Mango wine brewing method

Abstract

The invention relates to a mango wine brewing method, belongs to the technical field of fruit wine brewing processing, and aims to solve the technical problems that in traditional brewing, pectin cannot be continuously degraded due to rapid inactivation of pectinase in the initial stage of fermentation, and new turbid substances in the middle and later stages of fermentation are difficult to perceive in time and effectively remove. The method comprises the following steps: preparing slow-release compound enzyme microspheres, and continuously releasing pectinase in a fermentation process to realize primary clarification; monitoring the turbidity change of the fermentation liquor by using an online turbidity sensor, and automatically adding chitosan-based composite powder as a flocculating agent when the turbidity rises to a set amplitude; and then promoting aggregation and sedimentation of floccules through programmed temperature rise, and finally obtaining clarified wine liquid through low-temperature standing and separation. The method is mainly used for brewing production of mango wine, and the clarity, the stability and the aroma retention rate of wine liquid can be remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of fruit winemaking technology, specifically relating to a method for brewing mango wine. Background Technology

[0002] In the brewing process of mango wine, the treatment of pectin and the clarification of the wine are crucial steps affecting the quality and stability of the final product. Traditional brewing techniques typically add free pectinase before or at the beginning of fermentation to break down the pectin in the fruit pulp, reduce the viscosity of the mash, and increase the wine yield. However, in actual production, it has been found that mango fermentation mash is usually acidic and contains high levels of sugar. In such complex systems, the activity of free pectinase decreases rapidly, and its effective action time is much shorter than the entire fermentation cycle. Therefore, its degradation of the initial pectin in the raw materials is often incomplete. Pectin and its fragments that are not broken down in time persist during fermentation, becoming a potential matrix for the formation of complex turbidity later on. This is a significant reason for the early turbidity of the wine.

[0003] A further challenge lies in the fact that fermentation is not a static process. In the later stages of primary fermentation, active yeast metabolism and subsequent autolysis dynamically release new turbidities, such as mannoproteins and β-glucan, which are secondary turbidity sources. These substances interact with residual pectin from the earlier stages, forming a more stable and complex colloidal system. Traditional clarification processes often rely on experience, adding clarifying agents (such as gelatin, silica sol, or ordinary chitosan) at fixed points in time. This method lacks the ability to perceive the dynamic formation of turbidities in the fermentation broth in real time and cannot accurately capture the time window when secondary turbidity begins to form in large quantities. As a result, the timing of adding the clarifying agent is easily misaligned with the actual peak of turbidity formation: adding it too early results in insufficient target material, leading to wasted clarifying agent or even interference with fermentation; adding it too late, after the turbidity has formed a stable colloidal network structure, significantly reduces removal efficiency, making it difficult to guarantee clarification results.

[0004] Even when flocculants are added at the appropriate time, another common problem is the effectiveness of subsequent solid-liquid separation. In specific media such as mango wine, the flocs formed by simply adding flocculants and colloidal particles have a relatively loose structure and small particles. These flocs settle slowly and are difficult to completely remove during subsequent settling or centrifugation, which not only prolongs the production cycle but also causes the final product to become cloudy and lose stability during storage, affecting the product's shelf life and sensory quality.

[0005] In summary, several interrelated technical challenges in mango wine brewing are: how to match the action time of pectinase with the long fermentation cycle to achieve continuous enzymatic hydrolysis; how to accurately identify the timing when secondary turbidity begins to form in large quantities during the middle and late stages of fermentation and intervene accordingly; and how to make the formed flocs denser and easier to settle. Summary of the Invention

[0006] The purpose of this invention is to solve the following problems: Free pectinase added in the early stages of fermentation exhibits rapid activity decay in the complex acidic, sugar-containing system, failing to maintain its effect throughout the several-day fermentation cycle. This results in incomplete degradation of the initial pectin in the raw materials, leaving behind subsequent complex turbidity. In the middle and later stages of fermentation, yeast metabolism and autolysis dynamically generate new turbidity substances (secondary turbidity sources) such as mannoproteins and β-glucan. Traditional methods of adding these substances at fixed times cannot accurately detect this, leading to misaligned intervention and the formation of stable colloids from the secondary turbidity. In the fermentation broth, simply adding flocculants results in loose, fine flocs with slow settling speeds, making later separation difficult and leading to poor product storage stability.

[0007] To achieve the above objectives, the present invention provides a method for brewing mango wine with improved clarity, comprising the following steps: Step 1, Preparation of sustained-release complex enzyme microspheres: Pectinase is mixed with sodium alginate aqueous solution and added dropwise to calcium chloride solution to form gel microspheres with a diameter of 2-4 mm; the obtained gel microspheres are immersed in a 0.5%-1% konjac glucomannan solution for 10-20 minutes, then dried to a moisture content of 10%-15% to obtain sustained-release complex enzyme microspheres; wherein, the amount of pectinase added is 3000-5000 U per gram of sodium alginate. Step 2, Preparation of turbidity-responsive flocculant: Chitosan hydrochloride is dissolved in an acetic acid solution with a mass concentration of 0.5-1.5% to prepare a chitosan solution with a mass concentration of 1-3%; nano silica sol is added to the chitosan solution, the amount of nano silica sol added is 5%-10% of the mass of chitosan, and after stirring and mixing evenly, it is spray-dried to obtain chitosan-based composite powder; Step 3, Pre-fermentation treatment: Add the slow-release complex enzyme microspheres prepared in Step 1 to the pasteurized mango pulp with pH adjusted to 3.8-4.2. The addition amount is 10-20 grams of slow-release complex enzyme microspheres per liter of pulp. At the same time, inoculate with brewer's yeast at an inoculum size of 1×10⁻⁶. 6 -5×10 6 CFU / mL; Step 4, primary fermentation and first stage clarification: Primary fermentation is carried out at 18-22℃. During the fermentation process, the water in the pulp gradually penetrates into the slow-release complex enzyme microspheres, causing the konjac glucomannan layer to absorb water and swell. Pectinase begins to be slowly released and acts on the pectin substances in the pulp. Step 5, Second stage clarification: When the turbidity of the fermentation broth first shows an upward trend through the online turbidity sensor, and the increase exceeds 15%-25% of the initial turbidity, add the chitosan-based composite powder obtained in step 2 to the fermenter at a rate of 0.5-1.0 g / L of fermentation broth. Step 6, Third Stage Clarification: After adding chitosan-based composite powder, the fermentation system is subjected to programmed step heating and segmented heat preservation treatment, specifically including: first, raising the temperature from 18-22℃ to 24-26℃ and performing the first stage of heat preservation, then continuing to raise it to 27-28℃ and performing the second stage of heat preservation; the total duration of programmed heating and heat preservation is controlled within 3-6 hours; Step 7: After completing Step 6, restore the temperature of the fermentation system to 18-22℃ and continue fermentation until the sugar content stabilizes, thus ending the main fermentation. After letting all the fermentation liquid stand at 4-10℃ for 48-72 hours, separate the supernatant to obtain the clarified wine.

[0008] Further, in step 5, the online turbidity sensor collects the turbidity value of the fermentation broth once per minute and calculates its rate of change in real time. The criterion for the first appearance of an upward trend is: the average turbidity change rate of three consecutive collection cycles is greater than 0.5% per minute. After determining that an upward trend is met, chitosan-based composite powder is not added immediately, but monitoring continues until one of the following two conditions is met before addition is triggered: a) the cumulative increase in turbidity value from the starting point of the increase reaches 15%-25% of the initial turbidity; b) after the upward trend is confirmed, the cumulative time for the turbidity to continue to rise reaches 120-180 minutes. This solution addresses the specific technical problem of how to accurately and reliably identify the true turbidity rise signal representing the beginning of stable generation of secondary turbidity sources from complex background noise.

