A preparation method of tropical flavor carbonated beverage based on mixed bacteria cascade fermentation and aroma in-situ compensation

Abstract

This invention belongs to the field of carbonated beverage technology, and specifically discloses a method for preparing tropical-flavored carbonated beverages based on mixed-culture step fermentation and in-situ aroma compensation, including the following steps: (1) After pulping the fruit, water is added and mixed, and the mixture is first hydrolyzed by α-amylase at medium temperature, and then hydrolyzed by a compound enzyme to obtain enzymatically hydrolyzed clear juice; (2) Fermentation is carried out by inoculation, and fruit peel oligosaccharide fermentation liquid is added to obtain fermentation liquid; (3) After fermentation, the condensate is added back to the obtained fermentation liquid to obtain the added fermentation liquid, and then the esterification precursor compensation liquid is mixed into the added fermentation liquid to obtain the compensated fermentation liquid; (4) Composite wall slow-release microcapsules are aseptically added to the compensated fermentation liquid, carbon dioxide is charged, and the mixture is aseptically filled. This application solves the problem of aroma decay of tropical-flavored carbonated beverages from processing to shelf by compensating for aroma at both the production end and shelf end, and by using materials from passion fruit peel, yeast cells and other sources to form a step-by-step transfer of oligosaccharide-promoted bacteria, bacterial enzyme production, enzyme-released aroma, and aroma re-esterification.

Description

Technical Field

[0001] This invention belongs to the field of carbonated beverage technology, specifically relating to a method for preparing tropical-flavored carbonated beverages based on mixed-culture stepped fermentation and in-situ aroma compensation. Background Technology

[0002] Carbonated beverages are ready-to-drink drinks made by infusing carbon dioxide into liquids. The carbon dioxide releases upon sip, creating a refreshing, savory taste, making them a popular category for everyday consumption and as a accompaniment to meals. Tropical-flavored carbonated drinks are marketed based on the flavors of tropical fruits such as mango, pineapple, and passion fruit. The aromas of these fruits are composed of esters, terpenes, and norisoprene compounds, resulting in strong, distinctive aromas that are highly appealing to consumers. As consumer preferences shift from simply quenching thirst to seeking natural and nutritious options, fermented carbonated beverages made from fruit juice through microbial fermentation are gaining popularity. The fermentation process not only produces aromas but also generates organic acids, oligosaccharides, and various metabolites, resulting in a richer flavor profile and nutritional value compared to simply blended drinks, representing a direction for the upgrading of carbonated beverages.

[0003] Tropical-flavored fermented carbonated beverages face several unavoidable drawbacks in actual production. The characteristic aroma compounds of tropical fruits have low boiling points and are chemically reactive. Sterilization, blending, degassing, and carbonation during beverage processing all remove some of these volatile aromas, resulting in a significantly weaker aroma by the time the fermentation process is complete. Furthermore, these aroma compounds continue to hydrolyze and oxidize during the shelf life, leading to a noticeably weaker aroma compared to freshly made products. Fermentation for aroma production relies on microbial metabolism of sugars; more complete sugar metabolism results in a stronger aroma, but also increases ethanol levels. Carbonated beverages require very low ethanol content, creating a natural conflict between aroma production and alcohol limitation. Additionally, the fruit pulp particles retained to preserve the fruity texture tend to aggregate and settle during storage, forming flocculent precipitates that affect appearance and texture uniformity.

[0004] To address the aforementioned shortcomings, existing technologies have provided several solutions. For insufficient aroma, a common approach is to add edible flavorings to directly compensate for the intensity and complexity of the aroma. For a thin fermented flavor, some solutions utilize mixed fermentation with lactic acid bacteria and yeast, leveraging the metabolic differences between these two types of microorganisms to enrich the variety of flavor compounds. For excessive ethanol, common methods include diluting the fermentation broth, prematurely stopping fermentation before aroma development, and removing ethanol using membrane separation. For pulp sedimentation, thickeners such as pectin, xanthan gum, and sodium carboxymethyl cellulose are generally added to increase liquid viscosity and maintain particle suspension. Shelf-life quality is primarily ensured by increasing sterilization intensity and adding preservatives.

[0005] While these methods address existing shortcomings, they also introduce new problems. The aroma of added flavorings is inconsistent with the aroma produced during fermentation, resulting in a superficial fragrance that lacks the richness and fullness expected of fermented beverages, thus diminishing the natural selling point of fermented drinks. In mixed-culture fermentation with simultaneous inoculation of lactic acid bacteria and yeast, the two types of microorganisms compete for carbon and nitrogen sources, and acid and aroma production mutually restrain each other, leading to significant fluctuations in fermentation results across different batches and making flavor instability difficult. Dilution and premature termination of fermentation reduce ethanol levels while simultaneously weakening aroma compounds, resulting in a trade-off. Membrane separation for alcohol removal requires substantial equipment investment, making it unaffordable for small and medium-sized production. Adding thickeners stabilizes fruit pulp particles but also makes the drink sticky, affecting the refreshing taste, contradicting the refreshing positioning of carbonated drinks. More importantly, the aroma decay during shelf life is not being taken seriously. The industry's usual approach is to increase the initial amount of flavoring, using excessive amounts to counteract the decay. The beverages have a strong aroma when they leave the factory, but after a period of storage, they become bland. There is still no way to continuously replenish the aroma inside the finished product.

[0006] In summary, a method for preparing tropical-flavored carbonated beverages that allows for the full generation of aroma compounds during fermentation at low ethanol levels, compensates for lost aromas during production, continuously replenishes fading aromas from the finished product throughout its shelf life, and maintains stable suspension of fruit pulp particles without relying on external thickeners, is a pressing technical challenge in this field. Therefore, a method for preparing tropical-flavored carbonated beverages based on mixed-culture stepped fermentation and in-situ aroma compensation is needed. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, a method for preparing tropical-flavored carbonated beverages based on mixed-culture stepped fermentation and in-situ aroma compensation is provided.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing tropical-flavored carbonated beverages based on mixed-culture step fermentation and in-situ aroma compensation, the method comprising the following steps: (1) After pulping mango, pineapple and passion fruit, add water and mix. First, hydrolyze with α-amylase at medium temperature, then hydrolyze with compound enzyme, inactivate the enzyme, centrifuge to obtain clear juice, adjust the sugar content, sterilize, and obtain enzymatically hydrolyzed clear juice. (2) Inoculate the enzymatically hydrolyzed juice with *Lactobacillus plantarum*, *Lactobacillus paracasei* and *Leuconostoc mesenteroides* for fermentation; cool down, inoculate with co-domesticated ester-producing bacteria, and add pectin oligosaccharide fermentation broth to the fermentation broth to continue fermentation; cool down, mature, and obtain fermentation broth; (3) The fermentation process in step (2) is completely sealed. The tail gas at the top of the fermentation tank is condensed and collected. After the fermentation is completed, the condensate is added back to the fermentation liquid obtained in step (2) to obtain the added fermentation liquid. Then, the esterification precursor compensation liquid is mixed into the added fermentation liquid to obtain the compensation fermentation liquid. (4) After adjusting the sugar-acid ratio of the compensating fermentation broth obtained in step (3), aseptically add composite wall sustained-release microcapsules to the compensating fermentation broth, fill with carbon dioxide, and aseptically fill; the composite wall sustained-release microcapsules use aroma-stabilizing complex and β-glucosidase as core materials and dextran-sodium alginate-chitosan composite wall as wall material; the aroma-stabilizing complex is obtained by mixing and homogenizing glycoside-type flavor precursor concentrate and yeast-derived mannoprotein concentrate.

[0009] The preparation method of the glycoside-type flavor precursor concentrate is as follows: Take passion fruit peel, remove the outer hard shell and take the white peel layer, crush it, add water at a peel to water mass ratio of 1:5-8, add a compound enzyme composed of cellulase and pectinase at a mass ratio of 1:1-2, add 30-50 U / g of enzyme based on the peel mass, enzymatically hydrolyze at 45-50℃ for 60-90 min, inactivate the enzyme at 90℃ for 15 s, filter, and concentrate the filtrate under reduced pressure at 50-60℃ and vacuum degree of 0.08-0.09 MPa until the solid content is 20-30%, thus obtaining the glycoside-type flavor precursor concentrate.

[0010] The preparation method of the yeast-derived mannoprotein concentrate is as follows: Kluyveromyces martensii is inoculated into pineapple juice and cultured aerobically at 25-28℃ for 24-36h. The cells are collected by centrifugation, washed 1-2 times with sterile physiological saline, resuspended at a cell-to-water ratio of 1:5-10, and 2-4% salt by cell mass is added. The pH is adjusted to 4.5-5.5 with citric acid, and the cells are autolyzed at 45-50℃ for 12-24h. The supernatant is collected by centrifugation, and 95% edible ethanol is added until the final ethanol concentration in the system is 60-70% by volume. The mixture is allowed to stand at 2-6℃ for 8-12h, and the precipitate is collected by centrifugation. The precipitate is redissolved in water, desalted by ultrafiltration membrane with a molecular weight cutoff of 8-10kDa, and concentrated to a solid content of 15-25%. The resulting product is the yeast-derived mannoprotein concentrate.

[0011] The preparation method of the composite wall sustained-release microcapsules is as follows: Take the aroma-stabilized complex, add 30-50 U of β-glucosidase per gram of aroma-stabilized complex and mix well, add sodium alginate and fermentation-derived dextran concentrate, wherein the mass ratio of sodium alginate to aroma-stabilized complex is 2-4:100, and the mass ratio of fermentation-derived dextran concentrate to sodium alginate is 1:2-4. After stirring well, adjust the viscosity of the mixture to 150-400 mPa·s, and drop it into a calcium lactate aqueous solution, wherein the mass ratio of calcium lactate to water in the calcium lactate aqueous solution is 2-5:100. Solidify for 20-40 min, filter out, immerse in chitosan coating solution for 15-30 min, wash with water, and obtain the composite wall sustained-release microcapsules.

[0012] The mass ratio of glycoside-type flavor precursor concentrate to yeast-derived mannoprotein concentrate in the aroma-stabilizing complex is 2-4:1, the homogenization pressure is 20-30 MPa, and the homogenization is performed 2-3 times. The chitosan coating solution has a chitosan to 1% acetic acid solution mass ratio of 0.5-1.5:100, a chitosan deacetylation degree of not less than 85%, and a weight-average molecular weight of 50,000-200,000 Da. The preparation method of the fermentation source dextran concentrate is as follows: Leuconostoc mesenteroides is inoculated into a sucrose solution with a mass fraction of 8-12% and a viable count of 10^6-10^7 CFU / mL. Fermentation is carried out at 25-30℃ for 24-48 hours, and the bacteria are inactivated. Then, 95% edible ethanol is added until the final ethanol concentration is 60-70% by volume. The mixture is allowed to stand for alcohol precipitation, centrifuged to collect the precipitate, redissolved in water, and concentrated to a dextran content of 8-15 g / L to obtain the fermentation source dextran concentrate.

