Active probiotic soft sweets containing hyaluronic acid-alginic acid composite embedding system as well as preparation method and application of active probiotic soft sweets

Abstract

The invention discloses active probiotic soft sweets containing a hyaluronic acid-alginic acid composite embedding system as well as a preparation method and application thereof, and belongs to the technical field of functional food processing, the preparation method comprises the following steps: pre-inducing probiotics, preparing a composite gel core, constructing a hyaluronic acid adsorption shell, and mixing and forming; the pre-induction of the probiotics comprises the steps of activation and fermentation; the construction of the hyaluronic acid adsorption shell comprises the following steps: mixing soybean protein, citrus pectin and water, stirring at room temperature, adding hyaluronic acid, and stirring to obtain a coating layer solution; and mixing the cross-linked gel balls with the coating layer solution, stirring at room temperature, filtering, taking filter residues, washing, and freeze-drying to obtain the probiotic microcapsule. The prepared active probiotic soft sweets are high in viable organism survival rate and good in storage stability, have the efficacy of regulating constipation or improving skin and can tolerate the gastrointestinal tract environment, and special materials are not introduced in the preparation process.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of functional food processing, in particular to an active probiotic soft candy containing a hyaluronic acid-alginic acid complex embedding system, a preparation method and application thereof. BACKGROUND

[0002] As a food form with both pleasant taste and health function, probiotic soft candy has become an important development direction in the field of functional food. However, as active microorganisms, probiotics are easily inactivated in the processing (high-temperature boiling, acidic environment) of soft candy, storage (room temperature, water activity fluctuation) and human digestive tract (stomach acid, bile salt), which leads to substandard viable count of the final product and makes it difficult to exert the expected health efficacy.

[0003] In the prior art, the use of a single embedding material (such as sodium alginate) is a common protection method. For example, Chinese patent CN113875870B discloses an active probiotic soft candy and a preparation method thereof. The active probiotic soft candy is a soft candy in which Lactobacillus bulgaricus is embedded with sodium alginate, pectin and whey protein. This technology improves the survival rate of probiotics to a certain extent. However, this system has obvious deficiencies. First, the pectin-alginate network may still undergo acid-promoted hydrolysis, leading to structural disintegration and attenuation of the protective effect over time (the data shows that the viable count decreases by 6 orders of magnitude after 3 months) when the embedding structure is stored for a long time in a high-acid (pH 3.0-4.0) environment. Second, the introduction of whey protein enhances the gel, but may bring about an off-flavor and is not friendly to some vegetarian consumers.

[0004] Studies have shown that the introduction of trehalose and hyaluronic acid into calcium alginate gel can significantly improve the survival rate of Lactobacillus rhamnosus during freeze-drying and storage. This reveals the potential of synergistic protection of small molecule sugars and high molecular weight polysaccharides. However, this study only stays at the stage of freeze-dried powder and does not solve the application problem in soft candy, which is a complex system with high moisture, high sugar and high acid. If hyaluronic acid is directly used in soft candy, its high hydrophilicity and viscosity will seriously affect the texture and pouring forming of the soft candy.

[0005] Chinese patent CN120203154A discloses an active probiotic soft candy for improving intestinal health and enhancing immunity and a high-temperature preparation method thereof. This patent provides a preparation idea for a high-temperature-resistant Bacillus coagulans soft candy, which controls the spore state through "pre-activation" and "passivation" processes. However, this method is only suitable for Bacillus, and is not suitable for Lactobacillus and Bifidobacterium, which are more common but have poor acid resistance and are sensitive to heat and water activity.

[0006] In summary, the common problems and defects of the existing technology are as follows: Single protection dimension: Most technologies only rely on physical embedding, lack of strengthening strategies from the physiological state of probiotics themselves, leading to "inherent deficiency" of bacteria in subsequent stress.

[0007] Embedding materials are not resistant to complex environment: The single gel network is not stable in the long-term acidic environment of soft candy and digestive juice, and is easy to disintegrate.

[0008] Poor universality of process and formula: Many methods are only suitable for specific strains (such as spore-forming bacteria), or face cost, taste or regulatory restrictions due to the introduction of special materials (such as collagen, specific wall materials).

[0009] Single function: The technical solution only focuses on "protecting live bacteria", and fails to simultaneously give the product additional health value-added.

[0010] In summary, the existing technology lacks a comprehensive embedding solution that can be universally applied to various types of probiotics (especially non-sporulating bacteria) and can simultaneously resist the high temperature of soft candy processing, the acidic environment during storage, and the erosion of digestive juice. Therefore, developing a new type of composite embedding system with multiple protection functions and successfully integrating it into the soft candy matrix is a technical problem that needs to be solved in this field. SUMMARY

[0011] In view of the deficiencies of the prior art, the present application provides a kind of active probiotic soft candy containing hyaluronic acid-alginic acid composite embedding system, preparation method and application thereof, the active probiotic soft candy prepared has high survival rate of live bacteria, good storage stability, multiple functions, can resist gastrointestinal environment, and does not introduce special materials in preparation.

