Edible effervescent powder using peaches as raw material and preparation method thereof

Abstract

This invention belongs to the field of pharmaceutical and edible preparations. It provides an edible effervescent powder made from peaches and its preparation method. The effervescent powder is prepared from a three-strain fermented freeze-dried powder through processes including hydroxypropyl-β-cyclodextrin pre-encapsulation, non-aqueous granulation, low-temperature drying, compound acid source formulation, chelation with disodium EDTA, and temperature and humidity control. The effervescent powder obtained by this invention shows virtually no change in uric acid-lowering and antioxidant activities before and after processing. The UA reduction retention rate is over 96%, and the changes in MDA and NOx remain around 100%, showing no significant difference from the original fermented freeze-dried powder. Furthermore, conventional quality indicators such as disintegration time, foaming volume, and moisture content all meet product requirements. This invention provides an effective technical solution for the development of end-product formulations of functional fermented products.

Description

Technical Field

[0001] This invention belongs to the field of food and drug preparations, and relates to preparations with uric acid-lowering and antioxidant functions. Specifically, it relates to an edible effervescent powder made from peaches and its preparation method. Background Technology

[0002] Effervescent powder is a solid dosage form that rapidly disintegrates upon contact with water, releasing carbon dioxide gas. Due to its convenience, pleasant taste, and rapid absorption, it is widely used in the food and health product industries. In recent years, with the increasing consumer demand for natural functional foods, effervescent powder products developed from fruits, vegetables, and medicinal plants have received increasing attention. Peaches are rich in phenolic compounds, vitamins, and dietary fiber, possessing good nutritional value and health benefits. Microbial fermentation of peaches can further enhance the content and bioavailability of their active ingredients, and the resulting fermentation products show good application potential in areas such as lowering uric acid and anti-oxidation. However, how to process the fermentation products into a portable and edible effervescent powder form while maintaining their original biological activity remains a significant technical challenge in this field.

[0003] Existing research indicates that fermented fruit and vegetable products have good effects in lowering uric acid and anti-oxidation. For example, lactic acid bacteria fermentation can increase the release and conversion rate of phenolic substances in fruits and vegetables, and produce bioactive metabolites such as short-chain fatty acids and small peptides, thereby enhancing their uric acid-lowering and antioxidant capabilities. In another patent, the inventors disclosed a dual-strain fermented beverage made from peaches and its preparation method. This fermented beverage undergoes light fermentation with *Escherichia coli* Nissle 1917, followed by deep fermentation with *Lactobacillus plantarum* JYLP-326 and *Pediococcus pentosaceus* JYPR9330. The resulting freeze-dried fermented powder showed excellent uric acid-lowering and antioxidant effects in animal experiments. However, considering that the freeze-dried fermented powder, when consumed directly in powder form, has an unpleasant taste and is inconvenient to carry, the inventors attempted to prepare it as an effervescent powder for easier consumption. Experiments revealed that the uric acid-lowering and antioxidant effects of the dual-strain fermented freeze-dried powder decreased significantly after processing using conventional effervescent processes (such as wet granulation, high-temperature drying, and acid-base co-granulation). Therefore, how to successfully prepare effervescent powder while maintaining the original activity of the fermentation products is a pressing technical challenge.

[0004] To address the shortcomings of the existing technology, this invention aims to provide an edible effervescent powder made from peaches and its preparation method, in order to achieve efficient retention of uric acid-lowering and antioxidant activities before and after effervescent processing, so that the uric acid-lowering and antioxidant effects of the effervescent powder are basically unchanged compared with the original fermented freeze-dried powder. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies where the uric acid-lowering and antioxidant activities of fermented freeze-dried powders decrease significantly after being prepared into effervescent powders. This invention provides an edible effervescent powder made from peaches and its preparation method. The invention aims to optimize the fermentation strains and systematically improve the effervescent process, ensuring that the resulting effervescent powder exhibits virtually no change in uric acid-lowering and antioxidant activities before and after processing. Specifically, the uric acid (UA) reduction retention rate reaches over 95%, and the changes in MDA and NOx remain around 100%, showing no significant difference compared to the original fermented freeze-dried powder. Furthermore, this invention aims to ensure that the effervescent powder meets product requirements in terms of conventional quality indicators (appearance, pH, disintegration time, foaming volume, and moisture content), and possesses good formulation robustness and batch stability, providing a feasible technical solution for the development of functional fermented product formulations.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing edible effervescent powder using peaches as raw material includes the following steps: (1) Prepare the following raw materials: 28-32 parts by weight of three-strain fermented freeze-dried powder, 25.0-26.0 parts by weight of citric acid, 1.5-3.0 parts by weight of malic acid, 24.0 parts by weight of sodium bicarbonate, 11.0-14.5 parts by weight of mannitol, 3.0-3.5 parts by weight of hydroxypropyl-β-cyclodextrin, 1.5 parts by weight of ascorbic acid, 0.03-0.06 parts by weight of disodium EDTA, 1.5 parts by weight of sweet orange flavor, and 0.5 parts by weight of magnesium stearate; (2) Pre-encapsulation of yeast powder: 2.0~2.5 parts by weight of hydroxypropyl-β-cyclodextrin and three-strain fermentation freeze-dried powder in step (1) are placed in purified water and stirred at 35°C for 3 hours. Then, the mixture is dried under reduced pressure or freeze-dried at below 40°C to obtain hydroxypropyl-β-cyclodextrin-encapsulated yeast powder, which is then passed through an 80-mesh sieve. (3) Acid granulation: Mix the included baking powder obtained in step (2), citric acid, malic acid, half of the amount of mannitol, ascorbic acid, disodium EDTA and the remaining 0.5~1.5 parts by weight of hydroxypropyl-β-cyclodextrin evenly, granulate in a fluidized bed granulator with anhydrous ethanol as binder, dry at a temperature not higher than 42°C until the moisture content is not higher than 1.5%, and granulate through a 16-mesh sieve; (4) Alkali granulation: Sodium bicarbonate and the other half of mannitol are granulated with anhydrous ethanol as a binder. The drying temperature is not higher than 42°C, and the granules are sieved through a 16-mesh sieve. (5) Mixing and tableting: The acid granules obtained in step (3), the alkali granules obtained in step (4), sweet orange flavor and magnesium stearate are mixed for 5 minutes. Under the conditions of relative humidity not exceeding 20%, constant temperature of 21℃ and tableting mold temperature not exceeding 30℃, the mixture is pressed into tablets on a single punch tablet press with a pressure of 50~80 kN. The tablets are then pulverized through 40 mesh to obtain effervescent powder. The three-strain fermented freeze-dried powder is obtained by the following preparation method: using peach pulp as the fermentation substrate, Escherichia coli Nissle 1917 is inoculated sequentially for light fermentation, and Lactobacillus plantarum JYLP-326, Pediococcus pentosaceus JYPR9330 and Lactobacillus plantarum CGMCC 1.557 are inoculated sequentially for deep fermentation, followed by pasteurization, centrifugation, pH adjustment and freeze-drying.

[0007] Preferably, the preparation method of the three-strain fermented freeze-dried powder in step (1) specifically includes: manually cutting peach flesh into 1-2 mm cubes, adding chitosan acetate solution and loquat peel extract freeze-dried powder aqueous solution, the amount of chitosan and loquat peel extract freeze-dried powder added being 0.2% of the weight of peach flesh, to obtain fermentation substrate; inoculating Escherichia coli Nissle 1917, the inoculation amount being 2% of the substrate volume, and culturing at 37℃ and 100 rpm for 24 hours with shaking; then inoculating Lactobacillus plantarum JYLP-326, Pediococcus pentosaceus JYPR9330 and Lactobacillus plantarum CGMCC 1.557, the inoculation amount of each bacterium being 2% of the substrate volume, the inoculation volume ratio being 1:1:1, purging the headspace with high-purity nitrogen for 30 seconds and then incubating at 30℃ for 6 days; pasteurizing the culture, centrifuging to obtain the supernatant, and using 0.1 M Adjust the pH to 6.0 with NaHCO3, freeze-dry, and pass through an 80-mesh sieve.

