High-activity probiotic yoghurt protected by double-layer microcapsules as well as preparation method and application of high-activity probiotic yoghurt

Abstract

The invention belongs to the technical field of dairy product processing, and discloses high-activity probiotic yoghourt protected by double-layer microcapsules and a preparation method and application thereof, and the preparation method comprises the following steps: (1) preparing single-layer microcapsules; (2) preparing a double-layer microcapsule; and (3) preparation of yoghourt: homogenizing, sterilizing and cooling the raw milk (raw milk), adding the double-layer microcapsules and the leavening agent, and fermenting to obtain the yoghourt. The viable count of the probiotics in the yoghurt obtained by the invention can still be maintained at 106 CFU / mL or above after being stored for 25 days, and the water-holding capacity, the viscosity and the texture characteristics of the yoghurt are obviously superior to those of a control sample without microcapsules. The microcapsule can effectively protect the probiotics in the stomach environment, the release rate in the intestinal tract reaches up to 99%, and the survival rate and storage stability of the probiotics are remarkably improved. After the microcapsule is added into the yoghourt, the activity and the overall quality of the probiotics in the yoghourt are remarkably improved through double-layer microcapsule embedding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of dairy product processing, and in particular to a high-activity probiotic yogurt protected by double-layer microcapsules, as well as a preparation method and application thereof. BACKGROUND

[0002] The health benefits of live yogurt, such as regulating intestinal flora, enhancing immunity, and promoting nutrient absorption, have been widely recognized. The realization of these functions is highly dependent on the number of active probiotics contained when the yogurt is consumed. Generally, the industry believes that the number of live bacteria in probiotic yogurt should not be less than 10 6 CFU / g to exert basic physiological functions, and the number of live bacteria should reach or exceed 10 7 CFU / g to achieve significant and stable health effects. However, during the production, storage, transportation, sale, and consumption of yogurt, probiotics, especially lactobacillus strains sensitive to stress, are continuously subjected to heat processing, gastric acid, bile salts, and low-temperature anoxia, leading to a situation where the number of live bacteria decreases sharply in the early stage and slowly decays in the later stage during the shelf life. This results in the number of live bacteria approaching or even falling below the functional threshold at the end of the shelf life or even when consumed by consumers, severely weakening the claimed efficacy of the product. This is a core technical problem that has long plagued the yogurt industry and restricted the improvement of product power.

[0003] Microcapsule embedding technology is currently recognized as one of the most effective and practical methods for protecting bacterial viability. This technology aims to create a physical barrier and microenvironment for probiotics to isolate external adverse factors. The most commonly used method is the preparation of single-layer microcapsules by ion gelation with a sodium alginate-calcium chloride system. Although this method is simple and provides some physical protection, its porosity is large, and its blocking ability to hydrogen ions is limited. At the same time, its storage stability is poor, and the gel structure gradually degrades and shrinks in the acidic environment of yogurt and cold storage conditions, significantly reducing the protective performance over time. Previous studies have attempted to use sodium alginate, chitosan, and other materials for embedding, but there is still a problem of rapid decline in the number of live bacteria during the shelf life.

[0004] Therefore, there is a need in the art for a new microcapsule structure that can significantly enhance the protection of probiotics in yogurt, ensuring that the number of live bacteria remains stable at 10 7 CFU / g or above at the end of the standard 21-day shelf life, thereby solving the problems of declining lactobacillus number during the shelf life and the decline in the efficacy of live yogurt over time.

[0005] Through searches, no patent publications related to the present patent application have been found. SUMMARY

[0006] The present application aims to overcome the deficiencies in the prior art, and provide a double-layer microcapsule-protected high-activity probiotic yogurt and a preparation method and application thereof.

[0007] The technical solution adopted by the present application to solve its technical problems is: A preparation method of a double-layer microcapsule-protected high-activity probiotic yogurt, comprising the following steps: (1) Preparation of single-layer microcapsules: mix the probiotic bacterial suspension and the protein solution, then cross-link in vegetable oil by transglutaminase (TG enzyme) to form gel particles embedding the probiotics, wash with Ringer's solution to obtain single-layer microcapsules; (2) Preparation of double-layer microcapsules: pretreat the single-layer microcapsules prepared in step (1) in a sodium alginate solution, then transfer to a cross-linking environment containing calcium ions, so that the sodium alginate reacts with the calcium ions to prepare double-layer microcapsules embedded with calcium alginate; (3) Preparation of yogurt: mix raw milk, white granulated sugar, and optional compound stabilizers, homogenize, sterilize, cool, then add double-layer microcapsules and a starter culture to ferment and prepare the yogurt.