[0012] To solve the above technical problems, the technical solutions adopted by the present application are as follows: A preparation method of a kind of active probiotic soft candy containing hyaluronic acid-alginic acid composite embedding system, comprising: pre-inducing probiotics, preparing a composite gel core, constructing a hyaluronic acid adsorption shell, and mixing and forming; The pre-induced probiotics include: activation and fermentation. The activation activates the probiotics to obtain a probiotic seed solution with a concentration of 1×10 8 CFU / mL-5×10 8 CFU / mL. The probiotics are Bifidobacterium longum with strain number CICC 6198 and / or Lactobacillus rhamnosus with strain number CICC 6137, which are purchased through market channels. The fermentation inoculates the probiotic seed solution into a modified MRS liquid medium, anaerobically cultures at 37-38℃, centrifugally collects the bacteria, washes with physiological saline, and then resuspends in physiological saline to a concentration of 1×10 8CFU / mL-5x10 8 CFU / mL of probiotic bacteria liquid; In the fermentation, when the probiotic seed liquid is inoculated into the modified MRS liquid medium, the inoculation amount is 2-3%; When anaerobic culture is carried out at 37-38℃, the anaerobic culture time is 13-15h; The formula of the modified MRS liquid medium is: 9.5-10.5g peptone, 9.5-10.5g beef extract, 4.8-5.2g yeast extract, 1.9-2.1g diammonium hydrogen citrate, 1.9-2.1g potassium phosphate dibasic, 4.8-5.2g sodium acetate, 19-21g glucose, 15-16g trehalose, 50-60mL composite sodium hyaluronate solution, 0.9-1.1mL Tween 80, 0.55-0.6g magnesium sulfate, 0.24-0.26g manganese sulfate, 1000-1100mL water, pH 6.7-6.9; The preparation method of the composite sodium hyaluronate solution is: after mixing sodium hyaluronate and water, stirring until completely dissolved, adding calcium chloride, stirring for 40-60min, adding sodium L-glutamate, stirring for 40-60min, to obtain the composite sodium hyaluronate solution; In the preparation of the composite sodium hyaluronate solution, the amount ratio of sodium hyaluronate, water, calcium chloride, sodium L-glutamate is 1.5-1.6g:50-55mL:0.09-0.1g:5-5.5g; The number average molecular weight of the sodium hyaluronate is 10kDa; The preparation of the composite gel core includes: preparing a mixed glue solution, crosslinking; In the preparation of the mixed glue solution, sodium alginate, hyaluronic acid, citrus pectin, yeast beta-glucan and water are mixed, and then stirred at 75-80℃ until a uniform sol is formed, and then cooled to room temperature; In the preparation of the mixed glue solution, the mass ratio of sodium alginate, hyaluronic acid, citrus pectin, yeast beta-glucan and water is 1.5-1.6:1-1.1:1-1.2:1-1.2:95.5-98; The number average molecular weight of the hyaluronic acid is 300-500kDa; The crosslinking is carried out by stirring the mixed glue solution at a stirring speed of 200-400rpm at room temperature, then adding the probiotic bacteria liquid dropwise into the mixed glue solution, continuing to stir for 1-1.5h after the dropwise addition is completed, to obtain a mixed bacteria liquid; the mixed bacteria liquid is loaded into a syringe of an electrostatic droplet generation device, and is dropped into a composite calcium chloride aqueous solution by electrostatic droplet, while the composite calcium chloride aqueous solution is stirred at a stirring speed of 200-300rpm, and after the electrostatic droplet is completed, the stirring is continued for 30-50min, then filtration is carried out, the filter cake is taken, and a crosslinked gel ball is obtained; The volume ratio of the mixed glue solution to the probiotic bacterial solution is 9-10:1 in the crosslinking; The dropwise adding time of the probiotic bacterial solution is 30-60 min; The mass ratio of the mixed bacterial solution to the composite calcium chloride aqueous solution is 100:450-500; The inner diameter of the needle of the syringe in the electrostatic dropping is 0.4-0.5 mm; The voltage of the electrostatic drop generation device in the electrostatic dropping is 8 kV, and the needle drop speed is 14-15 drops / min; The composite calcium chloride aqueous solution is a mixed aqueous solution of calcium chloride and whey protein hydrolysate, wherein the mass fraction of calcium chloride is 3-4%, the mass fraction of whey protein hydrolysate is 1.4-1.5%, and the number average molecular weight of polypeptides in the whey protein hydrolysate is 1800; The pH of the PBS buffer is 7.2, and the molar concentration is 0.01 mol / L; In the construction of the hyaluronic acid adsorption shell, soybean protein, citrus pectin and water are mixed, stirred at a stirring speed of 200-500 rpm at room temperature for 40-60 min, hyaluronic acid is added, and stirred for 40-60 min to obtain a coating layer solution; the crosslinked gel ball is mixed with the coating layer solution, stirred at a stirring speed of 30-60 rpm at room temperature for 40-60 min, filtered, the filter residue is washed with water for 2-4 times, and freeze-dried to obtain the probiotic microcapsule; In the construction of the hyaluronic acid adsorption shell, the mass ratio of soybean protein, citrus pectin and water is 5.5-6:3.8-4:1000-1200; The mass ratio of soybean protein to hyaluronic acid is 5.5-6:10-11; The dosage ratio of the crosslinked gel ball to the coating layer solution is 50-60 g:1000-1200 mL; The temperature of the freeze-drying is -45℃ to -40℃, and the freeze-drying time is 28-30 h; In the mixing and molding, maltitol, gelatin, yeast beta-glucan and water are mixed, stirred at a stirring speed of 100-200 rpm at 70-80℃ for 40-60 min, boiled until the soluble solid content is 76-80%, cooled to 65-70℃, citric acid, strawberry essence and rice bran fatty alkanol are added, stirred at a stirring speed of 100-200 rpm for 30-50 min, cooled to 40-45℃, the probiotic microcapsule is added, stirred at a stirring speed of 100-200 rpm for 10-15 min, poured into a mold, cooled and shaped, dried to a moisture content of 17-19%, demolded, and the active probiotic soft candy is obtained; In the mixed molding process, the mass ratio of maltitol, gelatin, yeast β-glucan, and water is 30-32:8-9:2-2.2:60-65. The mass ratio of maltitol, citric acid, strawberry flavoring, and rice bran fatty alkyl alcohol is 30-32:0.5-0.6:1-1.1:0.02; The mass ratio of maltitol to probiotic microcapsules is 30-32:2-2.2.