[0009] Furthermore, in step 2, the preparation of the turbidity-responsive flocculant involves: the nano-silica sol is a surface-modified hydrophobic nano-silica sol, with a food-grade silane coupling agent as the surface modifier; the water contact angle of the modified nano-silica particles is 100°-130°; before the spray drying step, 0.5%-1.5% (by weight of chitosan) of β-cyclodextrin is added to the mixed solution of chitosan and nano-silica and stirred until dissolved. This solution addresses the technical problem of how to reduce the non-selective adsorption of valuable volatile aroma components (esters, terpenes, etc.) from mango wine during flocculation by chitosan-based composite powder (especially high-surface-area nano-silica), leading to flavor loss.

[0010] Furthermore, after fermentation initiation in step 3 and before online turbidity monitoring begins in step 5, a turbidity baseline calibration phase is established. This calibration phase involves continuously collecting 10-20 turbidity values ​​between the 4th and 8th hour after fermentation initiation, when the fermentation broth begins to stably produce bubbles. The median value is taken as the initial baseline turbidity value for that batch of fermentation. In subsequent step 5, this initial baseline turbidity value is used for calculation and evaluation. This solution addresses the technical problem of significant differences in initial turbidity of fruit pulp due to different mango varieties, ripeness, and crushing processes. Using the transient turbidity at fermentation initiation as the baseline for calculating the rise would result in batch-specific triggering conditions.

[0011] Furthermore, the slow-release composite enzyme microspheres prepared in step 1 also contain 0.1%-0.3% lysozyme in their sodium alginate calcium gel core; after fermentation in step 7 is completed and allowed to stand, and before separating the supernatant, a pectinate lyase solution with a mass concentration of 0.05%-0.1% is added to the fermenter, the amount added being 0.1%-0.2% of the fermentation liquid volume, and the mixture is gently stirred at 30-35℃ for 2-4 hours.

[0012] Furthermore, in step 2, during the preparation of the turbidity-responsive flocculant, the inlet air temperature of the spray dryer is controlled at 150-170℃, and the outlet air temperature is controlled at 70-90℃. The particle size distribution of the resulting chitosan-based composite powder is required to be: D10 ≥ 15μm, D50 between 40-70μm, and D90 ≤ 120μm. This solution addresses the technical problem that hydrophobic nano-silica exhibits poor dispersibility and is prone to agglomeration in hydrophilic systems, significantly reducing its synergistic effect. Simultaneously, the dissolution / dispersion rate of chitosan powder directly affects the initiation rate and uniformity of the flocculation reaction.

[0013] Furthermore, the programmed stepped heating and segmented heat preservation process in step 6 has the following temperature control procedure: First, the fermentation system temperature is uniformly increased from 18-22℃ to 24-26℃ within 30-45 minutes and maintained for 60-90 minutes; then, the temperature is further increased to 27-28℃ within 20-30 minutes and maintained for 60-90 minutes; after the maintenance phase, the temperature is uniformly reduced to below 22℃ within 60 minutes. This solution addresses the technical problem of reducing the thermal shock caused by the heating in step 6 (to 25-28℃) to the yeast community still undergoing post-fermentation, which could lead to abnormal fermentation or undesirable flavors.

[0014] Furthermore, in step 1, when preparing the slow-release composite enzyme microspheres, the mass concentration of the konjac glucomannan solution is 0.7%-0.9%; the soaking treatment is carried out at a temperature of 45-55℃ for 12-18 minutes; after soaking, the microspheres are dried in a circulating air environment at 40-50℃ until a dense coating forms on the surface and the moisture content drops to 10%-15%. The technical problem solved by this scheme is to ensure that the slow-release composite enzyme microspheres can achieve delayed initiation and stable release of pectinase in the specific environment of mango wine fermentation (cycle 5-10 days, pH 3.5-4.5), rather than rapid burst release or no release at all.

[0015] The beneficial effects of this invention are as follows: First, pectinase was encapsulated within a sodium alginate-calcium gel core by preparing slow-release composite enzyme microspheres coated with konjac glucomannan. This coating layer slowly swells in the aqueous environment of the fermentation broth, thus achieving delayed initiation and stable, continuous release of pectinase. This technique effectively extends the action time of pectinase from several hours to match the entire main fermentation cycle, ensuring continuous and thorough enzymatic hydrolysis of the continuously dissolved pectin from the early to the later stages of fermentation. This not only significantly reduces the turbid matrix formed by initial pectin residue, lowering the basic turbidity and viscosity of the fermentation broth, but also creates a better metabolic environment for yeast, thereby improving fermentation efficiency (increased alcohol content, reduced residual sugar).

[0016] Secondly, by introducing an online turbidity sensor to monitor the fermentation process in real time and setting intelligent triggering logic based on changing trends and cumulative amplitudes, the turbidity rise signal representing the beginning of stable formation of secondary turbidity sources such as yeast metabolites can be accurately and reliably identified. This technology overcomes the blindness of the traditional fixed-time-point addition method, achieving dynamic perception and precise response to turbidity events. It ensures that chitosan-based composite powder can be added in a timely manner within the critical time window when turbidity forms in large quantities but has not yet stabilized, greatly improving the targeting and timeliness of flocculation intervention. This solves the problem of incomplete clarification caused by misaligned intervention timing, resulting in a significant reduction in the final wine turbidity and high batch stability.

[0017] Furthermore, the prepared turbidity-responsive flocculant—chitosan-based composite powder—achieved multiple optimizations through the combination of hydrophobic nano-silica and β-cyclodextrin, and by controlling its particle size distribution. The hydrophobic nano-silica provided more adsorption sites and skeletal support, enhancing flocculation capacity and floc strength; β-cyclodextrin reduced the non-selective adsorption of volatile aroma components by the flocculant through inclusion complexation; and the optimized particle size ensured rapid and uniform dispersion of the powder. These combined techniques resulted in a flocculation process that was not only fast and efficient, producing denser flocs, but also maximally preserving the characteristic aroma of mango wine.

[0018] Finally, the programmed temperature increase following the addition of the flocculant, through a stepwise increase in system temperature, effectively reduced the liquid viscosity and increased the Brownian motion and collision probability of colloidal particles. This physical method strongly promoted the bridging effect of the flocculant and the further collision, aggregation, and growth of micro-flocs, resulting in larger and denser flocs. This directly accelerated the settling rate of the flocs, shortened the time required for subsequent clarification, and made the solid-liquid separation more thorough, yielding a clear wine with extremely low turbidity (28 NTU), while reducing aroma loss caused by prolonged settling or forced filtration.

[0019] In summary, the various technical means of this invention are interconnected and work together to solve a series of technical challenges, such as the continuous action of pectinase, accurate perception of turbidity events, efficient and low-loss flocculation, and rapid sedimentation and separation. Ultimately, the invention achieves a comprehensive and beneficial effect by improving the clarity, stability, aroma retention, and production controllability of mango wine.

[0020] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0021] Figure 1 The dissolution and release kinetics curves of the konjac glucomannan (KGM) coated sustained-release complex enzyme microspheres, chitosan and gelatin, and other coating materials of the present invention, in mango wine fermentation broth. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to examples, so that those skilled in the art can implement it based on the description.

