A strain of Kluyveromyces macrocarpa and its application in coffee bean degumming technology

Abstract

This invention discloses a strain of Kluyveromyces marxianus and its application in coffee bean degumming technology, belonging to the field of microbiology. The Kluyveromyces marxianus strain CI-06 was deposited on May 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 30564. The CI-06 strain possesses pectinase activity, and the pectinase it produces has high activity, which can be used in the biological degumming process of coffee to shorten degumming time and improve the quality of commercial coffee beans.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a strain of Kluyveromyces martensii and its application in coffee bean degumming technology, and especially to a strain of Kluyveromyces martensii CI-06 and its application in coffee bean degumming technology. Background Technology

[0002] Coffee is an important crop grown in many countries and one of the world's most popular beverages. There are two main methods for primary coffee processing: wet processing and dry processing. Yunnan, the main coffee-producing region, has abundant water resources and primarily uses wet processing. The processing mainly includes peeling, degumming, drying, grading, and packaging. Degumming is the most complex and prone to problems, significantly impacting coffee quality. Wet processing includes conventional wet processing and mechanical wet processing, the main difference being the degumming process. Coffee pectin is composed of sugars, enzymes, protopectin, and pectin, with galacturonic acid and methanol as its main components. It is highly hygroscopic and difficult to dry, so pectin is usually removed to accelerate drying. Conventional wet processing uses a traditional method: after peeling, the coffee is soaked in water, relying on the enzymes in the pectin itself for biological fermentation and degumming. This process is time-consuming and easily affected by weather and temperature. Furthermore, incomplete degumming can lead to residual mucus that becomes a breeding ground for microorganisms during the later storage and drying processes of coffee, causing secondary fermentation, resulting in off-flavors, and ultimately affecting the quality of the coffee. The degradation of mucus is closely related to the production of extracellular mucus-degrading enzymes, such as pectinase, by microorganisms. The removal of mucus is crucial for coffee drying, requiring the highest level of technical expertise, involving the most complex process, and being most prone to problems. Drying plays a vital role in coffee flavor; coffee consumers consider flavor more important than nutritional value. The quality of coffee degumming directly affects the storage effect and flavor of coffee, thus impacting the profits of the coffee industry.

[0003] Currently, the main degumming methods for coffee include natural fermentation degumming, chemical degumming, physical degumming, and microbial degumming; each has the following characteristics: (1) Natural fermentation degumming: It is uncertain when the fermentation endpoint will be reached, resulting in low coffee quality; (2) Chemical degumming: Acids and alkalis can easily destroy the flavor components of coffee and introduce inorganic ions, causing environmental pollution; (3) Physical degumming: After the coffee fruit is peeled, it is washed and the coffee beans are rubbed to remove the mucus, which can easily trigger secondary fermentation; (4) Enzymatic degumming: Enzymes have high requirements for environment, temperature, and pH value, and cannot be promoted in actual production; (5) Microbial degumming: In addition to rapid growth and reproduction and easy expansion of culture, microorganisms also have the advantages of accelerating the degradation of coffee mucus to form a good flavor.

[0004] The article "Research on the Impact of Enzymatic Degumming on Coffee Quality," published in the January 2019 issue of *Agricultural Product Processing*, revealed that enzymatic fermentation degumming has no adverse effects on coffee quality; on the contrary, it slightly outperforms traditionally fermented degummed coffee in terms of aroma, aftertaste, and balance. Furthermore, Chinese patent CN110305858A discloses an enzyme preparation and method for degumming fresh coffee cherries. This method uses an enzyme preparation composed of pectin esterase, pectin lyase, and polygalacturonase to improve degumming efficiency, reduce mold rate, and enhance coffee bean quality, while also reducing water consumption and being more environmentally friendly. Chinese patent CN114410489B discloses an anomalous Wickham yeast strain CAP5 and its applications. This strain exhibits good acid resistance and pectinase activity, and can not only degrade pectin to shorten degumming time and improve the quality of commercial coffee beans during biological degumming, but can also be used in the production of ethanol and fruit wine.

[0005] However, there are still few reports on coffee degumming strains, and practical applications are still in the experimental and demonstration stage. Therefore, it is necessary to develop more strains that can be used for degumming, promote the development of coffee bio-degumming technology, reduce the risk of coffee mold during drying and storage, and improve coffee flavor. Summary of the Invention

[0006] To achieve the above objectives, this invention provides a strain of Kluyveromyces marxianus CI-06, which is then inoculated into a fermentation tank containing peeled fresh coffee cherries to promote bio-degumming of coffee. The specific operation is as follows:

[0007] A strain of Kluyveromyces marxianus CI-06, isolated from fermented meat, a traditional fermented food from Yunnan, was deposited on May 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30564.

