High-temperature-resistant and gastric-acid-resistant probiotic soft sweets and preparation method thereof

Abstract

According to the high-temperature-resistant and gastric-acid-resistant probiotic soft sweets and the preparation method thereof, a three-layer composite embedding technology (an inner-layer sodium alginate-chitosan gel, a middle-layer whey protein-pectin composite film and an outer-layer HPMCP acid-resistant coating) and a step-by-step temperature control technology are adopted for synergistic effect, so that the high-temperature-resistant and gastric-acid-resistant probiotic soft sweets are prepared; the technical problem that probiotics are easy to inactivate in high-temperature processing and gastric acid environments is solved. Experimental data show that the viable count of the prepared probiotic soft sweets is still kept to be greater than or equal to 7.5 log CFU / g after the probiotic soft sweets are treated at the high temperature of 80 DEG C for 10 minutes, the survival rate of the probiotic soft sweets treated by simulated gastric juice (pH 2.0) for 2 hours is greater than or equal to 95%, the release rate of the probiotic soft sweets in simulated intestinal juice for 30 minutes reaches 92.5%, and the viable retention rate exceeds 87% after the probiotic soft sweets are stored at room temperature for 6 months. By optimizing the probiotic microcapsule structure and the matrix formula, the stability of the probiotics is remarkably improved, and a new technical scheme is provided for the development of the functional soft sweets.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of functional food processing, and specifically relates to a high-temperature-resistant and gastric-acid-resistant probiotic soft candy and a preparation method thereof. BACKGROUND

[0002] Probiotics are defined as "live microorganisms that have a beneficial effect on the health of the host when consumed in adequate amounts". A large number of studies have shown that probiotics can promote human health through various ways such as regulating intestinal flora balance, enhancing immune function, and improving metabolic health. However, the main challenge faced by probiotic products is how to maintain sufficient viable counts during processing, storage, and digestion.

[0003] Soft candy is a popular functional food carrier with good taste, convenient carrying, and high consumer acceptance. However, the high-temperature boiling step (usually over 100℃) in the traditional soft candy processing process can cause fatal damage to probiotics. Even if the post-addition process is used, the acidic environment (pH usually 3.0-4.5) and water activity in the soft candy still affect the stability of probiotics. In addition, the strong acidic environment (pH 1.5-3.0) in the human stomach and bile salts further reduce the survival rate of probiotics reaching the intestine.

[0004] Chinese patent application CN113875870A "Active probiotic soft candy and its preparation method" uses sodium alginate-pectin-whey protein composite gel to embed probiotics, which has a certain protective effect, but the high-temperature resistance is insufficient, and the viable count decreases by more than 90% after 80℃ treatment for 10 minutes. Chinese patent application CN119769596A "Preparation method of high-activity probiotic soft candy" uses a low-temperature process to prepare soft candy, which avoids high-temperature damage, but the product has poor shelf stability, and the protection problem in the stomach acid environment has not been solved.

[0005] Therefore, it is of great significance to develop a probiotic soft candy preparation technology that can resist high-temperature processing (60-85℃) and gastric acid environment (pH=1.8-2.5) without complex embedding equipment. SUMMARY

[0006] In view of the problems existing in the prior art, the present application provides a high-temperature-resistant and gastric-acid-resistant probiotic soft candy and a preparation method thereof, which uses a three-layer composite embedding technology (inner gel, middle composite film, and outer coating) and a step-by-step temperature control process to cooperatively prepare a high-temperature-resistant and gastric-acid-resistant probiotic soft candy. The purpose of the present application is to solve the technical problem that probiotics are easily inactivated in high-temperature processing and gastric acid environment, and to improve the stability of probiotics.

[0007] In order to achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0008] In one aspect, the present application provides a probiotic gummy, comprising a probiotic microcapsule, the probiotic microcapsule comprising an inner layer gel, a middle layer composite film and an outer layer coating, the outer layer coating comprising HPMCP.

[0009] In the traditional preparation process of probiotic gummy, only one layer (such as pure sodium alginate, pure whey protein) or two-layer structure (such as sodium alginate-whey protein) protection system is used, due to the lack of "precise enteric layer" and "hierarchical synergy", there are obvious short boards in terms of high temperature resistance and gastric acid resistance. In order to effectively provide the use effect of probiotic gummy and the utilization rate of probiotics, the present application innovatively proposes a three-layer composite structure of probiotic microcapsule embedding probiotics, through the synergistic effect between each layer of substances and the synergistic protection between different levels, the protection effect is multi-dimensionally strengthened, thereby improving the high temperature resistance and gastric acid resistance of probiotic gummy.

[0010] Firstly, in terms of gastric acid resistance, the outer layer HPMCP of the probiotic microcapsule does not dissolve in the gastric acid environment (pH<5.0), forming the first line of defense; the middle layer whey protein-pectin composite film further buffers the gastric acid, and the inner layer gel microspheres remain stable. The three-layer structure forms a multi-barrier of "enteric film + buffer film + gel core", solving the problem of easy penetration of traditional structure in gastric acid environment. Secondly, in terms of high temperature resistance, HPMCP is resistant to high temperature, and can maintain the integrity of the membrane structure during the heat processing of gummy, avoiding the excessive denaturation of the middle layer whey protein; at the same time, the middle layer composite film stabilizes the inner layer gel core, reducing swelling and probiotic leakage under high temperature, thereby significantly improving the survival rate of probiotics after heat processing. In addition, the pH responsiveness of HPMCP solves the problem of "inaccurate release" of traditional structure, ensuring that probiotics are only released in the intestinal tract, and improving the bioavailability of probiotics. At the same time, through the strong combination of the middle layer composite film with the inner layer and the outer layer, the stability of the overall structure is enhanced, reducing the leakage of probiotics due to membrane breakage.

