Kluyveromyces marxianus and application thereof in coffee bean degumming technology

Abstract

The invention discloses Kluyveromyces marxianus and an application thereof in a coffee bean degumming technology, and belongs to the field of microorganisms, the Kluyveromyces marxianus CI-06 strain is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on May 9, 2024, and the preservation number is CGMCC No. 30564. The CI-06 strain has pectinase activity, the pectinase produced by the CI-06 strain is high in activity, and the CI-06 strain can be used in the biological degumming process of coffee, and is used for shortening the degumming time and improving the quality of coffee commodity beans.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically relates to a strain of Kluyveromyces marxianus and application thereof in coffee bean degumming technology, in particular to a strain of Kluyveromyces marxianus CI-06 and application thereof in coffee bean degumming technology. Background Art

[0002] Coffee is an important crop grown in many countries and one of the most popular beverages in the world. There are two main methods for primary processing of coffee: wet processing and dry processing. The main coffee producing areas in Yunnan are relatively rich in water resources, and wet processing is the main method. The processing process mainly includes peeling, degumming, drying, grading, packaging, etc. Among them, the degumming process is the most complicated and most prone to problems. The quality of coffee will also be greatly affected. Wet processing includes ordinary wet processing and mechanical wet processing. The main difference is the degumming process. Coffee pectin is composed of substances such as sugar, enzymes, protopectin and pectin. The main components are galacturon and methanol. It has strong hygroscopicity and is not easy to dry. Therefore, in order to speed up the drying speed, pectin is usually removed. The ordinary wet method adopts the traditional method, that is, after the coffee is peeled, water is used as the medium, and the peeled coffee is soaked in water. The enzymes contained in the pectin itself are used for biological fermentation and degumming. It takes a long time and is easily affected by weather and temperature. Incomplete degumming will also cause the residual mucus to become a medium for microbial growth during the storage and drying of coffee, causing secondary fermentation, resulting in peculiar smell in the coffee, and ultimately affecting the quality of the coffee. The degradation of mucus is closely related to the production of extracellular mucus-degrading enzyme pectinase by microorganisms, and the removal of mucus is the key to coffee drying. It has the highest technical requirements, the most complex process, and is most prone to problems. Drying plays an important role in the flavor of coffee. Coffee consumers believe that its flavor is more important than its nutritional effect. The quality of coffee degumming directly affects the storage effect and flavor of coffee, and thus affects the profits of the coffee industry.

[0003] At present, the main methods for degumming coffee include natural fermentation degumming, chemical degumming, physical degumming and microbial degumming; each of which has the following characteristics: (1) Natural fermentation degumming: It is uncertain when the fermentation end point will be reached, and the coffee quality is low; (2) Chemical degumming: Acids and alkalis can easily destroy the flavor components of coffee, and introduce inorganic ions to cause environmental pollution; (3) Physical degumming: After the coffee fruit is peeled, it is washed and the mucus is removed by rubbing the coffee beans, which can easily cause secondary fermentation; (4) Enzymatic degumming: Enzymes have high requirements for the environment, temperature, and pH value, and cannot be promoted in actual production; (5) Microbial degumming: In addition to fast 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 "Study on the Effect of Enzymatic Degumming on Coffee Quality" published in the first issue of Agricultural Product Processing in January 2019 revealed that enzymatic fermentation degumming has no adverse negative effects on coffee quality. On the contrary, it is slightly better than traditional fermentation degumming coffee in terms of aroma, aftertaste and balance. Furthermore, Chinese patent CN110305858A-discloses an enzyme preparation and method for degumming fresh coffee fruit, which uses an enzyme preparation composed of pectinesterase, pectin lyase and polygalacturonase for degumming, thereby improving the degumming efficiency of coffee beans, reducing the mildew rate of coffee beans, and improving the quality of coffee beans. At the same time, it reduces water consumption and is more environmentally friendly. Chinese patent CN114410489B-discloses an abnormal Wickham yeast CAP5 strain and its application, which discloses an abnormal Wickham yeast CAP5 with good acid resistance and pectinase activity, which can not only degrade pectin for shortening the degumming time and improving the quality of commercial coffee beans in the process of biological degumming, but also can be used in the production of ethanol and the brewing of fruit wine.

