A method for preparing a fermented beverage featuring mulberry and monk fruit

Abstract

This invention discloses a method for preparing a mulberry-monk fruit fermented beverage, belonging to the field of microbial fermentation technology. The method includes raw material processing, alcoholic fermentation, acetic acid fermentation, and blending steps: Monk fruit is extracted using a combination of juice extraction and hot water countercurrent extraction; the extract is kept warm to improve clarity and inactivate enzymes; using mulberry juice and monk fruit juice as substrates, fruit wine yeast is inoculated for anaerobic fermentation; then, monk fruit juice and edible alcohol culture are added to adjust the alcohol content, followed by aerobic fermentation with *Acetobacter acetosporum* and acetic acid bacteria; finally, the fermented juice, glycoside-containing monk fruit juice, mulberry concentrate, and erythritol are blended to prepare the beverage. This invention fully utilizes mulberry and monk fruit resources, retains active ingredients such as polyphenols, anthocyanins, and polysaccharides, and produces a low-calorie, flavorful, and safe product suitable for industrial production, possessing both nutritional and health benefits.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and more specifically to a method for preparing a fermented beverage featuring mulberry and monk fruit. Background Technology

[0002] Mulberries, as functional berries, are rich in nutrients and have strong functional properties. However, due to their short harvest season, strong seasonality, and poor storage properties, deep processing of mulberries is beneficial to the development of the mulberry industry. Currently, the mulberry industry has entered a new stage of development, with a series of mulberry products emerging. However, due to the lack of mature processing technology, these products lack distinctive features. For example, mulberry vinegar often borrows fermentation processes from apple cider vinegar and regular vinegar, or even directly uses mulberry juice to dilute vinegar, resulting in products whose taste and quality fail to meet requirements. Therefore, how to fully utilize abundant mulberry resources and develop their deep processing has become an urgent issue to be addressed in the ecological construction of mulberry resources and the agricultural product processing industry.

[0003] Monk fruit is rich in triterpenoids, flavonoids, fatty acids, and other active ingredients, possessing high medicinal and edible value. Currently, in the development and utilization of monk fruit products, its triterpenoid component, mogroside, is the most important research subject. As a natural sweetener, it is 300 times sweeter than sucrose, while its calories are only 2% of sucrose. It boasts advantages such as low calories, non-toxicity, and antioxidant properties, making it a preferred sugar substitute for patients with diabetes, hypertension, and obesity. It has been widely used in the food, health food, and beverage industries. Currently, dried fruit remains the main product form of monk fruit on the market, belonging to primary processing products with low technological content. Deep processing of monk fruit to extract mogrosides presents problems such as low utilization rate, high cost, and easy loss of effective components. Therefore, focusing on the development and application research of new monk fruit series natural sweetener products, represented by high-purity mogroside extracts, is an important guarantee for the sustainable development of the monk fruit industry. The production and processing of monk fruit glycosides generates a large amount of byproducts rich in sugars and proteins, accounting for 80%-90% of the soluble solids in monk fruit. These byproducts contain abundant nutrients such as sugars, proteins, amino acids, and inorganic salts, as well as trace vitamins such as niacin, folic acid, pantothenic acid, vitamin B12, and biotin. By weight percentage of solids, these byproducts contain 50%-65% sugars, 8%-12% nitrogenous nutrients such as proteins and amino acids, and 6%-10% inorganic salts (especially rich in potassium and phosphorus). Their rich and diverse nutrients make them highly suitable for microbial fermentation. Currently, these byproducts are often neglected and disposed of as waste in wastewater treatment systems, leading to significant resource waste and placing immense pressure on wastewater treatment. How to transform these byproducts into valuable resources is a pressing issue that the monk fruit deep-processing industry needs to address to improve quality, reduce costs, and achieve environmental friendliness.

[0004] With the rapid development of the global health industry and the diversification of consumer demand for functional foods, natural, healthy, low-calorie functional beverages with unique flavors have become a market hotspot. Mulberries and monk fruit, as traditional medicinal and edible resources, contain not only abundant sugars but also rich active ingredients such as flavonoids, polyphenols, anthocyanins, polysaccharides, triterpenoids, and amino acids in their juice. These ingredients possess antioxidant, blood sugar-lowering, immune-enhancing, and anti-inflammatory effects, making them natural raw materials for developing fermented flavored beverages. Currently, the development of this type of end product still faces challenges such as low retention rate of active ingredients, poor flavor coordination, and insufficient stability in industrial production. Summary of the Invention

