.

[0003] Traditional coffee fermentation relies heavily on wild microbial communities in the natural environment. Due to the highly uncontrollable nature of environmental microorganisms, natural fermentation is easily affected by climate, temperature, humidity, and processing conditions, leading to an imbalance in the fermentation microecology. This not only results in significant batch-to-batch quality variations and poor flavor reproducibility, but also makes it highly susceptible to over-fermentation, producing a sour or earthy taste due to the excessive proliferation of spoilage bacteria or other microorganisms. To overcome the uncontrollability of natural fermentation, targeted inoculation fermentation technology is increasingly being applied to the processing of specialty coffee. Current technologies often use a single commercial yeast (such as a single brewer's yeast) for fermentation. While this can improve fermentation stability, the enzyme systems and metabolic pathways of a single strain are relatively limited, often only highlighting a single dimension of flavor (such as simple acidity or basic wine aroma), making it difficult to construct a complex flavor profile that is layered, aromatic, and full-bodied.

[0004] In recent years, with the increasing demand for specialty and blended coffees, combining coffee with uniquely flavored plant ingredients (such as flowers and fruits) has become a hot trend in innovation. Ancient tea tree flowers (the blossoms of the tea tree) are rich in tea polyphenols, flavonoids, amino acids, and unique volatile aromatic substances (such as linalool and geraniol), possessing a delicate high-boiling-point floral aroma and good antioxidant physiological activity. However, most current "tea flower flavored" coffees on the market are prepared using simple physical mixing methods, such as blending tea flowers with roasted coffee beans through a "scenting" process, or directly adding tea flower extract and syrup to the coffee liquid. These physical intervention methods have significant physical barriers and technical defects: not only is the flavor integration low, but the aroma is also superficial and easily evaporates during brewing; more importantly, physical blending cannot promote the release of macromolecular aroma precursors in tea flowers, making it difficult to achieve a deep biochemical fusion of the coffee's base flavor and the characteristic aroma of tea flowers. This results in a fragmented taste and a lack of aftertaste in the final product, and the active ingredients of the tea flowers are not efficiently biotransformed and utilized. In recent years, utilizing specific microorganisms to ferment green coffee beans to enhance targeted flavors has become a research hotspot for improving coffee quality. For example, patent document CN121136833A discloses the use of Torulaspora delbrueckii and staged fermentation with lactic acid bacteria to prepare lemongrass-flavored coffee. This flavored coffee attempts to integrate the plant aromas of the strain and plant matrix (citronella) into the coffee beans through secondary fermentation. However, this method lacks research on the fusion of the fermented aroma of the strain and the flavor of the plant matrix, and lacks accurate control over the stability and harmony of the flavor. Patent document CN121780333A proposes a method combining micro-germination treatment with golden bean fermentation to improve the fruity aroma of coffee. Although this technology can activate the enzyme system inside the coffee bean and enhance aroma precursors, the germination process causes changes in the physical structure of the coffee bean, and uneven heating is prone to occur during subsequent roasting. Moreover, the flavor it produces is mainly concentrated at the fruity level, lacking the ability to target specific aroma substances. Furthermore, patent document CN121570402A describes a technology for preparing active products using the co-fermentation of green tea powder and coffee, focusing on enhancing the soothing and antioxidant effects of the products. Although the application involves the mixed fermentation of multiple strains, its application is mainly geared towards the skincare field. The fermentation process involves coffee grinding and filtration, which cannot maintain the integrity of the green coffee beans and is difficult to process in the traditional sense of specialty coffee beans. The strain combination is also unlikely to produce high concentrations of aromatic esters and floral substances.

[0005] To effectively enrich floral compounds within green coffee beans, some studies have attempted to introduce plant-based ingredients during the fermentation stage. However, relying solely on natural microbial fermentation or single-strain yeast fermentation can lead to uncoordinated flavor profiles, making it difficult to release more aromatic precursors and form abundant esters. Floral compounds, acting as a "base aroma" to support and anchor the "high aroma" of tea flowers, can easily result in a thin flavor profile, or even mask the coffee's own excellent acidity due to improper fermentation.

[0006] Therefore, there is an urgent need in this field for a novel targeted co-fermentation technology that combines non-Saccharomyces yeast with Saccharomyces yeast, both possessing specific metabolic targeting properties. This technology utilizes ester-producing yeast to synthesize abundant esters, building a rich fruity base and enhancing body, while synergistically promoting the efficient fermentation and enhanced floral aroma production of floral yeast. This allows the bound aroma compounds from ancient tea tree flowers to be fully released and infiltrated into the green coffee beans. This has significant practical implications for solving the problems of flavor separation and low aroma retention in existing tea flower coffee, as well as for developing high-value-added, complex-flavor fermented coffees. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and to provide Pichia pastoris, Saccharomyces cerevisiae strains, their screening methods and applications.