[0013] The cooking temperature when the soluble solids content is 76-80% is 105-108℃. The cooling and shaping temperature is -5℃ to -3℃; The drying process, which involves drying to a moisture content of 17-19%, takes place at a temperature of 24-26°C and an environment with a relative humidity of 44-46%.

[0014] An active probiotic gummy prepared by the aforementioned preparation method.

[0015] Application of an active probiotic gummy prepared by the aforementioned method in solving problems of constipation and dull, dry skin.

[0016] The present invention provides a method for preparing active probiotic gummies containing a hyaluronic acid-alginic acid composite encapsulation system. By constructing a three-level protection system of "cell pre-induction - composite gel core - functional adsorption shell", the probiotics are protected step by step. Hyaluronic acid, an ingredient with clear health benefits, is compounded in the encapsulation system and the gummy matrix to achieve the functional integration of "probiotics+".

[0017] In the pre-induction of cells, a modified MRS medium containing trehalose and a compound sodium hyaluronate solution was used. Trehalose and hyaluronic acid are commonly used as cryoprotectants for bacteria. Trehalose has the function of stabilizing biofilm and protein structure, resisting stress and preserving freshness. Moreover, trehalose can penetrate into the cell interior and fill around active macromolecules such as proteins. When drying and losing water, the hydroxyl groups of trehalose can form hydrogen bonds with the polar groups of biomolecules, replacing the water molecules lost around the polar groups, thereby maintaining the stability of protein structure. The hydrogen bond between trehalose and the polar groups of phospholipids in the biofilm can prevent the membranes from getting close to each other due to dehydration, thereby inhibiting membrane fusion. Hyaluronic acid can form a stable glassy state on the cell surface, protecting the cells. However, the effects of trehalose and hyaluronic acid on improving the acid stress resistance of bacteria are limited. Therefore, the applicant added monosodium glutamate (MSG) and calcium ions. However, experiments showed that directly adding MSG, calcium ions, trehalose, and hyaluronic acid to MRS medium had limited effects and could not provide sustained acid stress resistance. This is because the cells have limited adsorption and fixation capacity for MSG, calcium ions, trehalose, and hyaluronic acid. Therefore, this invention first adds L-... Monosodium glutamate (MSG), calcium ions, and hyaluronic acid are mixed. Through the interaction between calcium ions, L-MSG, and hyaluronic acid, a complex is formed. During culture, cells first absorb trehalose, and then the complex formed by calcium ions, L-MSG, and hyaluronic acid coats the cell surface, protecting the cells. In an acidic environment, the calcium ions, L-MSG, and hyaluronic acid in the complex adsorb the acid. At the same time, the interaction between the complexes weakens, allowing cells to absorb L-MSG. L-MSG binds to protons, further reducing the impact of acid on the cells.

[0018] In the formation of the functional adsorption shell, soy protein and citrus pectin were first used. Soy protein and citrus pectin can act as emulsifiers for Pickering emulsions, forming micelles in water. Then, they are mixed with hyaluronic acid, which not only forms a film with hyaluronic acid, but the presence of hydrophobic groups can also improve the water resistance of the hyaluronic acid adsorption shell.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention avoids cost, taste, or regulatory limitations in the preparation of active probiotic gummies by not introducing special materials (such as collagen or specific wall materials). The prepared active probiotic gummies also have the following advantages: high live bacteria survival rate, with a live bacteria count reaching 1.1 × 10⁻⁶. 8 CFU / g -3.2×10 8CFU / g; good storage stability, after 30 days of storage at 37℃ and 75% relative humidity, the viable count retention rate is 89.2-93.5%, and after 90 days of storage at 37℃ and 75% relative humidity, the viable count retention rate is 74.1-79.8%; it can tolerate the gastrointestinal environment, after standing in artificial gastric fluid for 2 hours and then in artificial intestinal fluid for 2 hours, the viable count retention rate is 67.7-70.8%; it has multiple functions and has excellent ability to solve problems of difficult defecation and dull and dry skin. Detailed Implementation