[0008] Further, the probiotic bacterial suspension in step (1) is prepared by inoculating probiotics in MRS liquid medium, static culture, centrifugal collection of bacterial slurry, and resuspension with 0.85% physiological saline to obtain a bacterial suspension with a bacterial count of 10 9 -10 10 CFU / mL.

[0009] Further, the probiotics in step (1) are functional probiotics, including one or more of Bifidobacterium breve, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus casei, etc. in the Announcement of Bacterial Strains Allowed to be Used in Food.

[0010] Further, the protein in step (1) is sodium caseinate, whey protein, soybean protein isolate, etc. that can be cross-linked by TG enzyme, with a mass fraction of 10%.

[0011] Further, the vegetable oil in step (1) is one or more of soybean oil, sunflower oil, corn oil, and flaxseed oil.

[0012] Further, the specific preparation method of step (1) is: mix the bacterial suspension with a protein solution with a mass concentration of 10% at a volume ratio of 1:8-10, add transglutaminase (15-25 U / g of protein), mix the above mixture with vegetable oil at a volume ratio of 1:4-6, stir at 42°C and 600 rpm / min for 4-5 h to form gel particles, separate and wash with Ringer's reagent for 3 times to obtain single-layer microcapsules.

[0013] Further, the preparation method of the Ringer's solution in step (1) is as follows: 8.6 g of sodium chloride, 0.3 g of potassium chloride and 0.28 g of calcium chloride are dissolved in 1000 mL of distilled water to obtain the Ringer's solution.

[0014] Further, the mass fraction of the sodium alginate solution in step (2) is 1%.

[0015] Further, the calcium salt used in step (2) is selected from one or more of calcium chloride, calcium lactate, calcium phosphate and calcium carbonate, and the calcium ion concentration is 0.1 mol / L; the plant oil includes one or more of soybean oil, sunflower oil, corn oil and flaxseed oil.

[0016] Further, the double-layer microcapsule in step (2) has a complete spherical structure, with an average particle size of 383 μm and an embedding rate of 56.21%. Further, the specific preparation method of step (2) is as follows: the single-layer microcapsule is added to the 1% sodium alginate solution at a ratio of 15%-18% (w / v), and the gel particles are collected by centrifugation after stirring at 600 rpm / min for 30 min; the gel particles are resuspended in soybean oil containing 0.05% Tween 80 and 0.1 mol / L calcium ions, and the microcapsules are uniformly dispersed by stirring at an initial speed of 1000 rpm / min for 5 min, and then the speed is reduced to 700 rpm / min and maintained for 30 min; subsequently, the emulsion is destroyed by adding a solution containing 0.1 mol / L calcium ions, and the double-layer microcapsules are collected by centrifugation.

[0017] Further, the raw milk used in step (3) is fresh milk that meets the fresh milk purchase standards in China, which can be skimmed milk or partially skimmed milk.

[0018] Further, the amount of white granulated sugar added in step (3) is 5-7%, the amount of starter added is 0.004%-0.01%, and the amount of microcapsules added is 0.1%-0.2%. Further, the starter in step (3) is a conventional yogurt starter, including Streptococcus thermophilus and Lactobacillus bulgaricus.

[0019] Further, the specific preparation method of step (3) is as follows: white granulated sugar is added to the raw milk, and the mixture is stirred at 55°C and 700 rpm / min for 45 min to obtain a raw milk mixture; after homogenization, sterilization and cooling, the raw milk mixture is inoculated with the starter and the microcapsules for co-fermentation to prepare a high-activity probiotic yogurt protected by double-layer microcapsules.

[0020] Further, the fermentation temperature in step (3) is 40-45°C, the fermentation time is 6-8 h, and the fermentation is stopped when the pH reaches about 4.6.​​

[0021] The high-activity probiotic yogurt protected by the double-layer microcapsule is prepared by the preparation method.

[0022] The application of the preparation method in the preparation of yogurt.

