Coffee parchment fermented beverage and method of making same

Abstract

This invention provides a coffee parchment fermented beverage and its preparation method, belonging to the field of mixed-strain fermentation for fermented beverages. By utilizing a mixed-strain fermentation of *Saccharomyces bayanus* and *Kluyveromyces marxianus*, a coffee parchment solid fermented beverage is prepared. Through a clearly defined process of microbial isolation, purification, morphological observation, and molecular biological identification, two high-quality strains were obtained, and a mixed seed suspension was prepared. Based on this, the suspension was inoculated into Typica coffee parchment at an inoculum rate of 3-5% for fermentation and sterilization, resulting in a coffee parchment solid fermented beverage. This invention develops a novel product, filling a gap in the coffee product market. Furthermore, mixed-strain fermentation compensates for the insufficient flavor of single-strain fermentation and can increase the fermentation rate and reduce the fermentation cycle.

Description

Technical Field

[0001] This invention belongs to the food field, specifically relating to the preparation of fermented beverages by mixed-culture fermentation, and more specifically to a coffee parchment fermented beverage and its preparation method, particularly a coffee parchment fermented beverage and its preparation method prepared by mixed-culture fermentation using *Bacillus subtilis* and *Kluyveromyces masculinus*. Background Technology

[0002] Coffee beans come from coffee berries, coffee berries such as Figure 1 As shown, coffee cherries consist of the pericarp, pulp, parchment, silverskin, and coffee beans. The parchment is the light brown skin surrounding the coffee seed, also known as the inner skin. During coffee bean processing, an equal amount of coffee cherry shells and other byproducts are produced for every 1 kg of coffee beans, accounting for approximately 20% of the dry weight of the coffee, along with waste such as coffee grounds. Coffee cherry shells contain pectin, dietary fiber, protein, sugars, and polyphenols, among other active substances. They are high in carbohydrates (35-85%), soluble fiber (30.8%), minerals (3-11%), and protein (5-11%). They are also rich in insoluble dietary fiber and can be a source of phytochemicals in the food industry, such as tannins (5-9%) and anthocyanins (20%).

[0003] Aqueous extracts of coffee husks have recently been proposed as a source of safe food ingredients: rich in phytochemicals and antioxidant dietary fiber, they serve as a natural and sustainable source of antioxidants, alpha-glucosidase inhibitors, and colorants. Observed health-promoting properties suggest that coffee byproduct extracts may be used as functional food ingredients or supplements to reduce the risk of chronic diseases associated with oxidative stress and to control postprandial glucose levels. However, comprehensive utilization of coffee husk resources is currently not achieved; only about 15% of coffee husks are converted into fertilizer for soil fertilization in coffee plantations, while the remaining 85% are discarded or incinerated, resulting in both resource waste and environmental pollution, seriously threatening the ecological environment of coffee-producing areas.

[0004] Currently, many experts at home and abroad are dedicated to developing new ways to utilize coffee by-product resources, increasing their economic value, and achieving sustainable development of the industry. Summary of the Invention

[0005] To solve the above-mentioned technical problems, this invention transforms and utilizes parchment paper to prepare coffee parchment paper fermented beverages, as detailed below:

[0006] A coffee parchment fermented beverage is prepared by fermenting parchment paper that has been processed with honey, broken and rehydrated, with a mixed seed culture suspension. The mixed seed culture suspension is prepared by mixing Saccharomyces cerevisiae and Kluyveromyces masculinus in a 1:1 volume ratio.

[0007] A strain of Saccharomyces bayanus, strain CI-03, was deposited on May 9, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No: 30565, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0008] A strain of 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, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0009] A coffee parchment fermented beverage, consisting of fermented coffee parchment powder, prebiotics, and citric acid.

[0010] Furthermore, the fermented coffee parchment powder, prebiotics, and citric acid are formulated in a mass ratio of 100:1.5:0.4.

[0011] Furthermore, the prebiotic includes fructooligosaccharides and polydextrose, with a mass ratio of fructooligosaccharides to polydextrose of 2:1.

[0012] Furthermore, the beverage is a solid beverage.