[0013] The preparation method of the fruit peel oligosaccharide fermentation broth is as follows: Take the filtrate obtained after enzyme inactivation and filtration during the preparation of the glycoside-type flavor precursor concentrate, use the residual pectin and pectin degradation intermediates in the filtrate as substrates, add 10-20 U / g of pectinase according to the mass of the filtrate, perform a second enzymatic hydrolysis at 45-50℃ for 60-90 min, inactivate the enzyme, and concentrate to a solid content of 10-15%, which is the fruit peel oligosaccharide fermentation broth.

[0014] The preparation method of the esterification precursor compensating solution is as follows: Take the concentrated glycoside-type flavor precursor solution, add 20-40 U of β-glucosidase per gram of concentrated glycoside-type flavor precursor solution, enzymatically hydrolyze at 30-35℃ for 2-3 hours, raise the temperature to 90℃ and hold for 10 minutes to inactivate the enzyme, and cool to obtain the precursor hydrolysate; add glucose to the precursor hydrolysate to adjust the reducing sugar to 80-120 g / L, and adjust the yeast extract to precursor hydrolysate mass ratio of 0.5-1.5: Add 100g of yeast extract powder; and inoculate with *Lactobacillus plantarum*, with a viable count of 10^6-10^7 CFU / mL, ferment at 28-32℃ for 12-24h until pH 3.5-3.8; then inoculate with *Kluyveromyces martensii*, with a viable count of 10^6-10^7 CFU / mL, ferment at 20-25℃ with microaerobic fermentation for 24-48h, inactivate at 85℃ for 15s, and concentrate under reduced pressure to a solid content of 20-30% to obtain the esterification precursor compensating solution.

[0015] In step (1), the mass ratio of mango, pineapple, and passion fruit is 3-5:2-3:1-2, and the mass ratio of the pulp to water after pulping is 1:0.3-0.6; the medium-temperature α-amylase is added at 10-20 U / g based on the mass of the pulp, and enzymatic hydrolysis is performed at 60-65℃ for 30-45 min; the compound enzyme includes pectinase, cellulase, and hemicellulase, and the mass ratio of pectinase, cellulase, and hemicellulase is 3-5:1-2:1; the compound enzyme is added at 80-150 U / g based on the mass of the pulp, and enzymatic hydrolysis is performed at 45-50℃ for 90-150 min, followed by enzyme inactivation at 90℃ for 15 s; the sugar content is adjusted to 40-70 g / L with sucrose. In step (2), the enzymatically hydrolyzed juice is inoculated with *Lactobacillus plantarum*, *Lactobacillus paracasei*, and *Leuconostoc mesenteroides* at 25-28℃ and fermented for 12-18 hours until pH 3.8-4.0. The temperature is then lowered to 16-20℃, and a co-acclimated ester-producing bacteria agent is inoculated. A pectin oligosaccharide fermentation broth is added to the fermentation broth in 2-3 additions, with a cumulative addition amount of 8-15% of the enzymatically hydrolyzed juice mass. Fermentation continues for 24-36 hours until the ethanol content of the fermentation broth does not exceed 0.5% vol. The temperature is then lowered to 8-12℃ and matured for 48-72 hours to obtain the fermentation broth. The viable count of *Lactobacillus plantarum*, *Lactobacillus paracasei*, *Leuconostoc mesenteroides*, and the co-acclimated ester-producing bacteria agent is 10^6-10^7 CFU / mL. In step (3), the tail gas at the top of the fermenter is condensed at 0-4℃ and the condensate is collected; the amount of condensate added back is 1-3% of the mass of the fermentation liquid obtained in step (2); the mass ratio of the added fermentation liquid to the esterification precursor compensation liquid is 10-20:1. In step (4), the sugar-acid ratio is adjusted to 25-40:1 by using sucrose or glucose and citric acid to compensate the fermentation broth. The sugar-acid ratio is the ratio of the mass fraction of soluble solids to the mass fraction of total acid, and the total acid is calculated as citric acid. The amount of composite wall sustained-release microcapsules added is 0.3-0.8% of the mass of the compensated fermentation broth. Before carbon dioxide is charged, the compensated fermentation broth is cooled to 2-6℃, and the amount of carbon dioxide charged is 4.5-6.0 g / L.

[0016] The co-domesticated ester-producing bacterial agent is obtained by mixed subculturing of Kluyveromyces martensii and Saccharomyces cerevisiae. The specific culture method is as follows: Kluyveromyces martensii and Saccharomyces cerevisiae are mixed at a live cell ratio of 1-2:1 and inoculated into a culture medium composed of pineapple juice and passion fruit peel extract at a volume ratio of 2-4:1. The reducing sugar content of the culture medium is 60-80 g / L. The culture is subcultured at 22-26℃ for 3-5 generations, with each generation lasting 24 hours. The cells are collected by centrifugation after the last generation and resuspended in pineapple juice to a live cell count of 10^8-10^9 CFU / mL.

[0017] The preparation method of the passion fruit peel extract is as follows: take passion fruit peel, add water at a peel to water mass ratio of 1:5-8, extract at 60-70℃ for 60-90 minutes, filter and collect the filtrate to obtain the passion fruit peel extract.

[0018] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. This application employs a stepwise mixed-culture fermentation method for the enzymatic hydrolysis of mango, pineapple, and passion fruit juice. *Lactobacillus plantarum* and *Lactobacillus paracasei* initially produce acid, increasing the acidity of the fermentation broth. *Leuconostoc mesenteriae* utilizes sucrose to generate glucan. Subsequently, the mixture is cooled and inoculated with a co-acclimated ester-producing bacteria agent, followed by the addition of a fruit peel oligosaccharide fermentation broth. The oligosaccharides in this broth are not preferentially utilized by yeast but are selectively absorbed by lactic acid bacteria, promoting their continuous proliferation at low temperatures and the expression of β-glucosidase. This hydrolyzes the glycosidic-bonded aroma substances in the juice, releasing free aromas for further esterification by yeast. The low acidity and low temperature environment also suppresses the alcoholic fermentation intensity of the yeast. While aroma substances are fully generated, the ethanol content remains at a low level, balancing the requirements of rich aroma and low alcohol content in carbonated beverages.

[0019] 2. This application compensates for aroma at the production end. The fermentation process is completely closed. The condensate obtained by condensing the tail gas at the top of the fermentation tank is added back to the fermentation liquid, bringing back the esters and terpenes that escaped with the tail gas to the liquid phase. The esterification precursor compensation liquid is obtained by condensing the concentrated glycoside flavor precursor liquid through β-glucosidase release, lactic acid bacteria acid production, and yeast esterification in a step-by-step process. It has the same flavor as the added fermentation liquid. After being mixed in, it makes up for the loss of volatile aroma during sterilization, blending and carbonation. The resulting compensated fermentation liquid has a full and natural aroma and does not require the addition of external flavorings.

[0020] 3. This application compensates for aroma at the shelf end. The core material of the composite wall slow-release microcapsule, the aroma-stabilizing complex, is obtained by homogenizing a glycoside-type flavor precursor concentrate and a yeast-derived mannoprotein concentrate. The mannoprotein in the yeast-derived mannoprotein concentrate binds to aroma molecules through non-covalent interactions, slowing down their outward diffusion. The co-encapsulated β-glucosidase slowly hydrolyzes the glycoside-type flavor precursor concentrate during the shelf life, continuously releasing free aroma substances, forming a balance with the natural decay of aroma. The dextran-sodium alginate-chitosan composite wall provides reliable encapsulation, and the chitosan coating blocks the diffusion of polyphenols from the pulp into the capsule, protecting the activity of the β-glucosidase inside the capsule. The aroma of the beverage remains stable during the shelf life.

[0021] 4. The Leuconostoc mesenteroides of this application generates glucan in situ in the fermentation broth. The glucan increases the viscosity of the liquid phase and forms a hydration layer on the surface of the pulp particles and the composite wall slow-release microcapsules, which prevents particle aggregation and sedimentation. The system can be kept uniform without the addition of thickeners. At the same time, the glucan increases the adhesion and retention of carbon dioxide bubbles, resulting in a smooth and delicate taste in the beverage. Attached Figure Description

[0022] Figure 1 This is a process flow diagram of a method for preparing tropical-flavored carbonated beverages based on mixed-culture stepped fermentation and in-situ aroma compensation according to the present invention. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] In the specific embodiments of this application, the sources of various main raw materials are briefly described as follows: Mangoes, pineapples, and passion fruit, fresh fruits purchased from Guangxi Haijixing Agricultural Products International Logistics Center; The mesophilic α-amylase was purchased from Novozymes (China) Biotechnology Co., Ltd., model BAN 480L. Pectinase, purchased from Novozymes (China) Biotechnology Co., Ltd., model number Pectinex Ultra SP-L; Cellulase, purchased from Novozymes (China) Biotechnology Co., Ltd., model number Celluclast 1.5L; Hemicellulase, purchased from Novozymes (China) Biotechnology Co., Ltd., model number Pulpzyme HC; β-glucosidase, purchased from Ningxia Xiasheng Industrial Group Co., Ltd., model FDY-2206; Lactobacillus plantarum was purchased from the China Industrial Microbial Culture Collection Center, with accession number CICC 23137. Lactobacillus paracasei was purchased from the China Industrial Microbial Culture Collection Center, with accession number CICC6277. Leuconostoc mesenteroides was purchased from the China Industrial Microbial Culture Collection Center, with accession number CICC 23614. Kluyveromycin, purchased from China Industrial Microbial Culture Collection Center, accession number CICC1953; Saccharomyces cerevisiae was purchased from the China Industrial Microbial Culture Collection Center, with accession number CICC 1305; Yeast extract powder, purchased from Angel Yeast Co., Ltd., model FM802; Glucose, purchased from Xiwang Group Co., Ltd., food-grade glucose monohydrate; The sucrose was purchased from COFCO Sugar Holdings Co., Ltd., and was grade 1 white granulated sugar. Citric acid, purchased from Weifang Yingxuan Industrial Co., Ltd., is food-grade citric acid monohydrate. Sodium alginate, purchased from Qingdao Mingyue Seaweed Group Co., Ltd., food grade; Chitosan, purchased from Zhejiang Jinke Biochemical Co., Ltd., is food grade, with a degree of deacetylation of 90% and a weight-average molecular weight of 100,000 Da. Calcium lactate, purchased from Henan Jindan Lactic Acid Technology Co., Ltd., food grade; Acetic acid, purchased from Jiangsu Suopu (Group) Co., Ltd., food-grade glacial acetic acid; The technical solution of this application is as follows: A method for preparing tropical-flavored carbonated beverages based on mixed-culture stepped fermentation and in-situ aroma compensation, such as... Figure 1 As shown, the method includes the following steps: (1) After pulping mango, pineapple and passion fruit, add water and mix. First, hydrolyze with α-amylase at medium temperature, then hydrolyze with compound enzyme, inactivate the enzyme, centrifuge to obtain clear juice, adjust the sugar content, sterilize, and obtain enzymatically hydrolyzed clear juice. Tropical fruit processing has long suffered from low juice yield and viscous pulp, and conventional single pectinase treatment can only partially reduce viscosity. In step (1) of this application, a stepwise enzymatic hydrolysis is adopted. First, the fruit pulp is treated with mesophilic α-amylase at 60-65℃. The starch introduced by mango and pineapple has already begun to gelatinize and expand at high temperature. At this time, the mesophilic α-amylase acts on the α-1,4 glycosidic bonds inside the starch molecule, randomly cleaving it into short-chain dextrins. Dextrins no longer have the ability to recrystallize, thus avoiding starch turbidity during subsequent sterilization and storage from the source. Then, the temperature is lowered and a complex enzyme composed of pectinase, cellulase and hemicellulase is added. The pectinase hydrolyzes the galacturonic acid residues along the pectin backbone. The α-1,4 glycosidic bonds break down the long pectin chains, releasing the water from the slurry and reducing viscosity. Cellulase and hemicellulase degrade the β-1,4 glucan backbone and xylan side chains in the cell wall, respectively. Sugars, organic acids, and glycosidic aroma substances trapped inside the cells are then released into the liquid phase. The temperature settings for the two-stage enzymatic hydrolysis correspond to the activity range of their respective enzyme systems. The order of starch hydrolysis followed by cell wall hydrolysis avoids premature viscosity reduction of the colloid, which would cause starch granules to lose suspension support and partially settle to the bottom and gelatinize. The enzymatic hydrolysate obtained after centrifugation is clear, with a balanced sugar and acid content, and can be directly used as a fermentation substrate.