[0023] The advantages and positive effects obtained by the present application are as follows: 1. The method of the present application first crosslinks the protein in the vegetable oil by the transglutaminase to form a single-layer embedded probiotic microcapsule; then the microcapsule is pretreated in a sodium alginate solution and secondarily crosslinked in a calcium ion-containing environment to construct a double-layer microcapsule; after the raw milk is homogenized, sterilized and cooled, the double-layer microcapsule and the starter are co-fermented to prepare a high-activity probiotic yogurt protected by the double-layer microcapsule. The number of live probiotics in the yogurt obtained by the present application can be maintained at more than 10 6 CFU / mL after 25 days of storage, and the water holding capacity, viscosity and texture characteristics are significantly better than those of the control sample without the microcapsule. The microcapsule can effectively protect the probiotics in the stomach environment and has a release rate of up to 99% in the intestine, significantly improving the survival rate and storage stability of the probiotics. After being added to the yogurt, the double-layer microcapsule embedding significantly improves the activity of the probiotics in the yogurt and the overall quality of the yogurt.

[0024] 2. In the method of the present application, the probiotics are embedded by the emulsification method to form a microcapsule. The microcapsule has a double-layer structure: the inner layer is a network structure formed by the TG enzyme-catalyzed protein, and the outer layer is a protective layer composed of sodium alginate and calcium ions. The protection effect of the double-layer microcapsule on the probiotics is significantly better than that of the single-layer microcapsule and the free bacteria form, effectively improving the stability of the bacteria during processing and storage. The prepared microcapsule has a uniform particle size of about 383 μm and an embedding rate of 56.21%, and has good physicochemical properties and embedding efficiency.

[0025] 3. The probiotic double-layer microcapsule in the method of the present application has good gastrointestinal tolerance and storage stability. The microcapsule is stable in structure in the stomach environment and is not easily degraded, which can effectively protect the embedded probiotics; after entering the intestine, the probiotic activity release rate is high, reaching 99.31%. At the same time, the microcapsule shows excellent protection performance under low-temperature storage conditions, and the bacterial survival rate remains above 99% after 1 year of storage at 4℃ and -20℃, significantly prolonging the shelf life and use stability of the probiotics.

[0026] 4. In the preparation process of the yogurt, the method of the present application adds the starter and the microcapsule rich in probiotics at the same time to co-act on the fermentation system, which not only effectively improves the water holding capacity, viscosity and texture characteristics of the yogurt, but also significantly enhances the probiotic function. In addition, the preparation method also helps to improve the flavor quality and mouthfeel experience of the yogurt product.

[0027] 5. This invention utilizes casein and sodium alginate as wall materials to encapsulate lactic acid bacteria, protecting probiotics from the influence of the external environment, avoiding a large loss of live bacteria during processing and storage, and thus improving the probiotic effects of live bacteria yogurt. Attached Figure Description

[0028] Figure 1 This is an electron scanning microscope of the double-layer microcapsules in this invention; Figure 2 This is a survival diagram of the double-layered microcapsules in simulated gastric juice in this invention; Figure 3 This is a diagram showing the release of the bilayer microcapsules in simulated intestinal fluid according to the present invention; Figure 4 This is a stability diagram of the bilayer microcapsules in this invention at 4℃ and -20℃; Figure 5 This is a pH value diagram of the fermented milk in this invention; Figure 6 This is a water-holding capacity diagram of the fermented milk in this invention; Figure 7 This is a diagram showing the apparent viscosity of the fermented milk in this invention. Detailed Implementation

[0029] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0030] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0031] A highly active probiotic yogurt protected by double-layer microcapsules and its preparation method, including a microcapsule preparation step and a fermented milk preparation step.

[0032] I. Preparation of Microcapsules The preparation of the above-mentioned microcapsules includes two processes: the preparation of monolayer microcapsules and the preparation of bilayer microcapsules.

[0033] (I) Preparation of monolayer microcapsules The preparation of monolayer microcapsules involves mixing a probiotic suspension and a protein solution, adding TG enzyme, and stirring in vegetable oil at 42°C and 600 rpm / min for 4-5 hours to form monolayer gel particles.