[0023] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0024] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0025] All indicators in this invention were detected using standard or conventional analytical methods. Pectinase activity was determined using the DNS reducing sugar method: the reducing sugar produced by enzymatic hydrolysis of the pectin substrate reacted with DNS reagent, and colorimetric analysis was performed at a wavelength of 540 nm. Enzyme activity was calculated by comparing with a glucose standard curve, thus yielding the cumulative release rate. Turbidity monitoring and measurement were performed using a calibrated turbidimeter. During online monitoring, the sensor collected data at a set frequency; for offline measurement, the NTU value of the settled wine sample was directly read. The median was used to determine the initial reference turbidity. Particle size analysis of the chitosan-based composite powder was performed according to the national standard GB / T 19077 2024, using laser diffraction (dry dispersion). The instrument directly reported volume distribution parameters such as D10, D50, and D90. Alcohol content was determined using an alcohol meter method (referencing GB 5009.225): after distilling the wine sample, the alcohol content of the distillate was measured using an alcohol meter at a constant temperature of 20°C. Residual sugar content was determined by Fehling's reagent titration method (referring to GB5009.7-2016), and the result was expressed as glucose (g / L). Pectin residue was qualitatively determined using a rapid ethanol precipitation method: Take 10 mL of wine sample in a clean test tube, add 10 mL of anhydrous ethanol (wine sample to ethanol volume ratio 1:1), mix thoroughly, and let stand at room temperature (20-25℃) for 30 minutes. Observe whether obvious flocculent, filamentous, or gelatinous precipitates appear at the bottom of the test tube or the liquid surface interface. If no visible precipitate or only a slight, transient cloud-like appearance (dispersed upon shaking) is observed, it is considered negative (-); if stable and obvious flocculation or gelation occurs, it is considered positive (+), and recorded as (++) or (+++) depending on the amount of precipitation. The retention rate of characteristic aroma components was analyzed by headspace solid-phase microextraction combined with gas chromatography-mass spectrometry: characteristic aroma substances of mango were selected, and their retention percentages were calculated by comparing the chromatographic peak areas of the finished wine and the initial fruit pulp relative to the internal standard.

[0026] Example 1: Preparation of sustained-release complex enzyme microspheres This embodiment verifies the enzyme release performance of the prepared konjac glucomannan (KGM) coated slow-release composite enzyme microspheres (hereinafter referred to as composite microspheres) and compares them with conventional materials. It can solve the technical problems of premature inactivation and short action time of pectinase in mango wine fermentation, which cannot be matched with the long fermentation cycle.

[0027] Materials: Sodium alginate (food grade, viscosity 200±20 mPa·s), calcium chloride (analytical grade), konjac glucomannan (KGM, food grade, viscosity ≥15000 mPa·s), chitosan (degree of deacetylation ≥90%), gelatin (type A, Bloom 250), pectinase (derived from Aspergillus niger, enzyme activity 5000 U / g). Instruments: Electronic balance (accuracy 0.001g), thermostatic magnetic stirrer, syringe pump (equipped with 22G flat-tipped needle), laser particle size analyzer, forced-air drying oven, thermostatic shaking water bath, UV-Vis spectrophotometer, pH meter.

[0028] I. Preparation steps of Konjac glucomannan (KGM) coated microspheres: 1. Preparation of sodium alginate-pectinase mixture: Weigh 2.0g of sodium alginate and dissolve it in 98mL of deionized water at 45℃. Stir magnetically until completely dissolved and clear to obtain a 2% (w / v) sodium alginate solution. After cooling to room temperature, add 1.2g of pectinase powder (total enzyme activity 6000 U) and stir slowly at 4℃ for 2 hours to ensure uniform dispersion, avoiding the formation of bubbles.

[0029] 2. Calcium ion cross-linked gel microsphere molding: Prepare 200 mL of a 4% (w / v) calcium chloride solution as the curing solution. Using a syringe pump, add the above mixture dropwise into the calcium chloride solution under magnetic stirring at a rate of 20 mL / h through a 22G needle (approximately 10 cm above the liquid surface). After the addition is complete, continue gentle stirring to solidify for 40 minutes. Collect the microspheres through a 100-mesh sieve and gently rinse three times with deionized water to remove residual calcium ions and unfixed enzymes from the surface. The diameter of the wet gel microspheres was measured to be 3.2 ± 0.3 mm.

[0030] 3. Preparation and soaking of KGM coating solution: Weigh 0.8g of konjac glucomannan powder, dissolve it in 99.2mL of deionized water at 50℃, and stir continuously in a 50℃ water bath for 1 hour until a homogeneous, slightly viscous, transparent solution is formed, resulting in a KGM solution with a mass concentration of 0.8%.

[0031] 4. Coating treatment: Immerse all the obtained wet gel microspheres in the above-mentioned 50℃ KGM solution, and gently shake and soak in a 50℃ constant temperature water bath for 15 minutes to ensure that the microspheres are completely and uniformly coated. (Corresponding soaking temperature 45-55℃, time 12-18 minutes) 5. Drying and Shaping: The coated microspheres are sieved out, excess solution is drained, and they are laid flat on a drying tray and immediately placed in a 45℃ circulating air drying oven. After drying for 5 hours, they are removed, weighed, and samples are taken for testing. The moisture content of the microspheres is found to be 12%, and a smooth, dense, translucent coating has formed on the surface. At least 30 dried, intact microspheres are randomly selected and sectioned near the equator using a cryostat (or a manual blade) to obtain the microsphere cross-section. The sections are placed under an optical microscope, and the thickness of the coating layer is measured using a micrometer. The average value is taken as the coating layer thickness of this batch of microspheres, which is approximately 100 μm. (Corresponding to drying at 40-50℃, moisture content 10%-15%) II. Performance Testing Test method: Accurately weigh 1.00 g of dried slow-release complex enzyme microspheres (Example 1 group) and place them in a 150 mL stoppered conical flask. Add 100 mL of simulated fermentation broth (pH 3.8 citrate-disodium hydrogen phosphate buffer containing 12% glucose). Place the conical flask in a constant temperature shaker at 22 °C and 100 rpm.

[0032] Sampling and Measurement: Samples were taken at 0, 2, 4, 8, 12, 24, 36, 48, 72, and 96 hours. At each sampling point, the entire contents of the conical flask were rapidly filtered through a 0.45 μm filter membrane, and all filtrate was collected. The reducing sugar content in the filtrate was determined using the DNS method, converted to enzyme activity, and the cumulative release rate was calculated. Three replicates were prepared for each time point.

[0033] Comparative Example 1: Using conventional chitosan-coated microspheres To compare the effects of conventional coating materials, chitosan-coated microspheres were prepared as a comparative example. Except for the coating step, the preparation process of the sodium alginate-pectinase gel core was exactly the same as in Example 1. The coating solution was prepared by dissolving 2 grams of chitosan in 100 ml of a 1% (w / w) acetic acid solution. The wet microspheres were soaked at room temperature (25°C) for 15 minutes, and then dried at 40°C to constant weight. The performance testing methods were the same as in Example 1.

[0034] Comparative Example 2: Microspheres coated with low concentration of KGM A group of KGM-coated microspheres with low concentration parameters were prepared. A KGM solution with a concentration of only 0.3% (lower than the 0.7%-0.9% range in Example 1) was used as the coating solution. The soaking temperature was reduced to 35 degrees Celsius, the soaking time was shortened to 5 minutes, and high-temperature rapid drying was performed at 60 degrees Celsius. The performance testing methods were the same as in Example 1.

[0035] Comparative Example 3: Direct addition of free pectinase A control group without any encapsulation was set up. Pectinase powder with the same total enzyme activity (6000 U) as that encapsulated in the microspheres of Example 1 was added directly to 100 mL of simulated fermentation broth at the start of the experiment. Performance testing methods were the same as in Example 1.

[0036] Comparative Example 4: Conventional gelatin-coated microspheres To further compare the performance of conventional water-soluble coating materials, gelatin-coated microspheres were prepared as Comparative Example 4. Except for the coating step, the preparation process of the sodium alginate-pectinase gel core was exactly the same as in Example 1. The coating solution was prepared as follows: 5 grams of type A gelatin (Bloom strength 250) was weighed and added to 95 ml of deionized water at 50°C, and stirred until completely dissolved to obtain a 5% (w / w) gelatin solution. The wet gel microspheres were immersed in this gelatin solution and kept at 40°C for 10 minutes. Subsequently, the microspheres, along with the solution, were transferred to a 10°C environment and cooled for 30 minutes to allow the gelatin layer to fully gel. Finally, the microspheres were removed and dried to constant weight in a 35°C forced-air drying oven. The performance testing method was the same as in Example 1.