[0008] The present invention first provides Pichia kluyveri PB105, which was deposited at the China Center for Type Culture Collection on April 16, 2026, with accession number CCTCC No: M2026700.

[0009] We also provided Saccharomyces cerevisiae PB101, which was deposited at the China Center for Type Culture Collection on April 16, 2026, with accession number CCTCC No: M2026701.

[0010] The present invention also provides a microbial fermentation agent comprising Pichia kluyveri PB105 and Saccharomyces cerevisiae PB101.

[0011] Another object of the present invention is to provide a fermentation processing method for ancient tree camellia flavored coffee beans, the fermentation processing method comprising the following steps: S1. Pre-processing stage: 500g of green Catimor coffee beans, 35g of Yunnan Lincang Yongde ancient tree tea flower Pu'er tea tree white tea withered honey raw tea leaves, and 3g of Hubei Xianning freeze-dried osmanthus flowers; grind the selected amount of ancient tree tea flowers and osmanthus flowers in a grinder to 80-100 mesh, mix with the green Catimor coffee beans, put into a fermentation tank, seal, and sterilize with high pressure steam at 110℃ for 10 minutes. After sterilization, place on an ultra-clean workbench to cool naturally to room temperature for later use. S2. Bacterial Culture Preparation Stage: Pichia kluyveri PB105 and Saccharomyces cerevisiae PB101, which have undergone three generations of activation respectively, are centrifuged and then the cell concentration is adjusted to the desired level using sterile physiological saline. Take 1.5 ml of Pichia pastoris PB105 bacterial culture and 0.5 ml of Saccharomyces cerevisiae PB101 bacterial culture respectively, and add them to 200 mL of sterile water to prepare fermentation culture; S3. Fermentation stage: Mix the green coffee beans, ancient tree tea flower powder, osmanthus powder and fermentation liquid evenly, and ferment at 28-32℃ and 65-75% humidity for 20-26 hours, shaking once every 6 hours during the process. S4. Post-processing stage: Separate the fermented coffee beans from the powder, dry the coffee beans at 50-55℃ until the moisture content is 10%-12%, and then roast them to obtain ancient tree camellia flavored coffee beans.

[0012] A further technical solution of the present invention is: the sterilization in step S1 is high-pressure steam sterilization at 110°C for 10 minutes; The culture medium for activating the strains in step S1 is PDB medium for Kluyveromyces PB105 and Saccharomyces cerevisiae PB101.

[0013] A further technical solution of the present invention is: the PDB culture medium (1L) is: 12g potato extract powder, 20g glucose, distilled water to 1L, and 20.0g agar to form a solid culture medium (PDA culture medium).

[0014] Another object of the present invention is to provide an ancient tree camellia flavored coffee bean, prepared by the aforementioned fermentation processing method.

[0015] A further technical solution of the present invention is that it has at least one or more of the following flavors: tea flower fragrance, osmanthus fragrance, tea flavor, fruit fragrance, floral fragrance, and nut fragrance, and also has a sweetness similar to floral fragrance, fruit fragrance, and tea flavor.

[0016] This invention provides the combined application of Pichia kluyveri PB105 and Saccharomyces cerevisiae PB101 in the fermentation of green coffee beans; or The combined application of Pichia kluyveri PB105 and Saccharomyces cerevisiae PB101 in enhancing the fruity aroma of green coffee beans and reducing the bitterness of coffee.

[0017] The beneficial effects of this invention are as follows: By synergistically combining the ester-producing aroma characteristics of Kluyveromyces PB105 with the floral aroma-producing function of Saccharomyces cerevisiae PB101, and integrating flavor compounds from ancient tea and osmanthus, coffee beans acquire at least one or more complex flavors among tea, osmanthus, tea, and floral-fruity aromas. Bitterness is significantly reduced, fruit acidity is mellow, and the aftertaste is long-lasting. The flavor profile far surpasses that of single-strain fermentation or physically mixed products. The use of a directional inoculation compound fermenting agent replaces natural or single-strain fermentation, ensuring clear and controllable fermentation parameters, stable microbial community structure, and minimal flavor differences between batches of coffee beans with high reproducibility. This solves the core problem of unstable quality in traditional fermentation, making the fermentation process controllable and stable. The fermentation cycle is only 20-26 days. The fermentation time is significantly shorter than some traditional fermentation processes, and the pretreatment, fermentation, and processing steps are simple, requiring no complex equipment. This makes it suitable for standardized and industrialized production, reducing production costs and technical barriers, improving fermentation efficiency, simplifying the fermentation process, and innovating processing methods. Through microbial co-fermentation, the flavor substances of tangerine peel and the base flavor of coffee are biotransformed and deeply integrated, avoiding flavor separation problems caused by physical mixing. The flavor remains harmonious and unified from high temperature to medium and low temperature drinking, improving the degree of flavor integration. Attached Figure Description

