Preparation method of coffee mycelium film and application thereof

Abstract

This application relates to the field of vinegar processing technology, and more particularly to a method for preparing a coffee pectin-gelatin composite membrane and its application. The coffee pectin-gelatin composite membrane prepared by this method provides a suitable micro-aerobic environment for the fermentation system, while the slow-released active ingredients in the coffee pectin inhibit harmful bacteria in the environment (such as certain yeasts and molds), without significantly inhibiting the long-term domesticated kombucha lactic acid bacteria flora, thus constructing a "selective microbial barrier" at the fermentation interface. This strategy combines the high-value utilization of agricultural by-products with the regulation of the fermentation microecology.

Description

Technical Field

[0001] This application relates to the field of food engineering technology, and in particular to a method for preparing a coffee pectin-gelatin composite film and its application. Background Technology

[0002] With the increasing severity of environmental pollution caused by petroleum-based plastic packaging, the development of sustainable and biodegradable food packaging materials has become a research hotspot. Composite films based on biopolymers, due to their combination of the excellent properties of multiple components, have become an ideal alternative to traditional plastic packaging. Pectin, a natural polysaccharide rich in galacturonic acid residues, possesses excellent film-forming ability, biodegradability, non-toxicity, and gas barrier properties. Coffee pectin, in particular, derived from coffee industry byproducts, is not only abundant and low-cost but also possesses certain antibacterial activity. Using coffee pectin as a base material and adding other functional components to prepare composite films can fully realize its potential in the food packaging field, which is of great significance for solving the problem of plastic pollution and realizing the high-value utilization of coffee byproducts.

[0003] Currently, pectin-based composite films have made some progress in research and application. However, existing composite films still have many shortcomings. On the one hand, single-pectin films often suffer from insufficient mechanical strength, poor flexibility, and sensitivity to water vapor, making it difficult to meet the mechanical and barrier performance requirements of actual food packaging. On the other hand, the preparation process of existing composite films is relatively simple, usually just a simple mixing of pectin and reinforcing phases, lacking fine-tuning of the film-forming mechanism, inter-component interactions, and microstructure. This results in poor structural uniformity and performance stability of the composite films, limiting their large-scale production and commercial application.

[0004] Therefore, to address the problems of poor mechanical properties, unsatisfactory structural stability, and difficulty in controlling performance during the preparation process of existing pectin-based composite films, it is necessary to provide a novel coffee pectin composite film and its preparation method. By optimizing the composition and preparation process of the composite film, the aim is to prepare a composite film material that possesses excellent mechanical properties, good barrier properties, stable structure, and environmental friendliness. This will provide a high-performance, biodegradable, and green packaging solution for the food packaging industry and offer new technical support for the high-value utilization of coffee by-products. Summary of the Invention

[0005] To address or partially address the problems existing in related technologies, this application provides a method for preparing a coffee pectin-gelatin composite film, the method comprising the following steps: S1, Dry the coffee fruit peels, then grind and sieve them to obtain coffee powder; S2, the coffee powder obtained in step S1 is added to an 80% ethanol solution, treated in a water bath, and then filtered to obtain residue and filtrate. The residue is repeatedly filtered until the filtrate is colorless. The residue is collected as coffee pericarp alcohol-insoluble matter. S3, add the coffee pericarp alcohol insolubles obtained in step S2 to deionized water at a material-to-liquid ratio of 1:25-1:30 (g / mL), adjust the pH to 1.5 with hydrochloric acid, and then perform water bath extraction; after extraction, filter and collect the filtrate; S4. Concentrate the filtrate obtained in step S3 under reduced pressure to 1 / 3-1 / 4 of its original volume; after cooling, add anhydrous ethanol to the concentrate and let it stand at 4°C until precipitation is complete; collect the precipitate, wash it, and then dry it to constant weight and pulverize it to obtain coffee pectin. The volume ratio of the concentrated solution to anhydrous ethanol is 1:1.5; S5, dissolve gelatin in distilled water at 80°C, stir continuously for 30 minutes, then add glycerin accounting for 5% of the weight of gelatin; then add the coffee pectin obtained in S4 and stir for 2 hours to obtain a film-forming solution; The mass ratio of the gelatin to coffee pectin is 1:0.5-3; S6. The film-forming solution obtained in step S5 is cast onto the coating plate. After drying, the film is placed in an environment of 25°C and 50% relative humidity for 48 hours to obtain the coffee pectin-gelatin composite film.