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

[0006] To achieve the above object, the present invention provides a strain of Kluyveromyces marxianus CI-06, and inoculates the strain into a fermentation tank of peeled fresh coffee berries to promote coffee bio-degumming, and the specific operation is as follows:

[0007] A strain of Kluyveromyces marxianus, the Kluyveromyces marxianus CI-06 strain was isolated from Yunnan's traditional fermented food sour meat, and was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on May 9, 2024, with the deposit number: CGMCC No 30564.

[0008] Application of the Kluyveromyces marxianus CI-06 strain in coffee degumming preparation.

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

[0010] A coffee bean degumming method comprises the following steps:

[0011] S1, fresh coffee berries are washed and peeled and added into a fermentation tank, the filling volume of the fermentation tank does not exceed 80% of the fermentation tank capacity, and the fermentation tank is sealed with a breathable sealing film;

[0012] S2, inoculation, inoculating the activated Kluyveromyces marxianus CI-06 strain into a fermentation tank filled with peeled fresh coffee berries, the inoculation concentration of the Kluyveromyces marxianus yeast being 10 5 ~10 8 CFU / mL;

[0013] S3, fermentation and degumming. After inoculation, the fermentation tank is moved into a constant temperature and humidity room, the temperature is maintained at 28℃±2℃, the relative humidity is above 60%, and the degumming is completed after 24h-60h of fermentation.

[0014] Furthermore, the inoculation amount of Kluyveromyces marxianus 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] Working principle of the present invention: The present invention provides a Kluyveromyces marxianus CI-06 strain, which has pectinase activity. The pectinase activity produced by the CI-06 strain is high and is used for inoculating Kluyveromyces marxianus in the biological degumming process of coffee. The pectinase produced by the CI-06 strain reacts with the pectin of coffee beans to degrade the pectin and improve the mucus removal effect.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) A Kluyveromyces marxianus CI-06 strain with high pectinase production is provided, which fills the gap of degumming strains that can be used in coffee biodegumming technology;

[0019] (2) The CI-06 strain has pectinase activity. The pectinase activity it produces is high and can be used to produce pectinase or for coffee biodegumming, shortening the degumming time and improving the quality of commercial coffee beans; BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The flowchart of the application of Kluyveromyces marxianus in coffee bean production of the present invention;

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

[0022] Figure 3 is the standard curve of galacturonic acid;

[0023] Figure 4 The bar graph is a graph showing the effect of different inoculation amounts on pectinase activity according to the present invention;

[0024] Figure 5 It is a bar graph showing the effect of different fermentation temperatures on pectinase activity;

[0025] Figure 6 It is a bar graph showing the effect of different pH values ​​on pectinase activity;

[0026] Figure 7 The bar graph shows the effect of different fermentation time on pectinase activity;

[0027] Figure 8 A bar graph for orthogonal experimental verification of the present invention;

[0028] Fig. 9 This is a record of the coffee bean fermentation and degumming process and the coffee beans dried after 48 hours of fermentation. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following invention, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0030] Example 1: Screening and purification of strains

[0031] The dominant yeasts and lactic acid bacteria were screened from Yunnan fermented sour meat, Mouding fermented bean curd, Fuyuan pickled cabbage, Dali Binchuan grapes and Lancang coffee. 25 g of the sample was accurately weighed and placed in 225 mL of physiological saline (0.85% NaCl) for serial gradient dilution (10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6), 0.2 mL of each series of gradient dilutions were taken and spread on YPD solid medium and MRS medium respectively. Three parallel experiments were carried out for each gradient. According to the morphology, color, size, edge, smoothness and wetness of the colonies, a single colony was picked with a sterilized inoculation needle and inoculated on YPD solid medium and MRS medium. The colonies were cultured at 28°C±1°C and 37°C±1°C for 2 days respectively. After 3 to 5 times of separation and purification, the yeasts on YPD medium were numbered CI-06, CI-0 3 and LI-08, the lactic acid bacteria on MRS culture medium are numbered MF7-1, MF7-3, MF3-1, MF3-2, XT6-5, AI-08. Pick single colonies for YPD liquid and MRS broth culture in a 28±1℃ or 37±1℃ incubator until bacterial mud grows at the bottom of the test tube. Store in a -80℃ refrigerator with glycerol. The ratio of glycerol to bacterial suspension is 0.5:1 to 0.8:1. All physiological saline, test tubes, culture medium, and plates used must be sterilized at 121℃ for 20 min.