[0008] Application of the Kluyveromyces macrocarpa strain CI-06 in coffee degumming preparations.

[0009] Furthermore, the inoculation rate of the Kluyveromyces macrocarpa CI-06 strain during the coffee degumming process is 2%-10%.

[0010] A method for degumming coffee beans includes the following steps:

[0011] S1. After washing and peeling the fresh coffee cherries, add them to the fermentation tank. The filling volume of the fermentation tank shall not exceed 80% of the fermentation tank capacity, and the tank shall be sealed with a breathable sealing film.

[0012] S2, Inoculation: The activated Kluyveromyces macrocarpa strain CI-06 is inoculated into a fermentation tank containing peeled fresh coffee cherries at an inoculation concentration of 10. 5 ~10 8 CFU / mL;

[0013] S3, fermentation and degumming: After inoculation, the fermentation tank is transferred to a constant temperature and humidity chamber, where the temperature is maintained at 28℃±2℃ and the relative humidity is above 60%. Degumming is completed in 24-60 hours of fermentation.

[0014] Furthermore, the inoculation amount of Kluyveromyces martensii CI-06 strain in S2 is 4%.

[0015] Furthermore, the fermentation time in S3 is 48 hours, the fermentation temperature is 24°C, and the pH value is 6.

[0016] The working principle of this invention: This invention provides a strain of Kluyveromyces marxianus CI-06, which has pectinase activity. The pectinase produced by this strain has high activity and is used to inoculate Kluyveromyces marxianus into the bio-degumming process of coffee. The pectinase produced by this strain reacts with the pectin in coffee beans to degrade the pectin and improve the mucus removal effect.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] (1) A high-pectinase-producing strain of Kluyveromyces macrocarpa CI-06 was provided, filling the gap in degumming strains that can be used for coffee biodegumming technology.

[0019] (2) The CI-06 strain has pectinase activity, and the pectinase it produces has high activity. It can be used to produce pectinase or to degumm coffee, shorten the degumming time, and improve the quality of commercial coffee beans. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the application of Max Kluyveromycin in coffee bean production according to the present invention.

[0021] Figure 2 This is a colony diagram of the Kluyveromyces marxianus CI-06 strain of the present invention;

[0022] Figure 3 The standard curve for galacturonic acid;

[0023] Figure 4 This is a bar chart showing the effect of different inoculum amounts on pectinase activity according to the present invention.

[0024] Figure 5 A bar chart showing the effect of different fermentation temperatures on pectinase activity;

[0025] Figure 6 A bar chart showing the effect of different pH values ​​on pectinase activity;

[0026] Figure 7 A bar chart showing the effect of different fermentation times on pectinase activity;

[0027] Figure 8 This is a bar chart illustrating the orthogonal experimental verification of the present invention.

[0028] Figure 9 The images show the process of coffee bean fermentation and degumming, as well as a record of the coffee beans dried after 48 hours of fermentation. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, in the following descriptions, well-known structures and techniques have been omitted to avoid unnecessarily obscuring the concept of the invention.

[0030] Example 1: Screening, isolation and purification of strains

[0031] Dominant yeasts and lactic acid bacteria were screened from fermented meat in Yunnan, fermented bean curd in Mouding, pickled vegetables in Fuyuan, grapes from Binchuan in Dali, and coffee in Lancang. 25g of sample was accurately weighed and placed in 225mL of physiological saline (0.85% NaCl), and serially diluted (10... -2 10 -3 10 -4 10 -5 and 10 -6Take 0.2 mL of each serially diluted solution and spread it onto YPD solid medium and MRS medium, respectively. Perform three parallel experiments for each gradient. Based on the morphology, color, size, edge, smoothness, and moisture of the colonies, use a sterile inoculation needle to pick single colonies and inoculate them onto YPD solid medium and MRS medium. Incubate at 28℃±1℃ and 37℃±1℃ for 2 days, respectively. Perform 3–5 isolation and purification cycles to obtain yeast strains numbered CI-06 and CI-0 on YPD medium. Single colonies of lactic acid bacteria numbered MF7-1, MF7-3, MF3-1, MF3-2, XT6-5, and AI-08 on MRS medium were picked and cultured in YPD liquid and MRS broth at 28±1℃ or 37±1℃ until bacterial sludge grew at the bottom of the test tubes. Glycerol was stored at -80℃, with a glycerol to bacterial suspension ratio of 0.5:1 to 0.8:1. All physiological saline, test tubes, culture media, and plates used were sterilized at 121℃ for 20 min.