[0006] Furthermore, in step S2, the ratio of coffee powder to ethanol solution is 1:10 (g / mL).

[0007] Furthermore, in step S3, the water bath extraction temperature is 80-90℃ and the time is 90-120 min.

[0008] Furthermore, the concentration of gelatin in step S5 is 2% (g / ml).

[0009] Furthermore, the drying temperature in step S6 is 45°C, and the drying time is 24 hours.

[0010] On the other hand, this application also provides a method for preparing a coffee spawn film, the method comprising the following steps: After obtaining coffee pectin using the above preparation method, lactic acid bacteria agent is added; then it is coated on MRS medium for cultivation. During the cultivation process, the fermentation container is sealed with a coffee pectin-gelatin composite membrane obtained by the above preparation method; the amount of coffee pectin added is 0.4g; the amount of lactic acid bacteria agent added is 25% of the mass of coffee beans.

[0011] Furthermore, the culture temperature was 28°C, pH 7, and the time was 48 hours.

[0012] Furthermore, the lactic acid bacteria agent is a compound of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus fermentum, Lactococcus lactis, Pediococcus pentosus, Weisslerella paraintermedia, Lactobacillus bulgaricus, and Streptococcus thermophilus in a bacterial suspension volume ratio of 1:1:1:1:1:1:1:1:1.

[0013] On the other hand, this application also provides a method for preparing coffee vinegar, the method comprising adding coffee spawn obtained by the above method and fermenting to obtain coffee vinegar; the amount of coffee spawn added is 5%-10% of coffee beans.

[0014] On the other hand, this application also provides the application of the coffee pectin-gelatin composite film obtained by the above preparation method or the microbial film obtained by the above method in the preparation of coffee vinegar.

[0015] Beneficial effects 1. Traditional methods produce a linear flavor profile dominated by acetic acid, the complexity of which is limited by the metabolic capacity of the coffee bean extract and a single microbial strain. The fermentation of hulled coffee cherries yields sugars, pectin, organic acids (such as malic acid and tartaric acid), polysaccharides, and various microbial metabolites. Under the combined fermentation of the prepared coffee spore film, these substances can generate a far richer variety of esters, higher alcohols, ketones, and aldehydes than pure alcoholic fermentation. Combined fermentation may also produce a greater diversity of prebiotics, short-chain fatty acids, and microbial secondary metabolites.

[0016] 2. A natural biological preservative system was constructed: the symbiotic relationship of multiple microorganisms creates a competitive inhibitory environment, and the complex acidity of the products and dynamic pH changes can more effectively inhibit miscellaneous bacteria without relying on excessive sterilization or additives. Lactic acid bacteria are important functional microorganisms in the acetic acid fermentation process. They are the dominant bacterial group in acetic acid fermentation and an important biological source of organic acids such as acetic acid and lactic acid, as well as various flavor substances such as ethyl acetate and 2,3-butanedione.

[0017] 3. Improved raw material utilization and value: Transformed coffee pulp, which is usually discarded, into high-value-added products, creating a completely new value chain.

[0018] 4. The coffee pectin-gelatin composite membrane possesses certain air permeability and good biodegradability, making it a viable alternative to traditional plastic sealing materials and reducing environmental pollution. The coffee pectin-gelatin composite membrane plays a crucial role in this invention. Coffee pectin, derived from coffee byproducts, possesses unique polyphenolic residues that endow the composite membrane with additional bioactivity. While providing a suitable micro-aerobic environment for the fermentation system, the slowly released active ingredients in the coffee pectin inhibit harmful microorganisms in the environment (such as certain yeasts and molds) without significantly inhibiting the long-acclimated kombucha lactic acid bacteria flora, thus constructing a "selective microbial barrier" at the fermentation interface. This strategy of combining the high-value utilization of agricultural byproducts with fermentation microecological regulation is a significant feature distinguishing Example 2 from Comparative Example 1.

[0019] 5. Coffee pectin itself has antibacterial properties, which can inhibit harmful microorganisms from entering the fermentation system and ensure the health properties of the final product.

[0020] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0021] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings.

[0022] Figure 1 This is a technical route diagram for preparing coffee vinegar according to Example 2 of this application.

[0023] Figure 2 This is a flowchart showing the process parameters for preparing the bacterial film through single-factor experiments with different single factors.