[0025] (2) Inoculate the enzymatically hydrolyzed juice with *Lactobacillus plantarum*, *Lactobacillus paracasei* and *Leuconostoc mesenteroides* for fermentation; cool down, inoculate with co-domesticated ester-producing bacteria, and add pectin oligosaccharide fermentation broth to the fermentation broth to continue fermentation; cool down, mature, and obtain fermentation broth; In mixed-culture fermentation, simultaneous inoculation of lactic acid bacteria and yeast will lead to competition for carbon sources, and the production of acid and aroma will mutually restrain each other. Moreover, the more complete the sugar metabolism, the higher the ethanol content. In step (2) of this application, a step-by-step inoculation and gradient temperature control are adopted. In the first stage, *Lactobacillus plantarum*, *Lactobacillus paracasei*, and *Leuconostoc mesenteroides* are inoculated at 25-28℃. *Lactobacillus plantarum* and *Lactobacillus paracasei* convert glucose into lactic acid through glycolysis, and a small amount of acetic acid is produced by heterologous fermentation. The pH of the fermentation broth drops to 3.8-4.0 within 12-18 hours. *Leuconostoc mesenteroides* uses its extracellular glucan sucrase to transfer glucose units one by one to the primer to synthesize glucan mainly composed of α-1,6 glycosidic bonds, while releasing fructose into the liquid phase. The acidic environment and the glucan backbone are established simultaneously in this stage. Dextran is mainly synthesized in the first stage by catalyzed by dextran sucrase. After the temperature drops in the second stage, the activity of this enzyme decreases and the synthesis basically stops. The synthesized dextran remains in the liquid phase and continues to play a stabilizing and foam-holding role. The functional focus of the second stage shifts to the production of enzymes and aroma by lactic acid bacteria and the production of esters by yeast.

[0026] The second stage involves cooling the yeast to 16-20℃ and inoculating it with a co-acclimation agent for ester production. The low temperature and the acidity established in the previous stage work together to inhibit the activity of cell membrane transport proteins and key enzymes in glycolysis. This reduces the rate of glucose entering the cell and the flux of glycolysis, thus limiting ethanol production. However, acyltransferases, which catalyze ester synthesis, remain active under these conditions, allowing the binding reaction of acetyl-CoA with alcohols to continue. The metabolic focus of yeast shifts from alcohol production to ester production. Although the overall metabolic rate of yeast decreases at low temperatures, the acyltransferase-catalyzed esterification has low substrate concentration requirements, and the reaction is less affected by low temperatures. Furthermore, the low temperature reduces the volatilization and dissipation of already generated esters, allowing esters to accumulate rather than decrease. The galacturonic acid oligosaccharides in the added pectin oligosaccharide fermentation broth require specific permeable enzymes and pectin-degrading enzyme systems for transport and utilization. *Kluyveromyces martensii* and *Saccharomyces cerevisiae* lack this utilization pathway and do not compete with lactic acid bacteria for these oligosaccharides. Lactic acid bacteria, however, can selectively absorb these oligosaccharides, continuously proliferate at low temperatures, and express β-glucosidase. This enzyme hydrolyzes the glycosidic bonds of glycosidic aroma compounds in the clarified broth, releasing free aglycones such as linalool, geraniol, and β-ionone. These aglycones directly contribute floral and fruity aromas and also act as substrates for yeast esterification, generating corresponding terpene esters. Thus, the hierarchical transmission of oligosaccharides nourishing lactic acid bacteria, lactic acid bacteria producing enzymes, enzymes releasing aromas, and aroma re-esterification is closed. During the third stage of ripening at 8-12℃, the slow esterification of acids and alcohols at low temperatures makes the aroma more rounded, while residual proteins and polyphenol colloids aggregate and settle at low temperatures, naturally clarifying the fermentation broth.

[0027] The adaptability of microbial agents to raw materials directly affects the repeatability of fermentation. Ordinary microbial agents result in a long stagnation period after inoculation into high-acid and high-osmotic tropical fruit juices. The co-domesticated ester-producing microbial agent of this application mixes Kluyveromyces martensii and Saccharomyces cerevisiae at a live cell ratio of 1-2:1 and continuously passages them in a culture medium composed of pineapple juice and passion fruit peel extract. The acidity, sugar content, and flavor precursor composition of the culture medium are similar to the formal fermentation environment. Each generation is equivalent to a selection process, and the stress resistance traits such as cell membrane fatty acid composition and trehalose accumulation are gradually strengthened, shortening the stagnation period. Kluyveromyces martensii has a rich esterification enzyme system and rapid proliferation, and is responsible for the main aroma production. Saccharomyces cerevisiae is stress-resistant and maintains the basic fermentation structure when acidity and osmotic pressure fluctuate. The two yeasts form a stable ratio and division of labor during domestication, and the ester production behavior after inoculation into the fermentation broth is predictable, with consistent aroma profiles across different batches.

[0028] (3) The fermentation process in step (2) is completely sealed. The tail gas at the top of the fermentation tank is condensed and collected. After the fermentation is completed, the condensate is added back to the fermentation liquid obtained in step (2) to obtain the added fermentation liquid. Then, the esterification precursor compensation liquid is mixed into the added fermentation liquid to obtain the compensation fermentation liquid. A significant portion of the aroma generated during fermentation dissipates with the exhaust gas during sterilization, blending, degassing, and carbonation filling, a situation that conventional production passively bears. In step (3) of this application, the fermentation process is completely sealed. Esters and terpenes have low boiling points and high vapor pressures, rising with the carbon dioxide exhaust gas. The exhaust gas at the top of the tank is condensed at 0-4℃ and then re-condensed into a liquid phase. After fermentation, 1-3% of the fermentation liquid mass is added back, thus restoring this portion of aroma. The mixed esterification precursor compensation liquid makes up for the aroma gap caused by heat processing. The esterification precursor compensation liquid uses glycoside-type flavor precursor concentrate as raw material. It is first hydrolyzed by β-glucosidase to release aglycones, and then the temperature is raised to inactivate the enzyme, avoiding uncontrolled hydrolysis of residual enzymes in subsequent fermentation. After enzyme inactivation, glucose and yeast extract are added. The yeast extract provides amino acids. With the help of growth factors, *Lactobacillus plantarum* first metabolizes some glucose into lactic acid and acetic acid. The fermentation endpoint is controlled at pH 3.5-3.8 while retaining residual sugar, which accumulates organic acid substrates without making the environment too acidic. After inoculation with *Kluyveromyces martensii*, it uses glucose to produce ethanol, and uses ethanol and lactic acid and acetic acid in the system as substrates to synthesize ethyl acetate and ethyl lactate through acyl alcohol transferase catalysis. The isoamyl alcohol produced by the conversion of amino acids through the Ehrlich pathway combines with acetyl groups to form isoamyl acetate. Each type of ester in the compensation liquid has a clear production pathway and is homologous to the flavor of the main fermentation liquid. After mixing, the aroma is full and natural.

[0029] (4) After adjusting the sugar-acid ratio of the compensating fermentation broth obtained in step (3), aseptically add composite wall sustained-release microcapsules to the compensating fermentation broth, fill with carbon dioxide, and aseptically fill; the composite wall sustained-release microcapsules use aroma-stabilizing complex and β-glucosidase as core materials and dextran-sodium alginate-chitosan composite wall as wall material; the aroma-stabilizing complex is obtained by mixing and homogenizing glycoside-type flavor precursor concentrate and yeast-derived mannoprotein concentrate.

[0030] Aroma decay during shelf life is a more difficult problem to solve. Esters continue to hydrolyze in acidic liquid phases, and terpenes continue to oxidize. In step (4) of this application, composite wall sustained-release microcapsules are aseptically added to compensate for the loss of aroma. The core material aroma-stabilizing complex is homogenized from a glycoside-type flavor precursor concentrate and a yeast-derived mannoprotein concentrate. Mannoprotein is composed of sugar chains and peptides. The hydroxyl groups on the sugar chains form hydrogen bonds with aroma molecules, and the hydrophobic regions of the peptides hydrophobically associate with terpene molecules. The aroma molecules are reversibly anchored in the complex, and the diffusion rate from the core material outward is slowed down. The β-glucosidase co-embedded in the capsule slowly hydrolyzes the glycosidic bonds of the glycoside-type flavor precursor during shelf life, continuously releasing free aglycones. The release rate and the natural decay rate complement each other. The calcium ions provided by calcium lactate in the wall material crosslink with the guluronic acid blocks of sodium alginate to form an egg-box structured gel network. The fermentation-derived dextran concentrate is blended with sodium alginate to form the wall. Dextran molecules are interspersed in the sodium alginate gel network. The density and mechanical strength of the composite wall are better than those of the single sodium alginate wall. The resistance to leakage of the contents from the capsule increases accordingly. The chitosan coating utilizes its positive charge and film-forming properties to form a film on the spherical surface, blocking the diffusion of polyphenols from the pulp into the capsule and preventing polyphenols from binding with enzyme proteins and inactivating the enzymes. The slow-release function of the enzymes in the capsule is maintained throughout the shelf life.

[0031] In this invention, the condensate is re-added and the esterification precursor compensation liquid is re-mixed within the production system to complete the in-situ compensation of aroma. The composite wall sustained-release microcapsules complete the in-situ compensation of aroma within the finished product. The substances used for compensation in both cases originate from the fermentation system itself, without introducing any external flavorings.

[0032] Sedimentation and stratification of fruit pulp particles is a common problem in fruit-based beverages, and adding thickeners can sacrifice the refreshing taste of carbonated drinks. This application solves this problem by relying on dextran generated in situ by Leuconostoc mesenteroides in the fermentation broth. Dextran is a high-molecular-weight polysaccharide that increases the viscosity of the continuous phase after dissolution, slowing down the Brownian motion and gravitational sedimentation of the particles. Dextran segments adsorb onto the surface of fruit pulp particles and microcapsules to form a hydration layer. When particles are close together, steric hindrance is generated, and aggregation is inhibited. The system remains homogeneous without the addition of thickeners. Dextran and mannose protein in yeast-derived mannose protein concentrate can also adsorb onto the liquid film surface of carbon dioxide bubbles, delaying the aggregation and collapse of bubbles. As a result, the beverage has a long-lasting foam and a smooth texture after drinking.