[0018] Figure 1 Sensory evaluation and establishment of ester-producing and floral-producing strains (Figure) Figure 2 This is a graph showing the ester content of the strains, where, Figure 2 Figure a shows the results of ester production by the strain determined by the glycerol tributylate method; Figure 2 b is a graph showing the total ester content of the strain determined by the hydroxylamine-ferric chloride colorimetric method; Figure 3 Screening droplet heatmaps; Figure 4 Ancient tree tea flower fermentation process. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments: like Figure 1-4 As shown, This invention provides a strain of ester-producing Pichia pastoris, a strain of floral-scented yeast Saccharomyces cerevisiae, and a fermentation agent containing these strains, thereby providing a process-controllable and reproducible fermentation method for ancient tree camellia-flavored coffee beans, ultimately obtaining tangerine peel-flavored coffee beans with unique flavor, stable quality, and health benefits.

[0020] Two strains with clearly defined functions are provided: one is a Kluyveki yeast that produces esters and the other is a brewer's yeast that produces floral aromas. Their synergistic effect can achieve multiple benefits, such as enhancing floral and fruity aromas, reducing the original bitterness in green coffee beans, and improving flavor harmony. A microbial fermentation agent based on the above-mentioned strain is provided to provide a stable and controllable functional microbial community for coffee fermentation.

[0021] This invention provides a fermentation method for ancient tree camellia flavored coffee beans, which achieves a deep fusion of ancient tree camellia flavor and coffee base flavor, and solves the flavor separation problem caused by physical mixing. This product offers a coffee bean with an ancient tea tree flavor that is rich in floral and fruity aromas, low in bitterness, and has a long-lasting, tea-like aftertaste, satisfying consumers' demand for novel and complex flavored beverages.

[0022] This study provides information on the combined application of Pichia pastoris LB105 and Saccharomyces cerevisiae PB101 in the fermentation of green coffee beans, as well as their combined application in improving coffee flavor and reducing bitterness.

[0023] Strain identification and preservation 1. Kluyveromycin LB105 Classification and nomenclature: Pichia kluyveri PB105; Preservation information: Deposited on April 16, 2026 at the China Center for Type Culture Collection, accession number CCTCC No: M2026700; address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Strains were obtained from sun-dried Catimor coffee cherries from Menglian Tianyu Coffee Factory in Menglian County, Pu'er City, through isolation, purification, and screening. Functional characteristics: It has a prominent ability to produce ester-like aromatic compounds, which can significantly improve the smoothness of coffee flavor.

[0024] 2. Saccharomyces cerevisiae PB101 Classification and nomenclature: Saccharomyces cerevisiae PB101 Preservation information: Deposited on April 16, 2026 at the China Center for Type Culture Collection, accession number CCTCCNo: M2026701, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0025] Strain origin: Homologous to Pichia kluyveri PB105, both were obtained from sun-dried Catimor coffee cherries from Menglian Tianyu Coffee Factory in Menglian County, Pu'er City through isolation, purification, and screening.

[0026] Functional characteristics: It can metabolize to produce obvious floral and fruity aromas, giving coffee pleasant aromas such as floral, fruity, and wine-like notes, and can work synergistically with Pichia kluyveri PB105 to enrich the flavor profile.

[0027] Microbial fermentation agent A microbial fermentation agent comprises Pichia kluyveri PB105 and Saccharomyces cerevisiae PB101. In this microbial fermentation agent, the ratio of the two strains is optimized for the best fermentation effect, with a preferred volume ratio of Pichia kluyveri PB105 to Saccharomyces cerevisiae PB101 of 1.5:1.

[0028] The present invention provides a fermentation method for ancient tree camellia-flavored coffee beans, which includes four stages: pretreatment, bacterial culture preparation, fermentation, and roasting, as detailed below: Pre-processing stage: Mix green Catimor coffee beans with ancient tree tea flower powder and osmanthus powder ground to 80-100 mesh, sterilize, and cool to room temperature; select 500g of green Catimor coffee beans, 35g of Yunnan Lincang Yongde ancient tree tea flower Pu'er tea tree white tea withered honey raw tea leaves, and 3g of Hubei Xianning freeze-dried osmanthus flowers; grind the selected amount of ancient tree tea flowers and osmanthus flowers in a grinder to 80-100 mesh, mix with green Catimor coffee beans, put into a fermentation tank, seal with sealing film, sterilize with high pressure steam at 110℃ for 10 minutes, and place on a clean bench to cool naturally to room temperature for later use.

[0029] Inoculum preparation stage: Preserved Pichia kluyveri PB105 and Saccharomyces cerevisiae PB101 were inoculated into PDB medium and activated for three generations at 30℃ to obtain stable bacterial cultures. The activated cultures of both strains were centrifuged at 10,000 rpm for 10 minutes, and the supernatant was discarded. The cell concentration of both strains was adjusted to the appropriate level using sterile physiological saline. Take 1.5 mL of Kluyveromyces PB105 and 1 mL of Saccharomyces cerevisiae PB101 bacterial culture, add them to 200 mL of sterile water, shake well to prepare a compound fermentation culture.