[0024] Figure 3 These are colony morphology diagrams of microorganisms in kombucha. a) shows kombucha microorganisms cultured on MRS medium; b) shows kombucha microorganisms cultured on M17 medium.

[0025] Figure 4 It is the coffee spawn film prepared in Example 1.

[0026] Figure 5 These are pictures of the finished coffee vinegar products. The left picture shows the coffee vinegar produced using this method; the right picture shows the coffee vinegar produced using the traditional method. Detailed Implementation

[0027] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0028] Coffee pectin is a major component of coffee fruit processing byproducts (coffee peel), possessing excellent film-forming ability, biodegradability, non-toxicity, and certain antibacterial activity. Composite films prepared using coffee pectin as a substrate have broad application prospects in food packaging, fermentation engineering, and other fields. Especially in the processing of fermented products, using functional edible films as sealing materials for fermentation containers can not only regulate the micro-aerobic environment of the fermentation system but also achieve targeted regulation of the fermentation microecology through the slow release of active ingredients within the film. While there are a few reports on pectin-based composite films in existing research, most focus on commercially available pectin from fruits and vegetables. These raw materials undergo purification and lack natural active ingredients, making it difficult to impart selective antibacterial functions to the film materials. Furthermore, due to the unique origin of coffee pectin, residual polyphenols, chlorogenic acid, and other active substances in the raw material are easily lost during extraction, resulting in its application in composite film preparation not being systematically developed for a long time. A composite film preparation technology that uses coffee pectin as a functional component and considers both structural and microecological regulation has not yet been established. Therefore, developing a coffee pectin-based composite membrane that combines structural integrity with functional selectivity is of great significance for improving the quality and stability of fermented products.

[0029] Therefore, this application provides a method for preparing a coffee pectin-gelatin composite membrane. Using coffee processing byproducts as raw materials, and through optimized extraction and film-forming processes, the natural active ingredients in coffee pectin are preserved to the greatest extent. The prepared composite membrane exhibits excellent film-forming properties and mechanical strength, and can form a "selective microbial barrier" during fermentation, inhibiting contaminating bacteria without interfering with the metabolic activities of the target microbial community. This technology provides a new pathway for the high-value utilization of coffee byproducts and also provides functional material support for the regulation of the microecological environment in fermentation systems.

[0030] Unless otherwise specified, all reagents and materials used in the following examples were purchased from the market.

[0031] Example 1: Preparation of coffee pectin-gelatin composite film Coffee pectin is a component of the coffee fruit structure, located between the pulp and parchment. It primarily originates from the coffee fruit peel, a byproduct of coffee fruit processing. The peel accounts for approximately 40%-50% of the weight of fresh coffee fruit and is the largest waste product generated during wet coffee processing. Its pectin content can reach around 15% (dry basis), making it highly valuable for reuse. Coffee pectin is not ordinary commercial pectin. Due to the residual chlorogenic acid, caffeine, and polyphenols produced during the roasting process of raw coffee beans, coffee pectin, in addition to possessing common characteristics such as good film-forming ability, biodegradability, and non-toxicity, naturally possesses unique antioxidant and antibacterial activities. These bioactive substances derived from coffee itself make coffee pectin an ideal alternative to sustainable food packaging materials. Biopolymer films prepared using coffee pectin can not only serve as an environmentally friendly alternative to petroleum-based plastics but also hold promise for providing microecological protection for fermentation systems through the slow release of active substances.

[0032] 1. Preparation method of coffee pectin: S1. Collect fresh, ripe coffee cherries (Yunnan small-bean coffee). After washing, remove the coffee beans using a hulling machine to obtain fresh coffee cherry skins. Sun-dry the collected coffee cherry skins naturally for 2-3 days, or dry them in a 45℃ oven for 24 hours, until the moisture content drops below 20%. Grind the dried cherry skins using a grinder, pass them through an 80-mesh sieve to obtain coffee cherry skin powder, and seal and store for later use.

[0033] S2, weigh a certain amount of coffee pericarp powder and add it to an 80% (v / v) ethanol solution at a material-to-liquid ratio of 1:10 (g / mL). Heat under reflux in an 80℃ water bath for 30 minutes to remove monosaccharides, polyphenols, pigments, and some fat-soluble components from the pericarp. After treatment, filter the mixture. Repeat the above steps 2-3 times with the filter residue until the filtrate is essentially colorless. Finally, vacuum dry the filter residue at 40℃ to constant weight to obtain the ethanol-insoluble coffee pericarp. This step aims to enrich cell wall polysaccharides and improve the purity of subsequent pectin extraction.