[0037] Table 1. Comparison of release rate performance between Example 1 and Comparative Examples 1-4 like Figure 1 As shown in Table 1, Example 1 of this invention uses a 0.8% (w / w) konjac glucomannan (KGM) solution to coat the gel microspheres at 50°C, followed by gentle drying at 45°C. This process utilizes the density of KGM in its dry state and its unique property of slow swelling upon contact with water. Test results show that the resulting microspheres exhibit excellent slow-release characteristics in a simulated fermentation environment: the release rate is only about 3% in the initial 2 hours, effectively avoiding the impact and waste of enzymes in the complex environment at the beginning of fermentation; the release rate is about 57% after 24 hours, and reaches about 82% after 48 hours. This indicates that, through the dual effects of delayed on / off switching and diffusion control of KGM coating, the lifespan of pectinase is successfully extended from several hours to several days, allowing its release kinetics to be precisely matched with the 5-10 day main fermentation cycle of mango wine. This enables continuous degradation of pectin dissolved throughout the fermentation process, inhibiting the accumulation of turbidity at the source and achieving continuous clarification through simultaneous fermentation and degradation.

[0038] In contrast, Comparative Example 1 (chitosan coating) and Comparative Example 4 (gelatin coating) both exhibited rapid, sudden release, releasing the majority of enzyme activity within 2-4 hours. This is because chitosan dissolves rapidly in an acidic environment, while gelatin dissolves rapidly at a constant temperature, preventing the coating layer from forming an effective sustained-release barrier. Comparative Example 2 used a low-concentration (0.3%) KGM solution and short-term soaking at low temperature, resulting in a loose and non-dense coating layer structure. Although its release was slightly slower than traditional materials, it was still much faster than Example 1, with a release rate exceeding 95% after 24 hours, failing to achieve the sustained-release effect of this invention. Comparative Example 3 directly added free enzyme, which rapidly inactivated in the acidic, sugar-containing environment of simulated fermentation, providing no sustained effect. In summary, the data in Table 1 demonstrate that KGM and process parameters can prepare sustained-release composite enzyme microspheres with ideal sustained-release performance, resolving the contradiction between the short-lived action of pectinase and the need for sustained fermentation.

[0039] Example 2: Preparation of the chitosan-based composite powder (turbidity-responsive flocculant) of the present invention This embodiment prepares a chitosan-based composite powder for clarification during mango wine fermentation and verifies its advantages over conventional flocculants in terms of flocculation efficiency, sedimentation performance, and aroma retention.

[0040] I. Preparation Steps Raw material preparation: Chitosan hydrochloride with a deacetylation degree ≥85% and viscosity 50-200 mPa·s. Nano-silica sol with hydrophobic surface modification (using food-grade octyltriethoxysilane), solid content 20%, particle size 20-30 nm, and water contact angle approximately 115°. Food-grade β-cyclodextrin. Food-grade acetic acid, diluted to the required concentration.

[0041] The surface-modified hydrophobic nano-silica sol can be prepared as follows, which is part of this embodiment: The raw material is commercially available hydrophilic nano-silica sol (e.g., particle size 20-30 nm, solid content 20%). Octyltriethoxysilane is selected as the food-grade silane coupling agent. Modification reaction: 100 g of the above hydrophilic sol is placed in a three-necked flask equipped with a stirrer and reflux condenser, and heated to 60°C in a water bath and kept at a constant temperature. Under continuous stirring, 2.0 g of octyltriethoxysilane (10% of the SiO2 solid mass) is slowly added dropwise using a micro-injection pump, with the addition time controlled to be more than 1 hour. Curing and post-treatment: After the addition is complete, the reaction is continued at 60°C for 6 hours with stirring to allow the silane to fully hydrolyze and condense with the silanol groups on the silica surface. After the reaction, the product was cooled to room temperature and purified by dialysis with deionized water for 48 hours using an ultrafiltration membrane or dialysis bag with a molecular weight cutoff of 10 kDa to remove the reaction byproducts ethanol and unreacted silane coupling agent. The purified product was concentrated to a solid content of approximately 20% to obtain the desired hydrophobic nano-silica sol. Its hydrophobic properties were characterized by measuring the water contact angle of the prepared film: a small amount of sol was cast onto a clean glass slide, dried at 80°C, and the contact angle of deionized water on the membrane surface was measured using a contact angle meter via the static drop method; the result was 115°±5°.

[0042] Solution preparation and mixing: Measure 495 mL of deionized water, add 5.0 mL of glacial acetic acid, and stir to obtain an acetic acid solution with a mass concentration of approximately 1.0%. Weigh 10.0 g of chitosan hydrochloride powder and slowly add it to the above acetic acid solution. Stir magnetically at 25°C and 300 rpm for 4 hours until completely dissolved, obtaining a clear, slightly viscous chitosan solution with a mass concentration of approximately 2.0%. Add 0.1 g of β-cyclodextrin (1% of the chitosan mass) to the chitosan solution and continue stirring for 30 minutes until completely dissolved. Under continuous high-speed shearing (8000 rpm) stirring, slowly add 0.75 g (7.5% of the chitosan mass based on solid silica) of hydrophobic nano-silica sol. After the addition is complete, continue shearing and stirring for 1 hour to ensure uniform dispersion of the nano-silica and the formation of a stable composite mixture.

[0043] Spray drying molding: Transfer the above mixture to the feed tank of a small spray dryer. Set the key parameters for spray drying as follows: inlet air temperature: 165±5℃; outlet air temperature: 85±5℃; feed flow rate: 5 mL / min; atomization pressure: 0.3MPa; start the equipment for spray drying. Collect the powder from the bottom of the drying tower and the cyclone separator.

[0044] Post-processing: The collected powder was passed through a 100-mesh sieve to remove lumps. Using a laser particle size analyzer, the particle size distribution was measured according to GB / T19077 at an environment of 25±1℃ and 50±5% relative humidity, ultimately obtaining a free-flowing white to off-white chitosan-based composite powder. The particle size characteristics of the powder obtained in Example 1 were measured to be: D10 = 17.7 μm, D50 = 55.3 μm, D90 = 103.5 μm. See the table below for details: Table 2 Detailed data on particle size distribution Example 2 uses hydrophobically modified nano-silica and β-cyclodextrin to composite with chitosan. By controlling the inlet air temperature (165±5℃) and outlet air temperature (85±5℃) during spray drying, a chitosan-based composite powder with a specific particle size distribution was successfully prepared. The particle size data in Table 2 (D50=55.3μm, D90=103.5μm) show that the particle size of the powder is concentrated in the optimal range of 40-70μm, with extremely low content of large particles (>125μm) and fine powder (<10μm). The moderate particle size (D50≈55μm) ensures that the powder can be rapidly wetted and dispersed (dissolution time <60 seconds) and has sufficient mass to promote the subsequent sedimentation of flocs; at the same time, sufficient specific surface area ensures good adsorption activity.

[0045] II. Performance Comparison Test To verify the performance of the composite powder prepared in this embodiment (hereinafter referred to as the flocculant of this invention), the following comparative experiment was designed: 1. Test subject: The flocculant of this invention is the product of Example 2 above.

[0046] Comparative Example 5 (Ordinary chitosan powder): Chitosan hydrochloride was dissolved at the same concentration and spray-dried (inlet / outlet air temperature was the same as in Example 2), without the addition of nano silica and β-cyclodextrin.

[0047] Comparative Example 6 (excessively fine particle size powder): During preparation, the spray drying parameters (inlet air temperature 190℃, outlet air temperature 60℃) were changed to obtain powder with an excessively fine particle size (D50 = 22 μm).