[0030] Fermentation stage: Mix Katim coffee green beans, ancient tree camellia powder, osmanthus powder, and fermentation liquid evenly, and ferment at 28-32℃ and 65-75% humidity for 20-26 hours, shaking once every 6 hours during the process; pour the prepared fermentation liquid into the pre-treated fermentation tank containing coffee green beans, ancient tree camellia powder, and osmanthus powder, and shake thoroughly to ensure even contact between the coffee green beans, ancient tree camellia powder, osmanthus powder, and fermentation liquid; place the fermentation tank in a climate simulation incubator and ferment at 28-32℃ and 65-75% humidity for 20-26 hours, shaking once every 6 hours during the fermentation process, for a total of 4-5 times, to ensure uniform fermentation.

[0031] Post-processing stage: Separate the fermented coffee beans from the powder, dry the coffee beans at 50-55℃ until the moisture content is 10%-12%, and then roast them to obtain ancient tree camellia flavored coffee beans; after fermentation, separate the coffee beans from the dried tangerine peel powder and orange peel powder, and collect the fermented coffee beans; spread the separated coffee beans on the roasting rack and place them in an oven at 50-55℃ to dry until the moisture content of the coffee beans drops to 10%-12%, and then roast them to obtain ancient tree camellia flavored coffee products.

[0032] An ancient tree camellia-flavored coffee bean is prepared using the aforementioned fermentation process. This coffee bean possesses at least one or more complex flavor profiles selected from camellia, osmanthus, tea, fruit, floral, and nutty aromas, along with a sweetness reminiscent of floral, fruity, and tea notes. The inherent bitterness of coffee is significantly reduced, while it exhibits a mellow fruity acidity and a lingering sweetness. The flavor profile is rich, harmonious, and consistent, maintaining a stable ancient tree camellia flavor and floral-fruity notes regardless of whether it is consumed at high, medium, or low temperatures.

[0033] Application of strains The combined application of Pichia kluyveri PB105 and Saccharomyces cerevisiae PB101 in the fermentation of green coffee beans; or the combined application in enhancing coffee flavor, reducing bitterness, and improving the aftertaste of coffee.

[0034] Experimental culture medium and reagent formulations Potato Dextrose Broth (PDB) medium (1L) is prepared by adding 12g potato extract powder, 20g glucose, distilled water to 1L, and 20.0g agar to form a solid medium, which is potato dextrose agar (PDA).

[0035] Experimental equipment and related instruments Microbial screening equipment: MISS cell high-throughput single-cell microbial screening equipment; Fermentation equipment: YTQP-300B artificial climate chamber; Methods for isolation, purification and identification of bacterial strains Strains Isolation and Purification The screening of Pichia kluyveri PB105 and Saccharomyces cerevisiae PB101 in this invention: The samples were isolated and purified from fresh sun-dried coffee cherries collected from Menglian Tianyu Coffee Plantation in Menglian County, Pu'er City, Yunnan Province, China, as detailed below: Sample pretreatment: Mix the sample bag thoroughly, weigh 50g of fresh coffee fruit sample and put it into a 250mL Erlenmeyer flask, add 100mL of sterile physiological saline, crush the pulp and vortex for 5 minutes, place at 30℃ for 24h to prepare bacterial suspension; then transfer the coffee sample to a clean bench and let the bacterial suspension stand at room temperature for 10 minutes to settle, and collect the bacterial suspension using the five-point sampling method.

[0036] Plate counting: The bacterial suspension obtained after sample pretreatment and filtration was serially diluted with sterile physiological saline at 10¹, 10², 10³, and 10³. Dilute 120 μL of each sample onto a PDA plate for yeast isolation; incubate the PDB plates at 28°C for 24 hours, observe colony growth, calculate the total colony count based on the colony count on the plates, and dilute the samples to [a specific concentration]. The total number of colonies in the fresh fruit samples was approximately 162-200 single colonies, which met the criteria for high-throughput single-cell screening using MISS cells.

[0037] MISS cell isolation and culture: Based on the results of plate testing, the fresh coffee fruit samples were prepared into a 10⁴-fold bacterial suspension using sample bottles, generating consumable box 1—droplet tray 2. After incubation at 30℃ for 24 h, single-cell sorting was performed. A total of 2580 positive droplets were obtained from the coffee samples in PDB medium, which were inoculated into 96-well microplates, and 200 μL of PDB medium was added for expansion at 30℃. The selected single-cell strains were inoculated onto PDA solid medium for streak purification and amplification. Two replicates were used: one for strain identification, where genomic DNA was extracted using a kit, and PCR amplification was performed targeting the ITS sequence. The product was then sent for sequencing after passing agarose gel electrophoresis, and the sequencing results were compared against the NCBI database using BLAST. The other replicate was used for strain preservation in a 3:1 mixture of glycerol and water at -80°C.