[0020] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0021] Example 1 A method for preparing active probiotic gummies containing a hyaluronic acid-alginic acid complex encapsulation system, specifically comprising: Step 1. Pre-induction of probiotics: Step 1. Activation: One loopful of *Bifidobacterium longum* was streaked onto MRS solid medium and anaerobically cultured at 37°C for 24 hours. Single colonies with good growth were then picked and inoculated onto MRS liquid medium and anaerobically cultured at 37°C to achieve a bacterial concentration of 1×10⁻⁶. 8 CFU / mL probiotic seed liquid; The strain number of the Bifidobacterium longum is CICC 6198, which was obtained through commercial channels; The formulation of the MRS solid culture medium is as follows: 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium hydrogen citrate, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 20g glucose, 1mL Tween 80, 0.58g magnesium sulfate, 0.25g manganese sulfate, 18g agar powder, 1000mL water, pH 6.8. The formulation of the MRS solid culture medium is as follows: 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium hydrogen citrate, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 20g glucose, 1mL Tween 80, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1000mL water, pH 6.8. Step 2. Fermentation: Inoculate the probiotic seed culture at an inoculum rate of 2% into modified MRS liquid medium, and anaerobically culture at 37°C for 13 hours. Collect the bacterial cells by centrifugation, wash with physiological saline, and then resuspend in physiological saline to achieve a bacterial concentration of 1×10⁻⁶. 8 CFU / mL probiotic solution; The modified MRS liquid culture medium formula is as follows: 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium hydrogen citrate, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 20g glucose, 15g trehalose, 50mL compound sodium hyaluronate solution, 1mL Tween 80, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1000mL water, pH 6.8; The method for preparing the composite sodium hyaluronate solution is as follows: 1.5g of sodium hyaluronate and 50mL of water are mixed and stirred until completely dissolved. 0.09g of calcium chloride is added and stirred for 40min. 5g of L-sodium glutamate is added and stirred for 40min to obtain the composite sodium hyaluronate solution. The number-average molecular weight of the sodium hyaluronate is 10 kDa; Step 2. Preparation of the composite gel core: Step 1. Prepare the mixed adhesive solution: Sodium alginate, hyaluronic acid, citrus pectin, yeast β-glucan, and water were mixed in a mass ratio of 1.5:1:1:1:95.5 and stirred at 75°C until a uniform sol was formed. The mixture was then cooled to room temperature. The number-average molecular weight of the hyaluronic acid is 300 kDa; Step 2. Crosslinking: The mixed gel solution was stirred at 200 rpm at room temperature. Then, probiotic solution was added dropwise to the mixed gel solution, controlling the volume ratio of the mixed gel solution to the probiotic solution to be 9:1. The dropwise addition time of the probiotic solution was 30 min. After the dropwise addition was completed, stirring was continued for 1 h to obtain the mixed bacterial solution. The mixed bacterial solution was loaded into the syringe of the electrostatic drop generating device and electrostatically dropped into the composite calcium chloride aqueous solution, controlling the mass ratio of the mixed bacterial solution to the composite calcium chloride aqueous solution to be 100:450. At the same time, the composite calcium chloride aqueous solution was stirred at 200 rpm. During the electrostatic dropwise addition, the inner diameter of the syringe needle was controlled to be 0.4 mm, the voltage of the electrostatic drop generating device was controlled to be 8 kV, and the needle drop rate was 14 drops / min. After the electrostatic dropwise addition was completed, stirring was continued for 30 min. After filtration, the filter cake was collected to obtain cross-linked gel spheres. The composite calcium chloride aqueous solution is a mixed aqueous solution of calcium chloride and whey protein hydrolysate, wherein the mass fraction of calcium chloride is 3%, the mass fraction of whey protein hydrolysate is 1.4%, and the number average molecular weight of the polypeptides in the whey protein hydrolysate is 1800. The PBS buffer has a pH of 7.2 and a molar concentration of 0.01 mol / L; Step 3. Constructing the hyaluronic acid adsorption shell: Soy protein, citrus pectin, and water were mixed at a mass ratio of 5.5:3.8:1000 and stirred at 200 rpm for 40 min at room temperature. Hyaluronic acid was then added, and the mass ratio of soy protein to hyaluronic acid was controlled at 5.5:10. The mixture was stirred for another 40 min to obtain the coating layer solution. Cross-linked gel spheres were mixed with the coating layer solution at a volume ratio of 50 g:1000 mL and stirred at 30 rpm for 40 min at room temperature. The mixture was then filtered, and the residue was washed twice with water and freeze-dried at -45℃ for 28 h to obtain probiotic microcapsules. Step 4. Mixing and Molding: Maltitol, gelatin, yeast β-glucan, and water were mixed in a mass ratio of 30:8:2:60 and stirred at 100 rpm for 40 minutes at 70°C. The mixture was then cooked at 105°C until the soluble solids content reached 76%. The mixture was cooled to 65°C, and citric acid, strawberry flavor, and rice bran fatty alkyl alcohol were added. The mass ratio of maltitol, citric acid, strawberry flavor, and rice bran fatty alkyl alcohol was controlled at 30:0.5:1:0.02. The mixture was stirred at 100 rpm for 30 minutes and cooled to 40°C. Probiotic microcapsules were added, and the mass ratio of maltitol to probiotic microcapsules was controlled at 30:2. The mixture was stirred at 100 rpm for 10 minutes and poured into molds. The mixture was cooled and shaped at -4°C and then dried at 25°C and 45% relative humidity until the moisture content reached 17%. The mixture was then demolded to obtain active probiotic gummies.

[0022] This embodiment also provides an active probiotic gummies prepared by the aforementioned preparation method.