[0005] In view of this, the present invention provides a method for preparing a fermented beverage featuring mulberry and monk fruit. To achieve this objective, the present invention adopts the following technical solution: A method for preparing a fermented beverage featuring mulberry and monk fruit includes the following steps: Step 1: Raw material preparation and fermentation substrate formulation Preparation of monk fruit juice: Monk fruit glycosides were extracted by a combination of juice extraction and hot water countercurrent extraction, and then kept at 90°C for 1-2 hours after extraction. Preparation of mulberry juice: Take purplish-black mulberry fruits that are 7-9 ripe and prepare mulberry juice; Preparation: Weigh out mulberry juice and monk fruit purified concentrated juice and mix them in a solids ratio of 1:1. Add purified water to dilute to a sugar content of 15-17. Stir well and then sterilize. After sterilization, the fermentation substrate is obtained. Step 2: Alcoholic fermentation (2.1) Propagation of Candida albicans Activate 5% (by weight) of the initial fermentation substrate Candida albicans with 7% (by weight) of Luo Han Guo juice, and then carry out a second expansion culture. The expansion culture conditions are: shaker culture, speed 140 rpm, expansion time 24-30 h. (2.2) Activation of fruit wine yeast Inoculate fruit wine yeast at 0.02% of the total mass of the initial fermentation substrate. Before inoculation, let the inoculum stand for 15 minutes with fermentation substrate at 20 times the weight of the yeast and at a temperature of 35~37°C. (2.3) Inoculation and fermentation Monitor the temperature of the substrate in the alcohol fermentation tank and confirm that the temperature is between 26-30°C. Add the yeast liquid activated with the initial fermentation substrate and the expanded Candida ethanolica seeds to the fermentation tank through a closed connecting pipe. Start the material pump to circulate the material for 10 minutes to fully mix the fruit wine yeast with the substrate. Fermentation begins. Record the fermentation time. (2.4) Fermentation detection The fermentation process is anaerobic fermentation. The fermentation process is monitored in real time, and the fermentation temperature is controlled at 26-30°C. After 24 hours of fermentation, the alcohol content of the fermentation liquid is tested every 6 hours. When the alcohol content is greater than 5%, the fermentation is terminated. The alcohol fermentation time is 36-72 hours, and the pH value is 4.2-6.5. (2.5) Post-processing The fermented liquid that has been fermented is filtered through a 100-mesh sieve and put into a sterilization tank. Stirring and steam heating are turned on and the temperature is maintained at 75~80°C for 30 minutes. After the process is completed, the steam heating is turned off and the sterilization tank is kept sealed. Cold water is pumped into the jacket to cool the material to 34-37°C, and Luo Han Guo and mulberry fermented fruit wine is obtained. Step 3: Acetic acid fermentation (3.1) Preparation of fermentation substrate Adjust the alcohol content of the monk fruit and mulberry fermented fruit wine to 4.5%-6% and the pH value to 4.2-6.5. After completion, record the substrate volume and wait for inoculation with acetic acid fermentation. (3.2) Acetic acid bacteria expansion After sterilizing the Luo Han Guo and mulberry fermented fruit wine prepared in step (3.1), place it in a triangular conical flask, inoculate it with acetic acid bacteria, seal it with cotton plugs after inoculation, place it in a shaker, set the speed to 140 rpm and the shaker temperature to 34°C, and start the first-stage expansion culture. The first-stage expansion culture time is 24-36 hours. After the first-stage expansion, Luo Han Guo and mulberry fermented fruit wine was used as the fermentation substrate and placed in a fermentation tank. The acetic acid bacteria seed liquid from the first-stage expansion was inoculated into the fermentation substrate at 5% of the fermentation tank volume for a second-stage expansion. After inoculation, the fermentation tank was turned on and aerobic fermentation was carried out at a controlled temperature of 34-37°C with an aeration rate of 1.5-2.5 L / min. Samples were taken every 12 hours after fermentation started to test the total acid concentration. When the net acid concentration was ≥0.5 g / 100 ml and ≤1.0 g / 100 ml, the strain expansion was completed, and liquid active acetic acid bacteria were obtained. (3.3) Inoculation and fermentation Using monk fruit and mulberry fermented fruit wine as the fermentation substrate, a cultured liquid active acetic acid bacteria solution, accounting for 5%-7% of the substrate volume, is added to the fermentation tank. The fermentation tank temperature is set at 34-37°C, and the aeration rate is 2.5-3.5 m³ / h. 3 / h, take samples every 12h to test the total acid concentration and calculate the net acid concentration. When the net acid concentration is >3.0g / 100ml, take samples every 4h to test the total acid concentration and alcohol content. When the total acid content in the substrate no longer increases or the alcohol content is ≤0.5%, stop aeration immediately and terminate fermentation. (3.4) Post-processing After the fermentation of monk fruit and mulberry is terminated, the fermented fruit wine is sterilized, clarified and filtered to obtain mulberry and monk fruit fermented juice, which is stored at an ambient temperature of 0-10°C. Step 4: Preparation, sterilization, and bottling (4.1) Mixing and blending In a clean and ready mixing tank, add 30% purified water and 2% erythritol by weight, stir to dissolve, then add 6% fermented mulberry and monk fruit juice, monk fruit juice concentrate containing 3.5 wt% glycosides, 0.5% mulberry concentrate (solid weight), and purified water to make up to 100%. Then add 0.5%-0.8% diatomaceous earth filter aid by liquid volume. Control the temperature at 25-35°C, mix and stir for 60 minutes, then filter to obtain mulberry and monk fruit compound beverage. (4.2) Sterilization and filling After filtration and clarification, transfer the mulberry and monk fruit compound beverage to the feed tank of the sterilizer. After the sterilizer has finished sterilizing, reset the heating temperature of the sterilizer to 112~122°C and the flow rate to 0.8~1.3m. Open the circulation valve of the sterilizer, start the sterilizer, open the filling valve and cooling water, and start filling.

[0006] Preferably, the preparation of monk fruit juice in step one includes the following steps: Fresh monk fruit is processed using a crusher while preserving the integrity of the seeds. A screw extrusion press is used, with pressure controlled at 0.3-0.6 MPa and temperature at 20-25°C. The solid content of the extracted juice is 15-20%. The residue after juice extraction was continuously extracted for 90 minutes at 85°C using a tank-type dynamic countercurrent extraction unit with pure water as the solvent. The material-to-liquid ratio was 1:8-1:10 g / mL, the flow rate was 0.5-1.0 BV / h, and a 3-5 stage countercurrent structure was set.

[0007] Preferably, the preparation of mulberry juice in step one includes the following steps: Take purplish-black mulberry fruits that are 7-9 ripe, disinfect them by soaking them in ozone water or light salt water, rinse them with running water, drain them, and then juice them. Add 0.2%-0.3% pectinase to the extracted juice, enzymatically hydrolyze it at 30-37℃ for 3-5 hours, then coarsely filter it through a 20-mesh filter, finely filter it through a centrifuge or diatomaceous earth filter, homogenize it under high pressure at 15-20MPa, then degas it under vacuum at 60-80KPa to remove dissolved oxygen, and sterilize it to obtain mulberry juice.

[0008] Furthermore, a color-protecting treatment is carried out during the juicing process, which involves adding 0.1% vitamin C and 0.1% citric acid by weight of the mulberry or blanching at 60°C for 20 minutes; the juicing is performed using a screw juicer or a belt press.