[0033] The preparation method of the glycoside-type flavor precursor concentrate is as follows: Take passion fruit peel, remove the outer hard shell and take the white peel layer, crush it, add water at a peel to water mass ratio of 1:5-8, add a compound enzyme composed of cellulase and pectinase at a mass ratio of 1:1-2, add 30-50 U / g of enzyme based on the peel mass, enzymatically hydrolyze at 45-50℃ for 60-90 min, inactivate the enzyme at 90℃ for 15 s, filter, and concentrate the filtrate under reduced pressure at 50-60℃ and vacuum degree of 0.08-0.09 MPa until the solid content is 20-30%, thus obtaining the glycoside-type flavor precursor concentrate.

[0034] The preparation method of the yeast-derived mannoprotein concentrate is as follows: Kluyveromyces martensii is inoculated into pineapple juice and cultured aerobically at 25-28℃ for 24-36h. The cells are collected by centrifugation, washed 1-2 times with sterile physiological saline, resuspended at a cell-to-water ratio of 1:5-10, and 2-4% salt by cell mass is added. The pH is adjusted to 4.5-5.5 with citric acid, and the cells are autolyzed at 45-50℃ for 12-24h. The supernatant is collected by centrifugation, and 95% edible ethanol is added until the final ethanol concentration in the system is 60-70% by volume. The mixture is allowed to stand at 2-6℃ for 8-12h, and the precipitate is collected by centrifugation. The precipitate is redissolved in water, desalted by ultrafiltration membrane with a molecular weight cutoff of 8-10kDa, and concentrated to a solid content of 15-25%. The resulting product is the yeast-derived mannoprotein concentrate.

[0035] The preparation method of the composite wall sustained-release microcapsules is as follows: Take the aroma-stabilized complex, add 30-50 U of β-glucosidase per gram of aroma-stabilized complex and mix well, add sodium alginate and fermentation-derived dextran concentrate, wherein the mass ratio of sodium alginate to aroma-stabilized complex is 2-4:100, and the mass ratio of fermentation-derived dextran concentrate to sodium alginate is 1:2-4. After stirring well, if the viscosity of the mixture is too high, add drinking water to dilute it; if it is too low, add sodium alginate to thicken it. Adjust the viscosity of the mixture to 150-400 mPa·s, and drop it into a calcium lactate aqueous solution, wherein the mass ratio of calcium lactate to water in the calcium lactate aqueous solution is 2-5:100. Solidify for 20-40 min, filter out, immerse in chitosan coating solution for 15-30 min, wash with water, and obtain the composite wall sustained-release microcapsules.

[0036] The mass ratio of glycoside-type flavor precursor concentrate to yeast-derived mannoprotein concentrate in the aroma-stabilizing complex is 2-4:1, the homogenization pressure is 20-30 MPa, and the homogenization is performed 2-3 times. The chitosan coating solution has a chitosan to 1% acetic acid solution mass ratio of 0.5-1.5:100, a chitosan deacetylation degree of not less than 85%, and a weight-average molecular weight of 50,000-200,000 Da. The preparation method of the fermentation source dextran concentrate is as follows: Leuconostoc mesenteroides is inoculated into a sucrose solution with a mass fraction of 8-12% and a viable count of 10^6-10^7 CFU / mL. Fermentation is carried out at 25-30℃ for 24-48 hours, and the bacteria are inactivated. Then, 95% edible ethanol is added until the final ethanol concentration is 60-70% by volume. The mixture is allowed to stand for alcohol precipitation, centrifuged to collect the precipitate, redissolved in water, and concentrated to a dextran content of 8-15 g / L to obtain the fermentation source dextran concentrate.

[0037] The preparation method of the fruit peel oligosaccharide fermentation broth is as follows: Take the filtrate obtained after enzyme inactivation and filtration during the preparation of the glycoside-type flavor precursor concentrate, use the residual pectin and pectin degradation intermediates in the filtrate as substrates, add 10-20 U / g of pectinase according to the mass of the filtrate, perform a second enzymatic hydrolysis at 45-50℃ for 60-90 min, inactivate the enzyme, and concentrate to a solid content of 10-15%, which is the fruit peel oligosaccharide fermentation broth.

[0038] The preparation method of the esterification precursor compensating solution is as follows: Take the concentrated glycoside-type flavor precursor solution, add 20-40 U of β-glucosidase per gram of concentrated glycoside-type flavor precursor solution, enzymatically hydrolyze at 30-35℃ for 2-3 hours, raise the temperature to 90℃ and hold for 10 minutes to inactivate the enzyme, and cool to obtain the precursor hydrolysate; add glucose to the precursor hydrolysate to adjust the reducing sugar to 80-120 g / L, and adjust the yeast extract to precursor hydrolysate mass ratio of 0.5-1.5: Add 100g of yeast extract powder; and inoculate with *Lactobacillus plantarum*, with a viable count of 10^6-10^7 CFU / mL, ferment at 28-32℃ for 12-24h until pH 3.5-3.8; then inoculate with *Kluyveromyces martensii*, with a viable count of 10^6-10^7 CFU / mL, ferment at 20-25℃ with microaerobic fermentation for 24-48h, inactivate at 85℃ for 15s, and concentrate under reduced pressure to a solid content of 20-30% to obtain the esterification precursor compensating solution.

[0039] In step (1), the mass ratio of mango, pineapple, and passion fruit is 3-5:2-3:1-2, and the mass ratio of the pulp to water after pulping is 1:0.3-0.6; the medium-temperature α-amylase is added at 10-20 U / g based on the mass of the pulp, and enzymatic hydrolysis is performed at 60-65℃ for 30-45 min; the compound enzyme includes pectinase, cellulase, and hemicellulase, and the mass ratio of pectinase, cellulase, and hemicellulase is 3-5:1-2:1; the compound enzyme is added at 80-150 U / g based on the mass of the pulp, and enzymatic hydrolysis is performed at 45-50℃ for 90-150 min, followed by enzyme inactivation at 90℃ for 15 s; the sugar content is adjusted to 40-70 g / L with sucrose. In step (2), the enzymatically hydrolyzed juice is inoculated with *Lactobacillus plantarum*, *Lactobacillus paracasei*, and *Leuconostoc mesenteroides* at 25-28℃ and fermented for 12-18 hours until pH 3.8-4.0. The temperature is then lowered to 16-20℃, and a co-acclimated ester-producing bacteria agent is inoculated. A pectin oligosaccharide fermentation broth is added to the fermentation broth in 2-3 additions, with a cumulative addition amount of 8-15% of the enzymatically hydrolyzed juice mass. Fermentation continues for 24-36 hours until the ethanol content of the fermentation broth does not exceed 0.5% vol. The temperature is then lowered to 8-12℃ and matured for 48-72 hours to obtain the fermentation broth. The viable count of *Lactobacillus plantarum*, *Lactobacillus paracasei*, *Leuconostoc mesenteroides*, and the co-acclimated ester-producing bacteria agent is 10^6-10^7 CFU / mL. In step (3), the tail gas at the top of the fermenter is condensed at 0-4℃ and the condensate is collected; the amount of condensate added back is 1-3% of the mass of the fermentation liquid obtained in step (2); the mass ratio of the added fermentation liquid to the esterification precursor compensation liquid is 10-20:1; the condensate is measured according to the actual collection amount. If the collection amount is higher than 3% of the mass of the fermentation liquid obtained in step (2), it is added back at 3%; if it is lower than 1%, all the collected condensate is added back.

[0040] In step (4), the sugar-acid ratio is adjusted to 25-40:1 by using sucrose or glucose and citric acid to compensate the fermentation broth. The sugar-acid ratio is the ratio of the mass fraction of soluble solids to the mass fraction of total acid, and the total acid is calculated as citric acid. The amount of composite wall sustained-release microcapsules added is 0.3-0.8% of the mass of the compensated fermentation broth. Before carbon dioxide is charged, the compensated fermentation broth is cooled to 2-6℃, and the amount of carbon dioxide charged is 4.5-6.0 g / L.

[0041] The co-domesticated ester-producing bacterial agent is obtained by mixed subculturing of Kluyveromyces martensii and Saccharomyces cerevisiae. The specific culture method is as follows: Kluyveromyces martensii and Saccharomyces cerevisiae are mixed at a live cell ratio of 1-2:1 and inoculated into a culture medium composed of pineapple juice and passion fruit peel extract at a volume ratio of 2-4:1. The reducing sugar content of the culture medium is 60-80 g / L. The culture is subcultured at 22-26℃ for 3-5 generations, with each generation lasting 24 hours. The cells are collected by centrifugation after the last generation and resuspended in pineapple juice to a live cell count of 10^8-10^9 CFU / mL.

[0042] The preparation method of the passion fruit peel extract is as follows: take passion fruit peel, add water at a peel to water mass ratio of 1:5-8, extract at 60-70℃ for 60-90 minutes, filter and collect the filtrate to obtain the passion fruit peel extract. The preparation method of the pineapple juice is as follows: peel the pineapple, pulp it, press it, filter it, and sterilize the filtrate to obtain the pineapple juice.

[0043] In this invention, the white pericarp of passion fruit is broken down by cellulase and pectinase, releasing glycosides from the cells and concentrating them to obtain a glycoside-type flavor precursor concentrate. Using the residual pectin and pectin degradation intermediates in the filtrate as substrates, a pericarp oligosaccharide fermentation broth is obtained. One portion of pericarp raw material simultaneously provides aroma reserves and fermentation nutrients. Yeast cells are induced to autolyze by salt and citric acid, releasing mannose proteins from the cell walls into the liquid phase. After alcohol precipitation and enrichment, the broth is purified by ultrafiltration desalting. Leuconostoc mesenteroides ferments sucrose and is concentrated by alcohol precipitation to obtain a fermentation source dextran concentrate. Pericarp, microbial cells, and sugar sources are utilized layer by layer. All enzyme preparations and strains are food-grade. The preparation process only involves enzymatic hydrolysis, fermentation, and physical compounding, without chemical modification, ensuring both safety and industrial feasibility.

[0044] The present invention will be described in detail below through examples and comparative examples, but the scope of protection of the present invention is not limited to these examples. Unless otherwise specified, the chemical reagents and raw materials used in the following examples and comparative examples are all conventional commercially available products.

[0045] Example 1 Preparation of glycoside-type flavor precursor concentrate: Take passion fruit peel, remove the outer hard shell and take the white peel layer, crush it, add water at a peel to water ratio of 1:8, add a compound enzyme composed of cellulase and pectinase at a mass ratio of 1:1.5, add 30 U / g of enzyme based on the peel weight, enzymatically hydrolyze at 50℃ for 75 min, inactivate the enzyme at 90℃ for 15 s, filter, and reserve the filtrate. Take a portion of the filtrate and concentrate it under reduced pressure at 50℃ and a vacuum degree of 0.09 MPa until the solid content is 30%, which yields the glycoside-type flavor precursor concentrate, whose main components are glycosides with linalool, geraniol, and β-ionone as aglycones. Preparation of pericarp oligosaccharide fermentation broth: Take the above enzyme-inactivated and filtered filtrate, add 15 U / g of pectinase based on the filtrate weight, enzymatically hydrolyze a second time at 45℃ for 90 min, inactivate the enzyme, and concentrate until the solid content is 12%, which yields the pericarp oligosaccharide fermentation broth.