[0013] Furthermore, the fermented coffee parchment powder undergoes mixed-culture fermentation using a mixed seed culture suspension, wherein the mixed seed culture suspension is a 1:1 volume ratio of *Bacillus basilica* and *Kluyveromyces martensii*, with a concentration of 10. 8 CFU / mL.

[0014] A method for preparing a coffee parchment-based fermented beverage includes the following steps:

[0015] S1. Prepare a mixed seed culture suspension by mixing activated *Bacillus basilica* and *Kluyveromyces martensii* at a volume ratio of 1:1 to prepare a suspension with a concentration of 10. 8 A mixed seed culture suspension of CFU / mL;

[0016] S2, Material Selection: Select fresh Typica coffee cherries that are free from frost, microbial infection, and minor mechanical damage. After mechanically removing the skin, process them with honey to reduce the water content to 10-12%. After drying, remove the parchment paper and set it aside.

[0017] The honey processing method is a coffee bean processing method that falls between sun-drying and washing. It is also known as the pectin-containing sun-drying method. In the honey processing process, after the coffee cherries are harvested and sorted, the skin and pulp are removed, but some or all of the pectin is retained (this layer of pectin is called "honey" because it is sticky and has a high sugar content). Then, the coffee beans with pectin are sun-dried to reduce their moisture content to a certain level (10-12%).

[0018] S3, sieving: Pass the parchment paper through a 10-mesh sieve to remove impurities, garbage, etc.

[0019] S4, Crushing: Crush the sieved parchment using a high-speed blender;

[0020] S5, Rehydration: Wet the broken parchment with a spray until the surface is moist but without obvious water droplets;

[0021] S6, inoculation and fermentation: The activated bacterial solution is inoculated into the parchment paper rehydrated in S5 at an inoculation rate of 3-5%, a fermentation time of 12-15 hours, and a fermentation temperature of 28-32℃.

[0022] S7. After fermentation, lay the parchment paper flat on a tray in a 100℃ oven, about 0.5-1cm high, and dry for about 15 minutes until the surface is dry and no longer sticky. The drying is complete when the moisture content is below 7%.

[0023] S8, compound, is prepared by adding 100g of prebiotics to 1.5g of parchment paper. The prebiotics are composed of oligofructose and polydextrose in a mass ratio of 2:1. Citric acid is then added in a mass ratio of 0.4g:100g.

[0024] S9, canning sterilization, seals and sterilizes the compounded solid beverage, and the sterilization method can be pasteurization at 65-85℃ for 15-30 minutes;

[0025] S10, Brewing: Here is a brewing method: Take 20g of the finished product and place it in filter paper. Add 240ml-300ml of water at a powder-to-water ratio of 1:12-1.5, with a water temperature of 91-93℃.

[0026] Furthermore, the coffee cherries in S2 are Typica.

[0027] The *Bacillus basilica* in S1 is an endogenous microorganism isolated from the red grape soaking solution; *Kluyveromyces margaritifera* is an endogenous microorganism isolated from the sour meat soaking solution.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] ① Effectively utilize coffee parchment to develop new products, fill gaps in the coffee product market, reduce environmental pollution, and increase corporate revenue and consumer choice.

[0030] ② Compared to traditional honey processing, microbial fermentation can better utilize the pectin that the parchment paper could not fully decompose, and can also give it richer nutrients and a fuller flavor.

[0031] ③ Mixed-strain fermentation compensates for the lack of flavor in single-strain fermentation and can also increase the fermentation rate and reduce the fermentation cycle. Attached Figure Description

[0032] Figure 1 A structural hierarchy diagram of coffee beans;

[0033] Figure 2 This is a colony morphology diagram of *Bacillus basilica* CI-3 of the present invention;

[0034] Figure 3 This is a colony morphology diagram of Kluyveromyces Marcius CI-6 of the present invention;

[0035] Figure 4 The flowchart for preparing the coffee parchment fermented beverage according to the present invention is as follows;

[0036] Among them, 1-central cutting; 2-endosperm; 3-silver skin; 4-parchment (Endocarp); 5-pectin layer; 6-pulp (mesocarp); 7-outer skin (exocarp). Detailed Implementation

[0037] 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.