[0009] Furthermore, the sterilization is performed by pasteurization at 85-90℃ for 15-20 minutes, or ultra-high temperature instantaneous sterilization at 135-150℃ for 2-8 seconds.

[0010] Preferably, the disinfection in step one specifically involves: adding the mixed materials to the disinfection tank, turning on the stirring and steam heating, raising the temperature of the liquid in the tank to 85~90°C, maintaining the temperature for 60 minutes, and continuously turning on the stirring during the process; After disinfection, turn off the steam heating, keep the disinfection tank sealed, pump cold water into the jacket to cool the material to 26~30°C, and then transfer the material to a disinfected and ready-to-use alcohol fermentation tank.

[0011] Preferably, in step (2.3), the fermentation tank is kept sealed during the fermentation process, with only a one-way valve or an air barrier device with a water seal allowing gas to escape.

[0012] Preferably, the fermentation substrate requirements for the primary and secondary scale-up cultivation in step (3.2) are: alcohol content 3%-5%, pH value ≥4.80, and solids content 7.0%-9.0%.

[0013] Preferably, in step (3.4), pasteurization is used. The fermentation broth is transferred to a pasteurization tank, heating is turned on, and after the temperature rises to 75-80°C, it is kept warm for 30 minutes and then cooled to 40-50°C. Clarification is achieved by butterfly centrifugation or diatomaceous earth filtration.

[0014] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a method for preparing a mulberry-monk fruit fermented beverage, which has the following beneficial effects: Solving raw material processing problems, achieving efficient resource utilization, and promoting industrial upgrading: Addressing the issues of mulberry's poor storage life, immature deep processing technology, and low initial processing technology, high cost of glycoside extraction, and serious waste of by-products in monk fruit, this invention extends the mulberry fruit industry chain by scientifically fermenting and processing mulberries; and utilizes by-products from monk fruit glycoside extraction as fermentation raw materials to realize waste resource utilization, reduce sewage treatment pressure and raw material costs, and contribute to the sustainable development of both industries. Preserving the active ingredients of raw materials and enhancing the functionality of beverages: Using specific extraction and fermentation processes, the active ingredients and nutrients in mulberries and monk fruit are preserved to the maximum extent, giving the beverage multiple health benefits and meeting the needs of the health community. Optimize fermentation process to adapt to industrial production: Precisely control fermentation parameters to solve the problems of poor flavor and insufficient stability of similar beverages, improve product flavor and quality stability, and facilitate large-scale standardized production; Creating low-calorie specialty products to meet market trends: Using mogrosides and erythritol as sweeteners, the beverages are low in calories, suitable for specific groups, broadening the consumer base, and have broad market prospects. The technology is scientific, practical, cost-controllable, easy to promote, and has both economic and social benefits. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0016] Figure 1 The image shows the appearance of a fermented beverage featuring mulberry and monk fruit with different mulberry concentrates. Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1

[0019] The preparation method of mulberry-monk fruit fermented beverage includes the following steps: Step 1: Raw material processing Preparation of Luo Han Guo juice: The juice is extracted using a combination of juice extraction and hot water countercurrent extraction. The juice has good fluidity. After extraction, it is kept at 90°C for 1-2 hours to improve clarity, reduce turbidity, and ensure complete inactivation of proteases to control glycoside degradation. Juicing Process: Fresh monk fruit is processed using a crusher, ensuring the seeds remain intact to prevent the dissolution of oils and bitter substances. A screw press is then used, with pressure controlled at 0.3-0.6 MPa, operating at room temperature (20-25°C). The solid content of the extracted juice is approximately 15-20%. The juice is subsequently kept at 90°C for 1-2 hours to inactivate proteases and improve clarity.

[0020] Hot water countercurrent extraction: For the fruit pomace after juice extraction, a tank-type dynamic countercurrent extraction unit is used with pure water as solvent. The extraction is carried out continuously and dynamically for 90 minutes at 85°C. The material-liquid ratio is controlled at 1:8–1:10 (g / mL), the flow rate is 0.5-1.0 BV / h, and a 3-5 stage countercurrent structure is set. The extraction rate of effective components can reach 99.76%.

[0021] Mulberry Juice Preparation: Mulberry juice, rich in anthocyanins and other nutrients, offers both flavor and health benefits. Its processing focuses on color preservation and sterilization. ① Raw Material Pretreatment: Select 7-9 ripe, dark purple mulberries. Disinfect by soaking in ozone water or lightly salted water, then rinse and drain with running water to prevent microbial contamination. Color preservation is crucial during crushing and juicing. This can be achieved by adding a mixture of 0.1% vitamin C and 0.1% citric acid, or by blanching at 60℃ for 20 minutes to inactivate enzymes and prevent anthocyanin oxidation and browning. Using a screw press or belt press can achieve a juice yield of 66%-68%. ② Enzymatic Clarification Stage: Add 0.2%-0.3% pectinase and hydrolyze at 30-37℃ for 3-5 hours to reduce juice viscosity. Then, filter through a 20-mesh coarse filter, centrifuge, or diatomaceous earth filter to improve clarity. Finally, homogenize under 15-20 MPa high pressure to refine the pulp particles. ③ In the degassing and sterilization process, dissolved oxygen is removed by vacuum degassing at 60-80KPa. Sterilization can be performed by pasteurization at 85-90℃ for 15-20 minutes or ultra-high temperature instantaneous sterilization at 135-150℃ for 2-8 seconds.

[0022] According to the fermentation tank volume, the volume of the fermentation substrate should not exceed 70% of the fermentation tank volume. Weigh out mulberry juice and monk fruit purified concentrate according to the production plan, mix them in a 1:1 solids ratio, dilute with purified water to a Brix value between 15 and 17, stir well, take samples, and test the solids content and material volume.

[0023] Pasteurization (heat preservation): Before adding materials to the sterilization tank, confirm that it is in a fully sealed state. After adding materials, turn on the agitator and steam heating. When the temperature of the solution inside the tank reaches 85-90°C, confirm again that the sterilization tank is in a fully sealed state. Turn on the automatic temperature recorder to print the temperature record and record the start time. During the sterilization process, the temperature inside the tank is maintained between 85-90°C for 60 minutes in a sealed state, with the agitator running continuously throughout the process.