[0011] Further, the inner layer gel comprises sodium alginate, CaCl2 and optionally chitosan, β-cyclodextrin, and the middle layer composite film comprises a combination of whey protein and pectin.

[0012] The present application provides a three-layer composite structure of probiotic microcapsules, the inner layer of which is composed of sodium alginate, chitosan, beta-cyclodextrin and CaCl2. The sodium alginate and CaCl2 form a polyelectrolyte gel network through ionic cross-linking, which wraps the probiotics in the gel microspheres, provides a microenvironment buffer for the probiotics, and reduces the impact of temperature and pH changes on the cell structure. At the same time, chitosan and beta-cyclodextrin strengthen the encapsulation of the gel network on the probiotics, and enhance the compactness and stability of the gel network. The middle layer composite film is composed of whey protein and pectin. On the one hand, the hydrophobic group of whey protein combines with the hydrophilic group of the inner layer sodium alginate to form a dense protein-polysaccharide composite film, further reducing the penetration rate of gastric acid and high temperature to the core. On the other hand, the pectin forms a "synergistic gel" with the whey protein under acidic conditions, enhancing the mechanical strength of the middle layer and avoiding the over-swelling and disintegration of the inner layer gel in the stomach. The outer layer of HPMCP directly avoids the destruction of the inner layer gel microspheres and the middle layer composite film by gastric acid and high temperature. Through the synergistic effect between the substances in each layer structure, the protection effect of the probiotic microcapsules is significantly improved.

[0013] Further, the probiotic soft candy further comprises a gelling agent, a composite sugar matrix and an auxiliary material.

[0014] Further, the gelling agent comprises at least one of gelatin, gellan gum and pectin, the composite sugar matrix comprises at least one of maltitol, erythritol and glucose syrup, and the auxiliary material comprises at least one of citric acid, sodium citrate, food flavoring and inulin.

[0015] Further, the inner layer gel comprises 0.8-1.5 parts of sodium alginate, 2-4 parts of chitosan, 5-6.5 parts of beta-cyclodextrin and 1-2 parts of CaCl2, the gelling agent comprises 8-10 parts of gelatin, 0.5-1.2 parts of low-acyl gellan gum and 1.5-3 parts of pectin, the composite sugar matrix comprises 30-40 parts of maltitol, 15-20 parts of erythritol and 10-15 parts of glucose syrup, and the citric acid, sodium citrate, food flavoring and inulin are 0.5-1 part, 0.3-0.8 part, 0.2-0.5 part and 1-3 part, respectively.

[0016] Further, the probiotic in the probiotic microcapsules is Bacillus coagulans.

[0017] On the other hand, the present application provides a preparation method of probiotic soft candy, comprising the following steps:

[0018] Step (1): first prepare the inner layer gel microspheres, then dissolve the gel microspheres in a solution containing whey protein and pectin to obtain a middle layer composite film, and then coat an ethanol solution of HPMCP on the surface of the composite film to obtain probiotic microcapsules.

[0019] Step (2): Preparation of probiotic gummies.

[0020] Further, the preparation of the probiotic gummies in step (2) includes pre-activating the probiotic microcapsules, adding them to a high-temperature passivated gelling agent, and then adjusting the acidity and setting the final product.

[0021] Furthermore, the pre-activation temperature is 45°C, and the temperature is maintained for 25 minutes.

[0022] Furthermore, the high-temperature passivation involves first boiling to 78°C, then cooling to 65°C and holding at that temperature for 20 minutes.

[0023] Further, the preparation of the probiotic gummies in step (2) includes the following steps:

[0024] (1) Pre-activation: Dissolve the complex sugar matrix in water, add probiotic microcapsules, stir and keep warm to obtain a pre-activated solution;

[0025] (2) High temperature passivation: Mix the remaining complex sugar matrix with the gelling agent, boil it, cool it down and add the pre-activation solution, and keep it warm to obtain a high temperature passivation solution;

[0026] (3) Acid adjustment and shaping: After adding citric acid-sodium citrate buffer to the high-temperature passivation solution and mixing, add edible flavoring and inulin, pour the mixture into a mold and dry it to obtain the final product, probiotic soft candy.

[0027] To improve the heat resistance and stomach acid resistance of probiotic gummies, this invention also explored and improved the gummy preparation process. Regarding temperature settings in the preparation process, this invention adopts a step-by-step temperature control method: "pre-activation of probiotics at 45℃, followed by high-temperature inactivation, and finally, shaping at 60℃." First, the complex sugar matrix and probiotic microcapsules are mixed at 45℃. Through the synergistic effect of gentle temperature and nutrition, the probiotics are induced into a "stress-resistant state." Then, the gelling agent is boiled at 78℃ to fully dissolve gelatin, low-acyl gellan gum, and pectin. The temperature is then lowered to 65℃ before adding the probiotic pre-activation solution. 65℃ maintains the fluidity of the gelling agent while preventing probiotic inactivation due to high temperatures. Finally, shaping is achieved using a mold at 60℃. This gentle temperature promotes the formation of a dense and stable three-dimensional network in the gelling agent, building a physical protective barrier for the probiotics and reducing the impact of sudden temperature changes on the cells. By implementing step-by-step temperature control, the core contradiction between "high temperature required for gel formation" and "probiotics being afraid of high temperatures" is resolved, thereby comprehensively improving the heat resistance and stomach acid resistance of probiotics.