[0032] The YPD liquid culture medium consists of 10.0 g / L yeast extract, 20.0 g / L peptone, 20.0 g / L glucose, and pH 6.2±2; the YPD solid culture medium consists of 10.0 g / L peptone, 20.0 g / L glucose, 5.0 g / L yeast extract, 14.0 g / L agar, and pH 6.2±2.

[0033] MRS medium consists of 10.0 g / L peptone, 5.0 g / L beef meal, 4.0 g / L yeast extract, 20.0 g / L glucose, 1.0 mL Tween-80, 2.0 g / L K₂HPO₄·7H₂O, 5.0 g sodium acetate·3H₂O, 2.0 g triammonium citrate, 0.2 g magnesium sulfate (MgSO₄·7H₂O), 0.05 g manganese sulfate (MnSO₄·7H₂O), and 15.0 g agar. The final pH is... 6.2±2; MRS broth was prepared from casein digest 10.0 g / L, beef extract 10.0 g / L, yeast extract 4.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate (MgSO4·7H2O) 0.2 g / L, manganese sulfate (MnSO4·7H2O) 0.05 g / L, K2HPO4·7H2O 2.0 g / L, glucose 20.0 g / L, Tween-80 1.08 g / L, with a final pH of 5.7±2.

[0034] Example 2: Screening conditions for pectinase-producing strains

[0035] Strain activation: Yeast strains numbered CI-06, CI-03, and LI-08, and lactic acid bacteria strains numbered MF7-1, MF7-3, MF3-1, MF3-2, XT6-5, and AI-08 were activated. For each strain, two tubes of YPD liquid medium or MRS broth medium were activated, 10 mL per tube. After sterilization and cooling, 10-50 μL of glycerol preservation solution (preserved at -80℃ and brought to room temperature) were added. The culture was carried out at 28±1℃ or 37±1℃ until bacterial sludge grew. After mixing, the activated strain and the culture of the strain were obtained.

[0036] Initial screening of pectinase-producing strains

[0037] The purified strains were inoculated onto the primary screening medium and three parallel experiments were conducted. The cultures were incubated at 37±2℃ for 48 hours, stained with 10 g / L Congo red for 30 minutes, and then washed with 1 mol / L NaCl solution for 30 minutes to remove the stain. Strains that produced clear zones were selected, and the diameter of the clear zone and the colony diameter were measured using calipers. The ratio of clear zone diameter (R) to colony diameter (r) was compared, and strains with larger R / r values ​​were selected for secondary screening.

[0038] The primary screening medium consisted of: 8.0 g / L pectin, 0.01 g / L FeSO4·7H2O, 1.0 g / L K2HPO4, 0.5 g / L MgSO4, 3.0 g / L NaNO3, and 20.0 g / L agar powder, diluted to 1 L with distilled water, pH 6.0, and sterilized at 121℃ for 20 min. The pectin liquid medium was prepared without the addition of agar powder.

[0039] Secondary screening of pectinase-producing strains (determination of strain enzyme activity)

[0040] After activating the strains with high R / r values ​​obtained from the initial screening, they were cultured at 28±1℃ and 160r / min for 24h. Then, the culture activated for 24h was inoculated into fermentation medium (50 / 250mL) at a volume fraction of 4% and fermented at 28±1℃ and 160r / min for 48h. The enzyme activity of each strain was measured using the DNS method. The ratio of the transparent zone diameter to the colony diameter (R / r) was positively correlated with the enzyme activity of the produced pectinase. Generally speaking, as the (R / r) ratio increases, the enzyme activity of pectinase gradually increases. The specific steps are shown in the following sequence.

[0041] Select strains with the highest enzyme activity and store them in glycerol tubes at -80°C.

[0042] ① Strain activation

[0043] The numbered strains were activated on YPD and MRS solid medium plates and cultured at 28℃±1℃ and 37℃±1℃ until spores were grown.