[0024] After disinfection is complete, the steam heating is turned off, and the disinfection tank remains sealed. Cold water is pumped into the jacket for circulation, and the material is cooled to 26-30°C through the heat exchanger. Then, the material is transferred to a disinfected and ready-to-use alcohol fermentation tank.

[0025] Step 2: Alcoholic fermentation (2.1) Propagation of Candida albicans The activated Candida ethanol was used for secondary expansion culture with 7% (w / w) concentration of Luo Han Guo juice. The expansion conditions were: 7% Luo Han Guo juice, shaker culture, speed 140 rpm, expansion time 24-30 h.

[0026] (2.2) Activation of fruit wine yeast Inoculate the wine yeast at 0.02% (w / w) of the total initial fermentation substrate. Before inoculation, let the inoculum stand for 15 minutes with 20 times the weight of the yeast in fermentation substrate (at a temperature of 35~37°C).

[0027] Formula for calculating yeast quantity: Yeast quantity (kg) = Total amount of substrate for alcoholic fermentation (kg) × 0.02% (2.3) Inoculation and fermentation Monitor the temperature of the substrate in the alcohol fermentation tank and confirm that the temperature is between 26-30°C. Add the yeast liquid activated with the initial fermentation substrate (mulberry and monk fruit juice) and the expanded Candida ethanolica seeds to the fermentation tank through a sealed connecting pipe. Start the material pump to circulate the material for 10 minutes to ensure that the fruit wine yeast and substrate are fully mixed. Fermentation begins, and the fermentation time is recorded.

[0028] During fermentation, the fermentation tank is kept sealed, with only a one-way valve or an air barrier device with a water seal allowing gas to escape.

[0029] (2.4) Fermentation detection The entire fermentation process is anaerobic, with real-time monitoring and temperature control at 26-30°C. After 24 hours of fermentation, samples must be taken every 6 hours to test the alcohol content of the fermentation broth. Fermentation is terminated when the alcohol content exceeds 5%. The alcoholic fermentation time is 36-72 hours. The pH is kept natural (normal pH value is between 4.2 and 6.5).

[0030] (2.5) Heat treatment The fermented broth, after fermentation, is filtered through a 100-mesh sieve and introduced into a sterilization tank. Stirring and steam heating are then activated. When the temperature of the broth inside the tank reaches 75-80°C, the sterilization tank is reconfirmed to be fully sealed, and this process is maintained for 30 minutes with continuous stirring. After heat treatment, steam heating is turned off, and the sterilization tank remains sealed. Cold water is pumped into the jacket for circulation, and the material is cooled to 34-37°C via a heat exchanger. Samples are taken to test the alcohol content, pH, and other physicochemical properties of the fruit wine, as well as its taste. The material is then transferred to a sterilized and prepared acetic acid fermentation tank for inoculation and fermentation.

[0031] Step 3: Acetic acid fermentation (3.1) Preparation of fermentation substrate The alcohol content of the fermented mulberry fruit wine is controlled at 4.5%-6%, and the pH value is natural (natural pH value is between 4.2 and 6.5). After completion, the volume of the substrate is recorded. It is ready for inoculation with acetic acid fermentation. Generally, the higher the alcohol content consumed, the higher the concentration of acetic acid produced.

[0032] (3.2) Acetic acid bacteria expansion Acetic acid bacteria (Acetobacter acetospores and dried acetic acid bacteria) were inoculated into the pre-sterilized Luo Han Guo (monk fruit juice) fermentation substrate in an Erlenmeyer flask. The fermentation substrate requirements were: alcohol content 3%-5%, pH value ≥4.80, and solids content 7.0%-9.0%. After inoculation, the flask was sealed with a cotton plug and placed in a shaker. The shaker speed was set to 140 rpm and the shaker temperature to 34°C. The first-stage expansion culture was then started and recorded. The first-stage expansion culture time was 24-36 hours.

[0033] After the primary expansion, 5% by volume of the acetic acid seed culture from the primary expansion is inoculated into the fermentation substrate (mulberry and monk fruit wine) in a small self-priming fermenter for secondary expansion. The substrate for this secondary expansion is the same as that for the primary expansion. After inoculation, the fermentation equipment is started, and aerobic fermentation is carried out at a controlled temperature of 34-37°C with an aeration rate of 1.5-2.5 L / min. Samples are taken every 12 hours after fermentation starts to test the total acid concentration. When the net acid concentration is ≥0.5 g / 100 ml and ≤1.0 g / 100 ml, the culture is considered complete, and liquid active acetic acid bacteria are obtained. Net acid concentration (g / 100 ml) = current total acid concentration (g / 100 ml) - initial total acid concentration (g / 100 ml) (3.3) Inoculation and fermentation Add 5%-7% of the volume of the prepared liquid active acetic acid bacteria solution to the fermenter using a peristaltic pump. Set the upper and lower limits of the fermenter temperature control to 34-37°C, turn on the automatic heating and cooling devices, and set the defoamer to automatic mode at a frequency of 20Hz. Set the initial frequency of the aerator to 25Hz and turn on the acetic acid fermentation aerator. Adjust the aerator frequency according to the air flow rate value on the gas flow meter to control the aeration rate in the fermenter within the range of 2.5-3.5 m³ / h. Observe the liquid level in the tank through the sight glass. When a large number of small bubbles are seen rising to the surface and the liquid is boiling, it indicates that the aeration is normal.

[0034] Acetic acid fermentation is an aerobic fermentation process, and the air introduced should be filtered through a dedicated air filtration device.

[0035] (3.4) Fermentation monitoring Monitor the fermentation temperature to maintain it between 34-37°C. After fermentation starts, take samples every 12 hours to measure the total acid concentration and calculate the net acid concentration. When the net acid concentration is >3.0 g / 100 ml, take samples every 4 hours to measure the total acid concentration and alcohol content. When the total acid content in the substrate no longer increases or the alcohol content is ≤0.5%, immediately stop aeration and terminate the fermentation.