[0048] Comparative Example 7 (without β-cyclodextrin): Its preparation process was exactly the same as that of Example 2, including the use of the same source and amount of hydrophobic nano-silica sol (water contact angle 115°±5°), and the completely identical spray drying parameters (inlet air 165±5℃, outlet air 85±5℃). The only difference was that β-cyclodextrin was not added in the solution preparation and mixing steps.

[0049] 2. Testing Method: Solubility and dispersibility: Weigh 0.10 g of each sample from Example 2 and the comparative example, add it to 100 mL of simulated mango fermentation broth (containing 12% ethanol) at pH 3.8, stir magnetically, and record the time when the powder is completely dispersed without any visible particles.

[0050] Flocculation and sedimentation experiment: Three identical portions of turbid mango fermentation broth (turbidity approximately 150 NTU after primary fermentation) were taken, and the three flocculants mentioned above were added to each to a final concentration of 0.75 g / L. The mixture was stirred slowly at 50 rpm for 10 minutes at 25°C and then allowed to stand. The turbidity (NTU) of the supernatant was recorded at 5, 30, and 120 minutes after the start of settling, as well as the total time required for complete floc settling.

[0051] Aroma Adsorption Assessment: Isoamyl acetate (exhibiting banana and sweet fruit aromas) and terpineol (exhibiting floral and woody aromas) are commonly used key aroma active substances in mangoes and mango wines. This test selected these two as characteristic aroma components for adsorption loss assessment. In a clarified mango wine model with known contents of characteristic aroma substances (isoamyl acetate and terpineol), 0.75 g / L of the aforementioned flocculant was added. After treatment for 2 hours, the mixture was filtered. The peak areas of the aroma substances before and after treatment were measured using GC-MS, and the adsorption loss rate was calculated.

[0052] The test results are as follows: Table 3. Flocculant Test Results Table 3's performance comparison further reveals the synergistic effect of each component. Compared to Comparative Example 5 (ordinary chitosan), the flocculant of this invention (Example 2), due to the introduction of hydrophobic nano-silica, provides more adsorption sites and a network framework, resulting in faster flocculation (lower turbidity at 5 minutes), more thorough flocculation (turbidity of only 22 NTU at 120 minutes), and shorter settling time (150 minutes). Compared to Comparative Example 6 (powder with excessively fine particle size), the optimized particle size distribution of this invention effectively avoids the defects of fine powder being prone to agglomeration, difficult to disperse, and having loose flocs and slow settling. In addition, the addition of β-cyclodextrin plays a key role; its molecular cavity can encapsulate aroma substances, significantly reducing the aroma adsorption loss rate from ≥15% in Comparative Example 5 to ≤8%. In terms of flocculation performance, Comparative Example 7 is very close to Example 2 and significantly better than ordinary chitosan (Comparative Example 5). This proves that the addition of hydrophobic nano-silica is indeed the main factor in improving the physical properties of flocculation. However, regarding the key aroma retention indicator, while the aroma adsorption loss rate of Comparative Example 7 (≥12%) was better than that of ordinary chitosan (≥15%), it was significantly higher than that of Example 2 of this invention (≤8%). This difference clearly reveals the irreplaceable core role of β-cyclodextrin in reducing aroma adsorption. Hydrophobic nano-silica contributed to the improvement of aroma retention by reducing non-specific adsorption (compared to Comparative Example 5), but β-cyclodextrin, through its unique external hydrophilic and internal hydrophobic cavity structure, can actively encapsulate hydrophobic aroma molecules in mango wine, forming steric hindrance, thereby providing more efficient and selective protection. Therefore, the excellent overall effect of Example 2 stems from the synergistic effect of hydrophobic nano-silica and β-cyclodextrin: the former mainly optimizes the physical process of flocculation, while the latter specializes in protecting flavor substances. Therefore, the data in Tables 2 and 3 prove that the flocculant prepared by the particle size control and component compounding of the present invention is a special material that combines the advantages of rapid dispersion, efficient flocculation, easy sedimentation and low aroma absorption, and solves the problem that conventional clarifying agents are difficult to balance in terms of efficiency and aroma retention.

[0053] Example 3: Mango wine brewing using slow-release complex enzyme microspheres and multi-stage clarification. The mango wine brewing method of this embodiment includes three major steps: long-term enzymatic hydrolysis with slow-release complex enzyme microspheres, online turbidity-triggered flocculation, and programmed temperature enhancement. This systematically solves the problem of fermentation turbidity and yields mango wine with high clarity and high aroma retention.

[0054] I. Brewing Raw Materials and Initial Processing Select Kate mangoes of uniform ripeness, wash, peel, and extract the flesh. Blanch the flesh in boiling water for 2-3 seconds, then blend it into a homogeneous pulp using a pulping machine. Preferably, the pulp can also be pasteurized (at 65℃ for 20-25 minutes), followed by cooling to room temperature. Adjust the pH of the pulp to 4.0 using a citric acid solution; this environment is conducive to subsequent fermentation and inhibits unwanted microorganisms. The initial sugar content was measured to be 18°Bx.

[0055] II. Material Addition and Fermentation Add the following materials to the treated mango pulp. First, add 15 grams of the slow-release complex enzyme microspheres prepared in Example 1 at a rate of 15 grams per liter of pulp. Simultaneously, inoculate with activated Angel Yeast (strain RV002) at an inoculation density of 3 × 10⁶. 6 CFU / mL. After gently stirring to ensure the materials are evenly mixed, transfer the fermentation mash to a clean fermentation tank and start the primary fermentation at 20°C.

[0056] III. First Phase Clarification During the primary fermentation, the first stage of clarification occurs simultaneously. Water in the fermentation mash gradually permeates into the slow-release complex enzyme microspheres, and the outer konjac glucomannan (KGM) coating begins to slowly swell. Approximately 2-4 hours later, the inner sodium alginate calcium gel core begins to hydrate and release pectinase. This enzymatic hydrolysis process is stable and continuous, effectively degrading approximately 70% of the initial pectin dissolved from the mango raw material within the first 24-48 hours of fermentation. This significantly reduces the base viscosity and turbidity potential of the fermentation broth, creating favorable conditions for subsequent clarification.

[0057] IV. Second Phase Clarification As fermentation enters the mid-to-late logarithmic phase (approximately 36-48 hours after fermentation start), yeast metabolism becomes vigorous, and it begins to release large amounts of secondary turbidity precursors such as mannoproteins. At this time, the fermentation broth is continuously monitored using an online turbidity sensor (sampling frequency: 1 time / minute). The monitoring system makes judgments according to the set intelligent logic: when the system detects a continuous increase in turbidity, and the cumulative increase from the calibrated baseline value (measured at the 6th hour of fermentation) reaches 20%, it immediately triggers an addition command. Subsequently, the turbidity-responsive flocculant (chitosan-based composite powder) prepared in Example 2 of this invention is added to the fermenter at a dosage of 0.75 g / L of fermentation broth. This flocculant can rapidly adsorb, neutralize, and bridge colloidal particles in the fermentation broth, including residual pectin fragments, proteins, and yeast cells, forming visible flocculents.