[0044] ②Preparation of seed liquid

[0045] Under sterile conditions, activated bacterial spores were inoculated into 50 mL of pectin liquid culture medium and cultured at 28℃±1℃ and 160 rpm.

[0046] ③ Fermentation culture

[0047] Under sterile conditions, 1 mL of seed culture was inoculated into 50 mL of YPD and MRS liquid fermentation medium and cultured at 28℃±1℃ and 160 rpm.

[0048] ④ Preparation of crude enzyme solution:

[0049] The supernatant obtained after freezing and centrifuging the fermentation broth of the strain (4℃, 10000r / min for 5min) is the crude enzyme solution.

[0050] Solution preparation

[0051] (1) Preparation of citric acid-sodium citrate buffer solution (pH 5.0)

[0052] Preparation of Solution A: Accurately weigh 5.25g of citric acid and dilute with water to 250mL in a volumetric flask. Preparation of Solution B: Accurately weigh 7.75g of sodium citrate and dilute with water to 250mL in a volumetric flask. Preparation of the application solution: Weigh 41mL of Solution A and 59mL of Solution B using a graduated cylinder, mix them thoroughly, and then correct the pH using a pH meter to achieve a final pH of 5.0±0.05. Refrigerate for later use.

[0053] (2) Preparation of 3,5-dinitrosalicylic acid reagent (DNS)

[0054] Accurately weigh 1.575 g of 3,5-dinitrosalicylic acid, dissolve it in 65.5 mL of 2 mol·L⁻¹ NaOH solution, add it to 125 mL of 45.5 g of potassium sodium tartrate hot aqueous solution, then add 1.25 g of phenol and 1.25 g of sodium sulfite, and make up to 250 mL. Pour the solution into a brown reagent bottle and let it stand for one week before use.

[0055] (3) Preparation of 1% (w / v) pectin solution

[0056] Accurately weigh 1g of pectin powder, add an appropriate amount of citrate buffer, heat to dissolve, cool, and then bring the citrate buffer to a final volume of 100mL. Store in a 4℃ refrigerator for later use. The shelf life is no more than 5 days.

[0057] (4) 1 mg·mL⁻¹ galacturonic acid standard stock solution

[0058] Accurately weigh 0.020 g of galacturonic acid, dissolve it in water, and dilute it to a 20 mL volumetric flask. Store it in a refrigerator at 4°C for later use. The shelf life is no more than three days.

[0059] Galacturonic acid standard curve plotting

[0060] Take volumes of 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL, respectively, with a mass concentration of 1 mg·mL⁻¹. -1 Add D-galacturonic acid solution to a centrifuge tube, add deionized water to 1 mL, then add 1 mL of DNS reagent, mix thoroughly, boil for about 5 minutes, cool to room temperature, and dilute to volume with a 10 mL volumetric flask. Measure the absorbance using a spectrophotometer at a wavelength of 540 nm. Plot the absorbance on the ordinate and the amount of galacturonic acid on the abscissa. Then plot the galacturonic acid standard curve and the D-galacturonic acid standard curve. Calculate pectinase activity based on the curves. See... Figure 2 .

[0061] ⑤ Determination of pectinase activity in strains

[0062] Add 0.8 mL of pectin solution to a 50 mL colorimetric tube, heat in a 50 °C water bath for 5 min, add 0.2 mL of crude enzyme solution, heat in a 50 °C water bath for 30 min, add 3 mL of DNS reagent, boil in a water bath for 10 min, cool under running water, and dilute to 25 mL with pure water. Mix well, centrifuge at 10000 r / min for 5 min, and measure the OD value of the solution at 540 nm using a microplate reader. The control group consisted of inactivated crude enzyme solution. The pectinase activity was determined according to the galacturonic acid standard curve, and the results are shown in Table 1.

[0063] The formula for calculating enzyme activity is: S×D×1000 / (0.2×10).

[0064] In this formula, S represents the amount of galacturonic acid absorbed by the light measured on the standard curve; D represents the dilution factor of the enzyme solution; 1000 represents the conversion factor: mg to μg; 0.2 represents the amount of enzyme solution involved in the reaction, in mL; and 10 represents the enzyme reaction time, in min.

[0065] One unit of enzyme activity (U) is defined as the amount of enzyme consumed in 1 mL of crude enzyme solution to hydrolyze pectin and produce 1 μg of reducing sugar within a unit time of 1 min.

[0066] The results of the pectinase activity assay of the selected strains are as follows:

[0067] Table 1. Enzyme activity statistics of existing strains screened in the laboratory.