[0038] strain identification Fungal molecular identification (Pichia kluyveri PB105 and Saccharomyces cerevisiae PB101): DNA was extracted from the strains using a novel plant genomic DNA extraction kit. PCR amplification was performed using primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The PCR reaction system consisted of 45 μL Taq PCR Master Mix, 2.0 μL total DNA template, 2.0 μL ITS1, and 2.0 μL ITS4. 4.0 μL, total volume 25 μL; PCR reaction conditions were 98℃ pre-denaturation for 2 min, 35 cycles (including denaturation at 94℃ for 10 s, annealing at 54℃ for 10 s, extension at 72℃ for 10 s, and final extension at 72℃ for 5 min); the amplified products were sequenced after detection by 1% agarose gel electrophoresis, and the sequencing results were compared with BLAST sequences in the NCBI database.

[0039] Screening methods for ester-producing yeasts and aroma-producing yeasts: 1. Screening of ester-producing strains by olfactory test: According to GB / T 16291.1-2012 "General Guidelines for the Selection, Training and Management of Sensory Analysis Evaluators", the evaluation form for the ester aroma smelling test of yeast in PDA ferments should follow the principle of "Quantitative Descriptive Analysis" (QDA). By establishing consensus terminology and using a 9-point scale, the scientific quantification of fermentation flavor can be achieved.

[0040] Ethyl acetate standard reference sample: Prepare an ethyl acetate standard solution to a concentration of 10 μL / mL in a pure aqueous solution. This concentration range is considered to have a distinct "fruity / ester aroma" characteristic in sensory studies (it is generally believed that concentrations above 150 mg / L may produce an unpleasant sensation).

[0041] The sensory evaluation form is shown in Table 1.

[0042] 2. Tributyric acid glyceride method for plate analysis: Prepare identification medium (1% peptone, 0.5% yeast extract, 1% glyceryl tartrate emulsion, 2% agar, pH 6.0). Spot the pre-screened and purified strains onto plates using toothpicks and incubate at 30°C for 5–7 days. Measure the diameter of the clear zone (D) and the colony diameter (d) using calipers, and calculate the D / d ratio.

[0043] 3. Determination of total esters using the hydroxylamine-ferric chloride colorimetric method: Take 2 mL of the fermentation supernatant from the strain after three generations of culture, add 1 mL of alkaline hydroxylamine hydrochloride solution (a mixture of 2 M hydroxylamine hydrochloride and 3.5 M sodium hydroxide in equal volumes), and react at room temperature for 15 min. Add 1 mL of 4 M hydrochloric acid to adjust the pH, and finally add 1 mL of 0.37 M ferric chloride acidic solution. After color development, measure the absorbance at 525 nm. Plot a standard curve using ethyl acetate as the standard.

[0044] 3.1 Method for constructing a standard curve using ethyl acetate as a standard Objective: To establish a quantitative relationship (usually a linear relationship) between absorbance (A) and standard concentration (C), and obtain the regression equation A = kC + b, which is used to calculate the concentration of unknown samples.

[0045] 3.1.1 Accurately take a certain amount of ethyl acetate (1100ul) and dissolve it in ester-free ethanol.

[0046] 3.1.2 Transfer to a 100 mL volumetric flask and dilute to volume, then mix well. This solution is the standard stock solution, with a concentration of 1000 μg / mL.

[0047] A. Prepare six clean 50 mL volumetric flasks.

[0048] B. As shown in Table 1, accurately pipette different volumes of standard stock solution and add them to the respective solutions:

[0049] Table 1. Standard Working Fluid Preparation Table C. Perform the colorimetric reaction: Accurately pipette 2.0 mL of solution from each standard working solution (S1-S5) and blank tube, and place them into another set of clean colorimetric tubes or test tubes, strictly following the same operating procedure as the sample to be tested.

[0050] D. Absorbance Measurement: Preheat the spectrophotometer and zero it at 525 nm using a blank tube solution. Measure and record the absorbance (A) values ​​of the standard tubes S1 to S5 sequentially.

[0051] E. Plot a scatter plot on graph paper or using software, with the standard concentration (C, μg / mL) as the x-axis (X) and the measured absorbance value (A) as the y-axis (Y). Perform linear regression analysis on the data points to obtain the linear equation Y = kX + b and the correlation coefficient R².

[0052] k: Slope, representing sensitivity.

[0053] b: Intercept, which should ideally be close to 0.

[0054] R² should be ≥ 0.999, indicating a good linear relationship.

[0055] After screening using olfactory, tricresyl ester, and hydroxylamine-ferric chloride methods, strain PB105 was found to have a strong ester-producing ability, while strain PB101 had a significant floral aroma-producing ability. Subsequent sequence comparison revealed that strain PB105 was Pichia kluyveri and strain PB101 was Saccharomyces cerevisiae.