[0008] Preferably, the loquat peel extract freeze-dried powder in the loquat peel aqueous solution is prepared by the following method: dried loquat peel is added to distilled water at a weight-volume ratio of 1:10 and boiled for 30 minutes, filtered, and then freeze-dried to obtain loquat peel aqueous extract freeze-dried powder, which is then reconstituted with sterile water to prepare a 10% w / v aqueous solution before use.

[0009] Preferably, the amount of the three-strain fermented freeze-dried powder in step (1) is 32 parts by weight, the total amount of hydroxypropyl-β-cyclodextrin is 3.0 parts by weight, of which 2.5 parts by weight are used for pre-encapsulation of hydroxypropyl-β-cyclodextrin, and the remaining 0.5 parts by weight are hydroxypropyl-β-cyclodextrin.

[0010] Preferably, the air velocity of the fluidized bed granulation in steps (3) and (4) is 1.2 m / s.

[0011] Preferably, in step (1), the amount of citric acid is 26.0 parts by weight, the amount of malic acid is 2.0 parts by weight, and the amount of disodium ethylenediaminetetraacetate is 0.05 parts by weight.

[0012] Preferably, the process further includes a packaging step: encapsulating the obtained effervescent powder with aluminum foil blister packs in an environment with a relative humidity of less than 20%.

[0013] Preferably, the accelerated testing conditions for the packaging step are 40°C and 75% relative humidity for 3 months, during which the main component decreases by no more than 5%.

[0014] An edible effervescent powder made from peaches is prepared by the aforementioned method.

[0015] The aforementioned application of effervescent edible powder in the preparation of drugs or health products for lowering uric acid and / or antioxidation.

[0016] The beneficial effects of this invention are: This invention introduces *Lactobacillus plantarum* CGMCC 1.557 into a three-strain fermentation system. Without affecting the uric acid-lowering and antioxidant effects of the fermentation product, it induces the plant to secrete high-molecular-weight extracellular polysaccharides in situ. These polysaccharides are then pre-encapsulated with hydroxypropyl-β-cyclodextrin, granulated non-aqueously, dried at low temperature, combined with a compound acid source, chelated with disodium EDTA, and subject to strict temperature and humidity control, forming a multi-layered synergistic protection that achieves efficient retention of uric acid-lowering and antioxidant activities before and after effervescent processing. Experimental results show that the UA reduction retention rate of the effervescent powder in the embodiments of this invention reaches 96.8%~98.9%, and the changes in MDA and NOx are maintained between 98.6%~103.8%, showing no significant difference from the original fermented freeze-dried powder. In contrast, the UA reduction retention rate of conventional effervescent processes using two-strain fermented freeze-dried powder is only 32.8%~54.3%. Furthermore, the effervescent powder of this invention meets the product requirements in terms of conventional quality indicators such as disintegration time, foaming amount, and moisture content, providing a systematic strategy for the development of end formulations of functional fermented products by "improving the processing tolerance of fermented products and adapting the formulation process". Detailed Implementation

[0017] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are still within the scope of protection of the present invention.

[0018] The main raw materials and instruments used in the following embodiments and comparative examples of this invention: Main ingredients: Peaches: Locally available Longquan peaches; Loquat peel water extract: Dried loquat peel, boiled in distilled water at a weight-to-volume ratio of 1:10 for 30 minutes, filtered, and freeze-dried to obtain lyophilized loquat peel water extract powder, reconstituted with sterile water to prepare a 10% w / v aqueous solution before use; Chitosan: Deacetylation degree ≥85%, purchased from Sigma-Aldrich, dissolved in 1% v / v acetic acid solution to prepare a 2% w / v solution before use, and neutralized to pH=5.5 with sterile 1 mol / L sodium hydroxide solution before use; EcN inoculum: Escherichia coli Nissle 1917 (EcN), purchased from Shanghai Bohu Biotechnology Co., Ltd., cultured in LB medium at 37℃ to OD600 =1.0; *Lactobacillus plantarum* JYLP-326: purchased from Shandong Zhongke Jiayi Biotechnology Co., Ltd., preservation number CGMCC No. 18038, revived from a -80℃ glycerol stock bank (20% glycerol, -80℃), streaked on MRS agar plates, incubated at 30℃ for 48 h, single colonies were picked and inoculated into 50 mL MRS broth (100 mL Erlenmeyer flask), and incubated statically at 30℃ until OD600=2.0, as the inoculum for deep fermentation; *Pediococcus pentosaceus* JYPR9330: purchased from Shandong Zhongke Jiayi Biotechnology Co., Ltd., preservation number CGMCC No. 25554, revived and expanded using the same method as *Lactobacillus plantarum* JYLP-326; *Lactobacillus plantarum* CGMCC 1.557: purchased from Beijing Naphthalene Chemical Technology Co., Ltd., preservation number CGMCC 1.557, the resuscitation and expansion methods are the same as above, except that it is used in conjunction with the aforementioned two strains in the deep fermentation stage of the example; LB medium: Qingdao Haibo Biotechnology Co., Ltd., product number HB0128, prepared according to the instructions; MRS broth and MRS agar: Qingdao Haibo Biotechnology Co., Ltd., product numbers HB0386 and HB0385-1, prepared according to the instructions; anhydrous ethanol, glacial acetic acid, sodium hydroxide, sodium bicarbonate: analytical grade, Chengdu Kelong Chemical Co., Ltd.; citric acid, sodium bicarbonate: food grade, Henan Wanbang Industrial Co., Ltd., used after passing through an 80-mesh sieve; mannitol: food grade, Roquette, trade name PEARLITOL 25C, passed through an 80-mesh sieve; polyethylene glycol 6000: pharmaceutical grade, Liaoning Aoke Pharmaceutical Chemical Co., Ltd.; polyvinylpyrrolidone K30: pharmaceutical grade, BASF, trade name Kollidon 30; Hydroxypropyl-β-cyclodextrin: pharmaceutical grade, Xi'an Deli Biochemical Co., Ltd., degree of substitution 0.6~0.9, passed through 80 mesh sieve; Ascorbic acid: food grade, Shijiazhuang Weisheng Pharmaceutical Co., Ltd.; Disodium EDTA: analytical grade, Chengdu Kelong Chemical Co., Ltd.; Sweet orange flavor: food grade, Shenzhen Boton Flavoring Co., Ltd., item number BO-TO-1042; Magnesium stearate: pharmaceutical grade, Huzhou Zhanwang Pharmaceutical Co., Ltd., passed through 80 mesh sieve; Corn starch: food grade, Zhucheng Xingmao Corn Development Co., Ltd., prepared into 10% starch slurry as a water-based binder.

[0019] Main instruments: Electronic analytical balance, constant temperature shaker, ultra-clean workbench, anaerobic culture system (Mitsubishi Gas Chemical Co., Ltd.), fluidized bed granulator (Mini-Glabra), high-speed refrigerated centrifuge (Avanti J-26S XP), hot air circulating oven, high shear wet granulator, single punch tablet press, pH meter.

[0020] Fermented freeze-dried powder (double-strain fermented freeze-dried powder): (1) Preparation of fermentation substrate: First, wash the peaches with running tap water to remove dirt from the surface, then soak them in 70% (v / v) ethanol for 30 seconds for surface disinfection, and immediately rinse them three times with sterile distilled water; all subsequent operations are carried out in a clean bench; peel and pit the peaches aseptically, leaving only the pulp, and cut the pulp into uniform small cubes of about 1~2 mm by hand without adding water; add chitosan acetate solution and loquat peel aqueous solution, the weight of chitosan added is 0.2% of the weight of peach pulp, and the weight of loquat peel freeze-dried powder added is 0.2% of the weight of peach pulp; use the resulting mixture as the fermentation substrate; (2) Mild fermentation: The obtained fermentation substrate was inoculated with Escherichia coli Nissle 1917 that had been cultured overnight, with an inoculation amount of 2% (v / v) of the substrate volume, and placed in a constant temperature shaker at 37℃ and shaken at 100 rpm for 24 hours. (3) Deep fermentation: Inoculate the mildly fermented product with Lactobacillus plantarum JYLP-326 (inoculation amount is 2% v / v of substrate volume) and Pediococcus pentosacchari JYPR9330 (inoculation amount is 2% v / v of substrate volume). After inoculation, purge the headspace with high-purity nitrogen (≥99.99%) for 30 seconds to establish an anaerobic environment, and place it in a constant temperature incubator at 30℃ for 6 days for static culture; (4) The culture was first pasteurized, centrifuged at 4℃ and 8000rpm for 15 minutes and the supernatant was collected. The pH was adjusted dropwise with 0.1M NaHCO3 to 6.0, then freeze-dried, passed through an 80-mesh sieve, and sealed at -20℃ for later use.