[0034] According to one specific embodiment, the bacterial suspension is mixed with a 10% protein solution by volume ratio of 1:8-10, and glutamine transaminase (15-25 U / g of protein) is added. The above mixture is mixed with vegetable oil at a volume ratio of 1:4-6, stirred at 42°C and 600 rpm / min for 4-5 h to form gel particles. After separation, the gel particles are washed with Ringer's reagent for 3 times and collected for standby.

[0035] In the preparation of the single-layer microcapsules, the probiotics used include one or more of Bifidobacterium breve, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus casei, etc. which are in accordance with the "List of Bacteria Strains for Food" announcement. The number of bacteria in the bacterial suspension is 10 9 -10 10 CFU / mL.

[0036] In the preparation of the single-layer microcapsules, the protein wall material is sodium caseinate, whey protein, soybean protein isolate, etc. which can be cross-linked by TGase. The vegetable oil includes one or more of soybean oil, sunflower oil, corn oil, flaxseed oil, etc.

[0037] (II) Preparation of double-layer microcapsules In the preparation of the double-layer microcapsules, the prepared single-layer microcapsules are added to a sodium alginate solution, and calcium ions are used for cross-linking to prepare double-layer microcapsules.

[0038] According to one specific embodiment, the single-layer microcapsules are added to a 1% sodium alginate solution at a proportion of 15%-18% (w / v, mass concentration) and stirred at 600 rpm / min for 30 min. The gel particles are collected by centrifugation. The gel particles are resuspended in soybean oil containing 0.05% Tween 80 and calcium ions, and cross-linking reaction occurs under stirring. After adding a solution containing 0.1 mol / L calcium ions to destroy the emulsion, the double-layer microcapsules are collected by centrifugation.

[0039] In the preparation of the double-layer microcapsules, the calcium salt is selected from one or more of calcium chloride, calcium lactate, calcium phosphate, and calcium carbonate, and the concentration of calcium ions is 0.1 mol / L. The vegetable oil includes one or more of soybean oil, sunflower oil, corn oil, and flaxseed oil. During the preparation process, the gel particles are resuspended in soybean oil containing Tween 80 and calcium ions, and the microcapsules are uniformly dispersed by stirring at an initial speed of 1000 rpm / min for 5 min. Then, the speed is reduced to 700 rpm / min and maintained for 30 min to allow the sodium alginate outside to cross-link with Ca 2+ .

[0040] II. Preparation of fermented milk The fermented milk preparation method of the present invention can be carried out using conventional methods in the art. Specifically, the fermented milk preparation includes steps such as ingredient preparation, homogenization, sterilization, and fermentation.

[0041] The preparation steps include adding granulated sugar to the raw milk and stirring until well mixed; The homogenization and sterilization steps include homogenizing, sterilizing, and cooling the raw milk mixture; The fermentation step involves adding a starter culture and microcapsules to a cooled raw milk mixture for co-fermentation to prepare highly active probiotic yogurt protected by double-layer microcapsules.

[0042] According to one specific embodiment, the preparation of the above-mentioned fermented milk includes the following steps: (1) Ingredients: Mix raw milk, white sugar, and optional compound stabilizers at 52-56℃ and 600-800rpm / min for 35-55min; (2) Degassing: The raw milk is degassed; (3) Homogenization: The degassed raw milk is homogenized at a temperature of 55-65℃ and a pressure of 15-25MPa. (4) Sterilization: After homogenization, the raw milk is sterilized at a temperature of 93-95℃ for 5 minutes. (5) Cooling: Cool the sterilized mixture to 40-45℃; (6) Fermentation: Add starter culture and microcapsules to the cooled raw milk and ferment at 40-45℃ for 6-8 hours. Stop fermentation when the pH reaches about 4.6.

[0043] The raw milk used in the above-mentioned yogurt fermentation process is fresh milk that meets my country's standards for the purchase of raw milk, and can be skimmed or... Partially skimmed milk. According to a specific embodiment of the present invention, preferably, the milk used is fresh milk, or reconstituted milk made from milk powder; In the above yogurt fermentation process, the amount of white sugar added is 5-7%, the amount of starter culture added is 0.004%-0.01%, and microcapsules are used. The addition amount is 0.1%-0.2%; In the above-mentioned yogurt fermentation process, the starter culture is a conventional yogurt starter culture, including Streptococcus thermophilus and Lactobacillus bulgaricus.