[0068] strain number R / r strain enzyme activity AI-08 0.8 286.5 MF7-1 1.03 390.8 CI-03 0.92 322.6 LI-08 0.68 298.1 CI-06 1.2 412.9 MF7-3 1.01 386.1 MF3-1 0.94 364.3 MF3-2 0.76 256.8 XT6-5 0.51 194.6

[0069] Pectinase can cleave long-chain glycosidic bonds in pectin macromolecules, continuously exposing their reducing end groups. Therefore, the activity of pectinase can be evaluated by detecting the amount of reducing end groups generated by the degradation of long-chain glycosidic bonds. Strain CI-06 was selected for subsequent experiments and preserved in glycerol.

[0070] Based on the screening criteria, strain CI-06 was selected for subsequent experiments to identify strain CI-06:

[0071] After treating the CI-06 isolated strain with sodium hydroxide solution, 16S rRNA was extracted and amplified. The amplification system is as follows:

[0072] Further, PCR product sequencing is performed: after the PCR product passes the test, the target band is cut and purified and recovered, and the recovered product is used for Sanger sequencing.

[0073] (1) DNA extraction: TSINGKE Plant DNA Extraction Kit (General Type, Catalog No.: TSP101) was used.

[0074] S1. Add 200μL Buffer ATL and 20μL Proteinase K to the grinding tube, add half a spoonful of 3mm zirconium beads / one 5mm steel bead (depending on the state of the bacterial plate, zirconium beads are sufficient for bacteria, while steel beads are required for fungal hyphae and anaerobic bacteria), take a single colony from the bacterial plate into the grinding tube, and grind it at 60Hz for 2 minutes on an automatic grinder.

[0075] S2. Briefly centrifuge, add 200 μL Buffer AL, and mix thoroughly;

[0076] S3. Incubate in a 70℃ water bath for 15 min, shaking and mixing twice during the process. After lysis, centrifuge at 12000 rpm for 3 min, take 400 μL of supernatant and transfer it to a new deep well plate. Add 300 μL of Bufer BD and 20 μL of magnetic beads to the deep well plate and place it in station 2 of the extraction instrument.

[0077] S4. Take a new extraction plate and dispense 300 μL of Buffer BW1 into each well of the extraction instrument and place it at station 3. Dispense 75% ethanol into two plates with 500 μL wells and place them at stations 4 and 5 respectively. Dispense 100 μL / 80 μL / 60 μL of eluent into each well of the elution plate (dispense the corresponding volume according to the sample condition) and place it at station 6.

[0078] Note: Buffer BD and Buffer BW1 need to be diluted with a specified amount of anhydrous ethanol before use;

[0079] S5. Select the corresponding program, check the instrument status and extraction plate information, and then run it;

[0080] S6. After the run is complete, remove the elution plate for DNA concentration and electrophoresis gel detection, and store it at 4°C.

[0081] (2) PCR amplification: Amplification was performed using universal primers for bacterial identification. The amplification products were analyzed by agarose gel electrophoresis to determine whether the PCR product bands matched the target size, whether they were single, and whether there were any drag bands.

[0082] Table 2 Universal primers for bacterial species identification

[0083] Category name sequence Amplified sequence PCR length / bp bacteria 27F AGTTTGATCMTGGCTCAG 16S rDNA 1500 bacteria 1492R GGTTACCTTGTTACGACTT 16S rDNA 1500

[0084] (3) PCR product sequencing: After the PCR products passed the test, the target band was cut, purified, and recovered. The recovered products were then subjected to Sanger sequencing. The results are shown in Table 3. The PCR product sequencing results were compared and analyzed. The Sanger sequencing results were assembled using ContigExpress software, and inaccurate portions at both ends were removed. Furthermore, the assembled sequences were batch-aligned with nucleic acid databases using BLASTN (latest version v2.13). The latest version of the nt library was selected as the nucleic acid database. By comparing the nt library with the BLASTN database, the Accession Number of homologous sequences, species identification, and annotation could be obtained.

[0085] Table 3. Identification Results

[0086]

[0087] Morphological observation of isolates cultured on plates ( Figure 2 CI-06 is round, white, raised, transparent, with a smooth surface and neat edges.

[0088] Ultimately, based on the combined molecular biology and morphological results, CI-06 was identified as *Kluyveromyces marxianus*, and named *Kluyveromyces marxianus*. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number GDMCC No. 30564, deposited on May 9, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The sequence of this *Kluyveromyces marxianus* is shown in SEQ ID NO:1.