[0036] (3.5) Heat treatment Sterilize the fermented mulberry and monk fruit juice after fermentation has stopped: If heat treatment is used, the specific operation is to transfer the fermentation liquid to a pasteurizer, turn on the heating, and after the temperature rises to 75-80°C, keep it at that temperature for 30 minutes. After cooling to 40-50°C, put it into a barrel for storage or carry out the next step of filtration and clarification.

[0037] (3.6) Clarification and filtration The fermented mulberry and monk fruit juice is clarified by butterfly centrifugation or diatomaceous earth filtration. The processed fermented mulberry and monk fruit juice is then further processed into beverages or packaged into ton drums for storage at an ambient temperature of 0-10°C.

[0038] Step 4: Blending, sterilization, and bottling (4.1) Mixing and blending In a clean and ready mixing tank, add water equal to 30% of the expected weight. Add 2% erythritol according to the feed amount and stir until completely dissolved. Then, transfer 6% of the weighed mulberry and monk fruit fermented juice, monk fruit concentrate (containing 3.5% glycosides), 0.5% mulberry concentrate (solid weight), and 15kg of diatomaceous earth filter aid to the mixing tank. After completion, add the remaining purified water. Control the temperature at 25-35°C and mix for 60 minutes before performing combined filtration.

[0039] (4.2) Sterilization and filling After filtration and clarification, transfer the mulberry and monk fruit compound beverage to the sterilizer's feed tank. After the sterilizer has finished sterilizing, reset the sterilizer's heating temperature to 112~122°C and the flow rate to 0.8~1.3m. Open the sterilizer's circulation valve, start the sterilizer, open the filling valve and cooling water, and begin filling the material into acid-resistant aseptic bags. The filling specification is 15kg / bag or according to customer requirements. After filling, the low-acid top-feed water is packaged in polyethylene drums and temporarily stored in a cold storage (-4~8°C) for the next use.

[0040] Experimental Example I. Extraction and Determination of the Dosage of Monk Fruit Glycosides 1.1 Monk fruit extract Juice extraction test results showed that the juice had good fluidity and the juice from frozen fruit accounted for 1.56% of the fresh fruit, proving that the juice extraction effect was good. Juice diversion helps to reduce the total amount of sweet glycosides in the fruit residue, which is beneficial to improving the efficiency of hot water extraction. The relevant data of juice extraction are shown in Table 1.

[0041] Table 1. Data on juice output corresponding to different fruit-feeding speeds.

[0042] 1.2 Hot water countercurrent extraction: After squeezing the juice from the raw material of monk fruit, the pomace was extracted by hot water countercurrent extraction to achieve efficient extraction of glycosides. The residual amount of glycosides in the pomace was <0.01%, and the glycoside extraction yield was ≥99%. Specific data are shown in Table 2.

[0043] Table 2. Distribution of glycoside content (%) in the extraction tank after squeezing monk fruit juice.

[0044] 1.3 Heat preservation treatment of extract The extract was incubated at 90℃ for 1-2 hours. Centrifugation clarified the material significantly better than before incubation, with the turbidity of the centrifuged liquid decreasing by 21-61% after incubation. Furthermore, although the extract underwent high-temperature enzyme inactivation before incubation, it was not complete; after incubation, the protease was completely deactivated. This study demonstrates that the incubation process not only facilitates material clarification but also effectively controls the risk of glycoside degradation due to incomplete enzyme inactivation.

[0045] Table 3 Test Indicators of Materials Before and After Insulation

[0046] II. Flavor and Color Optimization of Mulberry-Monk Fruit Fermented Beverage Based on the strong acidity and slightly astringent taste of fermented mulberry and monk fruit juice, the flavor and color of monk fruit juice, mulberry juice, and sugar alcohols were optimized by selecting those containing 3.5% and those without 3.5%. The addition of monk fruit components mainly optimized the sweet-acid ratio and introduced active ingredients such as monk fruit glycosides. The addition of mulberry concentrate was to further enhance and optimize the flavor and color. Sugar alcohols can modify the sweetness of monk fruit glycosides, accelerating the sweetening process and making the sweetness more full-bodied. The proportions of fermented mulberry and monk fruit juice, monk fruit components, and erythritol were first determined using principal component analysis. Then, the scoring results for different amounts of mulberry concentrate added were obtained through gradient testing, as shown in Table 4.

[0047] Table 4. Overall Taste Evaluation of Mulberry and Monk Fruit Compound Beverages with Different Added Mulberry Concentrates

[0048] From the table and Figure 1 Effects of adding different mulberry concentrates on the overall taste and color of Luo Han Guo compound beverage: Adding 0.5% mulberry concentrate significantly improves the overall taste and makes the color contrast richer. The calorie value reaches a low-calorie level (less than 80kJ / 100ml). Therefore, the formula with 0.5% mulberry concentrate is the optimal one.

[0049] III. Processing Technology of Mulberry-Monk Fruit Fermented Beverage 3.1 Optimization of alcoholic fermentation process Alcohol was fermented using fruit wine yeast. Based on the optimized fermentation substrate (7.5% mulberry juice + 7.5% monk fruit juice + supplemented to 100%), 0.02% dried fruit wine yeast (commercial strain) was inoculated and fermented statically under anaerobic conditions at 26℃. The highest alcohol residue and corresponding time were measured. Table 5. Fermentation test results of fruit wine yeast Mulberry juice Luo Han Juice Purified water Maximum alcohol residue (mg / ml) Maximum alcohol residual time / h 7.5% 7.5% Make up to 100% 47.340 48 Optimized conditions for alcoholic fermentation: culture medium: 7% monk fruit juice; fermentation substrate: 7.5% mulberry juice + 7.5% monk fruit juice + water to make up to 100%; strain: fruit wine yeast; temperature: 26~28℃; fermentation method: static anaerobic fermentation.