[0032] The YPD liquid medium is composed 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 medium is composed 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 powder, 4.0 g / L yeast powder, 20.0 g / L glucose, 1.0 mL Tween-80, and K 2 HPO 4 .7H 2 O 2.0g / L, sodium acetate.3H 2 O 5.0g, ammonium citrate 2.0g, magnesium sulfate (MgSO 4 .7H 2 O) 0.2 g, manganese sulfate (MnSO 4 .7H 2 O) 0.05 g, agar 15.0 g, final pH 6.2 ± 2; MRS broth is composed of casein digest 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 4.0 g / L, ammonium citrate tribasic 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate (MgSO 4 .7H 2 O)0.2g / L, manganese sulfate (MnSO 4 .7H 2 O)0.05g / L,K 2 HPO 4 .7H 2O 2.0 g / L, glucose 20.0 g / L, Tween-80 1.08 g / L, final pH 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. Two tubes of YPD liquid culture medium or MRS broth culture medium were used for each strain for activation, with 10 mL in each tube. After sterilization and cooling, 10-50 μL of glycerol-preserved bacterial solution taken out of a -80°C refrigerator and cooled to room temperature was added. The culture was cultured at 28±1°C or 37±1°C to grow bacterial mud. After mixing, the activated strain and the culture of the strain were obtained.

[0036] Preliminary screening of pectinase-producing strains

[0037] The purified strains were inoculated onto the primary screening culture medium, and three parallel experiments were performed. The strains were cultured in a 37±2℃ incubator for 48h, stained with 10g / L Congo red stain for 30min, and then eluted with 1mol / L NaCl solution for 30min. The strains producing transparent zones were selected, and the diameters of the transparent zones and colonies were measured with a vernier caliper. The diameters of the transparent zones (R) and colonies (r) were compared, and strains with larger R / r values ​​were selected for rescreening.

[0038] The primary screening culture medium is: pectin 8.0g / L, FeSO 4 7H 2 O 0.01g / L, K 2 HPO 4 1.0g / L, MgSO 4 0.5g / L, NaNO 3 3.0g / L, agar powder 20.0g / L, distilled water to 1L, pH 6.0, sterilized at 121℃ for 20min. Pectin liquid medium is based on the initial screening medium without adding agar powder.

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

[0040] After activating the strains with larger R / r values ​​obtained in the initial screening, they were cultured at 28±1°C, 160r / min for 24h, and then the culture activated for 24h was inoculated into the fermentation medium (50 / 250mL) at a volume fraction of 4%, and fermented at 28±1°C, 160r / min for 48h. The enzyme activity of each strain was measured by 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 the pectinase gradually increases. The specific steps are shown in the following sequence numbers.

[0041] The strain with the highest enzyme activity was selected and stored in a -80°C refrigerator in glycerol tubes.

[0042] ① Strain activation

[0043] The above-numbered strains were activated on YPD and MRS solid culture medium plates and cultured at 28°C±1°C and 37°C±1°C until the strains grew spores.

[0044] ②Preparation of seed solution

[0045] Under a sterile environment, the activated strain spores were inoculated into 50 mL of pectin liquid culture medium and cultured at 28°C ± 1°C and 160 rpm.

[0046] ③ Fermentation culture

[0047] Under a sterile environment, 1 mL of seed solution was inoculated into 50 mL of YPD and MRS liquid fermentation medium and cultured at 28°C ± 1°C and 160 rpm.

[0048] ④ Preparation of crude enzyme solution:

[0049] The fermentation broth of the strain fermentation culture was subjected to refrigerated centrifugation (4°C, 10000r / min centrifugation for 5min) to obtain the supernatant as the crude enzyme solution.