[0058] To ensure the implementation of the aforementioned intelligent triggering logic, this embodiment employs an automatic control system based on a programmable logic controller (PLC) or an industrial computer (IPC). The hardware connection of this system is as follows: the analog signal output terminal (4-20 mA) of the online turbidity sensor is connected to the analog input module of the controller; the digital output module of the controller is connected to the solenoid valve or metering pump that drives the flocculant storage tank feeding valve. Its software control logic (algorithm) can be implemented and embedded in the controller through the following process: Data Acquisition and Initialization: The system reads the turbidity sensor current signal at a frequency of once per minute and converts it into a turbidity value (N). After completing the "turbidity reference calibration stage," the determined initial reference turbidity value (N0) is loaded into the system. Rate of Change Calculation and Initial Trend Judgment: The instantaneous rate of change of the current turbidity value (Nt) relative to the value of the previous minute (Nt-1) is calculated in real time. The system maintains a rate of change queue containing the most recent three acquisition cycles and calculates its average value (ΔN_avg). Upward Trend Confirmation: When ΔN_avg is greater than 0.5% per minute three times consecutively, the system determines that an upward trend has appeared for the first time and records the turbidity value at this moment as the starting point of the upward trend (N_start). Delayed Trigger and Dual Condition Judgment: After confirming the trend, the system starts two parallel timers / counters, but does not immediately trigger the addition: a) Amplitude Condition Monitor: Continuously calculates the cumulative upward amplitude (Nt - N_start) / N0 * 100%. When this value reaches a preset threshold (e.g., 20%), trigger condition a is met. b) Time Condition Monitor: Starts timing from the trend confirmation point. When the cumulative continuous upward time reaches a preset value (e.g., 150 minutes), trigger condition b is met. Action Execution: Once condition a or condition b is met, the controller immediately sends a pulse signal through the digital output module to open the feeding valve or start the metering pump, adding a fixed amount of chitosan-based composite powder at a preset flow rate and time. After completion, the system records and proceeds to the next process. This control logic can be implemented using ladder diagrams or structured text programming on commercially available PLCs, or it can be configured on IPCs using platforms such as LabVIEW and configuration software.

[0059] V. Third Stage Clarification After the flocculant is added and mixed evenly, the heating program is immediately initiated. The specific steps are as follows: Within 40 minutes, the fermenter temperature is rapidly increased from 20°C to 25°C and maintained at this temperature for 70 minutes; subsequently, within 25 minutes, the temperature is further increased to 27.5°C and maintained for another 75 minutes. This stepwise heating process significantly reduces the liquid viscosity, increases the Brownian motion of colloidal substances in the fermentation broth, disrupts the hydration layer on the surface of colloidal substances, affects the ionization of particle surfaces, and promotes the collision, aggregation, and growth of flocs, forming dense and easily settling flocs. After the heating phase, the system temperature is rapidly reduced and restored to 20°C within 60 minutes.

[0060] Programmed heating is achieved through an integrated temperature control system in the fermenter. This control system typically includes temperature sensors, an electric heating mantle / jacket circulating water heating system, cooling water valves, and a multi-segment programmable temperature controller or a temperature module controlled by a PLC. In practice, after the operator or automatic control system triggers the addition of flocculant, it immediately starts the preset heating program in the temperature controller or host computer software. This program can be set (taking the parameters of Example 3 as an example): First segment: Set the target temperature to 25℃, heating time to 40 minutes (i.e., uniformly rise from 20℃ to 25℃), and maintain it for 75 minutes after reaching the target temperature. Second segment: Set the target temperature to 27.5℃, heating time to 25 minutes, and maintain it for 75 minutes after reaching the target temperature. Third segment: Set the target temperature to 20℃, cooling time to 60 minutes (uniform cooling is achieved by automatically adjusting the opening of the cooling water valve). The temperature controller will automatically control the power output of the heating and cooling elements based on the real-time feedback from the temperature sensor using a PID (proportional-integral-derivative) algorithm, thereby accurately and automatically executing the above multi-segment temperature curves. This multi-segment PID control based on a programmable temperature controller or PLC is the standard configuration and control method for industrial fermenters.

[0061] VI. Fermentation Completion and Separation of Wine After completing the aforementioned multi-stage clarification intervention, the fermentation system continued secondary fermentation at 20°C until the residual sugar content stabilized below 4 g / L, indicating the end of primary fermentation. The entire fermentation cycle took approximately 7 days, about 3 days shorter than the traditional no-treatment process. After fermentation, all fermentation liquid was transferred to a 4°C cold storage tank and allowed to stand for 60 hours to mature. During this period, the intensified flocs and yeast sludge completely settled. Finally, the supernatant was carefully separated using a siphon method and then subjected to final fine filtration through a 0.45 μm membrane to obtain a clear and transparent mango wine.

[0062] The following comparative examples were set up to compare the effects with those of Example 3: Comparative Example 8: This example does not employ long-acting enzymatic hydrolysis with slow-release composite enzyme microspheres. It was used to verify whether long-acting enzymatic hydrolysis solved the problem of continuous pectin release in the early stages of fermentation. The technical solution was adjusted so that neither the slow-release composite enzyme microspheres of Example 1 were prepared nor added. Instead, an equal amount of free pectinase was directly added to the mango pulp at the start of fermentation. The online turbidity-triggered flocculation and programmed temperature enhancement steps were retained and were exactly the same as in Example 3.

[0063] The results showed that free pectinase acted rapidly in the early stages of fermentation, but its activity decreased sharply within 12-24 hours. In the later stages of fermentation, newly dissolved pectin and secondary turbidity accumulated in large quantities due to a lack of enzymatic hydrolysis. Because the basal turbidity accumulated faster and in greater quantities, the online turbidity sensor triggered the addition of flocculants earlier (around 24-30 hours of fermentation). The flocculants had to handle an excessively heavy and complex colloidal load, resulting in low clarification efficiency. The final wine had high turbidity (estimated >80 NTU), and the intense enzymatic hydrolysis in the early stages led to flavor imbalance. Therefore, later-stage flocculation alone could not compensate for the lack of enzymatic hydrolysis in the early stages.

[0064] Comparative Example 9: No online turbidity trigger was set to verify whether the real-time response triggering of turbidity events during multi-stage clarification based on this invention has a more significant effect than intervention at fixed time points. Technical adjustment: The sustained-release composite enzyme microspheres of Example 1 were used, but the online turbidity sensor was removed. Instead, the same dose of chitosan-based composite powder was added at a fixed fermentation time point (48 hours after fermentation start). The programmed temperature enhancement step was retained.

[0065] To fully evaluate the stability of fixed-time addition, five independent parallel brewing experiments were conducted in Comparative Example 9. The results showed that due to differences in raw materials or minor fluctuations in the fermentation process, the actual time of significant secondary turbidity formation was not fixed across different batches. Fixed-time addition led to a severe mismatch between the intervention timing and the actual turbidity event. In the five batches, the final turbidity results showed extremely high dispersion: two batches, due to addition too early (before sufficient turbidity formation), had turbidities of 35 NTU and 42 NTU, respectively; the other three batches, due to addition too late (after the turbidity had formed stable colloids), had turbidities of 78 NTU, 95 NTU, and 100 NTU, respectively. Therefore, the turbidity can be expressed as 35-100 NTU (fluctuation). This demonstrates that fixed-time triggering cannot adapt to the dynamic changes in the brewing process, resulting in highly unstable product quality. In contrast, the online turbidity triggering mechanism used in Example 2 adaptively captures the unique characteristics of each batch, ensuring high consistency and reliability of clarification effects across different batches (turbidity stabilized at around 28 NTU).

[0066] Comparative Example 10: No temperature-programmed enhancement step was performed. This was used to verify the effect of temperature-programmed enhancement on floc formation and accelerated sedimentation. Technical adjustment: The slow-release composite enzyme microspheres from Example 1 were used, and an online turbidity triggering mechanism was employed to add the flocculant. However, after adding the flocculant, the temperature program was not executed, and fermentation was maintained at a constant temperature of 20°C throughout.

[0067] The results showed that after adding the flocculant, the flocs formed slowly, were fine in size, and had a loose structure. The flocs settled slowly, and even after subsequent settling, a large number of fine suspended particles failed to settle completely, leading to separation difficulties and a large volume of lees. The final wine had poor clarity (turbidity expected >50 NTU), and the filtration process was heavily loaded, easily resulting in the loss of aroma compounds during filtration. This demonstrates that the heating step can physically alter colloidal behavior, which is crucial for achieving deep clarification.