[0046] Preparation of yeast-derived mannoprotein concentrate: Kluyveromyces martensii was inoculated into pineapple juice, which was obtained by peeling, pulping, pressing, and filtering pineapple. The juice was cultured aerobically at 25°C for 36 hours, and the cells were collected by centrifugation. The cells were washed twice with sterile physiological saline and resuspended at a cell-to-water ratio of 1:8. 2% salt by cell mass was added, and the pH was adjusted to 5.5 with citric acid. The cells were autolyzed at 48°C for 12 hours, and the supernatant was collected by centrifugation. 95% edible ethanol was added to bring the final ethanol concentration in the system to 70% by volume. The mixture was allowed to stand at 4°C for 8 hours, and the precipitate was collected by centrifugation. The precipitate was redissolved in water, desalted by ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and concentrated to a solid content of 20%. The resulting yeast-derived mannoprotein concentrate is mainly composed of mannoprotein with a weight average molecular weight of 20-200 kDa. Preparation of aroma-stabilizing complex: Glycoside-type flavor precursor concentrate and yeast-derived mannoprotein concentrate were mixed at a mass ratio of 2:1 and homogenized twice under high pressure at 30 MPa to obtain aroma-stabilizing complex.

[0047] Preparation of esterification precursor compensation solution: Take the concentrated glycoside-type flavor precursor, add 20U β-glucosidase per gram, hydrolyze at 35℃ for 2.5h, raise the temperature to 90℃ and hold for 10min to inactivate the enzyme, cool to obtain the precursor hydrolysate, add glucose to the precursor hydrolysate to adjust the reducing sugar to 80g / L, add yeast extract at a mass ratio of yeast extract to precursor hydrolysate of 1.5:100, inoculate with *Lactobacillus plantarum*, viable count 10^7 CFU / mL, ferment at 30℃ for 12h to pH 3.6, then inoculate with *Kluyveromyces martensii*, viable count 10^6 CFU / mL, microaerobic fermentation at 25℃ for 36h. Microaerobic fermentation is performed by sealing the fermentation vessel, maintaining headspace above the liquid surface, and introducing sterile air once every 12h at a rate of 0.08L per liter of fermentation liquid. Inactivate at 85℃ for 15s, concentrate under reduced pressure to a solid content of 20%, and obtain the esterification precursor compensation solution. Preparation of co-domesticated ester-producing bacteria: Kluyveromyces martensii and Saccharomyces cerevisiae were mixed at a live cell ratio of 2:1 and inoculated into a culture medium composed of pineapple juice and passion fruit peel extract at a volume ratio of 3:1. The passion fruit peel extract was prepared by adding water to passion fruit peel at a mass ratio of 1:6 (peel to water), extracting at 65℃ for 75 min, and filtering. The reducing sugar content of the culture medium was 60 g / L. The culture was subcultured at 26℃ for 4 generations, with each generation lasting 24 h. The cells were collected by centrifugation after the last generation and resuspended in pineapple juice to a live cell count of 10^8 CFU / mL.

[0048] Preparation of fermentation-derived dextran concentrate: Leuconostoc mesenteroides was inoculated into a 12% sucrose solution with a viable count of 10^7 CFU / mL, fermented at 30℃ for 24 h, inactivated at 85℃ for 15 s, and 95% edible ethanol was added until the final ethanol concentration was 70%. The mixture was allowed to stand for alcohol precipitation, centrifuged to collect the precipitate, redissolved in water, and concentrated to a dextran content of 12 g / L to obtain the fermentation-derived dextran concentrate.

[0049] Preparation of composite wall sustained-release microcapsules: Take the aroma-stabilized complex, add 30U of β-glucosidase per gram and mix well. Add sodium alginate and fermentation-derived dextran concentrate, with a mass ratio of sodium alginate to aroma-stabilized complex of 4:100 and a mass ratio of fermentation-derived dextran concentrate to sodium alginate of 1:3. After stirring, adjust the viscosity of the mixture to 150mPa·s and drop it into a calcium lactate aqueous solution, with a mass ratio of calcium lactate to water of 5:100. Solidify for 30min, filter out, and then immerse in a chitosan coating solution for 30min. The chitosan coating solution is prepared by adding chitosan to a 1% (v / v) acetic acid solution and stirring at room temperature for 30min until dissolved, with a mass ratio of chitosan to 1% (v / v) acetic acid solution of 0.5:100. Wash with water to obtain composite wall sustained-release microcapsules with a particle size of 1mm.

[0050] Main process: Peel and seed mango, pineapple, and passion fruit, then pulp them in a mass ratio of 8:5:3. Mix the pulp with water in a mass ratio of 1:0.3. Add 20 U / g of medium-temperature α-amylase based on the mass of the pulp, and enzymatically hydrolyze at 62℃ for 30 min. Cool down to 45℃, and add a compound enzyme consisting of pectinase, cellulase, and hemicellulase in a mass ratio of 5:2:1. Add 115 U / g of enzyme based on the mass of the pulp, and enzymatically hydrolyze for 150 min. Inactivate the enzyme at 90℃ for 15 s, centrifuge to obtain the clear juice, adjust the sugar content to 55 g / L with sucrose, sterilize, and obtain the enzymatically hydrolyzed clear juice. The enzymatically hydrolyzed juice was inoculated at 25°C with *Lactobacillus plantarum*, *Lactobacillus paracasei*, and *Leuconostoc mesenteroides*, with viable cell counts of 10^7 CFU / mL, 8 × 10^6 CFU / mL, and 6 × 10^6 CFU / mL, respectively. Fermentation was carried out for 18 hours until the pH reached 3.9. The temperature was then lowered to 16°C, and a co-acclimation ester-producing bacteria agent was inoculated with a viable cell count of 5 × 10^6 CFU / mL. A fruit peel oligosaccharide fermentation broth was added to the fermentation broth in three separate additions, with a cumulative addition amount of 12% of the enzymatically hydrolyzed juice mass. Fermentation was continued for 24 hours until the ethanol content of the fermentation broth reached 0.32% vol. The temperature was then lowered to 12°C, and the broth was allowed to mature for 60 hours to obtain the fermentation broth. The fermentation process is entirely closed. The exhaust gas from the top of the fermenter is condensed at 4°C, and the condensate is collected. After fermentation, 2% of the condensate (by weight of the fermentation liquid obtained in the previous steps) is added back to the fermentation liquid to obtain the back-fermented fermentation liquid. Then, the esterification precursor compensation liquid is mixed into the back-fermented fermentation liquid at a mass ratio of 10:1 to obtain the compensated fermentation liquid. The sugar-acid ratio of the compensated fermentation liquid is adjusted to 40:1 using sucrose and citric acid. 0.5% of the composite wall sustained-release microcapsules are suspended in a small amount of sterile water and aseptically added to the compensated fermentation liquid. The mixture is cooled to 2°C, charged with carbon dioxide to 6.0 g / L, and aseptically filled to obtain a tropical-flavored carbonated beverage.

[0051] Example 2 In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows: Preparation of glycoside-type flavor precursor concentrate: Take passion fruit peel, remove the outer hard shell and take the white peel layer, crush it, add water at a peel-to-water ratio of 1:5, add a compound enzyme composed of cellulase and pectinase at a mass ratio of 1:2, add 40 U / g of enzyme based on the peel weight, enzymatically hydrolyze at 45℃ for 90 min, inactivate the enzyme at 90℃ for 15 s, filter, and reserve the filtrate. Take a portion of the filtrate and concentrate it under reduced pressure at 55℃ and a vacuum degree of 0.08 MPa until the solid content is 20%, thus obtaining the glycoside-type flavor precursor concentrate, whose main components are glycosides with linalool, geraniol, and β-ionone as aglycones. Preparation of pericarp oligosaccharide fermentation broth: Take the above enzyme-inactivated and filtered filtrate, add 20 U / g of pectinase based on the filtrate weight, enzymatically hydrolyze a second time at 48℃ for 60 min, inactivate the enzyme, and concentrate until the solid content is 15%, thus obtaining the pericarp oligosaccharide fermentation broth.

[0052] Preparation of yeast-derived mannoprotein concentrate: Kluyveromyces martensii was inoculated into pineapple juice, which was obtained by peeling, pulping, pressing, and filtering pineapple. The juice was cultured aerobically at 26°C for 24 hours, and the cells were collected by centrifugation. The cells were washed once with sterile physiological saline and resuspended at a cell-to-water ratio of 1:10. 3% salt by cell mass was added, and the pH was adjusted to 4.5 with citric acid. The cells were autolyzed at 50°C for 18 hours, and the supernatant was collected by centrifugation. 95% edible ethanol was added to bring the final ethanol concentration in the system to 60% by volume. The mixture was allowed to stand at 6°C for 10 hours, and the precipitate was collected by centrifugation. The precipitate was redissolved in water, desalted by ultrafiltration membrane with a molecular weight cutoff of 8 kDa, and concentrated to a solids content of 25%. The resulting yeast-derived mannoprotein concentrate is mainly composed of mannoprotein with a weight average molecular weight of 20-200 kDa. Preparation of aroma-stabilizing complex: Glycoside-type flavor precursor concentrate and yeast-derived mannoprotein concentrate were mixed at a mass ratio of 3:1 and homogenized under high pressure at 20 MPa for 3 passes to obtain aroma-stabilizing complex.

[0053] Preparation of esterification precursor compensation solution: Take the concentrated glycoside-type flavor precursor solution, add 30U β-glucosidase per gram, enzymatically hydrolyze at 30℃ for 3h, heat to 90℃ and hold for 10min to inactivate the enzyme, cool to obtain precursor hydrolysate, add glucose to the precursor hydrolysate to adjust the reducing sugar to 100g / L, add yeast extract at a mass ratio of yeast extract to precursor hydrolysate of 0.5:100, inoculate with Lactobacillus plantarum, viable count 10^6 CFU / mL, ferment at 32℃ for 18h to pH 3.8, then inoculate with Kluyveromyces martensii, viable count 10^7 CFU / mL, microaericidal fermentation at 20℃ for 48h, inactivate at 85℃ for 15s, concentrate under reduced pressure to a solid content of 25%, and obtain esterification precursor compensation solution. Preparation of co-domesticated ester-producing bacteria: Kluyveromyces martensii and Saccharomyces cerevisiae were mixed at a live cell ratio of 1:1 and inoculated into a culture medium composed of pineapple juice and passion fruit peel extract at a volume ratio of 4:1. The passion fruit peel extract was prepared by adding water to passion fruit peel at a mass ratio of 1:5 (peel to water), extracting at 70℃ for 60 min, and filtering. The reducing sugar content of the culture medium was 70 g / L. The culture was subcultured at 22℃ for 5 generations, with each generation lasting 24 h. The cells were collected by centrifugation after the last generation and resuspended in pineapple juice to a live cell count of 5 × 10^8 CFU / mL.