[0023] Example 2 A method for preparing active probiotic gummies containing a hyaluronic acid-alginic acid complex encapsulation system, specifically comprising: Step 1. Pre-induction of probiotics: Step 1. Activation: One loopful of *Lactobacillus rhamnosus* was streaked onto MRS solid medium and anaerobically cultured at 38°C for 25 hours. Single colonies with good growth were then picked and inoculated onto MRS liquid medium and anaerobically cultured at 38°C to achieve a bacterial concentration of 1×10⁻⁶. 8 CFU / mL probiotic seed liquid; The strain number of the Lactobacillus rhamnosus is CICC 6137, which was obtained through commercial channels; The formulation of the MRS solid culture medium is as follows: 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium hydrogen citrate, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 20g glucose, 1mL Tween 80, 0.58g magnesium sulfate, 0.25g manganese sulfate, 18g agar powder, 1000mL water, pH 6.8. The formulation of the MRS solid culture medium is as follows: 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium hydrogen citrate, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 20g glucose, 1mL Tween 80, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1000mL water, pH 6.8. Step 2. Fermentation: Inoculate the probiotic seed culture at an inoculum rate of 3% into modified MRS liquid medium, and anaerobically culture at 38℃ for 15 h. Collect the bacterial cells by centrifugation, wash with physiological saline, and then resuspend in physiological saline to achieve a bacterial concentration of 1×10⁻⁶. 8 CFU / mL probiotic solution; The modified MRS liquid culture medium formula is as follows: 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium citrate, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 20g glucose, 16g trehalose, 60mL compound sodium hyaluronate solution, 1mL Tween 80, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1000mL water, pH 6.8; The method for preparing the composite sodium hyaluronate solution is as follows: 1.6g of sodium hyaluronate and 55mL of water are mixed and stirred until completely dissolved. 0.1g of calcium chloride is added and stirred for 60min. 5.5g of L-glutamate is added and stirred for 60min to obtain the composite sodium hyaluronate solution. The number-average molecular weight of the sodium hyaluronate is 10 kDa; Step 2. Preparation of the composite gel core: Step 1. Prepare the mixed adhesive solution: Sodium alginate, hyaluronic acid, citrus pectin, yeast β-glucan, and water were mixed in a mass ratio of 1.6:1.1:1.2:1.2:98 and stirred at 80°C until a uniform sol was formed. The mixture was then cooled to room temperature. The number-average molecular weight of the hyaluronic acid is 500 kDa; Step 2. Crosslinking: The mixed gel solution was stirred at 400 rpm at room temperature. Then, probiotic solution was added dropwise to the mixed gel solution, controlling the volume ratio of the mixed gel solution to the probiotic solution to be 10:1. The dropwise addition time of the probiotic solution was 60 min. After the dropwise addition was completed, stirring was continued for 1.5 h to obtain the mixed bacterial solution. The mixed bacterial solution was loaded into the syringe of the electrostatic drop generating device and electrostatically dropped into the composite calcium chloride aqueous solution, controlling the mass ratio of the mixed bacterial solution to the composite calcium chloride aqueous solution to be 100:500. At the same time, the composite calcium chloride aqueous solution was stirred at 300 rpm. During the electrostatic dropwise addition, the inner diameter of the syringe needle was controlled to be 0.5 mm, and the voltage of the electrostatic drop generating device was controlled to be 8 kV. The needle dropwise drop rate was 15 drops / min. After the electrostatic dropwise addition was completed, stirring was continued for 50 min. After filtration, the filter cake was collected to obtain cross-linked gel spheres. The composite calcium chloride aqueous solution is a mixed aqueous solution of calcium chloride and whey protein hydrolysate, wherein the mass fraction of calcium chloride is 4%, the mass fraction of whey protein hydrolysate is 1.5%, and the number average molecular weight of the polypeptides in the whey protein hydrolysate is 1800. The PBS buffer has a pH of 7.2 and a molar concentration of 0.01 mol / L; Step 3. Constructing the hyaluronic acid adsorption shell: Soy protein, citrus pectin, and water were mixed at a mass ratio of 6:4:1200 and stirred at 500 rpm for 60 min at room temperature. Hyaluronic acid was then added, and the mass ratio of soy protein to hyaluronic acid was controlled at 6:11. The mixture was stirred for another 60 min to obtain the coating layer solution. Cross-linked gel balls were mixed with the coating layer solution at a volume ratio of 60 g:1200 mL and stirred at 60 rpm for 60 min at room temperature. The mixture was then filtered, and the residue was washed four times with water and freeze-dried at -40℃ for 30 h to obtain probiotic microcapsules. Step 4. Mixing and Molding: Maltitol, gelatin, yeast β-glucan, and water were mixed in a mass ratio of 32:9:2.2:65 and stirred at 200 rpm for 60 minutes at 80°C. The mixture was then cooked at 108°C until the soluble solids content reached 80%. The mixture was cooled to 70°C, and citric acid, strawberry flavor, and rice bran fatty alkyl alcohol were added. The mass ratio of maltitol, citric acid, strawberry flavor, and rice bran fatty alkyl alcohol was controlled at 32:0.6:1.1:0.02. The mixture was stirred at 200 rpm for 50 minutes and cooled to 45°C. Probiotic microcapsules were added, and the mass ratio of maltitol to probiotic microcapsules was controlled at 32:2.2. The mixture was stirred at 200 rpm for 15 minutes and poured into molds. The mixture was cooled and shaped at -4°C and then dried at 25°C and 45% relative humidity until the moisture content reached 19%. The mixture was then demolded to obtain active probiotic gummies.

[0024] This embodiment also provides an active probiotic gummies prepared by the aforementioned preparation method.