[0068] Comparative Example 11: The traditional process of primary fermentation followed by single enzymatic clarification was used to compare the effectiveness of the multi-stage, process-oriented, and synergistic clarification scheme of Example 3 of this invention. Specific implementation steps: Mangoes of the same variety and maturity as in Example 3 were selected. After pulping and pasteurization (65℃, 30 minutes), the initial sugar content of the pulp was adjusted to 20°Bx with white sugar, and the pH was adjusted to 3.8 with citric acid. 0.06% (w / v) of brewing yeast (same strain as in Example 2) was added to the pulp. Primary fermentation was carried out at a constant temperature of 26℃ until the specific gravity of the fermentation liquid remained unchanged for 24 consecutive hours, at which point fermentation was considered complete. No functional microspheres were added during this process, and no online monitoring or intervention was performed. After primary fermentation, pectinase was added to the original wine for clarification. The addition amount was 0.05% (w / v), and enzymatic hydrolysis was carried out at 20℃ for 48 hours. After enzymatic hydrolysis, the precipitate was removed by centrifugation (4000 rpm, 15 minutes), and the resulting wine was filtered through a 0.45 μm membrane before bottling.

[0069] The results showed that the alcohol content (%vol) of Comparative Example 11 was approximately 10.5%, lower than that of Example 3. This is because the traditional process did not address the yeast metabolic stress caused by the accumulation of turbidity in the later stages of fermentation. The residual sugar (g / L) was approximately 6.5. The high residual sugar content at the fermentation endpoint also indicates that fermentation efficiency and thoroughness are limited by the turbid environment. The turbidity (NTU) was >120 because the added pectinase could only degrade a portion of soluble pectin, with extremely low degradation efficiency for stable complex colloids formed with proteins and polysaccharides during fermentation. Furthermore, static treatment could not provide the physical conditions for enhanced flocculation. Pectin residue testing was positive (++), and rapid testing (such as ethanol precipitation) showed a significant positive result, confirming a large amount of pectin or polysaccharide colloid residue. Characteristic aroma retention was ≤ 65%. The lack of flavor protection measures throughout the fermentation period, coupled with the reliance on centrifugation and filtration for endpoint treatment, resulted in a significant loss of volatile aroma components during the prolonged fermentation and drastic physical processing at the end of the fermentation process.

[0070] Comparative Example 12: The steps of multi-stage clarification were adjusted to verify the effect of the step of adding flocculant first and then enhancing temperature program in Example 3 of the present invention. Technical solution adjustment: The slow-release composite enzyme microspheres of Example 1 were used, retaining the online turbidity triggering mechanism. However, the step order was changed: after turbidity is triggered, the temperature program is executed first (to 27.5°C and maintained), and then the chitosan-based composite powder is added at the end of the temperature rise stage. After the addition, the high temperature is no longer specifically maintained, and the cooling and subsequent fermentation are directly started.

[0071] The results showed that the alcohol content (%vol) of the prepared wine was approximately 11.3%, the residual sugar (g / L) was approximately 4.2%, and the turbidity (NTU) was 72.

[0072] Pectin residue test was negative. Characteristic aroma retention: approximately 80%. Preheating without flocculant primarily accelerates yeast metabolism and molecular motion, but also makes some turbid colloidal particles more difficult to capture due to increased thermal motion. Adding the flocculant at the end of the high-temperature phase, just before the system enters the cooling phase, prevents the flocculant molecules and colloidal particles from colloidally colloiding, adsorbing, bridging, and growing at the optimal temperature. This results in small, loose flocs with poor settling properties. The final product has high turbidity, significantly reduced clarification, and a slight increase in aroma volatilization due to the prolonged high-temperature phase.

[0073] The performance parameters of Comparative Examples 8-12 and Example 3 are compared in the following table: Table 4. Comprehensive comparison of performance parameters between Comparative Examples 8-12 and Example 3 As can be seen, the method in Example 3, which employs a process of sustained-release complex enzyme microsphere long-term enzymatic hydrolysis (first stage), turbidity-triggered targeted flocculation (second stage), and programmed temperature enhancement (third stage), has significant effects.

[0074] First, the optimal performance of Example 3 in terms of alcohol content (11.8% vol) and residual sugar (3.2 g / L) demonstrates that the early slow-release enzymatic hydrolysis creates a cleaner, lower-viscosity fermentation environment for yeast, promoting metabolic efficiency. The outstanding performance of the core indicators turbidity (28 NTU) and aroma retention (92%) is the result of the synergistic effect of three stages: 1) The long-term effect of the slow-release complex enzyme microspheres continuously degrades pectin, laying a good foundation for clarification and reducing off-odors; 2) The online turbidity triggering mechanism ensures precise intervention at the critical moment of secondary turbidity outbreak, avoiding the timing mismatch and quality fluctuations of fixed-time addition (Comparative Example 9); 3) The programmed temperature rise significantly improves the aggregation degree and sedimentation rate of flocs, which is a key physical step to achieve deep clarification (compared to Comparative Example 10 without temperature rise, turbidity 58 NTU).

[0075] Comparative Example 8 (without microspheres) experienced initial control failure, leading to immense pressure in later processing and deterioration of both turbidity and aroma indicators. Comparative Example 11, using conventional processing methods, lagged significantly behind in all indicators, particularly exhibiting extremely high turbidity (>120 NTU) and extremely low aroma retention (65%), demonstrating the fundamental advantage of the process intervention of this invention over remedial measures. In particular, Comparative Example 12 (with adjusted step sequence) showed a decrease in effectiveness because the order of adding flocculant before heating produces a synergistic enhancing effect, while reversing the order disrupts this synergy, resulting in insufficient flocculation.

[0076] Example 4: Preparation and application of sustained-release composite enzyme microspheres with terminal self-degradation function This embodiment verifies the addition of lysozyme to slow-release composite enzyme microspheres and the treatment with pectinate lyase at the fermentation endpoint to address the long-term stability risk caused by functional microsphere residues and improve the process.

[0077] I. Preparation of Novel Sustained-Release Composite Enzyme Microspheres (Containing Lysozyme) Based on the optimized process of Example 1, the core formulation was upgraded. The specific steps are as follows: First, as described in Example 1, 2.0 g of sodium alginate was weighed and dissolved in 98 mL of deionized water. After the solution cooled, in addition to adding 1.2 g of pectinase, 0.006 g of lysozyme (enzyme activity ≥20000 U / mg) was added to the mixture, making the proportion of lysozyme to the dry weight of sodium alginate 0.3%. The mixture was slowly stirred at 4°C for 3 hours to ensure that the two enzymes were uniformly dispersed in the sodium alginate solution. The subsequent gel microsphere forming, konjac glucomannan coating, and drying steps were exactly the same as in Example 1, finally yielding a core-containing sustained-release composite enzyme microsphere, denoted as "self-degradable microspheres". The macroscopic morphology, particle size, and water content showed no significant difference from the product of Example 1.

[0078] II. Application of Brewing Technology The mango wine brewing process was identical to that of Example 3, including raw material processing, fermentation initiation, and three-stage clarification. The only difference was that the added microspheres were replaced with the "self-degradable microspheres" prepared in Example 4. After the primary fermentation, a clarified mixture of lees containing a large amount of microsphere precipitate was obtained. Subsequently, the terminal treatment of this invention was performed: a 0.08% pectinate lyase solution was added to the entire fermentation tank, at a concentration of 0.15% of the total fermentation liquid volume. The fermentation tank temperature was adjusted to 32°C, and the mixture was gently stirred at 40 rpm for 3 hours. This process aimed to utilize the specific cleavage effect of pectinate lyase on the sodium alginate molecular chains (β-D-mannuronic acid and α-L-guluronic acid) to systematically disintegrate the gel skeleton of the microspheres. After the treatment, the system was cooled to 4°C and allowed to stand for 72 hours to allow the completely disintegrated microsphere residues to fully co-sediment with other precipitates. Finally, the supernatant was separated to obtain the final product.