[0028] Furthermore, the pre-activated water temperature in (1) is 45°C, the water activity is 0.75-0.85, and the temperature is maintained for 25 min.

[0029] Furthermore, the high-temperature passivation cooking temperature of (2) is 78°C. After cooking for 5 minutes, the temperature is lowered to 65°C and the pre-activation liquid is added. The temperature is then maintained for 20 minutes.

[0030] Furthermore, the pH of the citrate-sodium citrate buffer solution used for acid adjustment and shaping in (3) is 4.2, the temperature of the mold is 60°C, and the drying conditions are 28°C and RH45% for 16 hours.

[0031] The present invention has the following beneficial effects:

[0032] 1. Through the synergistic design of "three-layer composite probiotic microcapsules + step-by-step temperature control", the bottleneck of traditional probiotic preparations in terms of processing tolerance and storage stability has been broken through, significantly improving the bioavailability and long-term stability of probiotics, and providing a new technical solution for the development of functional gummies.

[0033] 2. The composite encapsulation structure of inner gel, middle composite membrane and outer HPMCP coating is adopted. By utilizing the synergistic effect between the substances in each layer and the synergistic protection between layers, the heat resistance and stomach acid resistance of probiotic gummies are comprehensively improved, and the overall structural stability is enhanced.

[0034] 3. The step-by-step temperature control process of "pre-activation, high-temperature passivation, and final acid adjustment and shaping" is adopted. The temperature is set in stages and compounded according to the physicochemical properties of probiotics and the process requirements of other ingredients, which solves the technical problem that probiotics are easily inactivated in high-temperature processing and gastric acid environment. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to embodiments. It should be noted that the following embodiments are only used to explain and illustrate this invention and are not intended to limit this invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] Example 1: A heat-resistant and stomach-acid-resistant probiotic gummy

[0039] This embodiment provides a method for preparing heat-resistant and stomach-acid-resistant probiotic gummies, including the following steps:

[0040] (1) Preparation of probiotic microcapsules:

[0041] 1. Preparation of the inner gel: Dissolve 2.0 g sodium alginate and 5.0 g β-cyclodextrin in 100 mL of distilled water, add 10 mL of Bacillus coagulans powder (purchased from Shandong Siyang Biotechnology Co., Ltd., 200 billion CFU / g), and disperse the bacterial powder in the aqueous phase at a ratio of 1:15 (sodium alginate-β-cyclodextrin). Perform high-speed shearing (8000 rpm, 2 min). Use a syringe to drop the mixture into a solution containing 1.0% chitosan and 2.5% CaCl2 to form gel microspheres with a diameter of 1-2 mm.

[0042] 2. Dissolve 10 g of whey protein and 20 g of pectin separately in 100 mL of water, mix, and adjust the pH to 6.5. Immerse the inner layer gel microspheres in this solution and incubate at 50°C for 25 minutes to form a protein-pectin composite membrane.

[0043] 3. Using a fluidized bed coating device, an ethanol solution (8% concentration) of HPMCP is sprayed onto the surface of the middle layer microcapsules, controlling the coating weight gain to 10%, to obtain the final three-layer encapsulated probiotic microcapsules.

[0044] (2) Preparation of probiotic gummies:

[0045] S1. Pre-activation: Dissolve 20 g maltitol, 10 g erythritol and 8 g glucose syrup in 50 ml of 45℃ water (water activity 0.82), add 10 g probiotic microcapsules, stir at 300 rpm and keep warm for 25 min to obtain the pre-activated solution.

[0046] S2. High-temperature passivation: Mix 20 g of maltitol, 10 g of erythritol, 7 g of glucose syrup with a gelling agent (containing 10 g of gelatin, 1 g of low-acyl gellan gum and 2 g of pectin), cook to 78°C (76°Bx for solids), cool to 65°C, add pre-activation solution, and keep warm for 20 min to obtain high-temperature passivation solution.

[0047] S3. Acid Adjustment and Shaping: Add 5 ml of citric acid-sodium citrate buffer (pH=4.2) to the high-temperature passivation solution, then add 0.4 g of edible flavoring and 2 g of inulin. Mix for 5 min and pour into a mold (temperature 60℃). Dry at 28℃ and RH 45% for 16 hours to obtain a final product with a moisture content of 16%-18%.

[0048] The heat-resistant and stomach acid-resistant probiotic gummies prepared according to the above method were subjected to performance tests, specifically including the following aspects:

[0049] 1. Saliva resistance test:

[0050] The final product was placed in simulated saliva (an aqueous solution containing 100 U / mL α-amylase, pH 6.8-7.0), then incubated in a 37°C water bath with shaking at 150 rpm for 10 minutes. The total bacterial count was then measured. Results showed that the viable bacterial count decreased from 8.7 log CFU / g before treatment to 8.5 log CFU / g after treatment, with a survival rate of 97%.