[0025] Example 3 A method for preparing active probiotic gummies containing a hyaluronic acid-alginic acid complex encapsulation system, specifically comprising: Step 1. Pre-induction of probiotics: Step 1. Activation: One loopful of *Bifidobacterium longum* was streaked onto MRS solid medium and anaerobically cultured at 37°C for 24 hours. Single colonies with good growth were then picked and inoculated onto MRS liquid medium and anaerobically cultured at 37°C to achieve a bacterial concentration of 1×10⁻⁶. 8 CFU / mL of probiotic seed culture; one loop of *Lactobacillus rhamnosus* was inoculated into MRS solid medium and streaked, then anaerobically cultured at 37℃ for 24 h. Single colonies with good growth were picked and inoculated into MRS liquid medium, then anaerobically cultured at 37℃ to achieve a bacterial concentration of 1×10⁻⁶. 8 CFU / mL of probiotic seed solution No. 2; mix probiotic seed solution No. 1 and probiotic seed solution No. 2 at a volume ratio of 1:1 and stir at 30 rpm for 30 min at room temperature to obtain probiotic seed solution. The strain number of Bifidobacterium longum is CICC 6198, and the strain number of Lactobacillus rhamnosus is CICC6137. Both were purchased through commercial channels. The formulation of the MRS solid culture medium is as follows: 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium hydrogen citrate, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 20g glucose, 1mL Tween 80, 0.58g magnesium sulfate, 0.25g manganese sulfate, 18g agar powder, 1000mL water, pH 6.8. The formulation of the MRS solid culture medium is as follows: 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium hydrogen citrate, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 20g glucose, 1mL Tween 80, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1000mL water, pH 6.8. Step 2. Fermentation: Inoculate the probiotic seed culture at an inoculum rate of 2% into modified MRS liquid medium, and anaerobically culture at 37°C for 13 hours. Collect the bacterial cells by centrifugation, wash with physiological saline, and then resuspend in physiological saline to achieve a bacterial concentration of 1×10⁻⁶. 8 CFU / mL probiotic solution; The modified MRS liquid culture medium formula is as follows: 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium hydrogen citrate, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 20g glucose, 15g trehalose, 50mL compound sodium hyaluronate solution, 1mL Tween 80, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1000mL water, pH 6.8; The method for preparing the composite sodium hyaluronate solution is as follows: 1.5g of sodium hyaluronate and 50mL of water are mixed and stirred until completely dissolved. 0.09g of calcium chloride is added and stirred for 40min. 5g of L-sodium glutamate is added and stirred for 40min to obtain the composite sodium hyaluronate solution. The number-average molecular weight of the sodium hyaluronate is 10 kDa; Step 2. Preparation of the composite gel core: Step 1. Prepare the mixed adhesive solution: Sodium alginate, hyaluronic acid, citrus pectin, yeast β-glucan, and water were mixed in a mass ratio of 1.5:1:1:1:95.5 and stirred at 75°C until a uniform sol was formed. The mixture was then cooled to room temperature. The number-average molecular weight of the hyaluronic acid is 300 kDa; Step 2. Crosslinking: The mixed gel solution was stirred at 200 rpm at room temperature. Then, probiotic solution was added dropwise to the mixed gel solution, controlling the volume ratio of the mixed gel solution to the probiotic solution to be 9:1. The dropwise addition time of the probiotic solution was 30 min. After the dropwise addition was completed, stirring was continued for 1 h to obtain the mixed bacterial solution. The mixed bacterial solution was loaded into the syringe of the electrostatic drop generating device and electrostatically dropped into the composite calcium chloride aqueous solution, controlling the mass ratio of the mixed bacterial solution to the composite calcium chloride aqueous solution to be 100:450. At the same time, the composite calcium chloride aqueous solution was stirred at 200 rpm. During the electrostatic dropwise addition, the inner diameter of the syringe needle was controlled to be 0.4 mm, the voltage of the electrostatic drop generating device was controlled to be 8 kV, and the needle drop rate was 14 drops / min. After the electrostatic dropwise addition was completed, stirring was continued for 30 min. After filtration, the filter cake was collected to obtain cross-linked gel spheres. The composite calcium chloride aqueous solution is a mixed aqueous solution of calcium chloride and whey protein hydrolysate, wherein the mass fraction of calcium chloride is 3%, the mass fraction of whey protein hydrolysate is 1.4%, and the number average molecular weight of the polypeptides in the whey protein hydrolysate is 1800. The PBS buffer has a pH of 7.2 and a molar concentration of 0.01 mol / L; Step 3. Constructing the hyaluronic acid adsorption shell: Soy protein, citrus pectin, and water were mixed at a mass ratio of 5.5:3.8:1000 and stirred at 200 rpm for 40 min at room temperature. Hyaluronic acid was then added, and the mass ratio of soy protein to hyaluronic acid was controlled at 5.5:10. The mixture was stirred for another 40 min to obtain the coating layer solution. Cross-linked gel spheres were mixed with the coating layer solution at a volume ratio of 50 g:1000 mL and stirred at 30 rpm for 40 min at room temperature. The mixture was then filtered, and the residue was washed twice with water and freeze-dried at -45℃ for 28 h to obtain probiotic microcapsules. Step 4. Mixing and Molding: Maltitol, gelatin, yeast β-glucan, and water were mixed in a mass ratio of 30:8:2:60 and stirred at 100 rpm for 40 minutes at 70°C. The mixture was then cooked at 105°C until the soluble solids content reached 76%. The mixture was cooled to 65°C, and citric acid, strawberry flavor, and rice bran fatty alkyl alcohol were added. The mass ratio of maltitol, citric acid, strawberry flavor, and rice bran fatty alkyl alcohol was controlled at 30:0.5:1:0.02. The mixture was stirred at 100 rpm for 30 minutes and cooled to 40°C. Probiotic microcapsules were added, and the mass ratio of maltitol to probiotic microcapsules was controlled at 30:2. The mixture was stirred at 100 rpm for 10 minutes and poured into molds. The mixture was cooled and shaped at -4°C and then dried at 25°C and 45% relative humidity until the moisture content reached 17%. The mixture was then demolded to obtain active probiotic gummies.

[0026] This embodiment also provides an active probiotic gummies prepared by the aforementioned preparation method.

[0027] Comparative Example 1 The difference from Example 1 is that in step 2 of the pre-induced probiotics fermentation process, 1.5g of hyaluronic acid with a number average molecular weight of 10kDa is used instead of the compound sodium hyaluronate solution in the modified MRS liquid culture medium formulation.

[0028] The remaining technical solutions are consistent with those in Example 1.