[0050] 3.2 Optimization of Acetic Acid Fermentation Process 3.2.1 Screening of Acetobacter acetylcholine culture medium from cocoa beans Group A culture medium consisted of 1% glucose + 1% yeast extract + 3% edible alcohol; Group B culture medium consisted of 7% monk fruit juice + 3% edible alcohol. Both culture media were sterilized at 121℃ and 0.1 MPa for 15 min, cooled, and inoculated with two to three loops of the bacterial strain. The cultures were then incubated at 34℃ on a shaker at 140 rpm. Samples were taken to determine the cell density of the strains, and the data were recorded to obtain the optimal propagation time, inoculation period, and optimal culture medium. The results are shown in the table below: Table 6. Results of Acetobacter growth test on cocoa beans (OD600 value)

[0051] As can be seen from the test results of Acetobacter cocoa growth (OD600 value) in Table 6, there is no significant difference in the growth of Acetobacter cocoa in the two culture media (Group A and Group B). Given that the project mainly focuses on mulberry and monk fruit, Group B culture medium is preferred.

[0052] 3.2.2 Optimization of total acid (calculated as acetic acid) content through mixed fermentation of cocoa beans with Acetobacter and acetic acid bacteria After fermentation with fruit wine yeast (commercial strain), the mulberry and monk fruit wine was adjusted to 5% alcohol content and pH controlled within the range of 4.2-6.0. It was then sterilized at 121℃ and 0.1 MPa for 15 minutes, cooled to 34℃, and group C was inoculated with 6% *Acetobacter cassia* culture and fermented with aeration. Meanwhile, under the same fermentation substrate conditions, group D was inoculated with 3% *Acetobacter cassia* culture and 3% acetic acid bacteria culture solution and fermented with aeration. The following is a table showing the fermentation time and acid production: Table 7. Net Acid Production from Mixed Fermentation of Cocoa Beans with Acetobacter and Acetic Acid Bacteria

[0053] Table 7 shows the net acid production from the mixed fermentation of Acetobacter cocoa beans and acetic acid bacteria. It can be seen that the acid production capacity of the mixed fermentation of Acetobacter cocoa beans and acetic acid bacteria is much stronger than that of inoculating only Acetobacter cocoa beans. Therefore, the mixed fermentation method is preferred.

[0054] Optimized conditions for acetic acid fermentation: culture medium: 7% monk fruit juice + 3% edible alcohol; fermentation substrate: mulberry and monk fruit fermented fruit wine with an alcohol content of 4.5%~6.0% and a pH value of 4.2~6.0; fermentation temperature: 34℃~37℃; fermentation method: aerobic fermentation.

[0055] IV. Stability Study of Mulberry-Monk Fruit Fermented Beverage This study investigated the changes in various physicochemical indicators of pilot-scale samples of fermented mulberry and monk fruit juice and compound mulberry and monk fruit beverages under accelerated testing conditions over time, and tested their shelf life to provide a scientific basis for the production, packaging, storage, and transportation conditions of the products. Samples were stored in a stability test chamber at 35℃ and 75%±5% relative humidity for 6 months. Physicochemical indicators, sensory taste, and total microbial count were tested monthly. Initial and final samples were tested for all microbial parameters.

[0056] 4.1 Stability of Mulberry-Monk Fruit Fermented Beverage Table 8. Changes in accelerated experimental indicators of mulberry and monk fruit fermented beverages Storage time / month Total acid / % Turbidity Absorbance 440nm Transmittance 625nm pH Senses Total bacteria 0 4.08 2.63 0.283 94.77 3.88 qualified ＜1 1 4.20 5.35 1.030 89.56 3.84 qualified ＜1 2 4.12 3.45 1.164 81.38 3.76 qualified ＜1 3 4.10 3.65 1.542 77.59 3.93 qualified 1 4 4.09 3.25 1.583 73.13 3.90 qualified ＜1 5 4.05 8.17 1.850 64.74 3.86 qualified 1 6 4.01 10.84 1.933 66.85 3.83 qualified ＜1 Table 9. Accelerated Experimental Determination of Mulberry and Monk Fruit Fermented Beverages Storage time / month result Description and evaluation 0 qualified It has the unique aroma and flavor of this product. It tastes sour and has no off-flavors. 1 qualified It has the unique aroma and flavor of this product. It tastes sour and has no off-flavors. 2 qualified It has the unique aroma and flavor of this product. It tastes sour with a slightly burnt flavor. 3 qualified It has the unique aroma and flavor of this product. It tastes sour with a slightly bitter aftertaste, and the acidity is reduced. 4 qualified It has the unique aroma and flavor of this product. It has no off-odors. 5 qualified It has the unique aroma and flavor of this product. There is a slight burnt taste, with a stronger soy sauce flavor. 6 qualified It has the unique aroma and flavor of this product. Slightly bitter, with a slight burnt taste, and no off-odors. After 6 months of accelerated fermentation of mulberry and monk fruit juice: transmittance and pH showed a decreasing trend; turbidity and absorbance showed an increasing trend; total acidity showed a slight decreasing trend; total bacteria <1 CFU / g, meeting microbiological and taste standards. The accelerated test concluded, and the accelerated shelf life (40℃, 75% relative humidity) was 6 months. Based on the Q10 value calculation formula: for every 10K increase in temperature, the reaction rate generally increases to 2-4 times the original rate. Therefore, the shelf life of the sample at 30℃ is estimated to be 12-24 months; calculated at 2.5 times the original rate, the shelf life is 15 months. The shelf life at 20℃ is 37.5 months.

[0057] V. Functional and Safety Study of Mulberry-Monk Fruit Fermented Beverage 5.1 Functional Ingredients The fermented functional beverage developed was tested by a third-party testing agency and found to be rich in bioactive components such as total polyphenols, total anthocyanins, total polysaccharides, triterpenoids, and free amino acids.