[0044] The present invention will now be described in further detail with reference to specific embodiments.

[0045] Example 1 A preparation process for a probiotic-encapsulated fermented milk includes two processes: the preparation of probiotic microcapsules and the preparation of yogurt, specifically comprising the following steps: 1. Preparation of probiotic microcapsules (1) A certain amount of Bifidobacterium breve powder was inoculated into a test tube containing 5 mL of MRS liquid medium, and incubated at 37°C for 16 h. Three-zone streaking was performed on MRS solid medium, and incubated at 37°C for 48 h under anaerobic conditions. A single colony was picked and inoculated into MRS liquid medium, and incubated at 37°C for 16 h. The inoculum was then subcultured at a concentration of 2%, and the bacterial suspension was collected. 30 mL of the bacterial suspension was centrifuged at 8000 x g for 5 min, and the bacterial slurry was resuspended in 10 mL of 0.85% physiological saline. The bacterial count was 10 9 CFU / mL; (2) Sodium caseinate was dissolved in distilled water, and stirred at 600 rpm / min for 2 h at room temperature to prepare a 10% sodium caseinate solution. 18 mL of the sodium caseinate solution and 2 mL of the bacterial suspension were mixed, and TG enzyme was added to achieve an enzyme activity of 20 U / g protein. The mixture was stirred at 39°C for 4 h. After standing for 30 min, single-layer microcapsule particles in the oil phase were prepared; (3) The single-layer microcapsules were washed with Lin's reagent (0.86% sodium chloride, 0.03% potassium chloride, and 0.028% calcium chloride, all by mass percentage) three times, and stored for later use; (4) 15 g of the single-layer microcapsules were added to 100 mL of 1% sodium alginate solution, and stirred at 600 rpm / min for 30 min. The gel particles were collected by centrifugation at 2500 x g for 5 min; (5) The gel particles were added to 100 mL of soybean oil containing 0.05% Tween 80 and 0.1 mol / L calcium ions under the action of a magnetic stirrer, and stirred at 1000 rpm / min for 5 min, and then at 700 rpm / min for 30 min. Then, 200 mL of 0.1 mol / L calcium chloride solution was added, and the double-layer microcapsules were collected by centrifugation at 500 x g for 5 min.

[0046] 2. Preparation of probiotic microcapsule-rich yogurt (1) 92 g of raw cow milk and 7 g of white granulated sugar were mixed and stirred at 55°C and 700 rpm / min for 45 min to obtain a base material; (2) The base material was heated to 60°C and homogenized under a pressure of 20 MPa; (3) The homogenized base material was sterilized at 95°C for 5 min; (4) Cool the sterilized base material to 40-45℃, add 0.004g of starter (conventional yogurt starter, including Streptococcus thermophilus and Lactobacillus bulgaricus) and 1g of double-layer microcapsules for co-fermentation, ferment at 42℃ until the pH reaches below 4.6, and then stop fermentation; (5) After the fermented milk is broken and packaged, it is refrigerated at 4°C for post-maturation to obtain double-layer microcapsule yogurt rich in probiotics.

[0047] Example 2 The preparation process of a fermented milk rich in probiotic microcapsules includes two processes: the preparation of probiotic microcapsules and the preparation of yogurt, specifically comprising the following steps: 1. Preparation of probiotic microcapsules (1) Dissolve 0.1g of Bifidobacterium breve powder in 10mL of 0.85% physiological saline and mix well to obtain a bacterial suspension; (2) Sodium caseinate was dissolved in distilled water and stirred at 600 rpm / min for 2 h at room temperature to prepare a 10% sodium caseinate solution. 18 mL of sodium caseinate solution and 2 mL of bacterial suspension were mixed and TG enzyme was added to make the enzyme activity 20 U / g protein. The mixture was stirred at 39 °C for 4 h. After standing for 30 min, monolayer microcapsule particles in the oil phase were prepared. (3) Wash the monolayer microcapsules three times with Ringer's reagent (0.86% sodium chloride, 0.03% potassium chloride, 0.028% calcium chloride, all percentages above are mass percentages. Mix sodium chloride, potassium chloride, and calcium chloride evenly with distilled water to obtain Ringer's reagent). Store for later use. (4) Add 15g of monolayer microcapsules to 100mL of 1% sodium alginate solution, stir at 600rpm / min for 30min, centrifuge at 2500×g for 5min, and collect the gel particles. (5) Under the action of a magnetic stirrer, the above gel particles were added to 100 mL of soybean oil containing 0.05% Tween 80 and 0.1 mol / L calcium ions while stirring (1000 rpm / min). After stirring at 1000 rpm / min for 5 min, the mixture was stirred at 700 rpm / min for 30 min. Then, 200 mL of 0.1 mol / L calcium chloride solution was added, and the mixture was centrifuged at 500×g for 5 min to collect the double-layer microcapsules.