[0050] Solution preparation

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

[0052] Preparation of Liquid A: Accurately weigh 5.25g of citric acid and dissolve it to 250mL with water in a volumetric flask. Preparation of Liquid B: Accurately weigh 7.75g of sodium citrate and dissolve it to 250mL with water in a volumetric flask. Preparation of the application liquid: Use a measuring cylinder to weigh 41mL of Liquid A and 59mL of Liquid B, then mix them evenly, and then use a pH meter to correct the pH of the application liquid to 5.0±0.05, and refrigerate at low temperature 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 a hot aqueous solution of 45.5 g of potassium sodium tartrate, then add 1.25 g of phenol and 1.25 g of sodium sulfite, make up to 250 mL, pour it into a brown reagent bottle, and leave it for one week before use.

[0055] (3) Preparation of 1% (W / V) pectin solution

[0056] Accurately weigh 1 g of pectin powder, add appropriate amount of citric acid buffer, heat to dissolve, cool, then make up to 100 mL of citric acid buffer and place in a 4°C refrigerator for use. The shelf life shall not exceed 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 then add a fixed amount of solution into a 20 mL volumetric flask. Place it in a 4°C refrigerator for later use. The shelf life shall not exceed three days.

[0059] Galacturonic acid standard curve drawing

[0060] The volumes were 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL, respectively, and the mass concentration was 1 mg mL -1 D-galacturonic acid solution is added to a centrifuge tube, deionized water is added to 1 mL, and then 1 mL of DNS reagent is added. After thorough mixing, it is boiled for about 5 minutes, cooled at room temperature, and fixed to volume with a 10 mL volumetric flask. The absorbance is measured using a spectrophotometer at a wavelength of 540 nm. The ordinate is the measured absorbance, and the abscissa is the amount of galacturonic acid. Then a standard curve of galacturonic acid and a standard curve of D-galacturonic acid are drawn, and the pectinase activity is calculated based on the curve. Figure 2 .

[0061] ⑤ Determination of pectinase activity of strains

[0062] 0.8 mL of pectin solution was added to a 50 mL colorimetric tube, heated in a 50 ° C water bath for 5 min, 0.2 mL of crude enzyme solution was added, 50 ° C water bath for 30 min, 3 mL of DNS reagent was added, boiling water bath for 10 min, running water cooling, dilute to 25 mL with pure water, mixed evenly, centrifuged at 10000 r / min for 5 min, and the OD value of the solution was measured at 540 nm with an enzyme marker. The control group was an 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 calculation formula of enzyme activity is: S×D×1000 / (0.2×10)

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

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

[0066] The results of the determination of pectinase activity 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 the long-chain glycosidic bonds in pectin macromolecules to expose their reducing end groups. Therefore, the activity of pectinase can be evaluated by detecting the amount of reducing end groups produced by the degradation of long-chain glycosidic bonds by pectinase. The CI-06 strain was selected for subsequent experiments and stored in glycerol.

[0070] According to the screening conditions, strain CI-06 was selected for subsequent experiments and the CI-06 strain was identified:

[0071] After the CI-06 isolated strain was treated with sodium hydroxide solution, 16S rRNA was extracted from it for amplification. The amplification system was as follows:

[0072] Furthermore, the PCR product is sequenced: after the PCR product is detected to be qualified, the target band is cut and purified and recovered, and the recovered product is used for Sanger sequencing.

[0073] (1) DNA extraction: Use TSINGKE plant DNA extraction kit (universal type, product number: TSP101).

[0074] S1. Add 200 μL Buffer ATL and 20 μL Proteinase K to the grinding tube, add half a spoon of 3 mm zirconium beads or one 5 mm steel bead (depending on the status of the bacterial plate, bacteria can be added with zirconium beads, fungal hyphae and anaerobic bacteria need to add steel beads), take a single colony from the bacterial plate into the grinding tube, and grind it on an automatic grinder at 60 Hz for 2 minutes;

[0075] S2. Centrifuge briefly, add 200 μL Buffer AL, and mix thoroughly;

[0076] S3. Place in a 70℃ water bath for 15min, shake and mix twice, centrifuge at 12000rpm for 3min after lysis, take 400μL supernatant and transfer to a new deep-well plate, add 300μL Bufer BD and 20μL magnetic beads to the deep-well plate, and place it in station 2 of the extractor;

[0077] S4. Take a new extraction plate and dispense 300 μL of Buffer BW1 into each well and place it at station 3 of the extraction instrument. 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 elution into each well of the elution plate (dispense the corresponding volume according to the sample status) 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 completed, take out the elution plate for DNA concentration and electrophoresis gel detection and store it at 4°C.