[0051] 2. High temperature resistance test:

[0052] The final product was treated in an 80℃ water bath for 10 minutes, and the viable bacterial count was then measured. The results showed that the viable bacterial count was 8.7 log CFU / g before treatment and 7.8 log CFU / g after treatment, with a survival rate of 89.7%.

[0053] 3. Gastric acid resistance test:

[0054] The final product was placed in simulated gastric fluid (an aqueous solution containing 0.3% pepsin, pH=2.0) and treated with shaking (150 rpm) at 37°C for 2 hours. The viable bacterial count decreased from 8.7 log CFU / g before treatment to 8.4 log CFU / g after treatment, with a survival rate of 96.6%.

[0055] 4. Enteric release test:

[0056] After the final product was transferred into simulated small intestinal fluid (containing 0.1% trypsin, pH=6.8), the release rate reached 92.5% within 30 minutes; and it was completely released in simulated colonic fluid (pH=7.4) within 60 minutes.

[0057] 5. Storage stability:

[0058] Accelerated testing (37℃, RH75%) showed that the viable count was 7.2 log CFU / g after 3 months and 6.5 log CFU / g after 6 months, with a viable count retention rate of over 74%.

[0059] The viable cell retention rate was 94.6% after 6 months of storage under long-term stable room temperature conditions (25℃±2℃, RH60%±5%).

[0060] 6. Sensory evaluation:

[0061] The test was evaluated by 20 trained judges on a 9-point scale, with an average score of 8.3. The hardness was 16.5N, the elasticity was 0.82, and the taste was acceptable.

[0062] Example 2: Optimization of the three-layer structure of probiotic microcapsules

[0063] In order to improve the heat resistance and stomach acid resistance of probiotic gummies, thereby maintaining the high activity of probiotics, this invention explored and optimized the structure of probiotic microcapsules encapsulating probiotics, and investigated the differences in the effects of structural levels and material composition of probiotic microcapsules.

[0064] (1) Verification of the necessity of adopting a three-layer composite structure

[0065] This embodiment evaluated the differences in efficacy of probiotic gummies prepared using probiotic microcapsules with single-layer, two-layer, and three-layer encapsulation structures, respectively. The preparation methods for probiotic gummies based on the three encapsulation structures are as follows:

[0066] Probiotic Gummies I: Probiotic gummies I, which are encapsulated by only one layer of structure (i.e., cold-cured gel-chitosan), were prepared by referring to the Chinese patent application publication number CN119769596 A, entitled "A method for preparing highly active probiotic gummies".

[0067] Probiotic Gummies II: Probiotic gummies II with two-layer structure (i.e., a layer of sodium alginate-pectin and a layer of whey protein) were prepared by referring to the Chinese patent "An active probiotic gummies and their preparation method" with authorization publication number CN113875870B.

[0068] High-temperature and stomach-acid-resistant probiotic gummies: High-temperature and stomach-acid-resistant probiotic gummies with a three-layer composite structure were prepared using the method in Example 1.

[0069] The same high-temperature resistance test method and gastric acid resistance test method as in Example 1 were used to test the survival rate of probiotics in the three types of probiotic gummies under high temperature and gastric acid environments, as well as their release rate in small intestinal fluid. The test results are shown in Table 1. Survival rate (%) = (number of viable bacteria after treatment / initial number of viable bacteria) × 100%.

[0070] Table 1. Survival rate and release rate of probiotics in three types of probiotic gummies.

[0071]

[0072] As shown in Table 1, under high-temperature conditions, the survival rates of probiotics in probiotic gummies I, II, and the heat- and stomach-acid-resistant probiotic gummies were 32.4%, 45.6%, and 89.7%, respectively. In the stomach-acid environment, the survival rates were 25.5%, 78.9%, and 96.6%, respectively, and the release rates in intestinal fluid were 18.9%, 66.7%, and 92.5%, respectively. It can be observed that probiotics encapsulated in only one layer have very low survival rates under high-temperature and stomach-acid conditions, as well as low release rates in intestinal fluid. While the probiotics in probiotic gummies II, encapsulated in two layers, showed improved tolerance to stomach acid, they were still not heat-resistant. In contrast, the heat-resistant and stomach-acid-resistant probiotic gummies prepared in this invention maintain a probiotic survival rate of over 90% after high-temperature treatment and stomach acid digestion, with a release rate of 92.5% in intestinal fluid. This demonstrates that the three-layer composite encapsulation structure of the probiotic microcapsules designed in this invention effectively protects the probiotics within, improves their survival rate under high-temperature and stomach-acid conditions, and promotes their release in intestinal fluid. This is because the outer HPMCP layer of the three-layer composite probiotic microcapsules is insoluble in the stomach and heat-resistant, forming the "first line of defense." The middle whey protein-pectin composite membrane further buffers stomach acid, reducing swelling and probiotic leakage of the inner gel microspheres at high temperatures, thereby achieving precise release of probiotics in the intestines. This invention overcomes the bottleneck of insufficient tolerance in traditional probiotic gummies, significantly improving the efficacy of probiotics.