[0056] The table below shows the relevant sequences of strains Pichia kluyveri PB105 and Saccharomyces cerevisiae PB101.

[0057]

[0058] Core parameters of the compound fermentation process for ancient tree camellia flavored coffee A fermentation method for a tangerine peel flavored coffee uses Yunnan small-bean coffee-Catim washed-commercial beans; the fermentation strains used are Pichia kluyveri PB105 and Saccharomyces cerevisiae PB101. As mentioned above, the Pichia kluyveri PB105 and Saccharomyces cerevisiae PB101 strains were microbially preserved (preserved with glycerol at -80℃); the edible tea leaves used are ancient tea tree flowers from Yongde, Lincang, Yunnan, withered honey-processed Pu'er tea trees, and freeze-dried osmanthus flowers from Xianning, Hubei.

[0059] Example 1: Isolation, purification, and identification of the strain:

[0060] MISS cell isolation and culture: Based on the results from plate culture, a 10⁴-fold bacterial suspension was prepared from fresh coffee cherry samples into sample vials, generating consumable box 1—droplet tray 2. After incubation at 30℃ for 24 hours, single-cell sorting was performed. A total of 2580 positive droplets were obtained from the coffee samples in PDB medium, and then... The assay involved sorting and collecting 288 droplets, which were then inoculated into 96-well microplates. 100 μL of PDB medium was added to each well, and the plates were incubated at 30°C for 24 hours. The heatmap of the sorted droplets is shown below. Figure 3 As shown.

[0061] Sequence alignment results showed that a total of 29 representative microorganisms were screened, mainly including: PB101 Saccharomyces cerevisiae, PB102 Maudiozyma humilis, PB104 Pichia fermentans, PB105 Pichia kluyveri, PB106 Kurtzmaniella quercitrusa, PB108 Torulaspora delbrueckii, and PB109 Starmerella bacillaris, a fructrophile non-yeast yeast. Sensory evaluation results:

[0062] like Figure 1 As shown in Table 2, to accurately assess the volatile flavor potential of the candidate strains, the experimental team organized 12 sensory evaluators to participate in the olfactory test. To understand the ester aroma characteristics, diluted ethyl acetate standard was used as the reference flavor standard, and blind tests and multi-dimensional sensory scoring were conducted on the fermentation volatiles of the 7 target strains screened in the early stage.

[0063]

[0064] Table 2 Sensory Test Scoring Table 2. Sensory establishment of the core ester-producing strain PB105 (Pichia kluyveri) The olfactory results showed that strain PB105 (Pichia kluyveri) exhibited extremely prominent and pure ester aroma characteristics. Sensory evaluators unanimously agreed that its volatile aroma profile had a very high degree of flavor overlap with the ethyl acetate reference standard, with strong aroma penetration and no obvious off-odors, performing excellently in the "total ester aroma intensity" dimension. With its solid and mellow ester flavor base, this strain ranked second in overall preference among the seven evaluated strains, fully validating its application value as a core ester-producing target strain. Meanwhile, it is worth noting that strain PB101 (Saccharomyces cerevisiae) achieved the highest score (ranked first) in "overall preference" in this sensory evaluation. Unlike conventional Saccharomyces cerevisiae, strain PB101 released a highly distinctive complex floral and fruity aroma. Evaluators described its flavor profile as: an elegant, uplifting floral base interwoven with bright, sweet fruit notes, accompanied by an extremely pleasant, honey-like silky sweetness. This complex and sophisticated flavor profile perfectly matches the invention's goal of enhancing the "floral aroma and sweetness" of coffee, establishing its absolute advantage as a core floral aroma-targeting strain.

[0065] Single microorganisms often struggle to simultaneously achieve both extremely high concentrations of pure ester aromas and elegant, complex floral and fruity notes. Based on the aforementioned olfactory evaluation results, this invention creatively proposes a dual-strain synergistic strategy: combining *Pichia pastoris* (PB105), an excellent producer of ester aromas, with *Saccharomyces cerevisiae* (PB101), which dominates the release of advanced floral and fruity aromas. In the subsequent coffee fermentation process, PB105 is responsible for synthesizing a large amount of precursor esters to construct a rich flavor framework, while PB101 is responsible for transforming and imparting a high-pitched, sweet floral aroma to the coffee. This complementary strength lays the foundation for the microbial strains that enable the co-fermentation of coffee with a highly distinctive "ancient tree camellia flavor." Example 2: Results of ester production by the strain determined by the glyceryl tartrate method:

[0066] The results showed that there were significant differences in the ability of different strains to break down tricresyl ester.

[0067] High-yielding but poor-tasting strains not listed in the national "List of Microbial Strains that Can Be Used in Food" and "List of Microbial Strains that Can Be Used in Infant Food" or other legally regulated catalogs were eliminated: PB104 (Pichiafermentans Pasteurella) and PB106 (Kurtzmaniella quercitrusa). Despite their large hydrolytic transparent circle areas (120.10 mm² and 118.13 mm², respectively) and extremely high ester-producing potential, these two strains were excluded from the fermentation system based on pre-sensory assessment (PB104 has a strong, pungent odor) and (PB106 is not listed in the "List of Microbial Strains that Can Be Used in Food").