[0021] Fermented freeze-dried powder (three-strain fermented freeze-dried powder): (1) Preparation of fermentation substrate: First, wash the peaches with running tap water to remove dirt from the surface, then soak them in 70% v / v ethanol for 30 seconds for surface disinfection, and immediately rinse them three times with sterile distilled water; all subsequent operations are carried out in a clean bench; peel and pit the peaches aseptically, leaving only the pulp, and cut the pulp into uniform small cubes of about 1~2 mm by hand without adding extra water; add chitosan acetate solution and loquat peel aqueous solution, the weight of chitosan added is 0.2% of the weight of peach pulp, and the weight of loquat peel freeze-dried powder added is 0.2% of the weight of peach pulp; use the resulting mixture as the fermentation substrate; (2) Mild fermentation: The obtained fermentation substrate was inoculated with Escherichia coli Nissle1917 that had been cultured overnight at an inoculation amount of 2% v / v of the substrate volume and placed in a constant temperature shaker at 37℃ and shaken at 100 rpm for 24 hours. (3) Deep fermentation: Inoculate the mildly fermented product with Lactobacillus plantarum JYLP-326 (inoculation amount is 2% v / v of substrate volume), Pediococcus pentosaceus JYPR9330 (inoculation amount is 2% v / v of substrate volume) and Lactobacillus plantarum CGMCC 1.557 (inoculation amount is 2% v / v of substrate volume), with an inoculation volume ratio of 1:1:1; after inoculation, purge the headspace with high-purity nitrogen (≥99.99%) for 30 seconds to establish an anaerobic environment, and place it in a constant temperature incubator at 30℃ for 6 days to obtain the three-strain fermented product; (4) The three-strain fermentation product was first pasteurized, centrifuged at 4℃ and 8000 rpm for 15 minutes and the supernatant was collected. The pH was adjusted dropwise with 0.1 M NaHCO3 to 6.0 and then freeze-dried to obtain the three-strain fermentation freeze-dried powder. It was passed through an 80-mesh sieve and sealed at -20℃ for later use.

[0022] Example 1

[0023] Prepare the following raw materials: 30.0 g of three-strain fermented freeze-dried powder, 26.0 g of citric acid, 2.0 g of malic acid, 24.0 g of sodium bicarbonate, 12.0 g of mannitol, 3.0 g of hydroxypropyl-β-cyclodextrin (of which 2.0 g is used for pre-encapsulation of the yeast powder and 1.0 g is added to the acid granules), 1.5 g of ascorbic acid, 0.05 g of disodium EDTA, 1.5 g of sweet orange flavor, and 0.5 g of magnesium stearate; the solid components should be sieved through an 80-mesh sieve for later use.

[0024] Preparation method: 1. Pre-encapsulation of yeast powder: 30 g of three-strain lyophilized yeast powder and 2.0 g of hydroxypropyl-β-cyclodextrin were placed in purified water and reacted at 35°C with stirring for 3 hours; then dried under reduced pressure or lyophilized at below 40°C to obtain hydroxypropyl-β-cyclodextrin-encapsulated yeast powder, which was then passed through an 80-mesh sieve. 2. Acid Granulation: Mix the included baking powder, citric acid, malic acid, half of the mannitol (6.0 g), ascorbic acid (1.5 g), disodium EDTA (0.05 g), and the remaining hydroxypropyl-β-cyclodextrin (1.0 g) evenly. Granulate with anhydrous ethanol in a fluidized bed granulator. Dry at a temperature not exceeding 42℃ and a wind speed of 1.2 m / s until the moisture content is not higher than 1.5%. Then, granulate through a 16-mesh sieve. 3. Alkali granulation: Sodium bicarbonate and the remaining 6.0 g of mannitol are granulated with anhydrous ethanol in the same way. The drying temperature is not higher than 42℃, and the granules are sieved through a 16-mesh sieve. 4. Mixing and tableting: Mix the acid granules, alkali granules, sweet orange flavoring, and magnesium stearate for 5 minutes; the relative humidity of the tableting operation environment should not exceed 20%, the temperature should be constant at 21℃, and the temperature of the tableting mold should not exceed 30℃ to avoid frictional heat during pressing. Press the tablets into tablets on a single-punch tableting machine with a pressure of 50~80 kN, and pulverize them through a 40-mesh sieve to obtain effervescent powder. 5. Packaging: Aluminum foil blister packaging, with relative humidity below 20%; accelerated testing conditions: 40℃ / 75% relative humidity for 3 months, with a main component decrease of no more than 5% as the quality control target.

[0025] Example 2

[0026] Prepare the following raw materials: 32.0 g of three-strain lyophilized powder, 25.5 g of citric acid, 1.5 g of malic acid, 24.0 g of sodium bicarbonate, 11.0 g of mannitol, 3.0 g of hydroxypropyl-β-cyclodextrin (of which 2.5 g is used for pre-encapsulation of the yeast powder and 0.5 g is added to the acid granules), 1.5 g of ascorbic acid, 0.06 g of disodium EDTA, 1.5 g of sweet orange flavor, and 0.5 g of magnesium stearate; the solid components should be sieved through an 80-mesh sieve for later use.

[0027] Preparation method: 1. Pre-encapsulation of yeast powder: 32 g of three-strain lyophilized yeast powder and 2.5 g of hydroxypropyl-β-cyclodextrin were placed in purified water and reacted at 35°C with stirring for 3 hours; then dried under reduced pressure or lyophilized at below 40°C to obtain hydroxypropyl-β-cyclodextrin-encapsulated yeast powder, which was then passed through an 80-mesh sieve. 2. Acid Granulation: Mix the included baking powder, citric acid, malic acid, half of the mannitol (5.5 g), ascorbic acid (1.5 g), disodium EDTA (0.06 g), and the remaining hydroxypropyl-β-cyclodextrin (0.5 g) evenly. Granulate with anhydrous ethanol in a fluidized bed granulator. Dry at a temperature not exceeding 42℃ and a wind speed of 1.2 m / s until the moisture content is not higher than 1.5%. Then, granulate through a 16-mesh sieve. 3. Alkali granulation: Sodium bicarbonate and the remaining 5.5 g of mannitol are granulated with anhydrous ethanol in the same way. The drying temperature is not higher than 42℃, and the granules are sieved through a 16-mesh sieve. 4. Mixing and tableting: Mix acid granules, alkali granules, sweet orange flavoring, and magnesium stearate for 5 minutes; the relative humidity of the tableting operation environment should not exceed 20%, the temperature should be constant at 21℃, and the temperature of the tableting mold should not exceed 30℃ to avoid frictional heat during pressing. Press the tablets into tablets on a single-punch tableting machine with a pressure of 50~80 kN, and pulverize them through 40 mesh to obtain effervescent powder. 5. Packaging: Aluminum foil blister packaging, with relative humidity below 20%; accelerated testing conditions: 40℃ / 75% relative humidity for 3 months, with a main component decrease of no more than 5% as the quality control target.

[0028] Example 3

[0029] Prepare the following raw materials: 28.0 g of three-strain lyophilized powder, 25.0 g of citric acid, 3.0 g of malic acid, 24.0 g of sodium bicarbonate, 14.5 g of mannitol, 3.5 g of hydroxypropyl-β-cyclodextrin (of which 2.0 g is used for pre-encapsulation of the yeast powder and 1.5 g is added to the acid granules), 1.5 g of ascorbic acid, 0.03 g of disodium EDTA, 1.5 g of sweet orange flavor, and 0.5 g of magnesium stearate; the solid components should be sieved through an 80-mesh sieve for later use.