[0073] Furthermore, this invention employs a multi-layered composite structure with four or more layers to encapsulate probiotics, thereby obtaining probiotic microcapsules. The results showed that after high-temperature and gastric acid testing, the survival rate of probiotics encapsulated in the multi-layered structure was not significantly improved compared to the three-layered structure, and the preparation process was more complex and costly. Considering both efficacy and production cost, this invention preferably uses a three-layered composite structure for encapsulating probiotics, specifically probiotic microcapsules with an inner gel layer, a middle composite membrane layer, and an outer coating layer.

[0074] (2) Selection of materials for the inner gel layer

[0075] To ensure better synergy among the three layers and achieve optimal protective effects, this embodiment further investigated the composition of the inner, middle, and outer layers. First, the composition of the inner gel microspheres was examined. The inner gel was composed of the four material combinations shown in Table 2, with other preparation parameters remaining the same as in Example 1. Gummy candies with different inner gel compositions were obtained, and the products underwent high-temperature resistance, gastric acid resistance, and enteric release tests (simulating small intestinal fluid). The survival rate of probiotics under high temperature and gastric acid was calculated.

[0076] Table 2. Survival rate and release rate of probiotics using different inner gel layers.

[0077]

[0078] As shown in Table 2, after high-temperature and gastric juice treatment, the probiotic survival rate of the gummies in test group 1 still reached 90.2% and 96.9%, respectively, and the release rate in intestinal fluid reached 92.5%. In contrast, the probiotic survival rate in the gummies of the other three test groups was only above 70% and below 90%, and the release rate in intestinal fluid was less than 60%. This indicates that the inner gel composed of sodium alginate, β-cyclodextrin, chitosan, and CaCl2 can provide the best protective effect. The speculated reason is that the anionic polysaccharide of sodium alginate reacts with the CaCl2 of calcium chloride... 2+ A polyelectrolyte gel network is formed through ionic cross-linking, encapsulating probiotics within gel microspheres and isolating them from external oxygen and moisture. Simultaneously, the porous structure of the gel network provides a "microenvironment buffer" for the probiotics, reducing the impact of sudden temperature and pH changes on cell structure. Furthermore, the cationic polysaccharides provided by chitosan enhance the barrier's acid resistance and mechanical stability, while β-cyclodextrin further encapsulates the sodium alginate-chitosan gel, thereby strengthening the inner layer's protective effect on probiotics and improving the overall protective performance of the gel network. Other combinations of substances cannot achieve such excellent synergistic effects; therefore, the inner layer gel prepared using sodium alginate, chitosan, and CaCl2 simultaneously possesses excellent resistance to gastric acid, high temperatures, and stability.

[0079] (3) Material selection for the middle layer composite membrane

[0080] This embodiment further investigated the high-temperature resistance and gastric acid resistance of the middle layer composite membrane composed of different substances. The middle layer composite membrane was made of any combination of whey protein, soy protein isolate, pectin, and carrageenan. Other preparation processes were the same as in Example 1 (such as the preparation of the inner gel, the outer coating spraying, and the preparation of probiotic gummies). The obtained probiotic gummies were subjected to high-temperature resistance tests, gastric acid resistance tests, and enteric release tests (simulating small intestinal fluid), and the viable bacterial survival rate of the probiotics in high temperature and gastric acid was calculated.

[0081] Table 3. Survival rate and release rate of probiotics using different middle-layer composite membranes

[0082]

[0083] The results in Table 3 show that when whey protein and pectin were used in the middle layer composite membrane, the prepared gummies exhibited the best heat resistance and stomach acid resistance. The survival rate of probiotics in the gummies remained above 90% after high-temperature testing. However, when other compositions were used in the middle layer composite membrane, the survival rate of probiotics and their release rate in intestinal fluid were significantly reduced. Comparing the composition of these middle layer composite membranes, the difference in effectiveness can be attributed to the fact that the hydrophobic groups of whey protein can combine with the hydrophilic groups of the inner sodium alginate layer to form a dense protein-polysaccharide composite membrane. Simultaneously, the amino groups of whey protein can adhere tightly to the outer HPMCP coating through electrostatic interactions, thus improving the stability of the protective structure and further reducing the penetration rate of stomach acid and high temperatures into the core. Pectin, under acidic conditions, can enhance the mechanical strength of the middle layer, preventing excessive swelling and disintegration of the inner gel in the stomach. However, if soy protein isolate or carrageenan is used, the lack of specific ionic bonds limits the compatibility of the complex and reduces its effectiveness. Furthermore, using a combination of whey protein, pectin, and carrageenan may result in reduced effectiveness due to the molecular properties of carrageenan conflicting with the combination of whey protein and pectin. Therefore, whey protein and pectin are preferred for preparing the middle layer composite membrane.

[0084] (4) Selection of outer coating

[0085] This embodiment further investigated the differences in the effects of gummies prepared with different outer coatings. Three different outer coatings were used: hydroxypropyl methylcellulose phthalate (HPMCP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and cellulose acetate phthalate (CAP), as shown in Table 4. Other preparation parameters were the same as in Example 1. The prepared gummies were subjected to high-temperature resistance tests, gastric acid resistance tests, and enteric release tests according to the method in Example 1. The test results are shown in Table 4.