[0089] Example 3: Optimization of enzyme production fermentation process of strain CI-06

[0090] Prepare five sterilized fermentation tanks. After peeling the fresh coffee cherries, add 800g of peeled coffee beans to each fermentation tank and determine the pectinase activity using the same method as for strain-specific pectinase activity determination, except for the extraction of the crude enzyme solution from the fermented coffee. The only difference between this method and the strain-specific pectinase activity determination is the treatment of the crude enzyme solution.

[0091] Extraction of crude enzyme solution from fermented coffee:

[0092] Accurately weigh 6g of coffee sample and place it into a 50mL centrifuge tube. Then add 36mL of deionized water and use a grinder to grind the pectin on the surface of the coffee beans for 15min to completely remove it from the bean surface and dissolve it in water. Then use a syringe to draw 1mL of the ground liquid and centrifuge it in a high-speed refrigerated centrifuge at 4℃ and 10000r·min-1. Take the supernatant, which is the crude enzyme solution.

[0093] Single-factor experiment:

[0094] (1) Effect of inoculum quantity on pectinase activity

[0095] The fermentation time was fixed at 12 h, the fermentation temperature at 28 °C, and the initial pH at 3.0. The effects of inoculum concentrations (2%, 3%, 4%, 5%, 6%) on coffee degumming were investigated, and pectinase activity was measured. (See attached data.) Figure 3 .

[0096] (2) Effect of fermentation temperature on pectinase activity

[0097] The fermentation time was fixed at 12 h, the initial pH was 3.0, and the optimized inoculum size was used. The effects of fermentation temperature (20℃, 24℃, 28℃, 32℃, 36℃) on coffee degumming were investigated, and pectinase activity was measured. (See attached data.) Figure 4 .

[0098] (3) Effect of fermentation pH on pectinase activity

[0099] Using the optimized inoculum size, fermentation temperature, and fermentation time described above, the effect of initial pH (3.0, 4.0, 5.0, 6.0, 7.0) on coffee degumming was investigated, and pectinase activity was measured. (See attached data.) Figure 5 .

[0100] (4) Effect of fermentation time on pectinase activity

[0101] Using the optimized conditions of inoculum size, fermentation temperature, and initial pH 3.0 described above, the effects of fermentation time (0 h, 12 h, 24 h, 36 h, 48 h, 60 h) on coffee degumming were investigated, and pectinase activity was measured. (See attached data.) Figure 6 .

[0102] (5) Based on the results of the single-factor experiments above, an orthogonal design software was used to design a 4-factor, 3-level (see Table 4) L9(3) model. 4 Orthogonal experiments were conducted to optimize the culture conditions. The optimization results are shown in Table 4 below:

[0103] Table 4 Fermentation conditions for strain CI-06

[0104] level A. Fermentation time B. Fermentation temperature C Initial pH value D inoculation rate % 1 24 24 4 3 2 36 28 5 4 3 48 32 6 5

[0105] Table 5. Optimization results of orthogonal experiments on fermentation conditions for strain CI-06

[0106] Experiment No. A B C D Enzyme activity (U / mL) 1 1 1 1 1 203.5 2 1 2 2 2 283.7 3 1 3 3 3 196.5 4 2 1 2 3 248.5 5 2 2 3 1 264.3 6 2 3 1 2 238.1 7 3 1 3 2 342.6 8 3 2 1 3 316.2 9 3 3 2 1 322.7 <![CDATA[K1]]> 683.7 794.6 757.8 790.5 <![CDATA[K2]]> 750.9 864.2 854.9 864.4 <![CDATA[K3]]> 981.5 757.3 803.4 761.2 <![CDATA[k1]]> 227.9 264.9 252.6 263.5 <![CDATA[k2]]> 250.3 288.1 285.0 288.1 <![CDATA[k3]]> 327.2 252.4 267.8 253.7 R 99.3 35.6 32.4 34.4

[0107] As shown in Table 5 above, the enzyme activity of group A3B1C3D2 was 342.6 U / mL, which was significantly better than the enzyme activities of other groups. However, the optimal combination of K1, K2, and K3 was A3B2C2D2. Therefore, the optimal fermentation scheme for strain CI-06 needs to be verified through experiments. The verification results are as follows: Figure 7 The optimal solution was found to be A3B1C3D2, meaning the optimal enzyme production conditions for the strain were a fermentation time of 48 hours, a fermentation temperature of 24°C, an inoculum amount of 4%, and a pH of 6.