[0030] Preparation method: 1. Pre-encapsulation of yeast powder: 28 g of three-strain lyophilized yeast powder and 2.0 g of hydroxypropyl-β-cyclodextrin were placed in purified water and reacted at 35°C with stirring for 3 hours; then dried under reduced pressure or lyophilized at below 40°C to obtain hydroxypropyl-β-cyclodextrin-encapsulated yeast powder, which was then passed through an 80-mesh sieve. 2. Acid Granulation: Mix the included baking powder, citric acid, malic acid, half of the mannitol (7.25 g), ascorbic acid (1.5 g), disodium EDTA (0.03 g), and the remaining hydroxypropyl-β-cyclodextrin (1.5 g) evenly. Granulate with anhydrous ethanol in a fluidized bed granulator. Dry at a temperature not exceeding 42℃ and a wind speed of 1.2 m / s until the moisture content is not higher than 1.5%. Then, granulate through a 16-mesh sieve. 3. Alkali granulation: Sodium bicarbonate and the remaining 7.25 g of mannitol are granulated with anhydrous ethanol in the same way. The drying temperature is not higher than 42℃, and the granules are sieved through a 16-mesh sieve. 4. Mixing and tableting: Mix acid granules, alkali granules, sweet orange flavoring, and magnesium stearate for 5 minutes; the relative humidity of the tableting operation environment should not exceed 20%, the temperature should be constant at 21℃, and the temperature of the tableting mold should not exceed 30℃ to avoid frictional heat during pressing. Press the tablets into tablets on a single-punch tableting machine with a pressure of 50~80 kN, and pulverize them through 40 mesh to obtain effervescent powder. 5. Packaging: Aluminum foil blister packaging, with relative humidity below 20%; accelerated testing conditions: 40℃ / 75% relative humidity for 3 months, with a main component decrease of no more than 5% as the quality control target.

[0031] Comparative Example 1 Prepare the following raw materials: 30.0 g of double-strain fermented freeze-dried powder, 30.0 g of citric acid, 27.0 g of sodium bicarbonate, 10.0 g of sucrose, 5.0 g of lactose, 2.0 g of polyethylene glycol 6000, 1.5 g of sweet orange flavoring, and 0.5 g of magnesium stearate; pass the solid components through an 80-mesh sieve for later use.

[0032] Preparation method: 1. Mixing: Place the double-strain fermented freeze-dried powder, citric acid, sodium bicarbonate, sucrose, lactose and polyethylene glycol 6000 into a three-dimensional motion mixer and mix three times at 25 rpm for 10 minutes each time; 2. Wet granulation: Using 10% starch slurry as a binder, spray it into the mixed powder, granulate it in a high-shear wet granulator at 300 rpm for 3 minutes, and pass it through a 16-mesh sieve to obtain wet granules; 3. Drying: Place the wet granules in a hot air circulating oven and dry them at 60℃ until the moisture content is no more than 2%, which takes about 2 to 3 hours; 4. Granulation: The dried granules are granulated by passing them through a 16-mesh sieve; 5. Tableting and pulverizing: After granulation, add sweet orange flavoring and magnesium stearate, mix for 5 minutes, and compress into tablets with a pressure of 50~80 kN on a single punch tablet press. The tablet weight difference should be within ±5%. Further pulverize and pass through a 40-mesh sieve to obtain effervescent powder.

[0033] Comparative Example 2 Ingredients: 30.0 g of double-strain fermented freeze-dried powder, 28.0 g of citric acid, 25.0 g of sodium bicarbonate, 10.0 g of maltodextrin, 4.0 g of polyethylene glycol 6000, 2.0 g of polyethylene glycol 4000, 2.0 g of ascorbic acid, 1.5 g of sweet orange flavor, and 0.5 g of magnesium stearate; solid components are sieved through an 80-mesh sieve for later use.

[0034] Preparation method: 1. Melt coating: Weigh 4.0 g of polyethylene glycol 6000 from the formula, and add 2.0 g of polyethylene glycol 4000 as a melt medium supplement; place both in a 65℃ water bath to melt, then pour the mixed powder of double-fermented freeze-dried powder, citric acid, sodium bicarbonate and maltodextrin into the melted polyethylene glycol, and stir continuously for 5~8 minutes to make the coating uniform, then pour it into a -20℃ environment for rapid cooling and solidification; 2. Crushing: The solidified packaged block is crushed and passed through a 16-mesh sieve; 3. Mixing and tableting: Add ascorbic acid, 1.5 g of sweet orange flavoring and 0.5 g of magnesium stearate to the pulverized material, mix for 5 minutes, and then compress into tablets with a pressure of 50~80 kN on a single punch tablet press or pulverize through 40 mesh to obtain effervescent powder.

[0035] Comparative Example 3 Raw materials: 30.0 g of double-strain fermented freeze-dried powder, 25.0 g of citric acid, 3.0 g of malic acid, 24.0 g of sodium bicarbonate, 12.0 g of mannitol, 2.0 g of ascorbic acid, 0.05 g of disodium EDTA, 1.5 g of sweet orange flavor, and 0.5 g of magnesium stearate; solid components are sieved through an 80-mesh sieve for later use.

[0036] Preparation method: 1. Acid Granulation: Weigh 30.0 g of double-strain fermented freeze-dried powder, 25.0 g of citric acid, 3.0 g of malic acid, 6.0 g of mannitol, 2.0 g of ascorbic acid, and 0.05 g of disodium EDTA. Mix them evenly and spray granulation with a 1% PVP K30 anhydrous ethanol solution as a binder. Pass the granules through a 16-mesh sieve and dry them in a hot air circulating oven at 50°C until the moisture content is no higher than 3%. Then, granulate them through a 16-mesh sieve. 2. Alkali granulation: Weigh 24.0 g of sodium bicarbonate and 6.0 g of mannitol, granulate with 1% PVP K30 anhydrous ethanol solution in the same way, dry at 50℃, and granulate with 16 mesh. 3. Mixing and tableting: Mix acid granules, alkali granules, sweet orange flavoring, and magnesium stearate for 5 minutes, then compress them into tablets with a pressure of 50~80kN on a single punch tablet press or pulverize them through a 40-mesh sieve to obtain effervescent powder.

[0037] Comparative Example 4 Prepare the following raw materials: 30.0 g of three-strain fermented freeze-dried powder, 27.0 g of citric acid, 24.0 g of sodium bicarbonate, 13.0 g of mannitol, 2.0 g of hydroxypropyl-β-cyclodextrin, 1.0 g of ascorbic acid, 1.5 g of sweet orange flavor, and 0.5 g of magnesium stearate; pass the solid components through an 80-mesh sieve for later use.

[0038] Effervescent powder preparation method: 1. Acid Granulation: Mix the three-strain fermented freeze-dried powder, citric acid, half of mannitol (6.5 g), hydroxypropyl-β-cyclodextrin (2.0 g), and ascorbic acid (1.0 g) evenly. Use anhydrous ethanol as a binder. If necessary, add 1% PVP K30 anhydrous ethanol solution. Granulate in a fluidized bed granulator. Dry at a temperature not higher than 45℃ and a wind speed of 1.0~1.5 m / s until the moisture content is not higher than 2%. Sieve through a 16-mesh granulator.

[0039] 2. Alkali granulation: Sodium bicarbonate and the remaining 6.5 g of mannitol are granulated with anhydrous ethanol in the same way. The drying temperature is not higher than 45℃, and the granules are sieved through a 16-mesh sieve.

[0040] 3. Mixing and tableting: Mix acid granules, alkali granules, sweet orange flavoring, and magnesium stearate for 5 minutes, then compress them into tablets with a pressure of 50~80kN on a single punch tablet press or pulverize them through a 40-mesh sieve to obtain effervescent powder; the relative humidity of the operating environment should not exceed 30%, and the temperature should be 20~25℃.

[0041] 4. Packaging: Packaged in aluminum foil bags or blister packs in an environment with relative humidity below 20%.

[0042] Experimental Example 1 This experimental example examines the conventional properties of the effervescent powders obtained in each embodiment and comparative example, as detailed below.

[0043] 1. Samples: Effervescent powders of Comparative Examples 1-4 (D1-D4) and Effervescent powders of Examples 1-3 (S1-S3). Three batches were prepared in parallel for each group, and 20 samples were randomly selected from each batch for testing. The results are expressed as mean ± standard deviation.

[0044] 2. Testing Items and Methods 2.1 Appearance Visual inspection: Effervescent powder should be a dry, loose powder or granules with a uniform color, free from moisture absorption, clumping, and foreign matter. Take about 5 g and spread it evenly on white paper, then observe it under natural light.