[0048] 2. Preparation of probiotic-rich microcapsule yogurt (1) Mix 92g of raw milk and 7g of white sugar, and stir at 55℃ and 700rpm / min for 45min to obtain the base material; (2) Heat the base material to 60°C and homogenize it under a pressure of 20 MPa; (3) sterilize the homogenized base at 95℃ for 5 minutes; (4) cool the sterilized base to 40-45℃, add 0.004g of starter culture (regular yogurt starter culture, including Streptococcus thermophilus and Lactobacillus bulgaricus) and 1g of double-layer microcapsules for synergistic fermentation, and ferment at 42℃ until the pH is below 4.6, then stop fermentation; (5) after breaking the emulsion of the fermented milk, store it in a refrigerator at 4℃ for cold maturation to obtain a double-layer microcapsule yogurt rich in probiotics.

[0049] Comparative Example 1 This comparative example provides a yogurt preparation process, which is prepared in the same way as Example 1, except that only a starter culture is added during the preparation of the yogurt, without adding double-embedded probiotic microcapsules.

[0050] Comparative Example 2 This example provides a yogurt preparation process rich in probiotics, which is prepared in the same way as Example 1, except that double-embedded probiotic microcapsules are replaced by free probiotics during the preparation of the probiotic microcapsule yogurt.

[0051] The relevant tests are as follows: 1. In Example 1 and Example 2, single-layer microcapsules were prepared by TG enzyme cross-linked sodium casein, and double-layer microcapsules were prepared by coating the microcapsules with sodium alginate. Scanning electron microscopy found that the prepared double-layer microcapsules had a complete spherical structure (see Figure 1 ), with an average particle size of 383μm and an embedding rate of 56.21%. The present application successfully prepared double-layer microcapsules with regular structure, and through optimization of the preparation condition parameters, effectively embedded and protected Bifidobacterium breve.

[0052] The embedding rate was determined by placing 0.5g of microcapsules in 4.5mL of preheated to 37℃ disassembly liquid, incubating at 37℃ for 105min, gradient dilution, and selecting three consecutive appropriate dilutions for plate pouring. The embedding rate is represented by the ratio of the total number of microcapsule disassembly bacteria to the total number of bacteria in the prepared microcapsule bacterial suspension.

[0053] Further, the formula of the disassembly liquid is to dissolve 6.8g of potassium dihydrogen phosphate in 250mL of distilled water, then add 77mL of 0.2mol / L sodium hydroxide and 500mL of distilled water. Then add 10g of trypsin, mix, and adjust the pH to 6.8±0.1 with 0.2mol / L sodium hydroxide or 0.2mol / L hydrochloric acid, and dilute to 1000mL with distilled water, and filter sterilize with a 0.22μm filter membrane.

[0054] 2. Simulated in-vitro gastrointestinal digestion and storage stability experiments were performed on the double-layer microcapsules prepared in Examples 1 and 2.

[0055] (1) Simulated gastric conditions The test method was as follows: 0.5 g of microcapsules and 0.5 mL of B. breve bacterial suspension (bacterial content 10 9 CFU / mL) were placed in 4.5 mL of simulated gastric juice, vortexed to mix them well, and incubated in a 37°C constant-temperature shaking incubator at a speed of 150 r / min for 105 min. At 0, 30, 60, 90 and 120 min, the corresponding samples were taken out. The bacterial strain was released from the capsule solution, gradiently diluted and plated for counting, and the survival rate of the probiotic bacteria was determined.