[0081] (2) PCR amplification: Use universal primers for bacterial species identification to amplify, and analyze the amplified products by agarose gel electrophoresis to determine whether the PCR product bands are consistent with the target size, whether they are single, and whether there are any dragging bands.

[0082] Table 2 Universal primers for bacterial species identification

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

[0084] (3) PCR product sequencing: After the PCR product is qualified, the target band is cut and purified and recovered, and the recovered product is subjected to Sanger sequencing. The results are shown in Table 3. The PCR product test results are compared and analyzed, and the Sanger sequencing results are spliced ​​using the software ContigExpress, and the inaccurate parts at both ends are removed. Furthermore, the spliced ​​sequences are batch-blasted (latest version v2.13) to compare with the nucleic acid database. Among them, the latest version of the nt library is selected for the nucleic acid database. By blasting with the nt library, the Accession Number of the homologous sequence and the species identification and annotation can be obtained.

[0085] Table 3 Identification results

[0086]

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

[0088] Finally, based on the molecular biology and morphological results, CI-06 was identified as Kluyveromyces marxianus and named Kluyveromyces marxianus. It was deposited in the General Microbiological Center of China Microbiological Culture Collection Committee with the deposit number GDMCC No: 30564, the deposit date was May 9, 2024, and the deposit address was No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The sequence of the Kluyveromyces marxianus is shown in SEQ ID NO: 1.

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

[0090] Prepare 5 sterilized fermentation tanks, peel the coffee berries, put 800g of peeled coffee beans into each fermentation tank, and determine the pectinase activity in the same way as the strain enzyme activity determination, except for the extraction of the crude enzyme solution of the fermented coffee. The method for determining the pectinase activity here is different from that for determining the strain pectinase activity only in the treatment of the crude enzyme solution.

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

[0092] Accurately weigh 6 g of coffee sample and put it into a 50 mL centrifuge tube. Then add 36 mL of deionized water. Use a grinder to crush the pectin on the surface of the coffee beans for 15 minutes to completely separate it from the surface of the beans and dissolve it in water. Then use a syringe to absorb 1 mL of the crushed liquid and centrifuge it in a high-speed refrigerated centrifuge at 4°C, 10,000 r·min-1 to take the supernatant, which is the crude enzyme solution.

[0093] Single factor experiment:

[0094] (1) Effect of inoculation amount on pectinase activity

[0095] The fermentation time was fixed at 12 h, the fermentation temperature was 28 °C, and the initial pH was 3.0. The effect of inoculation amount (2%, 3%, 4%, 5%, 6%) on coffee degumming was investigated, and the pectinase activity was determined. 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 amount was used to investigate the effect of fermentation temperature (20°C, 24°C, 28°C, 32°C, 36°C) on coffee degumming, and the pectinase activity was determined. Figure 4 .

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

[0099] The above optimized conditions of inoculum amount, fermentation temperature and fermentation time were used to investigate the effect of initial pH (3.0, 4.0, 5.0, 6.0, 7.0) on coffee degumming, and the pectinase activity was determined. Figure 5 .

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

[0101] The above optimized conditions of inoculum amount, fermentation temperature and initial pH 3.0 were used to investigate the effect of fermentation time (0h, 12h, 24h, 36h, 48h, 60h) on coffee degumming, and the pectinase activity was determined. Figure 6 .

[0102] (5) Based on the above single factor test results, orthogonal design software was used to design 4 factors with 3 levels (see Table 4). 4 ) Orthogonal test was performed to optimize the culture conditions. The optimization results are shown in Table 4 below:

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

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

[0105] Table 5 Orthogonal optimization results of fermentation conditions of CI-06 strain

[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[K 1 ]]> 683.7 794.6 757.8 790.5 <![CDATA[K 2 ]]> 750.9 864.2 854.9 864.4 <![CDATA[K 3 ]]> 981.5 757.3 803.4 761.2 <![CDATA[k 1 ]]> 227.9 264.9 252.6 263.5 <![CDATA[k 2 ]]> 250.3 288.1 285.0 288.1 <![CDATA[k 3 ]]> 327.2 252.4 267.8 253.7 R 99.3 35.6 32.4 34.4