[0038] Example 1: Screening of Saccharomyces bayanus strain CI-03

[0039] Ripe Binchuan Red Globe grapes from Dali Prefecture, Yunnan Province, China were collected, placed in sterile sample bags, and quickly transferred to a low-temperature box for preservation. The samples were then transported to the laboratory after low-temperature storage.

[0040] Endogenous microorganisms were isolated from the maceration solution of red grapes. 10 ml of the maceration solution was weighed and placed in 90 mL of sterile physiological saline, then shaken and mixed thoroughly. The solution was serially diluted with sterile physiological saline, and 200 μL of the diluted solution was spread onto culture media. Yeast was cultured on yeast-extracted peptone-glucose agar plates at 28°C, and lactic acid bacteria were cultured on MRS agar plates at 37°C. Dominant yeast strains were selected based on colony morphology for further streaking isolation, obtaining single colonies. Isolates with consistent morphology were cryopreserved and identified.

[0041] Screening of Kluyveromyces marxianus CI-6 strain.

[0042] Sour meat, a traditional fermented food from Yunnan, China, was collected into sterilized sample bags and then quickly transferred to a low-temperature box for preservation. It was then transported to the laboratory under low-temperature conditions.

[0043] Endogenous microorganisms were isolated from the marinade of fermented meat. 10 ml of the marinade was weighed and placed in 90 mL of sterile physiological saline, then shaken to mix. The solution was serially diluted with sterile physiological saline, and 200 μL of the diluted solution was spread onto culture media. Yeast was cultured on yeast extract peptone glucose agar plates at 28°C, and lactic acid bacteria were cultured on MRS agar plates at 37°C. Dominant yeast strains were selected based on colony morphology for further streaking isolation, obtaining single colonies. Isolates with consistent morphology were cryopreserved and identified.

[0044] 2. Identification of strains

[0045] (1) DNA extraction: TSINGKE Plant DNA Extraction Kit (Universal) was used.

[0046] 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.

[0047] S2. Briefly centrifuge, add 200 μL Buffer AL, and mix thoroughly;

[0048] 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.

[0049] 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.

[0050] Note: Buffer BD and Buffer BW1 need to be diluted with a specified amount of anhydrous ethanol before use;

[0051] S5. Select the corresponding program, check the instrument status and extraction plate information, and then run it;

[0052] S6. After the run is complete, remove the elution plate for DNA concentration and electrophoresis gel detection, and store it at 4°C.

[0053] (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.

[0054] Table 1 Universal primers for bacterial species identification

[0055]

[0056] Table 2 ITS site PCR amplification system and amplification procedure

[0057]

[0058] (3) PCR product sequencing: After the PCR product passes the test, the target band is cut and purified and recovered. The recovered product is then used for Sanger sequencing.

[0059] Sequencing result alignment analysis: 1. Sanger sequencing results were assembled using ContigExpress software, and inaccurate portions at both ends were removed. 2. Batch alignment of the assembled sequences with BLASTN (latest version v2.13) against nucleic acid databases was performed. The latest version of the nt library was selected as the nucleic acid database. By performing BLASTN alignment with the nt library, the Accession Number of homologous sequences, species identification, and annotation could be obtained.

[0060] The results of the identification are as follows:

[0061] Table 3. Identification Results

[0062]

[0063] Morphological observation of the isolates cultured on plates revealed that strain CI-03 exhibited typical yeast characteristics. CI-03 colonies formed milky-white, round granules with a smooth surface. Gram staining of CI-03 was positive. Figure 2 CI-06 is round, white, raised, transparent, with a smooth surface and neat edges. Figure 3 ).

[0064] Finally, based on the combined molecular biology and morphological results, CI-03 was identified as *Saccharomyces bayanus*, and this *Saccharomyces bayanus* strain was deposited at the China General Microbiological Culture Collection Center (CGMCC). CI-03 was named *Saccharomyces bayanus*, with accession number CGMCC No: 30565, deposit date May 9, 2024, and deposit address No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The sequence of this *Saccharomyces bayanus* is shown in SEQ ID NO: 1. CI-6 was identified as *Kluyveromyces marxianus*, and named *Kluyveromyces marxianus*, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number GDMCC No: 30564, deposit date May 9, 2024, and deposit address No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The sequence of this *Kluyveromyces marxianus* is shown in SEQ ID NO: 2.