[0056] Further, the formula of the simulated gastric juice was as follows: 2.0 g of sodium chloride was dissolved in 7 mL of hydrochloric acid, and distilled water was added to make up to 1000 mL. The pH was adjusted to 1.2 with 0.1 mol / L hydrochloric acid, and pepsin was added to make the concentration 3.2 g / L. The solution was filtered through a 0.22 μm filter membrane to remove bacteria.

[0057] The survival of the probiotic bacteria in the simulated gastric juice is shown in Table 1 Figure 2 , where the survival amount of the probiotic bacteria at 0 min was taken as the benchmark. After 2 h in the simulated gastric juice, the viable count of the free bacteria decreased by 4.06 log CFU / g; the viable count of the activated single-layer microcapsules (bacterial powder was cultured in MRS medium and then embedded) decreased by 2.24 log CFU / g; the viable count of the unactivated single-layer microcapsules (bacterial powder was dispersed in physiological saline and then embedded) decreased by 2.25 log CFU / g; the viable count of the activated double-layer microcapsules (bacterial powder was cultured in MRS medium and then embedded) decreased by only 0.39 log CFU / g; and the viable count of the unactivated double-layer microcapsules (bacterial powder was dispersed in physiological saline and then embedded) decreased by only 0.38 log CFU / g. The results showed that the double-layer microcapsules of the present example were relatively stable in structure in the simulated gastric digestion environment, had strong resistance to gastric acid, could relatively effectively resist the erosion of gastric acid, maintained good stability and integrity of the microcapsules, and compared with the free bacteria and the single-layer microcapsules, the double-layer microcapsules could more effectively block the erosion of gastric acid on the probiotic bacteria, thereby significantly improving the survival rate of the probiotic bacteria in the harsh environment of the stomach.

[0058] (2) Simulated intestinal digestion conditions The test method was as follows: 0.5 g of microcapsules and 0.5 mL of B. breve bacterial suspension (bacterial content 10 9CFU / mL) were placed in 4.5 mL simulated intestinal fluid, vortexed to mix them evenly, and incubated in a 37℃ constant-temperature incubator at a speed of 150 r / min for 4 h. The culture was terminated in an ice-water bath, and the corresponding sample was taken out, gradiently diluted, and then plate-counted to determine the survival rate of the probiotic bacteria.

[0059] Further, the formula of the simulated intestinal fluid was as follows: 6.8 g of potassium dihydrogen phosphate was dissolved in 250 mL of distilled water, 77 mL of 0.2 mol / L sodium hydroxide and 500 mL of distilled water were added, 10 g of trypsin was added, and the pH was adjusted to 6.8±0.1 with 0.2 mol / L sodium hydroxide or 0.2 mol / L hydrochloric acid, and then the volume was made up to 1000 mL with distilled water, and the bacteria were removed by filtration with a 0.22 μm filter membrane.

[0060] The survival rate of the probiotic bacteria in the simulated intestinal fluid was as follows: Figure 3 , wherein the survival rate of the probiotic bacteria at 0 min was taken as the reference. After 4 h in the simulated intestinal fluid, the survival rate of the viable bacteria of the free bacteria was 92.56%; the survival rate of the viable bacteria released from the activated single-layer microcapsules (the bacteria powder was cultured in MRS medium and then embedded) was 94.60%; the survival rate of the viable bacteria released from the unactivated single-layer microcapsules (the bacteria powder was dispersed in physiological saline and then embedded) was 96.05%; the survival rate of the viable bacteria released from the activated double-layer microcapsules (the bacteria powder was cultured in MRS medium and then embedded) was 99.31%; and the survival rate of the viable bacteria released from the unactivated double-layer microcapsules (the bacteria powder was dispersed in physiological saline and then embedded) was 98.99%. The results show that the double-layer microcapsules of the embodiment can realize efficient and rapid release of the probiotic bacteria in the simulated intestinal environment. This property, combined with the excellent protection ability of the double-layer microcapsules in the gastric juice, jointly proves that the double-layer microcapsule system can realize the directional delivery function of the probiotic bacteria, i.e., the gastric protection and intestinal release, and greatly improves the bioavailability of the probiotic bacteria. As found by Wang (2025) et al., after 4 h in the simulated intestinal fluid, the survival rate of the probiotic bacteria in the nano calcium carbonate-sodium alginate microcapsules was 87.71%. However, compared with the probiotic bacteria microcapsules prepared by the method of the embodiment, there is still a significant gap.