[0107] From Table 5 above, we can see that A 3 B 1 C 3 D 2 The enzyme activity of the group was 342.6U / mL, which was significantly better than that of the other groups, but K 1 , K 2 and K 3 The best combination is A 3 B 2 C 2 D 2 Therefore, the optimal fermentation scheme of CI-06 strain needs to be verified by experiments. Figure 7 , the optimal solution is A 3 B 1 C 3 D 2 That is, the optimal enzyme production conditions of the strain are fermentation time 48h, fermentation temperature 24℃, inoculation amount 4% and pH value 6.

[0108] The optimal enzyme production conditions were used for fermentation degumming, as follows:

[0109] Prepare 5 sterilized fermentation tanks, put 800g of peeled coffee beans in each fermentation tank, and use the optimized fermentation conditions to degumming the coffee beans. Fermentation and degumming are carried out according to the conditions of 48h, fermentation temperature 24℃, inoculation amount 4%, pH value 6. Record the coffee bean conditions every 12h. The results are as follows: Figure 8 As shown, the enzyme activity and degumming rate were measured after 48 hours of fermentation, and compared with the uninoculated fermentation. Accurately weigh 100g of coffee beans and mucus attached to coffee beans in a fermentation tank, record the mass of each fermentation tank, put it into a climate simulation box for fermentation and culture for 48 hours, take out the fermentation tank and add 100mL of distilled water, record the mass of the water added to the fermentation tank and the coffee beans, put it in an ultrasonic cleaner for 15 minutes, and turn the fermentation tank upside down in a 30-mesh screen for 10 minutes (the coffee beans are still in the fermentation tank), and record the mass at this time. According to the calculation of pectin yield = post-fermentation mass / initial mass, see Table 6, and compare it with the uninoculated fermentation for 48 hours, and measure its enzyme activity, see Table 7.

[0110] Table 6 Degumming results of inoculation of CI-06

[0111] Group CI-06 fermentation 48h pectin yield / % Pectin yield after 48h fermentation without inoculation / % 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 determination results of inoculated and uninoculated CI-06 fermentation for 48h

[0113] CI-06 enzyme activity after 48h fermentation Uninoculated fermentation 48h enzyme activity 432.5 156.3 423.6 169.7 431.5 148.6 Average enzyme activity 429.2 158.20

[0114] (6) Sensory evaluation

[0115] The Specialty Coffee Association of America (SCAA) standard cupping test was used, and the sensory scoring table is shown in Table 8. (Note: The subjective attributes are complementary to each other, and the scores above 6-10 points are scored in increments of 0.25 points).

[0116] Table 8 Sensory Rating Table

[0117]

[0118] The coffee beans obtained after 48h of inoculation and the uninoculated coffee beans were scored according to Table 8 above, and beverages from each sample were prepared in the same way as the cup shape analysis. A single sample of 20ml was randomly provided to 10 coffee panelists in a 50ml plastic cup. The 10 coffee panelists absorbed the coffee and moved it in their mouths for 5 seconds, then swallowed and began to evaluate. Until no feeling after a maximum of 30 seconds. Each time the next sample was tasted, the subject rinsed his mouth with water, and the average score of the 10 coffee panelists was taken as the final score. The results are shown in Table 9.

[0119] Table 9 Sensory evaluation results

[0120] Member Number CI-06 fermentation 48h Uninoculated fermentation 48h 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] Prepare two sterilized fermentation tanks, put 1200g of peeled coffee beans in each fermentation tank, one of which is fermented with the optimal conditions of CI-06 strain (fermentation time 48h, fermentation temperature 24℃, inoculation amount 4% and pH value 6), and the other is fermented with uninoculated coffee. Take samples every 48h, take 200g each time, and take samples six times in total. Before 48h, they are all in the fermentation tank with breathable sealing film, and move to the climate simulation box after 48h. Accurately weigh 10g of ground coffee at each time point (remove the parchment paper), weigh 3g each time, put it in a constant weight aluminum box, and use HS153 halogen moisture meter to measure, heat and dry at 105℃ until the weight does not change within 90s, record the moisture content displayed by the instrument, and repeat three times for each sample. The results are shown in Table 10.