[0086] Table 4. Survival rate and release rate of probiotics encapsulated in different outer coatings

[0087]

[0088] As shown in Table 4, there was no significant difference in the survival rate of the three types of encapsulated probiotics in gastric juice, indicating that HPMCP, HPMCAS, and CAP can all form an insoluble enteric film in the stomach, preventing gastric acid and pepsin from eroding the middle and inner layers. However, the survival rate of HPMCP-encapsulated probiotics at high temperatures and their release rate in the intestine were significantly higher than the other two substances. This is because HPMCP has better heat resistance and its pH response range precisely matches the intestinal pH environment. Therefore, it is completely insoluble in the stomach and rapidly dissolves and releases probiotics after entering the small intestine, thus avoiding premature leakage in the stomach or delayed release in the intestine. In contrast, HPMCAS has a slightly wider pH response range and lower intestinal targeting, while CAP is prematurely hydrolyzed in the acidic environment of the stomach, leading to premature release of probiotics. Therefore, using HPMCP as the outer coating provides the best encapsulation protection.

[0089] (5) Ca 2+ Concentration optimization

[0090] To investigate Ca 2+ To investigate the effect of CaCl2 concentration on the performance of probiotic microcapsules, this example designed a single-factor experiment using CaCl2 concentration as the variable. Three gradients of CaCl2 concentrations (1.5%, 2.5%, and 3.5%) were set during the preparation of the inner gel layer of the probiotic microcapsules. Other preparation parameters remained consistent with Example 1, and each group was performed in triplicate (n=3). The probiotic mixture was dropped into a composite solution containing 1.0% chitosan and different concentrations of CaCl2, forming gel microspheres through ionic cross-linking. This process was used to construct microcapsules with different CaCl2 concentrations. 2+ Concentration of probiotic microcapsules. The hardness of the probiotic microcapsules was tested using a texture analyzer (TA.XT Plus). The test parameters were: test speed 1 mm / s, trigger force 5 g, and puncture depth 2 mm. The results are shown in Table 5.

[0091] Table 5 Different Ca 2+ Performance of probiotic microcapsules at different concentrations

[0092]

[0093] Note: Data are expressed as mean ± standard deviation (n=3).

[0094] As shown in Table 5, the statistical analysis revealed that the viable bacterial survival rate in the 2.5% CaCl2 concentration group (89.7±1.2%) was significantly higher than that in the 1.5% group (78.5±1.5%) and the 3.5% group (82.1±1.0%), and the difference was statistically significant (P<0.05). Further microcapsule hardness testing indicated that the gel microspheres formed in the 2.5% concentration group had a hardness of 12.3±0.5 N, exhibiting moderate mechanical strength; therefore, 2.5% CaCl2 is the optimal concentration. This is because, through Ca... 2+Coordination and cross-linking with chitosan molecular chains form a stable and appropriately permeable three-dimensional network. At this concentration, the microcapsules avoid the structural loosening caused by insufficient cross-linking at low concentrations (1.5%), while also preventing the microcapsule embrittlement caused by excessive cross-linking at high concentrations (3.5%). These results confirm that Ca... 2+ Concentration is a key parameter for regulating the microstructure of microcapsules and the encapsulation efficiency of probiotics. A CaCl2 concentration of 2.5% can achieve the best balance between cross-linking degree and mechanical properties, providing core experimental basis for the microencapsulation process of heat-resistant and gastric acid-resistant probiotic gummies.

[0095] (6) Optimization of sodium alginate and HPMCP concentrations

[0096] This embodiment further investigated the effect of sodium alginate and HPMCP concentration ratio on the performance of probiotic microcapsules. Four sodium alginate concentrations (0.5%, 1%, 1.5%, and 2%) were set during the inner gel preparation stage of the probiotic microcapsules, and four HPMCP concentrations (5%, 7%, 9%, and 10%) were set during the outer coating preparation stage. Other preparation parameters remained consistent with Example 1, thus preparing probiotic microcapsules with different ratios. The unencapsulated group was prepared by directly adding Bacillus coagulans powder to distilled water without any encapsulation treatment, resulting in an initial viable count of 9.8 log CFU / g. The encapsulation efficiency (EE) of each group of microcapsules was determined using a centrifugation-washing method. Specifically, 1.0 g of wet microcapsule sample was accurately weighed, dispersed in 50 mL of PBS buffer (0.1 M, pH 7.0), centrifuged at 2000 rpm for 10 minutes, and the supernatant was collected to determine the free bacterial count (N_free). Another equal amount of sample was crushed and the total bacterial count (N_total) was determined. The encapsulation efficiency was calculated using the formula: EE (%) = [ (N_total - N_free) / N_total ] × 100%. Simultaneously, the high-temperature resistance test was performed using the same method as in Example 1. The test results are shown in Table 6.

[0097] Table 6. Performance of probiotic microcapsules in different test groups

[0098]

[0099] Note: Gastric acid protection factor = number of viable bacteria in the treated group / number of viable bacteria in the unencapsulated group.

[0100] As shown in Table 6, compared with the control group containing a single layer of 1.5% sodium alginate, the introduction of different concentrations of HPMCP into the probiotic microcapsules significantly improved the encapsulation efficiency, probiotic survival rate, and gastric acid protection factor. When the concentration of sodium alginate was ≥1.0%, the encapsulation efficiency of the microcapsules exceeded 90% (P<0.05), and the survival rate of probiotics under high temperature conditions reached over 96%. Simultaneously, the HPMCP concentration was positively correlated with the gastric acid protection effect (Pearson r=0.934), and when the HPMCP concentration reached ≥9%, the survival rate of probiotics remained stable at over 97%. Therefore, considering both the microcapsule performance and composition, 1.5% sodium alginate and 9% HPMCP are the preferred formulations.