[0034] S3, This invention employs a dilute hydrochloric acid extraction method. Weigh the alcohol-insoluble matter prepared in step 2 and add it to deionized water at a material-to-liquid ratio of 1:25-1:30 (g / mL). Adjust the pH to 1.5 with 0.1 mol / L hydrochloric acid. Extract at a constant temperature and with stirring in a water bath at 80-90℃ for 90-120 minutes. After extraction, filter the solution while hot using double-layered gauze or filter paper, and collect the filtrate. The residue can be extracted once more, and the filtrates are combined.

[0035] S4. Concentrate the above filtrate under reduced pressure at 50°C to 1 / 3-1 / 4 of its original volume. After cooling, add 1.5 times the volume of anhydrous ethanol to the concentrate while stirring. A large amount of flocculent precipitate will be observed to precipitate. Let it stand overnight (12 hours) at 4°C until precipitation is complete. Collect the precipitate by suction filtration or centrifugation, and wash the precipitate 2-3 times successively with 75% ethanol and anhydrous ethanol to further remove residual pigments and free small molecule phenols. The final crude pectin product is dried in a vacuum drying oven at 40°C to constant weight, and then pulverized to obtain the active coffee pectin used in this invention.

[0036] 2. Preparation of coffee pectin-gelatin composite film Coffee pectin specifically refers to non-commercial crude pectin extracted from coffee cherry peels—a byproduct of fresh coffee cherry processing—and which retains the most active components of coffee (such as polyphenols and chlorogenic acid). Coffee cherry peels account for approximately 40%-50% of the weight of fresh coffee cherries and are the largest waste product generated during the wet coffee processing method. Their pectin content can reach around 15% (on a dry basis), making them highly valuable for reuse.

[0037] Coffee pectin-gelatin composite films were prepared using a solution casting method. 0.2 g of gelatin was dissolved in 20 ml of 80°C distilled water, and after continuous stirring for 30 minutes, glycerol (5% by weight of gelatin) was added as a plasticizer. Subsequently, 0.1-0.6 g of coffee pectin was added, and the mixture was stirred at 80°C for 2 hours to ensure uniform dispersion.

[0038] A control film without coffee pectin was prepared under the same conditions. Each portion of the film-forming solution (20 ml) was cast onto a flat polytetrafluoroethylene coated plate and dried at 45°C for 24 hours. After the dried film was peeled off, it was placed in an environment of 25°C and 50% relative humidity for 48 hours to obtain a coffee pectin-gelatin composite film for later use.

[0039] Example 2: Preparation of Coffee Vinegar 1. Activation and culture of Kombucha strains Kombucha is a functional fermented beverage made from tea leaves and sugar through fermentation by various microorganisms under the influence of a specific symbiotic flora (SCOBY). The fermentation process involves the synergistic metabolic activities of multiple microorganisms, including yeast, acetic acid bacteria, and lactic acid bacteria. Lactic acid bacteria, a core functional component of kombucha, is a microbial community with key probiotic properties. Through sugar metabolism, they produce lactic acid, short-chain fatty acids, and other bioactive metabolites. These metabolites play a crucial role in regulating the homeostasis of the host's gut microbiota, inhibiting the colonization of pathogens, enhancing immune function, and improving metabolic health.

[0040] Using kombucha symbiotic microbial film as a source of functional microorganisms, this method is applied to the fermentation of hulled coffee cherries. The aim is to develop a novel coffee vinegar product that combines the unique flavor and functional components of coffee with the health benefits of traditional vinegar through the synergistic effect of diverse microorganisms. This technological approach not only provides a new approach for the high-value utilization of coffee by-products but also offers practical reference for exploring the application of complex microbial communities in specialty fermented foods.

[0041] Weigh 8.0g of raw Pu-erh tea leaves, add 1.0L of hot water (90℃) and steep for 15 minutes. Filter the tea leaves, add 80g of sucrose to the tea soup and dissolve. Sterilize the sugary tea soup at 121℃ for 15 minutes. Dispense 250mL / bottle into 500mL sterile glass bottles and cool to room temperature. Inoculate with 2% (v / v) of bacterial stock solution from different origins and 3.2g / L of cellulose bacterial film. Seal the bottle mouth with sterile gauze and then incubate at 28℃ for 6 days to activate the kombucha. Store the activated kombucha at 4℃ for later use.