[0123] Table 10 Determination results of moisture content

[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 coffee degumming method comprising the following steps ( Figure 1 ):

[0126] S1, fresh coffee berries are washed and peeled and added into a fermentation tank, the filling volume of the fermentation tank does not exceed 80% of the fermentation tank capacity, and the fermentation tank is sealed with a breathable sealing film;

[0127] S2, inoculation, the concentration of 10 5 ~10 8 CFU / mL of activated Kluyveromyces marxianus CI-06 strain was inoculated into the fermentation tank containing peeled coffee cherries by spraying. The bacterial solution should evenly cover the surface of all coffee beans to ensure that every coffee bean can be exposed to the bacterial solution.

[0128] S3, fermentation and degumming, after inoculation, the fermentation tank is moved into a constant temperature and humidity chamber, the temperature is maintained at 28℃±2℃, the relative humidity is 60%, and the fermentation is carried out for 60h.

[0129] S31, the fermentation time is set to 60 hours. During the fermentation process, the temperature and humidity are checked every 12 hours, and the coffee beans are turned over to ensure uniform fermentation. The temperature, humidity and coffee bean status during the fermentation process are recorded, and the environmental parameters are adjusted in time to keep the temperature at 24℃±2℃ and the relative humidity above 60%.

[0130] S32, after the fermentation time reaches 60 hours, check the condition of the coffee beans to ensure that the fermentation is uniform and there is no odor.

[0131] S33, taking the fermented coffee beans out of the constant temperature and humidity chamber for subsequent processing (such as drying, removing parchment paper, roasting and grinding)

[0132] S4, drying, taking the fermented coffee beans out of the fermentation tank and placing them in a climate simulation box for drying until the moisture content is less than 12%.

[0133] S42, Drying preparation: Preheat the climate simulation chamber to the set drying conditions. Check whether the temperature and humidity control functions of the climate simulation chamber are normal.

[0134] Drying conditions of the climate simulation chamber:

[0135] Night 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 box to avoid stacking, turn on the climate simulation box, and start the drying process.

[0138] S44, Drying monitoring: During the drying process, check the temperature and humidity in the climate simulation box regularly to ensure that they are within the set range. Turn the coffee beans regularly to ensure uniform drying. Measure the moisture content of the coffee beans every 6 hours and stop drying when the moisture content is less than 12%.

[0139] It should be understood that the above specific embodiments of the present invention are only used for illustrative inventions or to explain the principles of the present invention, and do not constitute limitations on the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.

[0140]

[0141]

Claims

1. A strain of Kluyveromyces marxianus, the strain CI-06 was named Kluyveromyces marxianus, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 9, 2024, with the deposit number: CGMCC No 30564, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Application of the CI-06 strain according to claim 1 in a coffee degumming microbial preparation.

3. The use according to claim 2, wherein the inoculation amount of the CI-06 strain in the coffee degumming process is 2%-10%.

4. A coffee bean degumming method, characterized in that: The steps include: S1, fresh coffee berries are washed and peeled and added into a fermentation tank, the filling volume of the fermentation tank does not exceed 80% of the fermentation tank capacity, and the fermentation tank is sealed with a breathable sealing film; S2, inoculation, inoculating the activated CI-06 strain into a fermentation tank filled with peeled fresh coffee berries, the inoculation concentration of Kluyveromyces marxianus is 10 5 ~10 8 CFU / mL; S3, fermentation and degumming. After inoculation, the fermentation tank is moved into a constant temperature and humidity room, the temperature is maintained at 28℃±2℃, the relative humidity is above 60%, and the degumming is completed after 24h-60h of fermentation.

5. The coffee bean degumming method according to claim 4, characterized in that: The inoculum amount of CI-06 strain in S2 is 4%.

6. The coffee bean degumming method according to claim 4, characterized in that: The fermentation time in S3 is 48 hours, the fermentation temperature is 24° C., and the pH value is 6.

7. The coffee bean degumming method according to claim 4, characterized in that: The inoculation method of the CI-06 strain in S2 is spray inoculation.

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