[0029] Comparative Example 2 The difference from Example 1 is that in step three, "Constructing the Hyaluronic Acid Adsorption Shell," the use of soybean protein is omitted; that is, step three, "Constructing the Hyaluronic Acid Adsorption Shell," is replaced with: Citrus pectin and water were mixed at a mass ratio of 3.8:1000 and stirred at 200 rpm for 40 min at room temperature. Hyaluronic acid was then added, and the mass ratio of citrus pectin to hyaluronic acid was controlled at 3.8:10. The mixture was stirred for 40 min to obtain the coating layer solution. Cross-linked gel balls were mixed with the coating layer solution at a volume ratio of 50 g:1000 mL and stirred at 30 rpm for 40 min at room temperature. The mixture was filtered, and the residue was washed twice with water and freeze-dried at -45℃ for 28 h to obtain probiotic microcapsules.

[0030] The remaining technical solutions are consistent with those in Example 1.

[0031] Experimental Example 1 The number of live bacteria in the active probiotic gummies prepared in Examples 1-3 and Comparative Examples 1-2 was tested, and the results are as follows:

[0032] Experimental Example 2 The number of live bacteria in the active probiotic gummies prepared in Examples 1-3 and Comparative Examples 1-2 was tested. The active probiotic gummies prepared in Examples 1-3 and Comparative Examples 1-2 were then stored in an environment with a temperature of 37°C and a relative humidity of 75%. The number of live bacteria was measured after 30 days and 90 days of storage, respectively, and the live bacteria retention rate was calculated. The results are as follows:

[0033] Experimental Example 3 Tolerance tests were conducted on the active probiotic gummies prepared in Examples 1-3 and Comparative Examples 1-2, specifically: The number of live bacteria in the active probiotic gummies was tested. 50g of the active probiotic gummies were added to 500mL of artificial gastric fluid (pH 2.0, prepared according to the Chinese Pharmacopoeia method) and allowed to stand at room temperature for 2 hours. Then, 500mL of artificial intestinal fluid (pH 6.8, prepared according to the Chinese Pharmacopoeia method) was added and allowed to stand at room temperature for 2 hours. The number of live bacteria was then tested, and the live bacteria retention rate was calculated. The results are as follows:

[0034] Test Example 4 Sixty female subjects aged 30-35 years with constipation (less than 3 bowel movements per week) were selected and randomly divided into 6 groups of 10 subjects each, numbered 1-6. Groups 1-5 were the treatment group, and group 6 was the control group. The active probiotic gummies prepared in Examples 1-3 and Comparative Examples 1-2 were administered to subjects in groups 1-5 at a daily dose of 20g for 30 consecutive days. The improvement in constipation symptoms in groups 1-6 was statistically analyzed, and the results are as follows:

[0035] Experimental Example 5 Sixty female subjects aged 30-35 years with dull, dry skin were selected and randomly divided into six groups of 10 subjects each, numbered 1-6. Groups 1-5 were the treatment group, and group 6 was the control group. The skin stratum corneum moisture content of each subject in groups 1-6 was measured using a Corneometer@CM 825, and the average skin stratum corneum moisture content of each group was calculated. Then, the active probiotic gummies prepared in Examples 1-3 and Comparative Examples 1-2 were administered to subjects in groups 1-5, at a daily dosage of 20g for 30 consecutive days. The skin stratum corneum moisture content of each subject in groups 1-6 was measured again, and the average skin stratum corneum moisture content of each group was calculated. The rate of change in skin stratum corneum moisture content of groups 1-6 was also calculated. The results are as follows:

[0036] As can be seen from Examples 1-5, compared with the active probiotic gummies prepared in Example 1, the active probiotic gummies prepared in Comparative Example 1 have problems such as poor storage stability, poor tolerance to the gastrointestinal environment, and poor relief effect on constipation and dull and dry skin; the active probiotic gummies prepared in Comparative Example 2 have problems such as low number of live bacteria, poor storage stability, poor tolerance to the gastrointestinal environment, and poor relief effect on constipation and dull and dry skin.

Claims

1. A method for preparing active probiotic gummies containing a hyaluronic acid-alginic acid composite encapsulation system, characterized in that, include: Pre-induced probiotics were used to prepare a composite gel core, a hyaluronic acid adsorption shell was constructed, and the mixture was then molded. The pre-induced probiotics include: activation and fermentation; The fermentation process involves inoculating the probiotic seed culture into a modified MRS liquid medium, anaerobically culturing at 37-38°C, collecting the bacterial cells by centrifugation, washing, and resuspending them to form a probiotic culture. The modified MRS liquid culture medium is formulated as follows: 9.5-10.5g peptone, 9.5-10.5g beef extract, 4.8-5.2g yeast extract, 1.9-2.1g diammonium hydrogen citrate, 1.9-2.1g dipotassium hydrogen phosphate, 4.8-5.2g sodium acetate, 19-21g glucose, 15-16g trehalose, 50-60mL sodium hyaluronate solution, 0.9-1.1mL Tween 80, 0.55-0.6g magnesium sulfate, 0.24-0.26g manganese sulfate, 1000-1100mL water, pH 6.7-6.9; The method for preparing the composite sodium hyaluronate solution is as follows: sodium hyaluronate and water are mixed and stirred until completely dissolved, calcium chloride is added and stirred, L-sodium glutamate is added and stirred to obtain the composite sodium hyaluronate solution. To construct the hyaluronic acid adsorption shell, soybean protein, citrus pectin, and water were mixed and stirred at room temperature. Hyaluronic acid was then added and stirred to obtain a coating solution. Cross-linked gel spheres were mixed with the coating solution and stirred at room temperature. The mixture was then filtered, the residue was collected, washed, and freeze-dried to obtain probiotic microcapsules.