[0058] Table 10 Results of efficacy ingredient testing Serial Number Test Project Test data effect 1 Protein, g / 100ml 0.71 The framework and main substances that make up human tissues and organs play an important role in human life activities. 2 Total polyphenols, g / 100ml 0.09 It has the ability to regulate the expression of uric acid metabolism proteins and inhibit xanthine oxidase activity, and has potential preventive effects on cardiovascular diseases and gout. 3 Total anthocyanins, mg / 100ml 102.0 It has powerful antioxidant properties, which can eliminate free radicals in the body and prevent aging and related diseases; it can protect eyesight and prevent eye diseases such as macular degeneration and retinal diseases; it also has anti-inflammatory, anti-allergic, lipid-lowering, and blood sugar-lowering effects. 4 Crude polysaccharide, g / 100ml 0.13 Promotes the growth of bifidobacteria, inhibits pathogens, prevents constipation and bloating, promotes digestion, regulates gastrointestinal function, and enhances immunity. 5 Total triterpenes, g / 100ml 0.13 It has antioxidant, anti-inflammatory, immune-regulating, cardiovascular-health-improving, and nervous system-health-promoting effects and functions. 6 γ-Aminobutyric acid, mg / 100ml 51.51 It can increase the activity of glucosinolates in the brain, thereby promoting brain metabolism and repairing brain nerves, thus enhancing brain function; it can also inhibit the decarboxylation reaction of liver phosphate, thereby promoting liver and kidney health. 5.2 Safety Testing Acute oral toxicity tests and maximum tolerated dose tests of the beverage in mice were conducted according to the limits method of GB 15193.3-2014. The general characteristics, organ indices and biochemical indicators of the mice were comprehensively analyzed to evaluate the safety of the functional beverage.

[0059] The acute oral toxicity test employed the dose-limiting method. Twenty-six rats (half male and half female) were used. The test substance group (n=20) was administered the test substance by gavage at a dose of 19.95 g / kg body weight, while the control group (n=6) was administered an equal volume of purified water by gavage. The rats were observed for 14 days. During the observation period, no significant abnormalities were observed in either male or female animals, and weight gain was observed. Compared with the control group, the test substance group showed abnormalities in body weight at all measurement points. There were no statistically significant differences at any interval (P>0.05), and no deaths occurred. At the end of the experiment, all animals were grossly dissected; no abnormalities were observed visually, and organ indices were measured for evaluation.

[0060] Table 11 Results of Acute Oral Toxicity Test

[0061] Table 12 Results of Alanine aminotransferase (ALT) assay in 10% liver tissue homogenate from rats

[0062] Table 13 Results of Aspartate Transaminase (AST) assay in 10% rat liver tissue homogenate

[0063] Table 14 Results of Glutathione S-transferase (GST) assay in 10% rat liver tissue homogenate

[0064] Table 15 Results of protein assay in 10% rat liver tissue homogenate

[0065] Table 16. Detection of MDA content in 10% rat liver homogenate

[0066] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a fermented beverage featuring mulberry and monk fruit, characterized in that, Includes the following steps: Step 1: Raw material preparation and fermentation substrate formulation Preparation of monk fruit juice: Monk fruit glycosides were extracted by a combination of juice extraction and hot water countercurrent extraction, and then kept at 90°C for 1-2 hours after extraction. Preparation of mulberry juice: Take purplish-black mulberry fruits that are 7-9 ripe and prepare mulberry juice; Preparation: Weigh out mulberry juice and monk fruit purified concentrated juice and mix them in a solids ratio of 1:

1. Add purified water to dilute to a sugar content of 15-17. Stir well and then sterilize. After sterilization, the fermentation substrate is obtained. Step 2: Alcoholic fermentation (2.1) Propagation of Candida albicans Activate 5% (by weight) of the initial fermentation substrate Candida albicans with 7% (by weight) of Luo Han Guo juice, and then carry out a second expansion culture. The expansion culture conditions are: shaker culture, speed 140 rpm, expansion time 24-30 h. (2.2) Activation of fruit wine yeast Inoculate fruit wine yeast at 0.02% of the total mass of the initial fermentation substrate. Before inoculation, let the inoculum stand for 15 minutes with fermentation substrate at 20 times the weight of the yeast and at a temperature of 35~37°C. (2.3) Inoculation and fermentation Monitor the temperature of the substrate in the alcohol fermentation tank and confirm that the temperature is between 26-30°C. Add the yeast liquid activated with the initial fermentation substrate and the expanded Candida ethanolica seeds to the fermentation tank through a closed connecting pipe. Start the material pump to circulate the material for 10 minutes to fully mix the fruit wine yeast with the substrate. Fermentation begins. Record the fermentation time. (2.4) Fermentation detection The fermentation process is anaerobic fermentation. The fermentation process is monitored in real time, and the fermentation temperature is controlled at 26-30°C. After 24 hours of fermentation, the alcohol content of the fermentation liquid is tested every 6 hours. When the alcohol content is greater than 5%, the fermentation is terminated. The alcohol fermentation time is 36-72 hours, and the pH value is 4.2-6.

5. (2.5) Post-processing The fermented liquid that has been fermented is filtered through a 100-mesh sieve and put into a sterilization tank. Stirring and steam heating are turned on and the temperature is maintained at 75~80°C for 30 minutes. After the process is completed, the steam heating is turned off and the sterilization tank is kept sealed. Cold water is pumped into the jacket to cool the material to 34-37°C, and Luo Han Guo and mulberry fermented fruit wine is obtained. Step 3: Acetic acid fermentation (3.1) Preparation of fermentation substrate Adjust the alcohol content of the monk fruit and mulberry fermented fruit wine to 4.5%-6% and the pH value to 4.2-6.