[0045] 2.2 Solution pH Take 1 g of effervescent powder and add it to 20 mL of distilled water (37℃). After complete disintegration, measure the pH of the solution using a pH meter (FE28, Mettler Toledo). Repeat each group 3 times.

[0046] 2.3 Disintegration Time Limit The procedure was performed according to the 2020 edition of the Chinese Pharmacopoeia. Specifically, 1 g of effervescent powder was placed in a 250 mL beaker, and 200 mL of distilled water (37℃) was added. The time from contact with the water surface to complete disintegration (no visible particles) was recorded. Six samples were measured for each group, and the average value was taken.

[0047] 2.4 Foaming Amount According to the 2020 edition of the Chinese Pharmacopoeia, specifically, take 1 g of effervescent powder, place it in a 250 mL stoppered graduated cylinder, add 20 mL of distilled water (37℃), quickly seal the cylinder, record the maximum gas volume (mL), measure 6 samples for each group, and take the average value.

[0048] 2.5 Moisture Referring to the 2020 edition of the Chinese Pharmacopoeia, specifically, take about 1 g of effervescent powder, accurately weigh it, dry it at 105℃ to constant weight, and calculate the percentage loss (%). Measure 3 samples per group and take the average.

[0049] 3. Experimental Results and Analysis The experimental results are shown in Table 1.

[0050] Table 1

[0051] Based on the test results, all comparative examples (D1-D4) and examples (S1-S3) appeared as light yellow powders or granules, without severe clumping or foreign matter; the solution pH was between 4.0 and 4.5, indicating weak acidity suitable for oral administration; the disintegration time was between 41 and 108 seconds, meeting the requirement of ≤120 seconds for conventional effervescent powders; the foaming volume was between 11.8 and 21.5 mL, all ≥10 mL; and the moisture content was between 1.2 and 2.8%, all ≤3%. Therefore, all effervescent powders met the product requirements in terms of conventional quality indicators and possessed basic formulation usability.

[0052] Experiment Example 2 This experiment tested the uric acid-lowering and antioxidant properties of the effervescent powders obtained in each embodiment and comparative example.

[0053] 1. Reagents and Materials Preparation of potassium oxonate suspension: Accurately weigh 300 mg of potassium oxonate and place it in a 15 mL centrifuge tube. Add 0.5% sodium carboxymethyl cellulose carrier solution to a final volume of 10 mL. Vigorously disperse the suspension on a vortex mixer until no obvious particles are visible. Store in the dark for later use. Vortex again for 10 to 15 seconds before use.

[0054] Preparation of xanthine suspension: Accurately weigh 150 mg of xanthine and place it in a 15 mL centrifuge tube. Add 0.5% sodium carboxymethyl cellulose carrier solution to a final volume of 10 mL. Vigorously disperse the suspension on a vortex mixer until no obvious particles are visible. Store for later use. Vortex again for 10 to 15 seconds before use.

[0055] Preparation of allopurinol positive control suspension: Accurately weigh 10 mg of allopurinol and place it in a 15 mL centrifuge tube. First, dissolve it with a very small amount of 0.1 mol / L sodium hydroxide solution, and then make up to 10 mL with 0.5% sodium carboxymethyl cellulose carrier solution to achieve a final concentration of 1 mg / mL. Adjust the pH to 8.5.

[0056] Preparation of 0.5% sodium carboxymethyl cellulose carrier solution: Weigh 0.5 g of sodium carboxymethyl cellulose and place it in a 250 mL beaker. Add 100 mL of distilled water preheated to about 80 degrees Celsius and stir with a glass rod until completely dissolved and transparent. Then cool to room temperature and gel overnight in a 4-degree Celsius refrigerator. Set aside for use. When using, return to room temperature.

[0057] Preparation of reconstituted solution of lyophilized fermentation powder: Take appropriate amounts of lyophilized dual-strain fermentation powder and lyophilized triple-strain fermentation powder respectively, and reconstitute them with purified water to make the concentration of fermentation powder 30 mg / mL. Vortex for 30 seconds until completely dissolved. Prepare fresh solution before use.

[0058] Preparation of effervescent powder reconstituted solution: Take 1 g of each of the effervescent powders from Comparative Examples 1 to 4 and Examples 1 to 3, add 5 mL of warm water (37°C), vortex for 30 seconds until completely disintegrated and dissolved, and prepare fresh before use.

[0059] 2. Animal grouping and dosing regimen Male Kunming mice, weighing 22 to 25 grams, were selected and randomly divided into 11 groups of 10 mice each after 7 days of acclimatization. All mice were fasted for 12 hours before the experiment, but water was allowed. On the day of the experiment, the weight of each mouse was weighed to calculate the precise volume of drug administered.

[0060] The NC group served as the model control group. Mice in this group were first injected intraperitoneally with potassium oxonate suspension (300 mg / kg), and 30 minutes later, they were administered xanthine suspension (150 mg / kg) by gavage, along with an equal volume of 0.5% sodium carboxymethyl cellulose carrier solution.

[0061] The PC group served as the positive control group. Mice in this group were first injected intraperitoneally with potassium oxonate suspension (300 mg / kg), and 30 minutes later, they were administered xanthine suspension (150 mg / kg) by gavage, along with allopurinol suspension at a dose of 5 mg / kg.

[0062] Group B1 was the baseline group for the lyophilized double-fermented powder. Mice in this group were first injected intraperitoneally with potassium oxonate suspension (300 mg / kg), and 30 minutes later, they were administered xanthine suspension (150 mg / kg) by gavage, along with a reconstituted solution of the lyophilized double-fermented powder. The dosage was calculated based on the equivalent of the fermentation powder (150 mg / kg).

[0063] Group B2 was the baseline group for the three-strain fermented lyophilized powder. Mice in this group were intraperitoneally injected with potassium oxonate suspension (300 mg / kg), gavaged with xanthine suspension (150 mg / kg), and simultaneously gavaged with a reconstituted solution of the three-strain fermented lyophilized powder. The dosage was calculated based on the equivalent of the fermented powder (150 mg / kg).

[0064] Group D1 was the effervescent powder group of Comparative Example 1. Mice in this group were injected intraperitoneally with potassium oxonate suspension, gavaged with xanthine suspension, and simultaneously gavaged with reconstituted effervescent powder of Comparative Example 1. The dosage was calculated based on the equivalent of the fermentation powder, and the dosage and administration time of each suspension and fermentation method were the same as those of Group B1.

[0065] Group D2 was the control group of effervescent powder. Mice in this group were injected intraperitoneally with potassium oxonate suspension, gavaged with xanthine suspension, and simultaneously gavaged with a reconstituted solution of control group 2 effervescent powder. The dosage and administration time of each suspension and fermentation method were the same as those in Group B1.

[0066] Group D3 was the comparative example 3 effervescent powder group. Mice in this group were injected intraperitoneally with potassium oxonate suspension, gavaged with xanthine suspension, and simultaneously gavaged with comparative example 3 effervescent powder reconstituted solution. The dosage and administration time of each suspension and fermentation method were the same as those in Group B1.

[0067] Group D4 was the control group of effervescent powder 4. Mice in this group were injected intraperitoneally with potassium oxonate suspension, gavaged with xanthine suspension, and simultaneously gavaged with control group 4 effervescent powder reconstituted solution. The dosage and administration time of each suspension and fermentation method were the same as those of group B1.

[0068] Group S1 was the effervescent powder group of Example 1. Mice in this group were injected intraperitoneally with potassium oxonate suspension, gavaged with xanthine suspension, and simultaneously gavaged with reconstituted effervescent powder of Example 1. The dosage and administration time of each suspension and fermentation method were the same as those of Group B1.

[0069] Group S2 was the effervescent powder group of Example 2. Mice in this group were injected intraperitoneally with potassium oxonate suspension, gavaged with xanthine suspension, and simultaneously gavaged with reconstituted effervescent powder of Example 2. The dosage and administration time of each suspension and fermentation method were the same as those of Group B1.

[0070] Group S3 was the effervescent powder group of Example 3. Mice in this group were injected intraperitoneally with potassium oxonate suspension, gavaged with xanthine suspension, and simultaneously gavaged with reconstituted effervescent powder of Example 3. The dosage and administration time of each suspension and fermentation method were the same as those of Group B1.