[0101] Example 3: Optimization of Stepwise Temperature Control Process

[0102] To improve the heat resistance and stomach acid resistance of probiotic gummies, this embodiment further investigated the effect of temperature conditions during gummy preparation on probiotic activity, in order to reveal the combined mechanism of temperature control process and probiotic microcapsule structure on probiotic activity.

[0103] (1) The necessity of step-by-step temperature control

[0104] In this embodiment, probiotic microcapsules were first prepared using the same method as in Example 1. Then, probiotic gummies were prepared using the following three temperature control methods. Other parameters (such as the composition and proportion of complex sugar matrix, gelling agent, etc.) remained the same as in Example 1.

[0105] Method 1: Maintain a temperature of 65°C from the initial mixing of probiotic microcapsules and complex sugar matrix to the final shaping stage;

[0106] Method 2: The temperature is controlled at 45℃ during the pre-activation stage, 65℃ during the high-temperature passivation stage, and 65℃ during the acid adjustment and shaping stage.

[0107] Method 3: The temperature settings for the pre-activation stage, high-temperature passivation stage, and acid conditioning and shaping stage are the same as in Example 1.

[0108] The probiotic gummies prepared according to the above three methods were subjected to high temperature resistance test, gastric acid resistance test and enteric release test, and the test results are shown in Table 7.

[0109] Table 7. Probiotic survival rate and release rate under different temperature control processes

[0110]

[0111] The results in Table 7 show that the probiotic gummies prepared according to the stepwise temperature control method in Example 1, after high-temperature and gastric acid tests, still maintained a probiotic survival rate of over 89% and 96%, respectively, and a probiotic release rate in intestinal fluid of 92.5%, significantly higher than the survival and release rates of the probiotics obtained using the other two methods. This is because in the stepwise temperature control process, the probiotics are first pre-activated at 45°C to enhance their resistance, then the gel is fully dissolved at 80°C, and the pre-activation solution is added after cooling to 65°C. This temperature is precisely at the upper limit of the pre-activated probiotics' tolerance, which can stabilize the cell membrane structure and enzyme activity of the probiotics and reduce protein denaturation. Finally, the setting temperature of 60°C promotes the formation of a "dense and stable three-dimensional network" of the gelling agent, building a physical protective barrier for the probiotics. Therefore, by controlling the temperature in stages, the core contradiction between the need for high temperature for gel formation and the heat intolerance of probiotics is resolved, ultimately significantly improving the heat resistance and gastric acid resistance of the probiotics.

[0112] (2) Temperature optimization during the pre-activation stage

[0113] To determine the optimal temperature parameters for the pre-activation stage in the preparation of probiotic gummies, this embodiment employed a single-variable control method, setting pre-activation temperatures at three gradients: 40℃, 45℃, and 50℃. All other process parameters remained consistent with those in Example 1. Each group was replicated three times (n=3) to ensure the reliability of the results. The viable bacteria survival rate of the final probiotic gummies was then measured. The test results are shown in Table 8.

[0114] Table 8. Probiotic survival rate at different pre-activation temperatures

[0115]

[0116] As shown in Table 8, the viable bacteria survival rate in the 45℃ treatment group reached 92.3%, significantly higher than that in the 40℃ treatment group (85.6%) and the 50℃ treatment group (88.1%) (P<0.05). This indicates that the probiotic activity was highest and the effect was optimal under this temperature condition. This demonstrates that the 45℃ pre-activation temperature achieves a balance between probiotic metabolic activity and thermal stability—effectively activating the metabolic activity of probiotics (compared to the lower activation efficiency at 40℃) while avoiding heat damage caused by 50℃ (although the viable bacteria retention rate in the 50℃ group was higher than that in the 40℃ group, it was still significantly lower than that in the 45℃ group). Therefore, controlling the pre-activation stage temperature of probiotic gummies at 45℃ can significantly improve the efficacy of probiotic gummies.

[0117] Example 4: Saliva Influence Test

[0118] This invention further tests the saliva tolerance of the heat-resistant and gastric acid-resistant probiotic gummies prepared in Example 1 using an in vitro simulated saliva environment. The test method is as follows:

[0119] A simulated saliva solution containing α-amylase (100 U / mL) was prepared at pH 6.8-7.0. The probiotic gummies prepared according to Example 1 were cut into uniform small pieces, and two portions of approximately 0.5 g each were accurately weighed and placed in centrifuge tubes containing 10 mL of simulated saliva. The centrifuge tubes were placed in a 37°C water bath and shaken at 150 rpm. Samples were taken at 0, 1, 2, 3, 5, and 10 minutes. Immediately after sampling, the reaction was terminated by adding PBS buffer containing 0.1% (w / v) cysteine ​​hydrochloride. After appropriate dilution, the samples were plated on MRS agar plates and anaerobically incubated at 37°C for 48 hours. Colony forming units (CFU) were counted, and the survival rate was calculated: Survival rate (%) = (Number of viable bacteria after treatment / Initial number of viable bacteria) × 100%. The results are shown in Table 9.

[0120] Table 9. Number of probiotics surviving in simulated saliva at different time points.