[0054] Preparation of fermentation-derived dextran concentrate: Leuconostoc mesenteroides was inoculated into an 8% sucrose solution with a viable count of 10^6 CFU / mL, fermented at 30℃ for 36 h, inactivated, and then inactivated again at 85℃ for 15 s. 95% edible ethanol was added until the final ethanol concentration was 60%, and the mixture was allowed to stand for alcohol precipitation. The precipitate was collected by centrifugation, redissolved in water, and concentrated to a dextran content of 15 g / L to obtain the fermentation-derived dextran concentrate.

[0055] Preparation of composite wall sustained-release microcapsules: Take the aroma-stabilized complex, add 40U of β-glucosidase per gram and mix well. Add sodium alginate and fermentation-derived dextran concentrate, with a mass ratio of sodium alginate to aroma-stabilized complex of 2:100 and a mass ratio of fermentation-derived dextran concentrate to sodium alginate of 1:4. After stirring, adjust the viscosity of the mixture to 280mPa·s and drop it into a calcium lactate aqueous solution, with a mass ratio of calcium lactate to water of 2:100. Solidify for 40min, filter out, and then immerse in a chitosan coating solution for 15min, with a mass ratio of chitosan to 1% acetic acid solution of 1:100. Wash with water to obtain composite wall sustained-release microcapsules with a particle size of 1.2mm.

[0056] Main process: Peel and seed mango, pineapple, and passion fruit, then pulp them in a mass ratio of 5:3:2. Mix the pulp with water in a mass ratio of 1:0.45. Add 10 U / g of medium-temperature α-amylase based on the mass of the pulp, and hydrolyze at 65℃ for 38 min. Cool to 48℃, then add a compound enzyme consisting of pectinase, cellulase, and hemicellulase in a mass ratio of 3:1:1. Add 150 U / g of enzyme based on the mass of the pulp, and hydrolyze for 90 min. Inactivate the enzyme at 90℃ for 15 s, centrifuge to obtain the clear juice, adjust the sugar content to 70 g / L with sucrose, and sterilize to obtain the enzymatically hydrolyzed clear juice. The enzymatically hydrolyzed juice was inoculated at 26℃ with *Lactobacillus plantarum*, *Lactobacillus paracasei*, and *Leuconostoc mesenteroides*, with viable cell counts of 5×10^6 CFU / mL, 10^6 CFU / mL, and 10^7 CFU / mL, respectively. Fermentation was carried out for 12 hours until pH 4.0 was reached. The temperature was then lowered to 18℃, and co-acclimated ester-producing bacteria were inoculated with a viable cell count of 8×10^6 CFU / mL. A fruit peel oligosaccharide fermentation broth was added to the fermentation broth in two separate additions, with a cumulative addition amount of 15% of the enzymatically hydrolyzed juice mass. Fermentation was carried out for 30 hours until the ethanol content of the fermentation broth reached 0.26% vol. The temperature was then lowered to 8℃, and the broth was allowed to mature for 72 hours to obtain the fermentation broth. The fermentation process is entirely closed. The exhaust gas from the top of the fermenter is condensed at 0°C, and the condensate is collected. After fermentation, 3% of the condensate (by weight of the fermentation liquid obtained in the previous steps) is added back to the fermentation liquid to obtain the back-fermented fermentation liquid. Then, the esterification precursor compensation liquid is mixed into the back-fermented fermentation liquid at a mass ratio of 15:1 to obtain the compensated fermentation liquid. The sugar-acid ratio of the compensated fermentation liquid is adjusted to 25:1 using glucose and citric acid. 0.8% of the composite wall sustained-release microcapsules are suspended in a small amount of sterile water and aseptically added to the compensated fermentation liquid. The mixture is cooled to 4°C, charged with carbon dioxide to 4.5 g / L, and aseptically filled to obtain a tropical-flavored carbonated beverage.

[0057] Example 3 In this embodiment, the similarities to those in Embodiment 1 will not be repeated, and the differences are as follows: Preparation of glycoside-type flavor precursor concentrate: Take passion fruit peel, remove the outer hard shell and take the white peel layer, crush it, add water at a peel-to-water ratio of 1:6.5, add a compound enzyme composed of cellulase and pectinase at a mass ratio of 1:1, add 50 U / g of enzyme based on the peel weight, enzymatically hydrolyze at 48℃ for 60 min, inactivate the enzyme at 90℃ for 15 s, filter, and reserve the filtrate. Take a portion of the filtrate and concentrate it under reduced pressure at 60℃ and a vacuum degree of 0.09 MPa until the solid content is 25%, thus obtaining the glycoside-type flavor precursor concentrate, whose main components are glycosides with linalool, geraniol, and β-ionone as aglycones. Preparation of pericarp oligosaccharide fermentation broth: Take the above enzyme-inactivated and filtered filtrate, add 10 U / g of pectinase based on the filtrate weight, enzymatically hydrolyze a second time at 50℃ for 75 min, inactivate the enzyme, and concentrate until the solid content is 10%, thus obtaining the pericarp oligosaccharide fermentation broth.

[0058] Preparation of yeast-derived mannoprotein concentrate: Kluyveromyces martensii was inoculated into pineapple juice, which was obtained by peeling, pulping, pressing, and filtering pineapple. The juice was cultured aerobically at 28°C for 30 h, and the cells were collected by centrifugation. The cells were washed twice with sterile physiological saline and resuspended at a cell-to-water ratio of 1:5. 4% salt by cell mass was added, and the pH was adjusted to 5.0 with citric acid. The cells were autolyzed at 45°C for 24 h, and the supernatant was collected by centrifugation. 95% edible ethanol was added to bring the final ethanol concentration in the system to 65% by volume. The mixture was allowed to stand at 2°C for 12 h, and the precipitate was collected by centrifugation. The precipitate was redissolved in water, desalted by ultrafiltration membrane with a molecular weight cutoff of 9 kDa, and concentrated to a solids content of 15%. The resulting yeast-derived mannoprotein concentrate is mainly composed of mannoprotein with a weight average molecular weight of 20-200 kDa. Preparation of aroma-stabilizing complex: Glycoside-type flavor precursor concentrate and yeast-derived mannoprotein concentrate were mixed at a mass ratio of 4:1 and homogenized twice under high pressure at 25 MPa to obtain aroma-stabilizing complex.

[0059] Preparation of esterification precursor compensation solution: Take the concentrated glycoside-type flavor precursor solution, add 40U β-glucosidase per gram, enzymatically hydrolyze at 32℃ for 2h, heat to 90℃ and hold for 10min to inactivate the enzyme, cool to obtain precursor hydrolysate, add glucose to the precursor hydrolysate to adjust the reducing sugar to 120g / L, add yeast extract at a mass ratio of yeast extract to precursor hydrolysate of 1:100, inoculate with *Lactobacillus plantarum*, viable count 5×10^6 CFU / mL, ferment at 28℃ for 24h to pH 3.5, then inoculate with *Kluyveromyces martensii*, viable count 5×10^6 CFU / mL, microaericidal fermentation at 22℃ for 24h, inactivate at 85℃ for 15s, concentrate under reduced pressure to 30% solids content to obtain esterification precursor compensation solution. Preparation of co-domesticated ester-producing bacteria: Kluyveromyces martensii and Saccharomyces cerevisiae were mixed at a live cell ratio of 1.5:1 and inoculated into a culture medium composed of pineapple juice and passion fruit peel extract at a volume ratio of 2:1. The passion fruit peel extract was prepared by adding water to passion fruit peel at a mass ratio of peel to water of 1:8, extracting at 60℃ for 90 min, and filtering. The reducing sugar content of the culture medium was 80 g / L. The culture was subcultured at 24℃ for 3 generations, with each generation lasting 24 h. The cells of the last generation were collected by centrifugation and resuspended in pineapple juice to a live cell count of 10^9 CFU / mL. Preparation of fermentation-derived dextran concentrate: Leuconostoc mesenteroides was inoculated into a 10% sucrose solution with a viable count of 5 × 10^6 CFU / mL. Fermentation was carried out at 25℃ for 48 h, followed by inactivation at 85℃ for 15 s. 95% edible ethanol was added until the final ethanol concentration was 65%. The mixture was allowed to stand for alcohol precipitation, centrifuged to collect the precipitate, redissolved in water, and concentrated to a dextran content of 8 g / L to obtain the fermentation-derived dextran concentrate.

[0060] Preparation of composite wall sustained-release microcapsules: Take the aroma-stabilized complex, add 50U of β-glucosidase per gram and mix well. Add sodium alginate and fermentation-derived dextran concentrate, with a mass ratio of sodium alginate to aroma-stabilized complex of 3:100 and a mass ratio of fermentation-derived dextran concentrate to sodium alginate of 1:2. After stirring, adjust the viscosity of the mixture to 400 mPa·s and drop it into a calcium lactate aqueous solution, with a mass ratio of calcium lactate to water of 3.5:100. Solidify for 20 min, filter out, and then immerse in a chitosan coating solution for 22 min, with a mass ratio of chitosan to 1% acetic acid solution of 1.5:100. Wash with water to obtain composite wall sustained-release microcapsules with a particle size of 1.5 mm.

[0061] Main process: Peel and seed mango, pineapple, and passion fruit, then pulp them in a mass ratio of 3:2:1. Mix the pulp with water in a mass ratio of 1:0.6. Add 15 U / g of medium-temperature α-amylase based on the mass of the pulp, and hydrolyze at 60℃ for 45 min. Cool to 50℃, then add a compound enzyme consisting of pectinase, cellulase, and hemicellulase in a mass ratio of 8:3:2. Add 80 U / g of enzyme based on the mass of the pulp, and hydrolyze for 120 min. Inactivate the enzyme at 90℃ for 15 s, centrifuge to obtain the clear juice, adjust the sugar content to 40 g / L with sucrose, and sterilize to obtain the enzymatically hydrolyzed clear juice. The enzymatically hydrolyzed juice was inoculated at 28℃ with *Lactobacillus plantarum*, *Lactobacillus paracasei*, and *Leuconostoc mesenteroides*, with viable cell counts of 8×10^6 CFU / mL, 5×10^6 CFU / mL, and 10^6 CFU / mL, respectively. Fermentation was carried out for 15 h until the pH reached 3.8. The temperature was then lowered to 20℃, and a co-acclimated ester-producing bacteria agent was inoculated with a viable cell count of 10^7 CFU / mL. A fruit peel oligosaccharide fermentation broth was added to the fermentation broth in two separate additions, with a cumulative addition amount of 8% of the enzymatically hydrolyzed juice mass. Fermentation was carried out for 36 h until the ethanol content of the fermentation broth reached 0.35% vol. The temperature was then lowered to 10℃ and matured for 48 h to obtain the fermentation broth. The fermentation process is entirely closed. The exhaust gas from the top of the fermenter is condensed at 2°C, and the condensate is collected. After fermentation, 1% of the condensate (by weight of the fermentation liquid obtained in the previous steps) is added back to the fermentation liquid to obtain the back-fermented liquid. Then, the esterification precursor compensation liquid is mixed into the back-fermented liquid at a mass ratio of 20:1 to obtain the compensated fermentation liquid. The sugar-acid ratio of the compensated fermentation liquid is adjusted to 32:1 using sucrose and citric acid. 0.3% of the composite wall sustained-release microcapsules are suspended in a small amount of sterile water and aseptically added to the compensated fermentation liquid. The mixture is cooled to 6°C, charged with carbon dioxide to 5.2 g / L, and aseptically filled to obtain a tropical-flavored carbonated beverage.