2. The method for preparing active probiotic gummies containing a hyaluronic acid-alginic acid composite encapsulation system according to claim 1, characterized in that, The activation process involves activating the probiotics to obtain a bacterial concentration of 1×10⁻⁶. 8 CFU / mL - 5 × 10 8 CFU / mL probiotic seed liquid; The probiotics are Bifidobacterium longum with strain number CICC 6198 and / or Lactobacillus rhamnosus with strain number CICC 6137, both of which were purchased through commercial channels.

3. The method for preparing active probiotic gummies containing a hyaluronic acid-alginic acid composite encapsulation system according to claim 1, characterized in that, In the fermentation process, the inoculation amount of probiotic seed culture into the modified MRS liquid medium is 2-3%. When anaerobic culture is carried out at 37-38℃, the anaerobic culture time is 13-15h; The bacterial concentration of the probiotic solution is 1×10⁻⁶. 8 CFU / mL - 5 × 10 8 CFU / mL.

4. The method for preparing active probiotic gummies containing a hyaluronic acid-alginic acid composite encapsulation system according to claim 1, characterized in that, In the preparation of the composite sodium hyaluronate solution, the ratio of sodium hyaluronate, water, calcium chloride, and L-glutamate is 1.5-1.6g:50-55mL:0.09-0.1g:5-5.5g. The number-average molecular weight of the sodium hyaluronate is 10 kDa.

5. The method for preparing active probiotic gummies containing a hyaluronic acid-alginic acid composite encapsulation system according to claim 1, characterized in that, The preparation of the composite gel core includes: preparing a mixed gel solution and crosslinking it; The preparation of the mixed sol involves mixing sodium alginate, hyaluronic acid, citrus pectin, yeast β-glucan, and water, then stirring at 75-80°C until a uniform sol is formed, and then cooling to room temperature. The crosslinking process involves stirring the mixed adhesive solution at room temperature, then adding probiotic solution dropwise to the mixed adhesive solution. After the addition is complete, stirring continues to obtain a mixed bacterial solution. The mixed bacterial solution is then loaded into a composite calcium chloride aqueous solution via electrostatic drop-in, while the composite calcium chloride aqueous solution is stirred. After the electrostatic drop-in is complete, stirring continues, followed by filtration. The filter cake is then collected to obtain crosslinked gel spheres.

6. The method for preparing active probiotic gummies containing a hyaluronic acid-alginic acid composite encapsulation system according to claim 5, characterized in that, In the preparation of the mixed adhesive solution, the mass ratio of sodium alginate, hyaluronic acid, citrus pectin, yeast β-glucan, and water is 1.5-1.6:1-1.1:1-1.2:1-1.2:95.5-98. The number-average molecular weight of the hyaluronic acid is 300-500 kDa; In the crosslinking process, the volume ratio of the mixed adhesive solution to the probiotic solution is 9-10:1; The probiotic solution is added over a period of 30-60 minutes. The mass ratio of the mixed bacterial solution to the compound calcium chloride aqueous solution is 100:450-500; The inner diameter of the syringe needle used in electrostatic dripping is 0.4-0.5 mm; The voltage of the electrostatic droplet generating device in the electrostatic dripping process is 8kV, and the needle dripping speed is 14-15 drops / min. The composite calcium chloride aqueous solution is a mixed aqueous solution of calcium chloride and whey protein hydrolysate, wherein the mass fraction of calcium chloride is 3-4%, the mass fraction of whey protein hydrolysate is 1.4-1.5%, and the number average molecular weight of the polypeptides in the whey protein hydrolysate is 1800. The PBS buffer has a pH of 7.2 and a molar concentration of 0.01 mol / L.

7. The method for preparing active probiotic gummies containing a hyaluronic acid-alginic acid composite encapsulation system according to claim 1, characterized in that, In the constructed hyaluronic acid adsorption shell, the mass ratio of soybean protein, citrus pectin, and water is 5.5-6:3.8-4:1000-1200. The mass ratio of soybean protein to hyaluronic acid is 5.5-6:10-11; The ratio of cross-linked gel spheres to coating solution is 50-60g:1000-1200mL; The freeze-drying temperature is -45℃ to -40℃, and the freeze-drying time is 28-30h.

8. The method for preparing active probiotic gummies containing a hyaluronic acid-alginic acid composite encapsulation system according to claim 1, characterized in that, The mixing and molding process involves mixing maltitol, gelatin, yeast β-glucan, and water, stirring at 70-80°C, cooking until the soluble solids content reaches 76-80%, cooling to 65-70°C, adding citric acid, strawberry flavoring, and rice bran fatty alkyl alcohols, stirring, cooling to 40-45°C, adding probiotic microcapsules, stirring, pouring into a mold, cooling and shaping, drying until the moisture content reaches 17-19%, demolding, and obtaining active probiotic soft candy. In the mixed molding process, the mass ratio of maltitol, gelatin, yeast β-glucan, and water is 30-32:8-9:2-2.2:60-65. The mass ratio of maltitol, citric acid, strawberry flavoring, and rice bran fatty alkyl alcohol is 30-32:0.5-0.6:1-1.1:0.02; The mass ratio of maltitol to probiotic microcapsules is 30-32:2-2.2; The cooking temperature when the soluble solids content is 76-80% is 105-108℃. The cooling and shaping temperature is -5℃ to -3℃; The drying process, which involves drying to a moisture content of 17-19%, takes place at a temperature of 24-26°C and an environment with a relative humidity of 44-46%.

9. An active probiotic gummies prepared by the preparation method according to any one of claims 1-8.

10. The use of an active probiotic gummies prepared by any one of claims 1-8 in the preparation of products that have the effect of regulating constipation or improving skin texture.

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