[0042] 2. Isolation, purification and identification of microorganisms in kombucha The activated kombucha fermentation broth was shaken and mixed thoroughly, then serially diluted 10-fold with physiological saline to a final volume of 10. -7 The culture was then spread onto R2A agar plates. After incubation at 28°C for 72 hours to allow for sufficient colony growth, single colonies of different morphologies were selected under aseptic conditions and transferred to new MRS and M17 plates for purification until single-morphological colonies were obtained, thus preliminarily obtaining purified lactic acid bacteria strains. Following morphological identification, three passages of purification were performed to ensure the acquisition of pure strains. The obtained purified lactic acid bacteria strains were preserved using the glycerol preservation method.

[0043] 3. Molecular identification of strains The microbial composition of kombucha fermentation broth was analyzed using molecular biology methods. First, total DNA was extracted according to the instructions of the OMEGA EZNA™ Mag-Bind Soil DNA Kit. Universal bacterial primers Nobar_341F (CCTACGGGNGGCWGCAG) and Nobar_805R (GACTACHVGGGTATCTAATCC) were used. The reaction system consisted of: 15 μL of 2× Hieff® Robust PCR Master Mix, 1 μL of Bar-PCR primer F, 1 μL of Primer R, 10–20 ng of template DNA, and 30 μL of sterile water. Amplification conditions were: 94℃ pre-denaturation for 3 min; 5 cycles (94℃ denaturation for 30 s, 45℃ annealing for 20 s, 65℃ extension for 30 s); 20 cycles (94℃ denaturation for 20 s, 55℃ annealing for 20 s, 72℃ extension for 30 s); 72℃ extension for 5 min, followed by incubation at 10℃.

[0044] The amplified products were analyzed for fragment size by 2% agarose gel electrophoresis. PCR products meeting the requirements were sent to Sangon Biotech (Shanghai) Co., Ltd. for high-throughput sequencing. Sequence alignment analysis was performed using bioinformatics tools such as BLAST, and the species composition of the bacterial community was determined by combining the results with the NCBI database.

[0045] Table 1 Results of lactic acid bacteria strain screening 4. Preparation of bacterial suspension 200 μL of each of the eight strains listed in Table 1 after purification (combined in a volume ratio of 1:1:1:1:1:1:1:1) was spread onto MRS and M17 medium and cultured at 28°C for 72 h using the spread method. After the colonies had grown sufficiently, all the strains on the plate were scraped off to prepare a bacterial suspension for subsequent use.

[0046] 5. Preparation of bacterial film Based on preliminary experiments, we set five different factors—the amount of inoculant added as 25% of the weight of fresh coffee cherries, pH 7, temperature 28℃, fermentation time 48h, and coffee pectin addition amount 0.4g—to screen the process parameters for the preparation of the microbial film. The median values ​​were taken for the other factors.

[0047] After removing the outer skin of fresh specialty coffee cherries, lactic acid bacteria inoculants are added to prepare a microbial film. During the preparation process, the fermentation container is sealed with a coffee pectin-gelatin composite film. The prepared microbial film is weighed to determine the optimal single-factor level.

[0048] Table 2 Single-factor experimental design Depend on Figure 1 It can be seen that the bacterial film content is maximized when the bacterial agent addition is 25%, pH is 7, temperature is 28℃, time is 48h, and coffee pectin addition is 0.4g.

[0049] 6. Preparation of coffee vinegar Coffee vinegar is an acidic condiment or functional beverage made from coffee or coffee derivatives through microbial fermentation. Its core significance lies in achieving the high-value transformation of coffee resources (especially traditional waste coffee pulp) through targeted fermentation technology. This process not only creates a new sensory matrix that combines characteristic coffee flavor profiles with complex fermented acidity and aroma, but also constructs a sustainable circular economy path from agricultural byproducts to high-end fermented foods. This technology promotes the vertical extension of the coffee industry chain, enhancing industry resilience and overall profitability by creating high-value-added products.

[0050] Using the optimal process conditions determined by the above single-factor experiments (25% microbial agent addition, initial pH 7, fermentation temperature 28℃, fermentation time 48 h, and coffee pectin addition 0.4 g), coffee spawn was prepared using the method for preparing spawn in Example 2, and coffee vinegar was fermented using this coffee spawn.