[0108] Fermentation and degumming were carried out under optimal enzyme-producing conditions, as detailed below:

[0109] Prepare five sterilized fermentation tanks, placing 800g of peeled coffee beans in each tank. Fermentation is carried out under optimized conditions for degumming the coffee beans, following a 48-hour fermentation time, a fermentation temperature of 24℃, an inoculum rate of 4%, and a pH of 6. The coffee bean condition is recorded every 12 hours. The results are as follows: Figure 8 As shown, after 48 hours of fermentation, the enzyme activity and degumming rate were measured and compared with those of uninoculated fermentation. 100g of coffee beans and attached coffee bean mucus were accurately weighed into each fermentation tank, and the mass of each tank was recorded. The tanks were placed in a climate simulation chamber for 48 hours of fermentation. Afterward, 100mL of distilled water was added to each tank, and the mass of water and coffee beans was recorded. The tanks were then placed in an ultrasonic cleaner for 15 minutes, and then inverted over a 30-mesh sieve for 10 minutes (while the coffee beans remained in the tank). The mass at this point was recorded. Pectin yield was calculated as follows: post-fermentation mass / initial mass (see Table 6). The results were compared with those of uninoculated fermentation after 48 hours, and enzyme activity was measured (see Table 7).

[0110] Table 6. Degumming results after inoculation with CI-06 fermentation

[0111] Group CI-06 fermentation for 48 hours: pectin yield / % Pectin yield (%) after 48 hours of uninoculated fermentation 1 84.23 94.12 2 85.44 93.59 3 83.26 94.82 Average pectin yield 84.31 94.18

[0112] Table 7. Enzyme activity assay results of CI-06 after 48 hours of fermentation with and without inoculation.

[0113] CI-06 fermentation for 48 hours enzyme activity Enzyme activity after 48 hours of uninoculated fermentation 432.5 156.3 423.6 169.7 431.5 148.6 Average enzyme activity 429.2 158.20

[0114] (6) Sensory rating

[0115] The Specialty Coffee Association of America (SCAA) standard cupping method was adopted. The sensory rating table is shown in Table 8. (Note: Subjective attributes are complementary and compared with each other. Scores of 6-10 or above are scored in increments of 0.25 points.)

[0116] Table 8 Sensory Rating Table

[0117]

[0118] After 48 hours of fermentation with the inoculated bacteria, coffee beans and uninoculated coffee beans were obtained and sensory evaluated according to Table 8 above. Beverages from each sample were prepared in the same manner as the cup shape analysis. A single 20ml sample was randomly provided to 10 coffee group members in a 50ml plastic cup. Each of the 10 coffee group members inhaled the coffee, moved it in their mouth for 5 seconds, swallowed, and began evaluation. This continued until no sensation was felt after a maximum of 30 seconds. After each tasting of the next sample, the subjects rinsed their mouths with water. The average score from the 10 coffee group members was taken as the final score, and the results are shown in Table 9.

[0119] Table 9 Sensory rating results

[0120] Member number CI-06 fermentation for 48 hours Uninoculated fermentation for 48 hours 1 80.25 78.75 2 80 75.25 3 79.75 74.5 4 80.5 71.75 5 79.5 78.25 6 82 72.5 7 80.75 78 8 78.75 72.75 9 79.25 77.75 10 79.5 73 Average score 80.03 75.25

[0121] (7) Determination of moisture content

[0122] Two sterilized fermentation tanks were prepared, each containing 1200g of peeled coffee beans. One tank was fermented under optimal conditions using strain CI-06 (48h fermentation time, 24℃ fermentation temperature, 4% inoculum, and pH 6), while the other tank was used for uninoculated coffee fermentation. Samples were taken every 48 hours, 200g each time, for a total of six samplings. Before 48 hours, the samples were kept in a fermentation tank with a breathable sealing film, and after 48 hours, they were transferred to a climate simulation chamber. 10g of ground coffee (with parchment removed) was accurately weighed at each time point, and 3g was weighed for each measurement. The samples were placed in a constant-weight aluminum box and measured using an HS153 halogen moisture analyzer. The coffee was heated to 105℃ and dried until the weight no longer changed within 90s. The moisture content displayed on the instrument was recorded. Each sample was repeated three times. The results are shown in Table 10.