[0071] All test substances were reconstituted with purified water to the required concentration, and the gavage volume was 0.1 mL / 10 g body weight. The concentrations of the reconstituted solutions of the lyophilized fermentation powder and the effervescent powder were converted to the same amount based on the fermentation powder equivalent to ensure that the dosage was the same for each group.

[0072] 3. Sampling Two hours after modeling, during the peak uric acid window, blood was collected from all mice. The orbital venous plexus blood collection method was used. After mild anesthesia with isoflurane, approximately 0.3 to 0.5 mL of blood was collected by inserting a heparinized capillary glass tube into the orbital venous plexus. The blood was allowed to coagulate at room temperature for 30 minutes, then centrifuged at 3000 to 4000 rpm for 15 minutes at 4°C. The serum was separated, aliquoted, and stored at -80°C for later use.

[0073] 4. Detection indicators Serum uric acid was measured using a uricase peroxidase assay kit. Specifically, 10 μL of serum was mixed with 200 μL of working reagent, incubated at 37°C for 10 to 15 minutes, and the absorbance was read at 510 nm. The uric acid concentration was calculated based on the standard curve. The serum uric acid level in the model group should be more than twice that of the normal control group, i.e., not less than 240 μmol / L, to indicate successful model establishment.

[0074] Determination of serum malondialdehyde (MDA): The thiobarbituric acid method was used. Specifically, 100 μL of serum sample was taken, 1.0 mL of thiobarbituric acid chromogenic solution was added, and the mixture was heated in a 95°C water bath for 60 minutes. After cooling to room temperature with running water, 1.0 mL of n-butanol was added for extraction, vortexed for 30 seconds, and then centrifuged at 3000 rpm for 10 minutes. The upper organic phase was collected, and the absorbance was measured at a wavelength of 532 nm. The MDA concentration was calculated using a standard curve, with units of nmol / L.

[0075] Serum nitric oxide determination: The Griess method was used. Specifically, 100 μL of serum sample was taken and an equal volume of Griess reagent was added. This reagent contains 1% sulfonamide, 0.1% naphthylethylenediamine hydrochloride, and 2.5% phosphoric acid. After reacting at room temperature in the dark for 10 minutes, the absorbance was measured at a wavelength of 540 nm. The nitric oxide concentration was calculated using the sodium nitrite standard curve, and the unit is μmol / L.

[0076] 5. Data Calculation and Statistics Calculation of UA reduction retention rate: Using the serum UA reduction in the corresponding baseline group as a 100% baseline, the UA reduction retention rate for each effervescent powder group was calculated. Group B1 was used as the baseline reference for groups D1, D2, and D3, and group B2 was used as the baseline reference for groups D4, S1, S2, and S3. The formula for calculating the UA reduction retention rate is: UA reduction retention rate = 1 minus the difference between the mean UA value of the effervescent powder group and the mean UA value of the corresponding baseline group, divided by the difference between the mean UA value of the NC group and the mean UA value of the corresponding baseline group, then multiplied by 100%.

[0077] Calculation of MDA change rate: With the measured MDA value of the corresponding baseline group as 100%, calculate the MDA change rate of each group; the formula for calculating the MDA change rate is: the MDA change rate equals the mean MDA value of the experimental group divided by the mean MDA value of the corresponding baseline group, and then multiplied by 100%. NOx change rate calculation: The NOx change rate of each group is calculated with the measured NOx value of the corresponding baseline group as 100%. The formula for calculating the NOx change rate is: the NOx change rate is equal to the mean NOx value of the experimental group divided by the mean NOx value of the corresponding baseline group, and then multiplied by 100%.

[0078] All data are expressed as mean plus or minus standard deviation; comparisons among multiple groups were performed using one-way ANOVA with Tukey post-hoc test, and a p-value less than 0.05 was considered statistically significant.

[0079] 6. Experimental Results and Analysis Table 2

[0080] First, baseline data show no significant difference in the bulk activity of the two-strain fermented lyophilized powder (Group B1) and the three-strain fermented lyophilized powder (Group B2) in terms of uric acid reduction and antioxidant activity. The serum UA in Group B1 was 178±2 μmol / L, MDA was 7.4±0.3 nmol / mL, and NOx was 27.6±0.2 μmol / L; the corresponding values ​​in Group B2 were 176±3 μmol / L, 7.3±0.2 nmol / mL, and 27.4±0.3 μmol / L, respectively, with no statistically significant difference between the two groups (p>0.05). This phenomenon may be because the main contribution of CGMCC 1.557 lies in the in situ secretion of extracellular polysaccharides (EPS) to form a protective "armor" (see Experimental Example 3), rather than significantly enhancing the bulk uric acid-reducing or antioxidant activity of the fermentation broth. Therefore, the equivalence of bulk activity between B1 and B2 lays a fair baseline for subsequent comparisons of the effects of effervescent processing on the activity of the two types of fermented products.

[0081] Secondly, the data from Comparative Examples 1–3 (D1–D3) showed that the effervescent powder produced using the dual-strain fermentation freeze-dried powder via conventional effervescent processing exhibited a significant decrease in its uric acid-lowering and antioxidant activities. In group D1, serum UA increased to 296±18 μmol / L, with a UA reduction retention rate of only 36.6%, while the MDA change rate was as high as 131.1% and the NOx change rate reached 146.4%. The deterioration was even more severe in group D2, with serum UA reaching 303±20 μmol / L, a UA reduction retention rate of only 32.8%, and MDA and NOx change rates increasing to 135.1% and 151.5%, respectively. In group D3, the activity retention improved somewhat, with a UA reduction retention rate of 54.3%, and MDA and NOx change rates of 119.2% and 133.6%, respectively, but it was still significantly worse than baseline (p<0.01). These three comparative studies share the common characteristic of using dual-strain fermented freeze-dried powder and employing conventional effervescent processes—whether water-based bonding + 60°C drying, PEG melt encapsulation at 65°C, or ethanol granulation + 50°C drying—none of these methods effectively protected the heat-sensitive and acid-base-sensitive active ingredients in the fermented product. The lack of an extracellular polysaccharide protective layer in dual-strain fermented freeze-dried powder exposes the active ingredients directly to the high-temperature, humid, and acid-base reaction environments during processing, leading to irreversible degradation of uric acid-lowering and antioxidant substances.

[0082] Comparative Example 4 (D4) used a three-strain fermented lyophilized powder, but the effervescent process was adjusted. The serum UA in this group was 229±14 μmol / L, with a UA reduction retention rate of 71.8%, and MDA and NOx change rates of 109.6% and 118.2%, respectively. Although the activity retention rate of D4 was significantly higher than that of D1-D3 (p<0.01), it was still significantly lower than the baseline of B2 (p<0.05), indicating that even with the use of the three-strain fermented lyophilized powder, without the proprietary process of this patent, it is still difficult to achieve efficient retention of active ingredients. The results of D4 reveal a key fact: although the EPS secreted in situ by CGMCC 1.557 during the three-strain fermentation process can provide some protection for the active ingredients (significantly improved compared to the two-strain unprotected method), this protective effect is limited. EPS mainly forms complexes with phenols and small molecule metabolites through hydrogen bonds, playing a physical buffering role in processing. However, in the absence of exogenous inclusion protection (such as HP-β-CD pre-inclusion) and metal ion chelating agents (EDTA), some active ingredients may still be lost due to thermal degradation, acid-base pre-reaction, or the action of residual polyphenol oxidase.

[0083] In contrast, the effervescent powders of Examples 1-3 (S1-S3) showed excellent effects in lowering uric acid and antioxidant activity that were almost consistent with the baseline. In Group S1, serum UA was 180±3 μmol / L, with a UA reduction retention rate of 97.9%, and MDA and NOx change rates of 101.4% and 102.2%, respectively, showing no significant difference from the baseline of B2 (p>0.05). The results of Groups S2 and S3 were highly consistent with S1, with UA reduction retention rates of 98.9% and 96.8%, respectively, and MDA and NOx change rates between 98.6% and 103.8%. The differences between the three groups were no greater than 2 percentage points, and none were significantly different from B2.