[0065] Example 2: A coffee parchment fermented beverage

[0066] A coffee parchment fermented beverage is a solid beverage prepared by mixing fermented coffee parchment powder, prebiotics, and citric acid in a mass ratio of 100:1.5:0.4. The prebiotics are composed of fructooligosaccharides and polydextrose, with a fructooligosaccharide:polydextrose ratio of 2:1. Therefore, the solid beverage is prepared by mixing 100g of coffee parchment powder, 1g of fructooligosaccharides, 0.5g of polydextrose, and 0.4g of citric acid.

[0067] The aforementioned fermented coffee parchment powder was subjected to mixed-culture fermentation using a mixed seed culture suspension, wherein the mixed seed culture suspension consisted of *Bacillus basilica* and *Kluyveromyces martensii* mixed at a 1:1 volume ratio to a final concentration of 10. 8 A mixed seed culture suspension of CFU / mL.

[0068] Example 3: A method for preparing a coffee parchment fermented beverage, specifically including the following steps:

[0069] S1. Prepare a mixed seed culture suspension by mixing activated *Bacillus basilica* and *Kluyveromyces martensii* at a volume ratio of 1:1 to prepare a suspension with a concentration of 10. 8 A mixed seed culture suspension of CFU / mL;

[0070] Bacillus subtilis (CI-3) and Kluyveromyces martensii (CI-6) stored in glycerol tubes at -80℃ were inoculated into YPD liquid medium and cultured in a constant temperature shaking incubator at 28±2℃ at a speed of 180±20 rpm for 24-36 h, activating two generations.

[0071] S2, Material Selection: Select fresh Typica coffee cherries that are free from frost, microbial infection, and minor mechanical damage. After mechanically removing the skin, process them with honey and then remove the parchment paper after drying.

[0072] (1) Pick and remove immature, shriveled, diseased, pest-infested, and foreign objects, and select fresh coffee cherries that are free from frost, microbial infection, and minor mechanical damage for later use.

[0073] (2) Cleaning, sorting and peeling

[0074] Wash the harvested coffee cherries with clean water, remove any floating cherries and foreign objects, and clean the remaining coffee cherries thoroughly. Spread them out to drain the water until no water droplets are visible to the naked eye. Then peel the coffee cherries (without washing them) to obtain fresh coffee beans.

[0075] (3) Honey treatment and shelling

[0076] When the coffee beans obtained from honey processing are dried to a moisture content of 10-12%, they are dehulled to obtain parchment paper, which can be used as a raw material for fermented beverages.

[0077] (4) Preparation of coffee parchment fermented beverage

[0078] S3, sieving: Pass the parchment paper through a 10-mesh sieve to remove impurities, garbage, etc.

[0079] S4, Crushing: Crush the sieved parchment using a high-speed blender;

[0080] S5, Rehydration: Wet the broken parchment with a spray until the surface is moist but without obvious water droplets;

[0081] S6, inoculation and fermentation: The activated bacterial solution is inoculated at an inoculation rate of 3-5%, a fermentation time of 12-15 hours, and a fermentation temperature of 28-32℃ into the parchment paper rehydrated in S5 for 30-36 hours.

[0082] S7. After fermentation, lay the parchment paper flat on a tray in a 100℃ oven, about 0.5-1cm high, and dry for about 15 minutes until the surface is dry and no longer sticky. The drying is complete when the moisture content is below 7%.

[0083] S8, compound, add prebiotics to the above-mentioned dry parchment powder at a ratio of 1.5g / 100g, the ratio of fructooligosaccharide to polydextrose in the prebiotics is 2:1, and add citric acid to the above-mentioned dry parchment powder at a ratio of 0.4g / 100g;

[0084] S9, canning sterilization, seals and sterilizes the compounded solid beverage, and the sterilization method can be pasteurization at 65-85℃ for 15-30 minutes;

[0085] S10, Brewing: Here is a brewing method. Take 20g of the finished product and place it in filter paper. Brew it at a powder-to-water ratio of 1:12-15, that is, add 240ml-300ml of water at a temperature of 91-93℃.