[0051] The specific operation is as follows: Fresh, ripe specialty coffee cherries are skinned to obtain coffee beans. The pulp is washed, drained, and placed in a sterile fermentation container. 5%-10% of the bacterial film prepared in step 5 (based on the weight of the coffee beans) is added. The initial pH is adjusted to 7.0, and the mixture is incubated at a constant temperature of 28°C for 48 hours. During this period, samples are taken every 24 hours to test the total acid content. Fermentation is terminated when the total acid content stabilizes and reaches the expected acidity. The fermentation liquid is transferred to a sealed container and allowed to mature at 4°C for 7 days. After centrifugation or filtration to remove sediment and bacterial residue, a clear coffee vinegar product is obtained. Testing shows that this coffee vinegar is dark brown or amber in color, clear and bright, with a rich coffee aroma and a mellow vinegar fragrance.

[0052] Comparative Example 1: Preparation of Traditional Coffee Vinegar In this embodiment, coffee spawn is not added during the fermentation of vinegar. Instead, 5%-10% of the coffee bean mass of the spawn is added for fermentation. The remaining steps are the same as in Example 2, and traditional coffee vinegar is prepared.

[0053] Total acidity: The total acidity was determined by potentiometric titration, referring to the national standard GB / T 5009.41-2003 "Analytical Methods for the Hygienic Standard of Vinegar". The total acidity was calculated as acetic acid.

[0054] Total sugar: Fehling's reagent method was used.

[0055] Alcohol content: The density bottle method was used, referring to the method in the national standard GB / T 1503-2006 "General Analytical Methods for Wines and Fruit Wines".

[0056] Total phenols: Pipette 2.0 mL of sample into a test tube, using distilled water as a blank control, and add 0.2 mL of Folin-Ciocalteu's reagent. Mix well and incubate at room temperature for 3 min. Then add 2.0 mL of 7.5% Na₂CO₃ solution and react in the dark for 1.5 h. Measure the absorbance at 765 nm. Total phenol content is expressed as gallic acid, and the unit for total phenol content in the sample is mg / mL.

[0057] Total flavonoids: Take 5 mL of sample, add 0.3 mL of NaNO2 (50 g / L), let stand at room temperature for 6 min, add 0.3 mL of AlCl3 solution (100 g / L), let stand at room temperature for 6 min, then add 4 mL of NaOH solution (40 g / L), and dilute to 10 mL with 80% methanol. Measure the absorbance at 510 nm. A blank control is prepared using 80% methanol solution instead of the sample. Total flavonoid content is calculated as rutin, and the unit for total flavonoid content in the sample is mg / mL.

[0058] Caffeine content: determined according to GB 5009.139-2014 "Determination of Caffeine in Food". Microbiological indicators were determined in accordance with GB / T 4789.21—2003.

[0059] Sensory evaluation: The scoring method is shown in Table 3.

[0060] Table 3 Sensory rating table for coffee vinegar Table 4 Comparison of coffee vinegar prepared with microbial film and traditional coffee vinegar As can be seen from the data in Table 4, the total acidity of the coffee vinegar in Example 2 is higher than that of traditionally produced coffee vinegar because lactic acid bacteria fermentation produces lactic acid, which works synergistically with acetic acid produced by acetic acid bacteria. The acid profile is also more complex, resulting in a smoother taste.

[0061] Total sugar: Using hulled fresh coffee cherries, the natural sugars in the pulp (such as sucrose and pectin) provide a richer carbon source for microorganisms. The residual sugar contains complex components such as oligosaccharides, while the traditional method only relies on coffee bean extract + exogenous sugars, resulting in a simpler residual sugar content.

[0062] Alcohol content: The coffee vinegar produced by microbial fermentation (heterogeneous fermentation) and acetic acid bacteria work together to consume alcohol, resulting in a significantly lower alcohol residue than the traditional method (where only acetic acid bacteria consume alcohol), making it more suitable for people who are sensitive to alcohol.

[0063] Total phenols and total flavonoids: The pulp and skin of fresh coffee cherries are the main accumulation sites of polyphenols and flavonoids. Coffee vinegar made using mycelium releases and retains these components through fermentation, while the traditional method only uses coffee beans (the phenol content in beans is much lower than that in pulp).

[0064] Caffeine: Caffeine is mainly found in the endosperm of coffee beans. Both processes can effectively extract it, so the difference in indicators is small, and it can be used as a stable marker for product traceability.