[0084] The reason why the present invention can achieve essentially no loss of activity before and after effervescent processing may be due to the synergistic effect of three layers of protection: The first layer is the EPS secreted in situ by CGMCC 1.557 during the fermentation of the three bacteria, which forms a hydrogen bond complex with the active ingredient in advance, providing basic processing tolerance; The second layer is HP-β-CD pre-encapsulation, which encapsulates chlorogenic acid, catechins and small molecule metabolites through hydrophobic cavities to form a stable inclusion complex. Even if the EPS complex is partially dissociated in subsequent processing, the encapsulated active ingredient is still in a "protective chamber"; The third layer is the strictly controlled process parameters - non-aqueous granulation avoids acid-base pre-reactions caused by water, the drying temperature ≤42℃ falls within the safety window of heat-sensitive metabolites, malic acid buffers the initial dissolution pH, EDTA chelates residual copper ions to inhibit the activity of polyphenol oxidase, the tableting mold temperature is controlled at ≤30℃ to eliminate frictional heat, and the packaging environment RH≤20% eliminates moisture absorption pre-reactions during storage. These three layers of protection form a progressive defense system, ensuring the integrity of the active ingredients throughout the entire processing chain.

[0085] In summary, the experimental results in Table 2 clearly reveal the following patterns: the activity of the dual-strain fermented lyophilized powder decreased significantly under conventional effervescent processes (UA reduction retention rate 32.8%~54.3%); the activity of the triple-strain fermented lyophilized powder improved under process downgrading conditions but still showed significant loss (UA reduction retention rate 71.8%); only the triple-strain fermented lyophilized powder of this patent, combined with a dedicated effervescent process, can achieve highly efficient retention of uric acid-lowering and antioxidant activities (UA reduction retention rate ≥96.8%, MDA and NOx change rates both close to 100%), and this effect exhibits good formulation robustness. These results provide a systematic strategy for the development of functional fermented product formulations: "improved fermented product processing tolerance + formulation process adaptation".

[0086] Experimental Example 3 This experiment investigates the extracellular polysaccharides in lyophilized powders fermented with two strains and lyophilized powders fermented with three strains, in order to support the mechanism mentioned in Experiment 2.

[0087] 1. Determination of extracellular polysaccharide content (phenol-sulfuric acid method) The content of extracellular polysaccharides in lyophilized powder fermented by three bacteria was determined using the phenol-sulfuric acid method. This method is based on the hydrolysis of polysaccharides in concentrated sulfuric acid to form monosaccharides, which are then dehydrated to form furfural derivatives. The furfural derivatives react with phenol to form an orange-yellow compound with a characteristic absorption at 490 nm.

[0088] 1.1 Reagent Preparation: Glucose standard (Sigma, catalog number G8270) was dried to constant weight at 105℃. 10.0 mg was accurately weighed and placed in a 100 mL volumetric flask, dissolved in distilled water, and diluted to the mark to obtain a 100 μg / mL glucose standard stock solution. Before use, this stock solution was diluted to 10, 20, 40, 60, 80, and 100 μg / mL. Phenol solution was prepared by mixing 80 g of analytical grade phenol with 20 g of distilled water to prepare an 80% phenol solution, which was stored protected from light. Before use, it was diluted with distilled water to a 5% phenol solution. Concentrated sulfuric acid was analytical grade, with a density of 1.84 g / mL.

[0089] 1.2 Sample Pretreatment: Accurately weigh approximately 50 mg of the three-strain fermentation lyophilized powder and place it in a 10 mL centrifuge tube. Add 5 mL of distilled water, vortex for 30 seconds to disperse evenly, and centrifuge at 8000 rpm for 15 minutes at 4℃. Collect the supernatant. Add anhydrous ethanol to the supernatant to a final volume fraction of 80%, mix well, and let stand at 4℃ for 12 hours to precipitate the polysaccharide. Centrifuge at 8000 rpm for 15 minutes, discard the supernatant, and wash the precipitate twice with 80% ethanol, centrifuging after each wash. Vacuum dry the precipitate at 40℃ to constant weight to obtain crude polysaccharide. Redissolve the crude polysaccharide in distilled water to 1.0 mL as the sample solution to be tested.

[0090] 1.3 Operating Steps: Take 1.5 mL centrifuge tubes and set up standard tubes, sample tubes, and blank tubes respectively. Add 0.5 mL of glucose standard working solution to the standard tube, 0.5 mL of the sample solution to be tested to the sample tube, and 0.5 mL of distilled water to the blank tube. Add 0.5 mL of 5% phenol solution to each tube, mix well, and then quickly add 2.5 mL of concentrated sulfuric acid and mix well. Let stand at room temperature for 30 minutes, and measure the absorbance value at 490 nm. Plot a standard curve with glucose concentration as the abscissa and absorbance as the ordinate, and perform linear regression. Substitute the sample absorbance into the standard curve to calculate the polysaccharide content. The result is expressed as the number of milligrams of extracellular polysaccharide contained in each gram of lyophilized fermentation powder (mg / g). Each group is repeated 3 times, and the mean value is taken.

[0091] 2. Determination of extracellular polysaccharide molecular weight (HPLC-RI method) The weight-average molecular weight of extracellular polysaccharides was determined using high performance liquid chromatography-refractive index detection (HPLC-RI).

[0092] Instruments and chromatographic conditions: A high-performance liquid chromatograph (Agilent 1260 Infinity II) equipped with a differential refractive index detector (RI) and a TSK-GEL G5000PWxl gel chromatography column (7.8 mm × 300 mm, particle size 7 μm) was used. The mobile phase was 0.1 mol / L phosphate buffer (pH 7.0), the flow rate was 0.5 mL / min, the column temperature was 35 °C, and the injection volume was 20 μL.

[0093] Standards: Pullulan series standards (Shodex, molecular weight range 5.9×10³ ~ 7.86×10³) 5 The sample (Da) was prepared into a 1 mg / mL solution using the mobile phase, filtered through a 0.22 μm filter membrane, and injected. The retention time was recorded, and a standard curve was plotted using the logarithm of molecular weight versus the retention time.

[0094] Sample preparation: Dissolve and dilute the crude polysaccharide (extracted by the same phenol-sulfuric acid method) in the mobile phase to 1 mg / mL, filter through a 0.22 μm filter membrane, and inject 20 μL. Record the chromatogram and calculate the weight-average molecular weight (Mw) based on the standard curve.

[0095] 3. Determination of monosaccharide composition of extracellular polysaccharides (PMP derivatization-HPLC method) The monosaccharide composition of extracellular polysaccharides was determined by 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization combined with high performance liquid chromatography.

[0096] Polysaccharide hydrolysis: Take about 5 mg of crude polysaccharide and place it in an ampoule. Add 1 mL of 2 mol / L trifluoroacetic acid (TFA), seal the ampoule with nitrogen, and hydrolyze at 110℃ for 4 hours. After cooling, dry the ampoule with nitrogen at 40℃, add 1 mL of methanol, and dry it again. Repeat this process three times to remove all TFA. Dissolve the residue in 0.5 mL of 0.3 mol / L NaOH solution to obtain the hydrolysis product solution.

[0097] PMP derivatives: Take 100 μL of the hydrolysis product solution, add 100 μL of 0.5 mol / L PMP methanol solution, mix well, and react in a water bath at 70℃ for 60 minutes; after cooling, add 100 μL of 0.3 mol / L HCl solution to neutralize, then add 1 mL of chloroform for extraction, vortex for 30 seconds, centrifuge at 3000 rpm for 5 minutes, discard the organic phase, and repeat the extraction three times; filter the aqueous phase through a 0.22 μm filter membrane for analysis.

[0098] Monosaccharide standards: Standard monosaccharides (Sigma) such as mannose, galactose, glucose, arabinose, rhamnose, and xylose were prepared into 1 mg / mL solutions with distilled water and subjected to PMP derivatization using the same method.

[0099] Chromatographic conditions: The high-performance liquid chromatograph (Agilent 1260 Infinity II) was equipped with a UV detector at a wavelength of 250 nm. The column was a Zorbax Eclipse XDB-C18 column (4.6 mm × 250 mm, particle size 5 μm). Mobile phase A was 0.1 mol / L phosphate buffer (pH 7.0), and mobile phase B was acetonitrile. The gradient elution program was as follows: 0–10 min, phase B 16%; 10–30 min, phase B 16%–20%; 30–40 min, phase B 20%–25%; 40–45 min, phase B 25%–16%. The flow rate was 1.0 mL / min, the column temperature was 30 °C, and the injection volume was 20 μL.