[0068] The core target strain, PB105 (Pichia kluyveri), a target ester-producing strain, achieved an ester-producing zone area of ​​104.72 mm². Among safe fermentation strains with ester-producing capabilities, this further confirms PB105's excellent ability to produce ester-like flavor compounds. Simultaneously, the target strain PB101 (Saccharomyces cerevisiae) also produced a transparent circle of 89.19 mm², indicating that it not only dominates the release of high-grade floral and fruity aromas but also possesses a certain foundation for ester production and forms a good flavor synergy with PB105 in complex fermentation. Example 3: Determination of total ester content in bacterial strains using the hydroxylamine-ferric chloride colorimetric method:

[0069] The sensory properties of volatile flavor compounds largely depend on the physicochemical basis of microbial secondary metabolism. The total ester content in the fermentation supernatant of each candidate strain was accurately and quantitatively analyzed using the hydroxylamine-ferric chloride colorimetric method. The results showed that the total ester synthesis capacity of non-Saccharomyces cerevisiae populations was significantly better than that of Saccharomyces cerevisiae. Among them, strain PB104 (Pichia fermentans) had the highest total ester concentration, reaching 4584.44 μg / mL, while the target floral and fruity aroma-producing strain PB101 (Saccharomyces cerevisiae) produced 2748.40 μg / mL, lower than non-Saccharomyces cerevisiae, but still showing the necessary basic ester production characteristics.

[0070] The "masking effect" of negative flavors means that high biomass ester metabolism does not directly equate to a positive sensory experience. Olfactory sensory data showed that strain PB104 had extremely high ester yields, but the fermentation process was accompanied by a strong, pungent odor, a strong sour taste, and a musty, yeast-autolysis smell. These undesirable fermentation byproducts strongly "masked" the target ester aroma, severely disrupting flavor harmony and placing this strain at the lowest level in overall sensory preference. Furthermore, although PB106 (Kurtzmaniella quercitrusa) exhibited superior ester metabolism levels (total ester content of 4442.07 μg / mL), such microorganisms are not yet included in the national "List of Microbial Strains that Can Be Used in Food" and "List of Microbial Strains that Can Be Used in Infant Food," and have been excluded due to potential biosafety restrictions and regulatory risks. In summary, considering the high ester yield, sensory flavor evaluation, and compliance with food safety regulations, PB105 (Pichia kluyveri) achieved the highest total ester accumulation target of 4334.07 μg / mL, with extremely high sensory matching of pure ester aroma and full compliance with biosafety characteristics, and was selected as the best candidate fermentation strain.

[0071] PB105 exhibits a high yield and pure ester aroma, while leading the dual-strain fermentation strategy in synergy with PB101, which releases intense floral and fruity aromas as a "flavor enhancer." This synergy successfully overcomes the problem of single aroma and odor masking in traditional fermentation, achieving a directional, efficient, and safe reshaping of ancient tree camellia flavors in fermented coffee beans. Example 4: Core parameters of the compound fermentation process for ancient tree camellia flavored coffee:

[0072] As shown in the figure, to achieve the above objectives, this invention provides a method for preparing tangerine peel flavored coffee beans based on the co-fermentation of Kluyveromyces and Saccharomyces cerevisiae, characterized by the following steps: This invention provides a fermentation method for tangerine peel flavored coffee, which is achieved through four stages: bacterial culture preparation, pre-fermentation preparation (pretreatment), fermentation, and post-treatment. Inoculum preparation stage: Preserved Pichia kluyveri PB105 and Saccharomyces cerevisiae PB101 were inoculated into PDB medium and activated for three generations at 30℃ to obtain stable bacterial cultures. The activated cultures of both strains were centrifuged at 8000 rpm for 10 minutes, and the supernatant was discarded. The cell concentration of both strains was adjusted to the appropriate level using sterile physiological saline. Take 1.5 mL of Kluyveromyces PB105 and 1 mL of Saccharomyces cerevisiae PB101 bacterial culture, add them to 200 mL of sterile water, shake well to prepare a compound fermentation culture.

[0073] Pre-processing stage: Mix the green Catimor coffee beans with ancient tree tea flower powder and osmanthus powder ground to 80-100 mesh, sterilize, and then cool to room temperature; Select 500g of green Catimor coffee beans, 35g of Yunnan Lincang Yongde ancient tree tea flower Pu'er tea tree white tea withered honey raw tea leaves, and 3g of Hubei Xianning freeze-dried osmanthus flowers; Select a certain amount of ancient tea flowers and osmanthus flowers and grind them in a grinder to 80-100 mesh. Mix them with green Catimor coffee beans and put them into a fermentation tank. Seal the tank with sealing film and sterilize it with high-pressure steam at 110℃ for 10 minutes. After sterilization, place it on a clean bench and let it cool naturally to room temperature before use.