[0065] Traditional methods produce a linear flavor profile dominated by acetic acid, the complexity of which is limited by the metabolic capacity of the coffee bean extract and a single microbial strain. Fermentation of hulled coffee cherries yields sugars, pectin, organic acids (such as malic acid and tartaric acid), polysaccharides, and various microbial metabolites. This application utilizes a screened kombucha lactic acid bacteria community to cultivate a microbial membrane in a microaerobic environment constructed from a coffee pectin-gelatin composite membrane, achieving targeted and enhanced fermentation. Under the combined fermentation of the prepared microbial membrane, these bacteria can generate far more esters, higher alcohols, ketones, and aldehydes than pure alcoholic fermentation. Combined fermentation may also produce a more diverse range of prebiotics, short-chain fatty acids, and microbial secondary metabolites.

[0066] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing a coffee pectin-gelatin composite film, characterized in that, The preparation method includes the following steps: S1, Dry the coffee fruit peels, then grind and sieve them to obtain coffee powder; S2, the coffee powder obtained in step S1 is added to an 80% ethanol solution, treated in a water bath, and then filtered to obtain residue and filtrate. The residue is repeatedly filtered until the filtrate is colorless. The residue is collected as coffee pericarp alcohol-insoluble matter. S3, add the coffee pericarp alcohol insolubles obtained in step S2 to deionized water at a material-to-liquid ratio of 1:25-1:30 (g / mL), adjust the pH to 1.5 with hydrochloric acid, and then perform water bath extraction; after extraction, filter and collect the filtrate; S4. Concentrate the filtrate obtained in step S3 under reduced pressure to 1 / 3-1 / 4 of its original volume; after cooling, add anhydrous ethanol to the concentrate and let it stand at 4°C until precipitation is complete; collect the precipitate, wash it, and then dry it to constant weight and pulverize it to obtain coffee pectin. The volume ratio of the concentrated solution to anhydrous ethanol is 1:1.5; S5, dissolve gelatin in distilled water at 80°C, stir continuously for 30 minutes, then add glycerin accounting for 5% of the weight of gelatin; then add the coffee pectin obtained in S4 and stir for 2 hours to obtain a film-forming solution; The mass ratio of the gelatin to coffee pectin is 1:0.5-3; S6. The film-forming solution obtained in step S5 is cast onto the coating plate. After drying, the film is placed in an environment of 25°C and 50% relative humidity for 48 hours to obtain the coffee pectin-gelatin composite film.

2. The preparation method according to claim 1, characterized in that, In step S2, the ratio of coffee powder to ethanol solution is 1:10 (g / mL).

3. The preparation method according to claim 1, characterized in that, In step S3, the water bath extraction temperature is 80-90℃ and the time is 90-120 min.

4. The preparation method according to claim 1, characterized in that, The concentration of gelatin in step S5 is 2% (g / mL).

5. The preparation method according to claim 1, characterized in that, The drying temperature in step S6 is 45°C, and the drying time is 24 hours.

6. A method for preparing coffee spawn film, characterized in that, The method includes the following steps: After obtaining coffee pectin by the preparation method according to claim 1, lactic acid bacteria agent is added; then it is coated on MRS medium for cultivation. During the cultivation process, the fermentation container is sealed with a coffee pectin-gelatin composite membrane obtained by the preparation method according to any one of claims 1-5; the amount of coffee pectin added is 0.4g; the amount of lactic acid bacteria agent added is 25% of the mass of coffee beans.

7. The method according to claim 6, characterized in that, The culture was conducted at a temperature of 28°C, pH 7, for a duration of 48 hours.

8. The method according to claim 6, characterized in that, The lactic acid bacteria agent is a compound of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus fermentum, Lactococcus lactis, Pediococcus pentosus, Weisslerella paraintermedia, Lactobacillus bulgaricus, and Streptococcus thermophilus in a bacterial suspension volume ratio of 1:1:1:1:1:1:1:1:

1.

9. A method for preparing coffee vinegar, characterized in that, The preparation method includes adding the coffee spawn prepared by the method of claim 8 and fermenting it to obtain coffee vinegar; the amount of coffee spawn added is 5%-10% of the coffee beans.

10. The application of a coffee pectin-gelatin composite film obtained by the preparation method according to any one of claims 1-5 or a microbial film obtained by the method according to claims 6-8 in the preparation of coffee vinegar.

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