[0086] Example 4: Preparation of coffee parchment fermented beverage using different fermentation processes

[0087] 1. The specific operation is as follows: According to the preparation method of Example 3, the effects of different strains on the quality of coffee parchment fermented beverages were studied by changing the strains introduced into the fermentation tank.

[0088] Group 1: Fermentation using a single strain of *Bacillus basilica* CI-3;

[0089] Group 2: Fermentation using a single strain of Kluyveromyces masculinus CI-6;

[0090] Group 3: Mixed-culture fermentation according to Example 3;

[0091] Group 4: No inoculation treatment was performed; this group served as the control group.

[0092] 2. Relevant indicators were measured for the above groups.

[0093] (1) Moisture content determination: The moisture content of the parchment paper from groups 1 to 4, after fermentation for 36 hours, was determined by drying. The determination method is as follows: An HS153 halogen moisture analyzer was used. 2g of coffee sample was accurately weighed and placed in a constant-weight aluminum box. The sample was heated to 105℃ and dried until the weight no longer changed within 90 seconds. The moisture content displayed by the instrument was recorded. Each sample was repeated three times. The moisture content determination results for different groups are shown in Table 4 below.

[0094] Table 4 Moisture content of different groups

[0095] Group Moisture content % Group 1 (CI-3) 6.41±0.42 Group 2 (CI-6) 6.24±0.37 Group 3 (Mixed Bacteria) 6.52±0.23 Group 4 (Control) 6.55±0.47

[0096] Moisture content determination showed that the moisture content of each group was below 7%, which is in line with the technical requirements for solid beverages.

[0097] (2) Sensory inspection

[0098] Prepare a 50ml sample according to the label instructions, pour it into a colorless, transparent container, place it in a bright place, and observe its state, color, aroma, and taste. The sensory evaluation criteria are shown in Table 5.

[0099] Table 5 Sensory Evaluation Table for Coffee Parchment Fermented Beverage

[0100]

[0101] Table 6 Sensory ratings for different groups

[0102] Group Sensory rating Group 1 (CI-3) 81.31±1.52 Group 2 (CI-6) 82.41±0.51 Group 3 (Mixed Bacteria) 84.63±0.36 Group 4 (Control) 76.58±2.44

[0103] Sensory evaluation was conducted on the coffee parchment fermented beverage samples of each group according to the sensory evaluation table in Table 5, and the sensory scores are shown in Table 6. The parchment samples fermented with single strains CI-3 and CI-6 respectively scored higher than the blank group without strains, which shows the effectiveness of the strains in the fermentation of coffee parchment fermented beverages. The highest score of the mixed-strain fermentation in the third group shows that the mixed-strain fermentation technology proposed in this patent application is beneficial to the fermentation of coffee parchment fermented beverages.

[0104] (3) Protein content was determined according to the method specified in GB5009.5.

[0105] Table 7 Protein content of different groups

[0106] Group Protein content % Group 1 (CI-3) 7.56±0.35 Group 2 (CI-6) 7.33±0.52 Group 3 (Mixed Bacteria) 6.77±0.21 Group 4 (Control) 8.44±0.42

[0107] According to the national standard GB5009.5, the protein content of the samples from the four groups was measured. The protein content of the mixed-culture fermented samples was lower than that of the other three groups. It is speculated that the mixed-culture fermentation process makes better use of protein macromolecules to break them down into amino acids, which are then used to form flavor compounds through other pathways.

[0108] (4) Caffeine, determined according to the method specified in GB / T5009.139.

[0109] Table 8. Caffeine content in different groups

[0110] Group Caffeine content % Group 1 (CI-3) 0.18±0.05 Group 2 (CI-6) 0.19±0.02 Group 3 (Mixed Bacteria) 0.21±0.07 Group 4 (Control) 0.23±0.03

[0111] The caffeine content of the four samples was determined according to GB / T5009.139. As shown in the table, the fermentation process of the two strains had little effect on caffeine, so mixed fermentation also had little effect on caffeine.