[0074] Fermentation stage: After pretreatment, place the green coffee beans, ancient tree tea flower powder, and osmanthus powder into a fermentation tank, then pour in the prepared fermentation liquid and shake thoroughly to ensure that the green coffee beans, ancient tree tea flower powder, and osmanthus powder are evenly in contact with the fermentation liquid. Place the fermentation tank in a climate simulation incubator and ferment for 20-26 hours at 28-32℃ and 65-75% humidity. Shake the tank once every 6 hours during fermentation, for a total of 4-5 times, to ensure uniform fermentation.

[0075] Post-processing stage: After fermentation, the coffee beans are separated from the dried tangerine peel powder and orange peel powder, and the fermented coffee beans are collected. The separated coffee beans are spread out on the roasting rack and placed in an oven at 50-55℃ to dry until the moisture content of the coffee beans drops to 10%-12%. After roasting, a coffee product with the flavor of ancient tree camellia can be obtained.

[0076] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions conceived without inventive effort should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A Pichia pastoris strain, characterized in that, This strain is *Kluyveromyces* (Kluyveromyces). Pichia kluyveri PB105 was deposited at the China Center for Type Culture Collection on April 16, 2026, with accession number CCTCC No: M2026700.

2. A strain of *Saccharomyces cerevisiae*, characterized in that, This strain is *Saccharomyces cerevisiae*. Saccharomyces cerevisiae PB101 was deposited at the China Center for Type Culture Collection on April 16, 2026, with accession number CCTCC No: M2026701.

3. A microbial fermentation agent, characterized in that, It comprises the Kluyveromyces PB105 as described in claim 1 and / or the Saccharomyces cerevisiae PB101 as described in claim 2, and combinations thereof.

4. A fermentation and processing method for ancient tree camellia-flavored coffee beans based on the strains or fermenting agents of claims 1-3, characterized in that, The fermentation processing method includes the following steps: S1. Pre-processing stage: By weight ratio, 500 parts of green Catimor coffee beans, 35 parts of Yunnan Lincang Yongde ancient tree tea flower Pu'er tea tree white tea withered honey raw tea leaves, and 3 parts of Hubei Xianning freeze-dried osmanthus flowers are ground to 80-100 mesh in a grinder. After mixing with the green Catimor coffee beans, the mixture is placed in a fermentation tank, sealed, and sterilized by high-pressure steam at 110℃ for 10 minutes. After sterilization, it is placed in a clean bench and allowed to cool naturally to room temperature before use. S2, Bacterial Culture Preparation Stage: Kluyveromyces PB105 and Saccharomyces cerevisiae PB101, which have undergone three generations of activation, were centrifuged and then the cell concentration was adjusted to 7 × 10⁻⁶ cells / mL with sterile physiological saline. 7 CFU / mL - 2 × 10 8 CFU / mL, take 1.5ml of Kluyveromyces PB105 bacterial culture and Saccharomyces cerevisiae PB101 bacterial culture respectively, and add them to 200mL of sterile water to prepare fermentation culture; S3. Fermentation stage: Mix green Catim coffee beans, ancient tree tea flower powder, and osmanthus powder, then mix them evenly with the fermentation liquid. Ferment for 20-26 hours at a temperature of 28-32℃ and a humidity of 65-75%, shaking the mixture once every 6 hours during the process. S4. Post-processing stage: Separate the fermented coffee beans from the powder, dry the coffee beans at 50-55℃ to a moisture content of 10%-12%, and then roast them to obtain ancient tree camellia flavored coffee beans.

5. The fermentation method according to claim 4, characterized in that, The sterilization in step S1 is high-pressure steam sterilization at 110°C for 15 minutes. The culture medium used to activate the strains in step S1 is PDB medium for both Kluyveromyces PB105 and Saccharomyces cerevisiae PB101.

6. The fermentation method according to claim 5, characterized in that, The PDB culture medium is prepared by adding 12g of potato extract powder and 20g of glucose to 1L of distilled water, and then adding 20.0g of agar to make a solid culture medium.

7. A type of ancient tree camellia-flavored coffee bean, characterized in that, It is prepared by the fermentation processing method according to any one of claims 4-6.

8. The ancient tree camellia flavored coffee bean according to claim 7, characterized in that, It has at least one or more of the following flavors: tea flower, osmanthus, tea, fruit, floral, and nut, and also has a sweetness that combines floral, fruity, and tea-like notes.

9. The application of the Kluyveromyces PB105 of claim 1 and the Saccharomyces cerevisiae PB101 of claim 2 in the fermentation of green coffee beans.

10. The application of the Kluyveromyces PB105 of claim 1 and the Saccharomyces cerevisiae PB101 of claim 2 in improving coffee flavor and reducing coffee bitterness.

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