[0062] Comparative Example 1 The process is basically the same as in Example 1, except that no fruit peel oligosaccharide fermentation solution is added to the fermentation broth. The rest of the process is the same as in Example 1. At the end of fermentation, the ethanol content of the fermentation broth is 0.52% vol.

[0063] Comparative Example 2 The process is basically the same as in Example 2, except that instead of inoculating with co-domesticated ester-producing bacteria, it is inoculated with a mixed culture of Kluyveromyces martensii and Saccharomyces cerevisiae with equal viable cell counts that has not been co-domesticated. The rest of the process is the same as in Example 2. At the end of fermentation, the ethanol content of the fermentation broth is 0.44% vol.

[0064] Comparative Example 3 The process is basically the same as in Example 1, except that the condensate is not added back to the fermentation broth after fermentation, and the esterification precursor compensation solution is not mixed in. The rest of the process is the same as in Example 1.

[0065] Comparative Example 4 The process is basically the same as in Example 3, except that no composite wall sustained-release microcapsules are added after preparation. The rest of the process is the same as in Example 3.

[0066] Comparative Example 5 The process is basically the same as in Example 2, except that the core material of the composite wall sustained-release microcapsule is a glycoside-type flavor precursor concentrate and β-glucosidase, that is, the glycoside-type flavor precursor concentrate is used instead of the aroma-stabilizing complex. The rest of the process is the same as in Example 2.

[0067] Comparative Example 6 The process is basically the same as in Example 1, except that Leuconostoc mesenteroides is not inoculated during fermentation, and an equal amount of water is used instead of the concentrated dextran fermentation source in the preparation of the composite wall sustained-release microcapsules. The rest of the process is the same as in Example 1, and the ethanol content of the fermentation broth is 0.49% vol at the end of fermentation.

[0068] Performance Test Results and Analysis The finished beverages prepared in each embodiment and comparative example were used as test samples. After bottling, all samples were placed under the same conditions for 24 hours to allow the system to equilibrate, and then the following measurements were performed. Each sample was measured in triplicate, and the average value was taken. The test results are detailed in Table 1. The specific test methods are as follows: Determination of total volatile aroma components: Take 8 mL of the finished beverage and place it in a 20 mL headspace vial. Add 2 g of sodium chloride, tighten the cap, and place it in a 50℃ water bath for equilibration for 20 min. Insert the aged divinylbenzene / carbon molecular sieve / polydimethylsiloxane composite extraction head into the headspace vial and perform headspace adsorption at 50℃ for 40 min. Immediately after removal, insert it into the gas chromatograph injection port and desorb at 250℃ for 5 min. Perform gas chromatography-mass spectrometry analysis using a DB-WAX capillary column. The column temperature is programmed, starting at 40℃ and holding for 3 min, then increasing to 230℃ at 5℃ / min and holding for 10 min. The carrier gas is high-purity helium. The mass spectrometer ion source is an electron impact source with an electron energy of 70 eV and a scan range of 35-350 amu. The detected components are qualitatively identified by spectral library search. The content of each component is calculated using the corresponding external standard curve. The sum of the contents of each component is the total volatile aroma components. Immediately after sampling, a total aroma component was measured to determine the total aroma component of the new product. Each sample was then divided into 500mL polyester bottles, with the top space not exceeding 5% of the bottle's internal volume. After sealing, the bottles were stored at 25°C in the dark for 90 days. After the storage period, the total aroma component was measured again under the same conditions. The ratio of the total aroma component after storage to the total aroma component of the new product, expressed as a percentage, is the aroma retention rate.

[0069] Centrifugation sedimentation rate determination: Take 50 mL of the shaken finished beverage, accurately measure it and inject it into a 50 mL stoppered graduated centrifuge tube, centrifuge at 3000 r / min for 15 min, take it out and let it stand for 2 min, read the volume corresponding to the sedimentation layer at the bottom of the centrifuge tube, and the ratio of the sedimentation volume to the sample volume of 50 mL, expressed as a percentage, is the centrifugation sedimentation rate.

[0070] Foam half-life determination: Place the finished beverage in a 4℃ refrigerator for more than 4 hours. Take 200mL of sample and inject it rapidly along the inner wall of a 500mL graduated cylinder within 5 seconds. Start timing immediately after injection and read the initial height of the foam layer. Observe and record the time required for the height of the foam layer to decay to half of the initial height, in seconds. This is the foam half-life.

[0071] As shown in Table 1, the total aroma components in all three examples were between 452 and 486 μg / L, and all comparative examples were lower than the 452 μg / L in Example 2. Comparative Example 1, without the addition of pericarp oligosaccharide fermentation broth, had a total aroma component concentration of 358 μg / L. This indicates that the oligosaccharides in the pericarp oligosaccharide fermentation broth, acting as a selective carbon source for lactic acid bacteria, supported the proliferation of lactic acid bacteria and the continuous expression of β-glucosidase in the second stage of low-temperature fermentation. Without this supply, the release of glycosidic aroma compounds in the enzymatically hydrolyzed liquid decreased, resulting in a reduction in yeast-esterilizable substrates and insufficient aroma accumulation at the fermentation endpoint. Simultaneously, the ethanol content in Comparative Example 1 reached 0.52% vol, exceeding the 0.5% vol limit. This is because the lactic acid bacteria utilized less sugar, leaving more residual sugar for yeast to undergo alcoholic fermentation. This, conversely, confirms that fermentation promotion and alcohol limitation are two results on the same chain. Comparative Example 2 used un-co-acclimated mixed bacterial solution instead of co-acclimated ester-producing bacterial agent. The total aroma component was 381 μg / L, which was lower than 452 μg / L in Example 2. Un-acclimated yeast adapts slowly to high acid and high osmotic pressure environment, and the ester production period is delayed. The effective ester production time in the low temperature section is compressed, indicating that the co-acclimation treatment is not a formal change, but a practical means to ensure the efficiency of low temperature ester production.

[0072] From the perspective of production-side compensation, Comparative Example 3, which did not add back condensate or mix in esterification precursor compensation liquid, had a total aroma component of 326 μg / L, which was the lowest among all samples. Compared with Example 1's 486 μg / L, the difference was more than 30%. This difference is on the order of magnitude of the loss of volatile aroma during sterilization, blending, and carbonation. This indicates that the two pathways of adding back condensate and mixing back esterification precursor compensation liquid together constitute the compensation at the production end. Moreover, the flavor of the esterification precursor compensation liquid is the same as that of the main fermentation liquid, and its addition does not introduce the abruptness of added flavorings. From the perspective of shelf-storage compensation, the difference in aroma retention rate is more telling than the difference in the total aroma of newly manufactured products. The aroma retention rate of Examples 1-3 is 73-81%, while Comparative Example 4, without the addition of composite wall sustained-release microcapsules, has a retention rate of only 46%, showing the fastest decay. Comparative Example 3, lacking compensation liquid, has a retention rate of 52%. Comparative Example 5, whose core material does not contain yeast-derived mannoprotein concentrate, has a retention rate of 61%, falling between Examples 2 and Comparative Example 4. This gradient indicates that the slow hydrolysis of β-glucosidase within the capsule provides incremental release, and the non-covalent binding of mannoprotein to aroma molecules suppresses the release rate. The combination of these two factors matches the release rhythm with the natural decay rhythm of the aroma. Comparative Example 5, with only enzyme remaining and lacking the constraint of mannoprotein, experiences rapid release in the early stages but becomes unsustainable in the later stages, resulting in a lower retention rate than the Examples. Comparative Example 6 has an aroma retention rate of 66%, with an intact core material and sustained-release structure. However, the lack of fermentation-derived glucan concentrate in the wall material reduces the encapsulation strength of the composite wall, leading to accelerated leakage of substances within the capsule during the later stages of storage, and consequently, a decline in the retention rate.

[0073] In terms of stability and taste, the centrifugal sedimentation rate of the examples was 0.6-0.9%, and the foam half-life was 39-52 s. Comparative Example 6, without inoculation with *Leuconostoc mesenteroides* and without fermentation-sourced dextran concentrate in the wall material, saw its centrifugal sedimentation rate increase to 4.2% and its foam half-life decrease to 24 s. Comparative Example 6 simultaneously lacked both in-situ generated dextran and wall material dextran, resulting in dual instability of the fruit pulp particles and microcapsules. The aggregated particles also carried the liquid phase into the sedimentation layer, further amplifying the sedimentation volume. Therefore, the difference from the examples was greater than other single-variable comparative examples, directly demonstrating that the in-situ generated dextran by *Leuconostoc mesenteroides* is the main force maintaining the suspension of fruit pulp particles and foam retention. Its ethanol content of 0.49% vol was also higher than in Example 1, because the absence of *Leuconostoc mesenteroides* diverting sucrose allowed the yeast to obtain more fermentable sugars. The centrifugation sedimentation rate of Comparative Examples 1-5 was 1.1-1.5%, and the foam half-life was 32-38 s, both slightly inferior to the corresponding examples. The fermentation process of Comparative Examples 3, 4, and 5 was consistent with the corresponding examples, with small differences. This was mainly due to the slight stabilizing effect of the fermentation source polysaccharides and microcapsule wall material dextran introduced by the esterification precursor compensation liquid on the system. Comparative Example 1 showed relatively larger differences due to limited lactic acid bacteria proliferation, and Comparative Example 2 showed relatively larger differences due to yeast metabolic disorders. Overall, the examples were superior to the comparative examples in all four dimensions of total aroma, aroma retention rate, stability, and foam retention. Moreover, the shortcomings of each comparative example precisely corresponded to the functions of the removed technical features, indicating that the beneficial effects of this application are the result of the superposition of the effects of stepped fermentation, fermentation broth addition, in-situ compensation, and composite wall microcapsules, and not a single accidental result.