[0121]

[0122] As shown in Table 9, after 10 minutes of salivary amylase digestion, the number of viable bacteria in both groups decreased from the initial 9.0 log CFU / g to 8.5 log CFU / g, with a survival rate of approximately 95%. The in vitro validation results demonstrate that during normal eating (usually a few minutes to a dozen minutes), saliva has little impact on the activity of probiotics in gummies; the activity of probiotics does not significantly decrease after chewing and amylase digestion.

[0123] Example 5: Long-term stability test

[0124] This invention further designed orthogonal experiments to investigate the interactive effects of storage temperature (4℃, 25℃, 40℃), relative humidity (45%RH, 60%RH, 75%RH), and packaging materials (aluminum foil composite film, PET / AL / PE, ordinary OPP) on the activity and stability of probiotics in the gummies. The heat-resistant and gastric acid-resistant probiotic gummies prepared in Example 1 were stored according to the five storage conditions listed in Table 10, with five parallel samples (n=5) in each group. Samples were taken every 3, 6, and 12 months to test viable cell count, water activity, and textural properties. The viable cell count was determined according to GB 4789.35-2023 using the plate count method (MRS medium, anaerobic culture at 37℃ for 48 h); water activity was measured using an Aqualab 4TE water activity meter at 25℃. The test results are shown in Table 10.

[0125] Table 10 Changes in viable bacterial count and water activity under different storage conditions

[0126]

[0127] According to the results in Table 10, under storage conditions of 40℃, the water activity exceeded 0.75 after 12 months, and the viable bacteria retention rate only reached about 70%. Under storage conditions of 25℃, the viable bacteria retention rate reached over 82% after 12 months, but the effect was still not as good as the storage method of 4℃, 45%RH, and aluminum foil. Using aluminum foil packaging at 4℃, the viable bacteria retention rate reached 97.6% after 12 months. Therefore, the storage method of 4℃, 45%RH, and aluminum foil is preferred.

[0128] In addition, the shelf-life stability of the product was tested. The probiotic gummies prepared in Example 1 were stored under the two conditions shown in Table 11, with 5 parallel samples (n=5) in each group. Samples were taken at 0, 3, 6, and 12 months to detect the viable bacteria count (method as before) and water activity.

[0129] Table 11 Stability data under different storage conditions

[0130]

[0131] The results showed that the probiotic gummies prepared in this invention exhibited excellent stability under long-term storage conditions, with a viable bacteria retention rate still above 85% after 12 months. Accelerated test data further verified the product's tolerance under harsh conditions, and its trend was in good agreement with that of long-term tests, which can be used to predict the product's shelf life.

[0132] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any modifications and alterations made by those skilled in the art without departing from the spirit and scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A probiotic soft candy, characterized in that, The probiotic microcapsule comprises an inner layer gel, a middle layer composite film and an outer layer coating, wherein the outer layer coating comprises HPMCP.

2. The probiotic gummy of claim 1, wherein, The inner layer gel comprises sodium alginate, CaCl2 and optionally chitosan and β-cyclodextrin, and the middle layer composite film comprises a combination of whey protein and pectin.

3. The probiotic gummies as described in claim 2, characterized in that, The probiotic gummy further comprises a gelling agent, a complex sugar base and an auxiliary material.

4. The probiotic gummies as described in claim 3, characterized in that, The gelling agent comprises at least one of gelatin, gellan gum and pectin, the complex sugar base comprises at least one of maltitol, erythritol and glucose syrup, and the auxiliary material comprises at least one of citric acid, sodium citrate, food flavor and inulin.

5. The probiotic gummies as described in claim 4, characterized in that, The inner layer gel comprises 0.8-1.5 parts of sodium alginate, 2-4 parts of chitosan, 5-6.5 parts of β-cyclodextrin and 1-2 parts of CaCl2, the gelling agent comprises 8-10 parts of gelatin, 0.5-1.2 parts of low acyl gellan gum and 1.5-3 parts of pectin, the complex sugar base comprises 30-40 parts of maltitol, 15-20 parts of erythritol, 10-15 parts of glucose syrup, the citric acid is 0.5-1 part, the sodium citrate is 0.3-0.8 part, the food flavor is 0.2-0.5 part and the inulin is 1-3 parts.

6. The probiotic gummies as described in claim 5, characterized in that, The probiotic in the probiotic microcapsule is Bacillus coagulans.

7. A method of preparing a probiotic soft candy, characterized by, The method comprises the following steps: Step (1): first, prepare the inner layer probiotic gel microspheres, then dissolve the gel microspheres in a solution containing whey protein and pectin to obtain the middle layer composite film, and then coat the ethanol solution of HPMCP on the surface of the composite film to obtain the probiotic microcapsule; Step (2): preparation of the probiotic gummy.

8. The method of claim 7, wherein, The preparation of the probiotic gummy in step (2) comprises the following steps: after pre-activation of the probiotic microcapsule, the probiotic microcapsule is added to the high-temperature inactivated gelling agent, and then the acid is adjusted and shaped to obtain the final product.

9. The method of claim 8, wherein, The pre-activation temperature is 45℃, and the incubation time is 25 min.

10. The method of claim 8, wherein, The high-temperature inactivation is first cooked to 78℃, then cooled to 65℃, and then incubated for 20 min.

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