[0100] Calculation: Qualitative analysis was performed using the retention time of each monosaccharide standard, and the molar percentage of each monosaccharide was calculated using the peak area normalization method.

[0101] Table 3

[0102] Table 4

[0103] The data in Tables 3 and 4 show that the average content of extracellular polysaccharides in the three-strain fermented freeze-dried powder is 9.2–9.8 mg / g, and the weight-average molecular weight is approximately 1.12 × 10⁻⁶. 6 The monosaccharide molar ratio of Da was mannose:galactose:glucose = 5.5:7.4:1.0, and there were no significant differences in content, molecular weight, and monosaccharide composition among different batches (p>0.05), indicating that CGMCC 1.557 had stable EPS production performance in the three-strain fermentation system. No extracellular polysaccharides were detected in the dual-strain fermented lyophilized powder (content was below the detection limit of 0.1 mg / g). The EPS content of each group (B2, D4, S1~S3) of the three-strain fermented lyophilized powder was not less than 8 mg / g.

[0104] The EPS data in Tables 3 and 4 provide direct molecular evidence to explain the differences in the activity retention rates of the groups in Table 2 in Experimental Example 2. The dual-strain fermented freeze-dried powder contains no EPS (B1 and D1~D3). Therefore, during effervescent processing, the active ingredients are exposed to high temperature, humidity, and acidic / alkaline environments. The UA reduction retention rate of D1~D3 is only 32.8%~54.3%, while the changes in MDA and NOx are significantly increased. The EPS content in the triple-strain fermented freeze-dried powder is consistently above 9 mg / g (B2, D4, S1~S3). During processing, EPS can provide basic protection for the active ingredients through hydrogen bonding. Therefore, the UA reduction retention rate of D4 (triple-strain but with process downgrade) is increased to 71.8%, significantly better than D1~D3. However, while the EPS content of D4 was similar to that of S1-S3 (9.1-9.8 mg / g), the UA reduction retention rate of S1-S3 was as high as 96.8%-98.9%, while that of D4 was only 71.8%. This difference indicates that the protective effect of EPS has an upper limit—EPS alone is insufficient to achieve lossless retention of activity. It must work synergistically with HP-β-CD pre-encapsulation, EDTA chelation, malic acid compounding, and strict process control to increase the activity retention rate to over 95%. There was no significant correlation between the EPS content and activity retention rate of S1-S3 (r=0.15, p>0.05), further demonstrating that when the pre-encapsulation and strict process control are in place, individual differences in EPS content are no longer a limiting factor for activity retention.

Claims

1. A method for preparing edible effervescent powder using peaches as raw material, characterized in that, Includes the following steps: (1) Prepare the following raw materials: 28-32 parts by weight of three-strain fermented freeze-dried powder, 25.0-26.0 parts by weight of citric acid, 1.5-3.0 parts by weight of malic acid, 24.0 parts by weight of sodium bicarbonate, 11.0-14.5 parts by weight of mannitol, 3.0-3.5 parts by weight of hydroxypropyl-β-cyclodextrin, 1.5 parts by weight of ascorbic acid, 0.03-0.06 parts by weight of disodium EDTA, 1.5 parts by weight of sweet orange flavor, and 0.5 parts by weight of magnesium stearate; (2) Pre-encapsulation of yeast powder: 2.0~2.5 parts by weight of hydroxypropyl-β-cyclodextrin and three-strain fermentation freeze-dried powder in step (1) are placed in purified water and stirred at 35°C for 3 hours. Then, the mixture is dried under reduced pressure or freeze-dried at below 40°C to obtain hydroxypropyl-β-cyclodextrin-encapsulated yeast powder, which is then passed through an 80-mesh sieve. (3) Acid granulation: Mix the included baking powder obtained in step (2), citric acid, malic acid, half of the amount of mannitol, ascorbic acid, disodium EDTA and the remaining 0.5~1.5 parts by weight of hydroxypropyl-β-cyclodextrin evenly, granulate in a fluidized bed granulator with anhydrous ethanol as binder, dry at a temperature not higher than 42°C until the moisture content is not higher than 1.5%, and granulate through a 16-mesh sieve; (4) Alkali granulation: Sodium bicarbonate and the other half of mannitol are granulated with anhydrous ethanol as a binder. The drying temperature is not higher than 42°C, and the granules are sieved through a 16-mesh sieve. (5) Mixing and tableting: The acid granules obtained in step (3), the alkali granules obtained in step (4), sweet orange flavor and magnesium stearate are mixed for 5 minutes. Under the conditions of relative humidity not exceeding 20%, constant temperature of 21℃ and tableting mold temperature not exceeding 30℃, the mixture is pressed into tablets on a single punch tablet press with a pressure of 50~80 kN. The tablets are then pulverized through 40 mesh to obtain effervescent powder. The three-strain fermented freeze-dried powder is obtained by the following preparation method: using peach pulp as the fermentation substrate, Escherichia coli Nissle 1917 is inoculated sequentially for light fermentation, and Lactobacillus plantarum JYLP-326, Pediococcus pentosaceus JYPR9330 and Lactobacillus plantarum CGMCC 1.557 are inoculated sequentially for deep fermentation, followed by pasteurization, centrifugation, pH adjustment and freeze-drying.

2. The preparation method according to claim 1, characterized in that, The preparation method of the three-strain fermented freeze-dried powder in step (1) specifically includes: manually cutting peach flesh into 1-2 mm cubes, adding chitosan acetate solution and loquat peel extract freeze-dried powder aqueous solution, the amount of chitosan and loquat peel extract freeze-dried powder added is 0.2% of the weight of peach flesh, to obtain fermentation substrate; inoculating Escherichia coli Nissle 1917, the inoculation amount is 2% of the substrate volume, and culturing at 37℃ and 100 rpm for 24 hours with shaking; then inoculating Lactobacillus plantarum JYLP-326, Pediococcus pentosaceus JYPR9330 and Lactobacillus plantarum CGMCC 1.557, the inoculation amount of each strain is 2% of the substrate volume, the inoculation volume ratio is 1:1:1, purging the headspace with high-purity nitrogen for 30 seconds and then incubating at 30℃ for 6 days; pasteurizing the culture, centrifuging to obtain the supernatant, and using 0.1 M Adjust the pH to 6.0 with NaHCO3, freeze-dry, and pass through an 80-mesh sieve.

3. The preparation method according to claim 2, characterized in that, The loquat peel extract freeze-dried powder in the loquat peel aqueous solution is prepared by the following method: dried loquat peel is added to distilled water at a weight-volume ratio of 1:10 and boiled for 30 minutes. After filtration, it is freeze-dried to obtain loquat peel aqueous extract freeze-dried powder. Before use, it is reconstituted with sterile water to prepare a 10% w / v aqueous solution.

4. The preparation method according to claim 1, characterized in that, The amount of the three-strain fermented freeze-dried powder used in step (1) is 32 parts by weight, and the total amount of hydroxypropyl-β-cyclodextrin is 3.0 parts by weight, of which 2.5 parts by weight are used for pre-encapsulation of hydroxypropyl-β-cyclodextrin, and the remaining 0.5 parts by weight are hydroxypropyl-β-cyclodextrin.

5. The preparation method according to claim 1, characterized in that, The air velocity for fluidized bed granulation in steps (3) and (4) is 1.2 m / s.

6. The preparation method according to claim 1, characterized in that, In step (1), the amount of citric acid used is 26.0 parts by weight, the amount of malic acid used is 2.0 parts by weight, and the amount of disodium ethylenediaminetetraacetate used is 0.05 parts by weight.

7. The preparation method according to claim 1, characterized in that, It also includes a packaging step: the resulting effervescent powder is encapsulated in aluminum foil blister packs in an environment with a relative humidity of less than 20%.

8. The preparation method according to claim 7, characterized in that, The accelerated testing conditions for the packaging step are 40°C and 75% relative humidity for 3 months, during which the main component decreases by no more than 5%.

9. An edible effervescent powder made from peaches, characterized in that, Prepared by the method according to any one of claims 1 to 8.

10. The use of the edible effervescent powder according to claim 9 in the preparation of uric acid-lowering and / or antioxidant drugs or health products.

Images