[0074] Table 1 Analysis of Test Results Example 1 486 78 0.7 48 Example 2 452 73 0.9 39 Example 3 469 81 0.6 52 Comparative Example 1 358 57 1.5 36 Comparative Example 2 381 60 1.3 32 Comparative Example 3 326 52 1.1 37 Comparative Example 4 437 46 1.2 38 Comparative Example 5 441 61 1.4 33 Comparative Example 6 445 66 4.2 24 The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing tropical-flavored carbonated beverages based on mixed-culture stepped fermentation and in-situ aroma compensation, characterized in that, The method includes the following steps: (1) After pulping mango, pineapple and passion fruit, add water and mix. First, hydrolyze with α-amylase at medium temperature, then hydrolyze with compound enzyme, inactivate the enzyme, centrifuge to obtain clear juice, adjust the sugar content, sterilize, and obtain enzymatically hydrolyzed clear juice. (2) Inoculate the enzymatically hydrolyzed juice with *Lactobacillus plantarum*, *Lactobacillus paracasei* and *Leuconostoc mesenteroides* for fermentation; cool down, inoculate with co-domesticated ester-producing bacteria, and add pectin oligosaccharide fermentation broth to the fermentation broth to continue fermentation; cool down, mature, and obtain fermentation broth; (3) The fermentation process in step (2) is completely sealed. The tail gas at the top of the fermentation tank is condensed and collected. After the fermentation is completed, the condensate is added back to the fermentation liquid obtained in step (2) to obtain the added fermentation liquid. Then, the esterification precursor compensation liquid is mixed into the added fermentation liquid to obtain the compensation fermentation liquid. (4) After adjusting the sugar-acid ratio of the compensating fermentation broth obtained in step (3), aseptically add composite wall sustained-release microcapsules to the compensating fermentation broth, fill with carbon dioxide, and aseptically fill; the composite wall sustained-release microcapsules use aroma-stabilizing complex and β-glucosidase as core materials and dextran-sodium alginate-chitosan composite wall as wall material; the aroma-stabilizing complex is obtained by mixing and homogenizing glycoside-type flavor precursor concentrate and yeast-derived mannoprotein concentrate.

2. The method for preparing a tropical-flavored carbonated beverage based on mixed-culture step fermentation and in-situ aroma compensation according to claim 1, characterized in that, The preparation method of the glycoside-type flavor precursor concentrate is as follows: Take passion fruit peel, remove the outer hard shell and take the white peel layer, crush it, add water at a peel to water mass ratio of 1:5-8, add a compound enzyme composed of cellulase and pectinase at a mass ratio of 1:1-2, add 30-50 U / g of enzyme based on the peel mass, enzymatically hydrolyze at 45-50℃ for 60-90 min, inactivate the enzyme at 90℃ for 15 s, filter, and concentrate the filtrate under reduced pressure at 50-60℃ and vacuum degree of 0.08-0.09 MPa until the solid content is 20-30%, thus obtaining the glycoside-type flavor precursor concentrate.

3. The method for preparing a tropical-flavored carbonated beverage based on mixed-culture stepped fermentation and in-situ aroma compensation according to claim 1, characterized in that, The preparation method of the yeast-derived mannoprotein concentrate is as follows: Kluyveromyces martensii is inoculated into pineapple juice and cultured aerobically at 25-28℃ for 24-36h. The cells are collected by centrifugation, washed 1-2 times with sterile physiological saline, resuspended at a cell-to-water ratio of 1:5-10, and 2-4% salt by cell mass is added. The pH is adjusted to 4.5-5.5 with citric acid, and the cells are autolyzed at 45-50℃ for 12-24h. The supernatant is collected by centrifugation, and 95% edible ethanol is added until the final ethanol concentration in the system is 60-70% by volume. The mixture is allowed to stand at 2-6℃ for 8-12h, and the precipitate is collected by centrifugation. The precipitate is redissolved in water, desalted by ultrafiltration membrane with a molecular weight cutoff of 8-10kDa, and concentrated to a solid content of 15-25%. The resulting product is the yeast-derived mannoprotein concentrate.

4. A method for preparing tropical-flavored carbonated beverages based on mixed-culture step fermentation and in-situ aroma compensation according to any one of claims 1-3, characterized in that, The preparation method of the composite wall sustained-release microcapsules is as follows: Take the aroma-stabilized complex, add 30-50 U of β-glucosidase per gram of aroma-stabilized complex and mix well, add sodium alginate and fermentation-derived dextran concentrate, wherein the mass ratio of sodium alginate to aroma-stabilized complex is 2-4:100, and the mass ratio of fermentation-derived dextran concentrate to sodium alginate is 1:2-4. After stirring well, adjust the viscosity of the mixture to 150-400 mPa·s, and drop it into a calcium lactate aqueous solution, wherein the mass ratio of calcium lactate to water in the calcium lactate aqueous solution is 2-5:

100. Solidify for 20-40 min, filter out, immerse in chitosan coating solution for 15-30 min, wash with water, and obtain the composite wall sustained-release microcapsules.

5. The method for preparing a tropical-flavored carbonated beverage based on mixed-culture stepped fermentation and in-situ aroma compensation according to claim 4, characterized in that, The mass ratio of glycoside-type flavor precursor concentrate to yeast-derived mannoprotein concentrate in the aroma-stabilizing complex is 2-4:1, the homogenization pressure is 20-30 MPa, and the homogenization is performed 2-3 times. The chitosan coating solution has a chitosan to 1% acetic acid solution mass ratio of 0.5-1.5:100, a chitosan deacetylation degree of not less than 85%, and a weight-average molecular weight of 50,000-200,000 Da. The preparation method of the fermentation source dextran concentrate is as follows: Leuconostoc mesenteroides is inoculated into a sucrose solution with a mass fraction of 8-12% and a viable count of 10^6-10^7 CFU / mL. Fermentation is carried out at 25-30℃ for 24-48 hours, and the bacteria are inactivated. Then, 95% edible ethanol is added until the final ethanol concentration is 60-70% by volume. The mixture is allowed to stand for alcohol precipitation, centrifuged to collect the precipitate, redissolved in water, and concentrated to a dextran content of 8-15 g / L to obtain the fermentation source dextran concentrate.

6. A method for preparing tropical-flavored carbonated beverages based on mixed-culture step fermentation and in-situ aroma compensation according to claim 1 or 2, characterized in that, The preparation method of the fruit peel oligosaccharide fermentation broth is as follows: Take the filtrate obtained after enzyme inactivation and filtration during the preparation of the glycoside-type flavor precursor concentrate, use the residual pectin and pectin degradation intermediates in the filtrate as substrates, add 10-20 U / g of pectinase according to the mass of the filtrate, perform a second enzymatic hydrolysis at 45-50℃ for 60-90 min, inactivate the enzyme, and concentrate to a solid content of 10-15%, which is the fruit peel oligosaccharide fermentation broth.

7. A method for preparing tropical-flavored carbonated beverages based on mixed-culture step fermentation and in-situ aroma compensation according to claim 1 or 2, characterized in that, The preparation method of the esterification precursor compensating solution is as follows: Take the concentrated glycoside-type flavor precursor solution, add 20-40 U of β-glucosidase per gram of concentrated glycoside-type flavor precursor solution, enzymatically hydrolyze at 30-35℃ for 2-3 hours, raise the temperature to 90℃ and hold for 10 minutes to inactivate the enzyme, and cool to obtain the precursor hydrolysate; add glucose to the precursor hydrolysate to adjust the reducing sugar to 80-120 g / L, and adjust the yeast extract to precursor hydrolysate mass ratio of 0.5-1.5: Add 100g of yeast extract powder; and inoculate with *Lactobacillus plantarum*, with a viable count of 10^6-10^7 CFU / mL, ferment at 28-32℃ for 12-24h until pH 3.5-3.8; then inoculate with *Kluyveromyces martensii*, with a viable count of 10^6-10^7 CFU / mL, ferment at 20-25℃ with microaerobic fermentation for 24-48h, inactivate at 85℃ for 15s, and concentrate under reduced pressure to a solid content of 20-30% to obtain the esterification precursor compensating solution.

8. The method for preparing a tropical-flavored carbonated beverage based on mixed-culture step fermentation and in-situ aroma compensation according to claim 1, characterized in that, In step (1), the mass ratio of mango, pineapple, and passion fruit is 3-5:2-3:1-2, and the mass ratio of the pulp to water after pulping is 1:0.3-0.6; the medium-temperature α-amylase is added at 10-20 U / g based on the mass of the pulp, and enzymatic hydrolysis is performed at 60-65℃ for 30-45 min; the compound enzyme includes pectinase, cellulase, and hemicellulase, and the mass ratio of pectinase, cellulase, and hemicellulase is 3-5:1-2:1; the compound enzyme is added at 80-150 U / g based on the mass of the pulp, and enzymatic hydrolysis is performed at 45-50℃ for 90-150 min, followed by enzyme inactivation at 90℃ for 15 s; the sugar content is adjusted to 40-70 g / L with sucrose; In step (2), the enzymatically hydrolyzed juice is inoculated with *Lactobacillus plantarum*, *Lactobacillus paracasei*, and *Leuconostoc mesenteroides* at 25-28℃ and fermented for 12-18 hours until pH 3.8-4.

0. The temperature is then lowered to 16-20℃, and a co-acclimated ester-producing bacteria agent is inoculated. A pectin oligosaccharide fermentation broth is added to the fermentation broth in 2-3 additions, with a cumulative addition amount of 8-15% of the enzymatically hydrolyzed juice mass. Fermentation continues for 24-36 hours until the ethanol content of the fermentation broth does not exceed 0.5% vol. The temperature is then lowered to 8-12℃ and matured for 48-72 hours to obtain the fermentation broth. The viable count of *Lactobacillus plantarum*, *Lactobacillus paracasei*, *Leuconostoc mesenteroides*, and the co-acclimated ester-producing bacteria agent is 10^6-10^7 CFU / mL. In step (3), the tail gas at the top of the fermenter is condensed at 0-4℃ and the condensate is collected; the amount of condensate added back is 1-3% of the mass of the fermentation liquid obtained in step (2); the mass ratio of the added fermentation liquid to the esterification precursor compensation liquid is 10-20:

1. In step (4), the sugar-acid ratio is adjusted to 25-40:1 by using sucrose or glucose and citric acid to compensate the fermentation broth. The sugar-acid ratio is the ratio of the mass fraction of soluble solids to the mass fraction of total acid, and the total acid is calculated as citric acid. The amount of composite wall sustained-release microcapsules added is 0.3-0.8% of the mass of the compensated fermentation broth. Before carbon dioxide is charged, the compensated fermentation broth is cooled to 2-6℃, and the amount of carbon dioxide charged is 4.5-6.0 g / L.

9. The method for preparing a tropical-flavored carbonated beverage based on mixed-culture step fermentation and in-situ aroma compensation according to claim 1, characterized in that, The co-domesticated ester-producing bacterial agent is obtained by mixed subculturing of Kluyveromyces martensii and Saccharomyces cerevisiae. The specific culture method is as follows: Kluyveromyces martensii and Saccharomyces cerevisiae are mixed at a live cell ratio of 1-2:1 and inoculated into a culture medium composed of pineapple juice and passion fruit peel extract at a volume ratio of 2-4:

1. The reducing sugar content of the culture medium is 60-80 g / L. The culture is subcultured at 22-26℃ for 3-5 generations, with each generation lasting 24 hours. The cells are collected by centrifugation after the last generation and resuspended in pineapple juice to a live cell count of 10^8-10^9 CFU / mL.

10. A method for preparing a tropical-flavored carbonated beverage based on mixed-culture stepped fermentation and in-situ aroma compensation according to claim 9, characterized in that, The preparation method of the passion fruit peel extract is as follows: take passion fruit peel, add water at a peel to water mass ratio of 1:5-8, extract at 60-70℃ for 60-90 minutes, filter and collect the filtrate to obtain the passion fruit peel extract.

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