[0112] 5. Total sugar

[0113] The total sugar content of parchment paper powder was determined using the sulfuric acid-phenol method. Accurately weigh 0.5 g of sample, wash with distilled water, and dilute to volume in a 10 mL volumetric flask. After thorough mixing, centrifuge at 3000 rpm for 15 min, and take 2 mL of the supernatant to dilute to a 100 mL volumetric flask. Take 2 mL of the diluted sample solution, add 1 mL of 5% phenol and 5 mL of 98% concentrated sulfuric acid to a test tube, allow to react for 30 min, and measure the absorbance at 490 nm. Simultaneously, using glucose as a standard, plot the absorbance value on the ordinate and the glucose concentration on the abscissa to calculate the total sugar content of coffee at different processing points.

[0114] Table 9 Total sugar content of different groups

[0115] Group Total sugar content (mg / g) Group 1 (CI-3) 56.12±1.33 Group 2 (CI-6) 54.13±1.63 Group 3 (Mixed Bacteria) 56.88±2.21 Group 4 (Control) 63.42±1.55

[0116] The total sugar content of the four groups was determined by the above method. The total sugar content determines the sweet taste. The process of microbial fermentation uses sugar as a nutrient and metabolizes it to produce various flavor substances. As shown in Table 9, the total sugar content of the blank group was the highest, followed by the mixed group. In terms of sensory evaluation, the sweet and sour taste of mixed fermentation is more harmonious, while the taste of the blank group is relatively simple, so the sensory evaluation score is not high.

[0117] 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.

[0118] Sequence list information:

[0119] DTD Version: V1_3

[0120] Filename: A coffee parchment fermented beverage and its preparation method.xml

[0121] Software Name: WIPO Sequence

[0122] Software version: 2.1.0

[0123] Generation Date: 2024-08-13

[0124] Basic Information:

[0125] Current application / applicant file name: 1253000043120331X7

[0126] Applicant's Name or Title: Yunnan Agricultural University

[0127] Applicant's name or title / language:zh

[0128] Applicant's Name or Title / Latin Name: Yunnan Agricultural University

[0129] Invention Title: A Coffee Parchment Fermented Beverage and Its Preparation Method (zh)

[0130] Total number of sequences: 2

[0131] sequence:

[0132] Serial Number (ID): 1

[0133] Length: 832

[0134] Molecular type: DNA

[0135] Feature location / qualifier:

[0136] -source,1..832

[0137] >mol_type,genomic DNA

[0138] >organism,Saccharomyces bayanus

[0139] residues:

[0140] taaagtcgta acaaggtttc cgtaggtgaa cctgcggaag gatcattaaa gaaatttaat 60

[0141] aattttgaaa atggattttt ttgttttggc aagagcatga gagcttttac tgggcaagaa 120

[0142] gacaagagat ggagagtcca gccgggcctg cgcttaagtg cgcggtcttg ctaggcttgt 180

[0143] aagtttcttt cttgctattc caaacggtga gagatttctg tgcttttgtt ataggacaat 240

[0144] taaaaccgtt tcaatacaac acactgtgga gttttcatat ctttgcaact ttttctttgg 300

[0145] gcattcgagc aatcggggcc cagaggtaac aaacacaaac aattttatct attcattaaa 360

[0146] tttttgtcaa aaacaagaat tttcgtaact ggaaatttta aaatattaaa aactttcaac 420

[0147] aacggatctc ttggttctcg catcgatgaa gaacgcagcg aaatgcgata cgtaatgtga 480

[0148] attgcagaat tccgtgaatc atcgaatctt tgaacgcaca ttgcgcccct tggtattcca 540

[0149] gggggcatgc ctgtttgagc gtcatttcct tctcaaacat tctgtttggt agtgagtgat 600

[0150] actctttgga gttaacttga aattgctggc cttttcattg gatgtttttt ttccaaagag 660

[0151] aggtttctct gcgtgcttga ggtataatgc aagtacggtc gttttaggtt ttaccaactg 720

[0152] cggctaatct ttttttatac tgagcgtatt ggaacgttat cgataagaag agagcgtcta 780

[0153] ggcgaacaat gttcttaaag ttgacctcca atcaggtagg agtacccgct ga 832

[0154] Sequence number (ID): 2

[0155] Length: 717

[0156] Molecular type: DNA

[0157] Feature location / qualifier:

[0158] -source,1..717

[0159] >mol_type,genomic DNA

[0160] >organism,Kluyveromyces marxianus

[0161] Residues:

[0162] agtaaaagtc gtaacaaggt ttccgtaggt gaacctgcgg aaggatcata aagattatga 60

[0163] atgaatagat tactggggga atcgtctgaa caaggcctgc gcttaattgc gcggccagtt 120

[0164] cttgattctc tgctatcagt tttctatttc tcatcctaaa cacaatggag ttttttctct 180

[0165] atgaactact tccctggaga gctcgtctct ccagtggaca taaacacaaa caatattttg 240

[0166] tattatgaaa aactattata ctataaaatt taatattcaa aactttcaac aacggatctc 300

[0167] ttggttctcg catcgatgaa gaacgcagcg aattgcgata tgtattgtga attgcagatt 360

[0168] ttcgtgaatc atcaaatctt tgaacgcaca ttgcgccctc tggtattcca gggggcatgc 420

[0169] ctgtttgagc gtcatttctc tctcaaacct ttgggtttgg tagtgagtga tactcgtctc 480

[0170] gggttaactt gaaagtggct agccgttgcc atctgcgtga gcagggctgc gtgtcaagtc 540

[0171] tatggactcg actcttgcac atctacgtct taggtttgcg ccaattcgtg gtaagcttgg 600

[0172] gtcatagaga ctcataggtg ttataaagac tcgctggtgt ttgtctcctt gaggcatacg 660

[0173] gctttaacca aaactctcaa agtttgacct caaatcaggt aggagtaccc gctgaac 717

[0174] END

Claims

1. A method for preparing a coffee parchment-based fermented beverage, characterized in that, Includes the following steps: S1. Prepare a mixed seed culture suspension by mixing activated *Bacillus basilica* and *Kluyveromyces martensii* at a volume ratio of 1:1 to prepare a suspension with a concentration of 10. 8 A mixed seed culture suspension of CFU / mL; The bay yeast ( Saccharomyces bayanus The strain CI-03 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 9, 2024, with the accession number CGMCC No: 30565. The Max Kluyveromyces ( Kluyveromyces marxianus The strain CI-06 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 9, 2024, with accession number CGMCC No. 30564. S2, Material Selection: Select fresh Typica coffee cherries that are free from frost, microbial infection, and mechanical damage. After mechanically removing the skin, they undergo honey treatment and are dried before removing the parchment paper for later use. S3, sieving: Pass the parchment paper through a 10-mesh sieve to remove impurities and debris; S4, Crushing: Crush the sieved parchment using a high-speed blender; S5, Rehydration: Wet the broken parchment with a spray until the surface is moist but without obvious water droplets; S6, inoculation and fermentation: The activated mixed seed culture suspension is inoculated at an inoculation rate of 3-5%, fermentation time is 30-36 hours, and fermentation temperature is 28-32℃. The mixture is then inoculated into the parchment paper rehydrated in S5 for fermentation. S7. After fermentation, lay the parchment paper flat on a tray in a 100℃ oven to a height of 0.5-1cm and dry for 15 minutes until the surface is dry and no longer sticky. The drying process is complete when the moisture content is below 7%. S8, compound, add prebiotics according to the mass ratio of dried coffee parchment powder after fermentation: prebiotics: citric acid = 100:1.5:0.4, the prebiotics are composed of oligofructose and polydextrose in a mass ratio of 2:1, and then add citric acid according to the corresponding mass ratio; S9, canning sterilization: The compounded solid beverage is sealed and packaged and sterilized. The sterilization method is pasteurization at 65-85℃ for 15-30 minutes.

2. A coffee parchment fermented beverage prepared according to the method of claim 1.

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