[0123] Table 10 Results of Moisture Content Determination

[0124] Fermentation time / h CI-06 fermentation moisture content / % Moisture content of uninoculated fermentation / % 0 68.96 68.00 48 54.87 54.60 96 45.03 48.25 144 32.35 32.41 192 23.99 25.95 240 13.78 18.92 288 8.39 10.20

[0125] Example 4: A method for degumming coffee, comprising the following steps ( Figure 1 ):

[0126] S1. After washing and peeling the fresh coffee cherries, add them to the fermentation tank. The filling volume of the fermentation tank shall not exceed 80% of the fermentation tank capacity, and the tank shall be sealed with a breathable sealing film.

[0127] S2, inoculation, with a concentration of 10 5 ~10 8 CFU / mL activated Kluyveromyces macrocarpa CI-06 strain was inoculated into a fermentation tank containing peeled coffee cherries using a spray inoculation method. During spraying, the bacterial solution needs to evenly cover the surface of all coffee beans to ensure that each coffee bean can come into contact with the bacterial solution.

[0128] S3, fermentation and degumming: After inoculation, the fermenter is transferred to a constant temperature and humidity chamber, where the temperature is maintained at 28℃±2℃ and the relative humidity at 60%, and fermentation lasts for 60 hours.

[0129] S31, fermentation time set to 60 hours. During fermentation, temperature and humidity are checked every 12 hours, and the coffee beans are turned to ensure even fermentation. Temperature, humidity, and coffee bean condition are recorded during fermentation, and environmental parameters are adjusted in a timely manner to maintain a temperature of 24℃±2℃ and a relative humidity of over 60%.

[0130] S32: After 60 hours of fermentation, check the condition of the coffee beans to ensure that the fermentation is uniform and there are no off-odors.

[0131] S33: The fermented coffee beans are removed from the temperature and humidity controlled chamber for further processing (such as drying, removing parchment paper, roasting, and grinding).

[0132] S4, Drying: Remove the fermented coffee beans from the fermentation tank and place them in a climate simulation chamber to dry until the moisture content is less than 12%.

[0133] S42, Drying Preparation: Preheat the climate simulation chamber to the set drying conditions. Check that the temperature and humidity control functions of the climate simulation chamber are working properly.

[0134] Drying conditions for the simulation chamber:

[0135] Nighttime conditions: Temperature 18℃±2℃, Humidity 60%±2%.

[0136] Daytime conditions: Temperature 28℃±2℃, Humidity 40%±2%.

[0137] S43, Drying process: Spread the fermented coffee beans evenly on the drying tray of the climate simulation chamber, avoiding piling up. Turn on the climate simulation chamber to start the drying process.

[0138] S44, Drying Monitoring: During the drying process, regularly check the temperature and humidity inside the climate simulation chamber to ensure they remain within the set range. Periodically turn the coffee beans to ensure even drying. Measure the moisture content of the coffee beans every 6 hours until the moisture content is less than 12%, at which point drying should cease.

[0139] It should be understood that the specific embodiments described above are merely illustrative of the invention or for explaining the principles of the invention, and do not constitute a limitation thereof. Therefore, any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and scope of the invention should be included within the protection scope of the invention. Furthermore, the appended claims are intended to cover all variations and modifications falling within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries.

Claims

1. A method for degumming coffee beans, characterized in that, Includes the following steps: S1. After washing and peeling the fresh coffee cherries, add them to the fermentation tank. The filling volume of the fermentation tank shall not exceed 80% of the fermentation tank capacity, and the tank shall be sealed with a breathable sealing film. S2, Inoculation: The activated CI-06 strain is inoculated into a fermentation tank containing peeled fresh coffee cherries. CI-06 is named *Kluyveromyces martensii*. Kluyveromyces marxianus The *Kluyveromyces martensii* strain was deposited on May 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30564, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The inoculum concentration of *Kluyveromyces martensii* was 10-1. 5 ~10 8 CFU / mL, inoculation amount of the strain 4%; S3, Fermentation and Degumming: After inoculation, the fermenter is transferred to a constant temperature and humidity chamber and fermented for 48 hours at a relative humidity of over 60%, a fermentation temperature of 24℃, and a pH of 6 to complete degumming.

2. The coffee bean degumming method according to claim 1, characterized in that, The inoculation method for the CI-06 strain in S2 is spray inoculation.

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