[0079] III. Stability Performance Verification and Comparative Analysis To verify the effectiveness of Example 4, the final product of Example 4 was bottled and subjected to an accelerated storage test for 32 weeks at a constant temperature and in the dark at 25°C. Turbidity was measured periodically, and the lees sediment was examined at the end of the test.

[0080] 1. Microscopic morphological observation of wine lees After the final treatment was completed and the mixture was allowed to stand, the lees precipitate of Example 4 showed a uniform and fine flocculent appearance. Under a microscope, the complete spherical microsphere structure could no longer be identified, indicating that its gel network had been effectively degraded.

[0081] 2. Long-term turbidity stability of the wine The long-term storage turbidity tracking data is shown in Table 5 below. The product of Example 4 exhibited extremely high turbidity stability throughout the entire 32-week observation period, with its turbidity consistently remaining near its initial value (approximately 30 NTU) with minimal fluctuations, and the wine remained consistently clear and transparent. This indicates that the risk of post-turbidity caused by microsphere residues has been fundamentally eliminated.

[0082] Table 5 Long-term stability performance of mango wine The lysozyme in the core of the self-degradable microspheres in Example 4 acts slowly in the internal environment of the microspheres during the later stages of fermentation, slightly weakening the gel network or providing an entry point for subsequent processing; while the final pectinate lyase treatment is the key step. This enzyme can specifically recognize and cleave the glycosidic bonds in sodium alginate molecules, thereby completely disintegrating the gel skeleton of the microspheres and fundamentally eliminating the risk of releasing residual substances due to physical rupture. Therefore, the product exhibits excellent long-term turbidity stability.

[0083] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for brewing mango wine, characterized in that, Includes the following steps: Step 1, Preparation of sustained-release complex enzyme microspheres: Pectinase is mixed with sodium alginate aqueous solution and added dropwise to calcium chloride solution to form gel microspheres with a diameter of 2-4 mm; the obtained gel microspheres are immersed in a 0.5%-1% konjac glucomannan solution for 10-20 minutes, then dried to a moisture content of 10%-15% to obtain sustained-release complex enzyme microspheres; wherein, the amount of pectinase added is 3000-5000 U per gram of sodium alginate. Step 2, Preparation of turbidity-responsive flocculant: Chitosan hydrochloride is dissolved in an acetic acid solution with a mass concentration of 0.5-1.5% to prepare a chitosan solution with a mass concentration of 1-3%; nano silica sol is added to the chitosan solution, the amount of nano silica sol added is 5%-10% of the mass of chitosan, and after stirring and mixing evenly, it is spray-dried to obtain chitosan-based composite powder; Step 3, Pre-fermentation treatment: Add the slow-release complex enzyme microspheres prepared in Step 1 to the pasteurized mango pulp with pH adjusted to 3.8-4.

2. The addition amount is 10-20 grams of slow-release complex enzyme microspheres per liter of pulp. At the same time, inoculate with brewer's yeast at an inoculum size of 1×10⁻⁶. 6 -5×10 6 CFU / mL; Step 4, primary fermentation and first stage clarification: Primary fermentation is carried out at 18-22℃. During the fermentation process, the water in the pulp gradually penetrates into the slow-release complex enzyme microspheres, causing the konjac glucomannan layer to absorb water and swell. Pectinase begins to be slowly released and acts on the pectin substances in the pulp. Step 5, Second stage clarification: When the turbidity of the fermentation broth first shows an upward trend through the online turbidity sensor, and the increase reaches 15%-25% of the initial turbidity, add the chitosan-based composite powder obtained in step 2 to the fermenter at a rate of 0.5-1.0 g / L of fermentation broth. Step 6, Third Stage Clarification: After adding chitosan-based composite powder, the fermentation system is subjected to programmed step heating and segmented heat preservation treatment, specifically including: first, raising the temperature from 18-22℃ to 24-26℃ and performing the first stage of heat preservation, then continuing to raise it to 27-28℃ and performing the second stage of heat preservation; the total duration of programmed heating and heat preservation is controlled within 3-6 hours; Step 7: After completing Step 6, restore the temperature of the fermentation system to 18-22℃ and continue fermentation until the sugar content stabilizes, thus ending the main fermentation. After letting all the fermentation liquid stand at 4-10℃ for 48-72 hours, separate the supernatant to obtain the clarified wine.

2. The method according to claim 1, characterized in that, In step 5, the online turbidity sensor collects the turbidity value of the fermentation broth once per minute and calculates its rate of change in real time; The criteria for determining the first appearance of an upward trend are: the average turbidity change rate is greater than 0.5% per minute for three consecutive collection cycles; after determining that an upward trend is met, chitosan-based composite powder is not added immediately, but monitoring continues until one of the following two conditions is met before addition is triggered: a) The turbidity value increases cumulatively from the point where it begins to rise, reaching 15%-25% of the initial turbidity; or, b) After the upward trend is confirmed, the cumulative time for the turbidity to continue rising reaches 120-180 minutes.

3. The method according to claim 1, characterized in that, In step 2, the preparation of the turbidity-responsive flocculant is as follows: the nano-silica sol is a surface-modified hydrophobic nano-silica sol, and the surface modifier is a food-grade silane coupling agent. The water contact angle of the modified nano-silica particles is 100°-130°. Before the spray drying step, 0.5%-1.5% of β-cyclodextrin by mass of chitosan is added to the mixed solution of chitosan and nano-silica and stirred to dissolve.

4. The method according to claim 2, characterized in that, After fermentation is started in step 3 and before online turbidity monitoring begins in step 5, a turbidity baseline calibration phase is set up. The baseline calibration phase is as follows: 4-8 hours after fermentation starts, when the fermentation broth begins to stably produce bubbles, 10-20 turbidity values ​​are continuously collected, and the median is taken as the initial baseline turbidity value for this batch of fermentation. In the subsequent step 5, the initial baseline turbidity value is calculated and judged.

5. The method according to claim 4, characterized in that, The sustained-release composite enzyme microspheres prepared in step 1 also contain 0.1%-0.3% lysozyme in their sodium alginate calcium gel core. After fermentation in step 7 is completed and allowed to stand, and before separating the supernatant, a pectinate lyase solution with a mass concentration of 0.05%-0.1% is added to the fermenter at a volume of 0.1%-0.2% of the fermentation liquid volume, and the mixture is gently stirred at 30-35°C for 2-4 hours.

6. The method according to claim 3, characterized in that, In step 2, when preparing the turbidity-responsive flocculant, the inlet air temperature of the spray dryer is controlled at 150-170℃ and the outlet air temperature is controlled at 70-90℃. The particle size distribution of the obtained chitosan-based composite powder is required to be: D10 ≥ 15μm, D50 between 40-70μm, and D90 ≤ 120μm.

7. The method according to claim 1, characterized in that, The programmed step heating and segmented heat preservation process in step 6 has the following temperature control procedure: First, the fermentation system temperature is uniformly increased from 18-22℃ to 24-26℃ within 30-45 minutes and maintained for 60-90 minutes; then, the temperature is further increased to 27-28℃ within 20-30 minutes and maintained for 60-90 minutes; after the maintenance phase, the temperature is uniformly reduced to below 22℃ within 60 minutes.

8. The method according to claim 1, characterized in that, In step 1, when preparing the sustained-release complex enzyme microspheres, the mass concentration of the konjac glucomannan solution is 0.7%-0.9%; the soaking treatment is carried out at a temperature of 45-55℃ for 12-18 minutes; after soaking, the microspheres are dried in a circulating air environment at 40-50℃ until a dense coating is formed on the surface of the microspheres and the water content is reduced to 10%-15%.

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