5. After completion, record the substrate volume and wait for inoculation with acetic acid fermentation. (3.2) Acetic acid bacteria expansion After sterilizing the Luo Han Guo and mulberry fermented fruit wine prepared in step (3.1), place it in a triangular conical flask, inoculate it with acetic acid bacteria, seal it with cotton plugs after inoculation, place it in a shaker, set the speed to 140 rpm and the shaker temperature to 34°C, and start the first-stage expansion culture. The first-stage expansion culture time is 24-36 hours. After the first-stage expansion, Luo Han Guo and mulberry fermented fruit wine was used as the fermentation substrate and placed in a fermentation tank. The acetic acid bacteria seed liquid from the first-stage expansion was inoculated into the fermentation substrate at 5% of the fermentation tank volume for a second-stage expansion. After inoculation, the fermentation tank was turned on and aerobic fermentation was carried out at a controlled temperature of 34-37°C with an aeration rate of 1.5-2.5 L / min. Samples were taken every 12 hours after fermentation started to test the total acid concentration. When the net acid concentration was ≥0.5 g / 100 ml and ≤1.0 g / 100 ml, the strain expansion was completed, and liquid active acetic acid bacteria were obtained. (3.3) Inoculation and fermentation Using monk fruit and mulberry fermented fruit wine as the fermentation substrate, a cultured liquid active acetic acid bacteria solution, accounting for 5%-7% of the substrate volume, is added to the fermentation tank. The fermentation tank temperature is set at 34-37°C, and the aeration rate is 2.5-3.5 m³ / h. 3 / h, take samples every 12h to test the total acid concentration and calculate the net acid concentration. When the net acid concentration is >3.0g / 100ml, take samples every 4h to test the total acid concentration and alcohol content. When the total acid content in the substrate no longer increases or the alcohol content is ≤0.5%, stop aeration immediately and terminate fermentation. (3.4) Post-processing After the fermentation of monk fruit and mulberry is terminated, the fermented fruit wine is sterilized, clarified and filtered to obtain mulberry and monk fruit fermented juice, which is stored at an ambient temperature of 0-10°C. Step 4: Preparation, sterilization, and bottling (4.1) Mixing and blending In a clean and ready mixing tank, add 30% purified water and 2% erythritol by weight, stir to dissolve, then add 6% fermented mulberry and monk fruit juice, monk fruit juice concentrate containing 3.5 wt% glycosides, 0.5% mulberry concentrate (solid weight), and purified water to make up to 100%. Then add 0.5%-0.8% diatomaceous earth filter aid by liquid volume. Control the temperature at 25-35°C, mix and stir for 60 minutes, then filter to obtain mulberry and monk fruit compound beverage. (4.2) Sterilization and filling After filtration and clarification, transfer the mulberry and monk fruit compound beverage to the feed tank of the sterilizer. After the sterilizer has finished sterilizing, reset the heating temperature of the sterilizer to 112~122°C and the flow rate to 0.8~1.3m. Open the circulation valve of the sterilizer, start the sterilizer, open the filling valve and cooling water, and start filling.

2. The method for preparing a mulberry-monk fruit fermented beverage according to claim 1, characterized in that, The preparation of monk fruit juice in step one includes the following steps: Fresh monk fruit is processed using a crusher while preserving the integrity of the seeds. A screw extrusion press is used, with pressure controlled at 0.3-0.6 MPa and temperature at 20-25°C, resulting in a juice solids content of 15-20%. The residue after juice extraction was continuously extracted for 90 minutes at 85°C using a tank-type dynamic countercurrent extraction unit with pure water as the solvent. The material-to-liquid ratio was 1:8-1:10 g / mL, the flow rate was 0.5-1.0 BV / h, and a 3-5 stage countercurrent structure was set.

3. The method for preparing a mulberry-monk fruit fermented beverage according to claim 1, characterized in that, The preparation of mulberry juice in step one includes the following steps: Take purplish-black mulberry fruits that are 7-9 ripe, disinfect them by soaking them in ozone water or light salt water, rinse them with running water, drain them, and then juice them. Add 0.2%-0.3% pectinase to the extracted juice, enzymatically hydrolyze it at 30-37℃ for 3-5 hours, then coarsely filter it through a 20-mesh filter, finely filter it through a centrifuge or diatomaceous earth filter, homogenize it under high pressure at 15-20MPa, then degas it under vacuum at 60-80KPa to remove dissolved oxygen, and sterilize it to obtain mulberry juice.

4. The method for preparing a mulberry-monk fruit fermented beverage according to claim 3, characterized in that, Color protection is carried out during the juicing process by adding 0.1% vitamin C and 0.1% citric acid by weight of mulberry or blanching at 60℃ for 20 minutes; the juicing is carried out using a screw juicer or a belt press.

5. The method for preparing a mulberry-monk fruit fermented beverage according to claim 3, characterized in that, The sterilization process involves pasteurization at 85-90℃ for 15-20 minutes or ultra-high temperature instantaneous sterilization at 135-150℃ for 2-8 seconds.

6. The method for preparing a mulberry-monk fruit fermented beverage according to claim 1, characterized in that, The disinfection process described in step one is as follows: add the mixture to the disinfection tank, turn on the stirring and steam heating, and keep the temperature of the liquid in the tank at 85~90°C for 60 minutes, with stirring continuously during the process; After disinfection, turn off the steam heating, keep the disinfection tank sealed, pump cold water into the jacket to cool the material to 26~30°C, and then transfer the material to a disinfected and ready-to-use alcohol fermentation tank.

7. The method for preparing a mulberry-monk fruit fermented beverage according to claim 1, characterized in that, In step (2.3), the fermentation tank is kept sealed during the fermentation process, with only a one-way valve or an air barrier device with a water seal allowing gas to escape.

8. The method for preparing a mulberry-monk fruit fermented beverage according to claim 1, characterized in that, The fermentation substrate requirements for the primary and secondary scale-up cultivation in step (3.2) are: alcohol content 3%-5%, pH value ≥4.80, and solids content 7.0%-9.0%.

9. The method for preparing a mulberry-monk fruit fermented beverage according to claim 1, characterized in that, In step (3.4), pasteurization is used. The fermentation broth is transferred to a pasteurization tank, heating is turned on, and the temperature is raised to 75-80°C. After holding at the temperature for 30 minutes, it is cooled to 40-50°C. Clarification is achieved by butterfly centrifugation or diatomaceous earth filtration.

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