[0061] (3) Storage stability experiment of the double-layer microcapsules The test method was as follows: the probiotic bacteria double-layer microcapsules were divided into sealed vials and stored at -20℃ and 4℃, and plate counting was performed every month to determine the viable bacteria count.

[0062] The storage stability results of the double-layer microcapsules were as follows: Figure 4The microcapsules at both storage temperatures showed good survival of probiotic bacteria during the 12-month storage period. Specifically, the viable count decreased by 0.041 Log CFU / g after storage at 4°C, and only 0.032 Log CFU / g after storage at -20°C. The results showed that the double-layer microcapsule structure of the present embodiment could provide long-term effective protection for the embedded probiotic bacteria, significantly delaying the loss of viable bacteria during storage. At the same time, the data further confirmed that lower storage temperature (such as -20°C) was more conducive to maintaining the activity of probiotic bacteria in the microcapsules and the stability of the product. As Chavarri (2010) et al. found that the viable count of Bifidobacterium microencapsulated by chitosan decreased by 0.35 log CFU / ml after 28 days of refrigeration (4°C). However, compared with the probiotic microcapsules prepared by the method of the present embodiment, there is still a significant gap.

[0063] 3. The pH, water holding capacity, texture and apparent viscosity of the yogurt prepared in Examples 1 and 2 were measured.

[0064] (1) pH measurement The change of pH of the yogurt during the storage period is shown in Figure 5During the 21-day storage period, the pH values of all groups showed a downward trend, indicating that the system was undergoing a continuous acidification process. However, Example 1 and Example 2 showed significant technical effects superior to the comparative examples in terms of efficient initial acid-producing capacity and maintaining system pH stability. At the beginning of storage (day 1), the pH values of all experimental groups were at a similar level (about 4.2-4.3), ensuring the consistency of the subsequent comparison baseline. The pH values of Comparative Example 1 and Comparative Example 2 continued to decline from day 1 to day 21, from 4.28 to 4.19 (Comparative Example 1) and from 4.30 to 4.18 (Comparative Example 2), respectively, with a significant decrease. In contrast, the present examples showed better pH stability. In particular, Example 1, with an initial pH value of 4.22, only slightly decreased to 4.11 on day 21, with a change much smaller than the comparative examples, and the initial pH value of 4.22 was significantly lower than the comparative examples, with the Bifidobacterium breve and the starter under the protection of microencapsulation exerting rapid and synergistic acid-producing effects in the early stage. Example 2 also showed excellent stability. This enhanced pH stability is of crucial positive significance for maintaining product activity, extending shelf life, and ensuring use efficiency. The results show that, compared with the comparative examples, the use of the technical solutions of the present examples not only ensures the initial effectiveness of the product, but also significantly improves the stability and reliability of the product during the storage period, producing a synergistic technical effect. At the same time, by comparing Example 1, Comparative Example 1, and Comparative Example 2, it can be seen that the starter and the double-layer microcapsule in the method of the present application have a synergistic effect, which can synergistically improve the relevant properties of the fermented milk rich in probiotic microcapsules prepared, especially the starter and the double-layer microcapsule with a mass ratio of 0.004:1 have a significant synergistic effect, which can significantly synergistically improve the relevant properties of the fermented milk rich in probiotic microcapsules prepared.

[0065] (2) Water holding capacity determination The test method is as follows: After the yogurt is ripened, about 20 g is taken into a 50 mL centrifuge tube. Centrifuge at 4000 x g for 20 min at 4°C, discard the supernatant and invert the centrifuge tube for about 5 min. The total mass of the sample and the centrifuge tube after centrifugation m1; the mass of the centrifuge tube m2; the mass of the sample m. The water holding capacity is represented by the ratio of the mass of the sample after centrifugation to the mass of the sample.

[0066] The change of yogurt water holding capacity during the storage period is shown in Figure 6During the 21-day storage period, Example 1 and Example 2 showed significant technical effects in maintaining the water holding capacity stability of the yogurt, which was significantly better than the comparative examples without using the technology. By the 21st day, the water holding capacity of Comparative Example 1 and Comparative Example 2 had decreased to 65.57% and 63.57%, respectively, indicating that a large amount of whey was separated from the system structure. In contrast, Example 1 remained at 70.63%, while Example 2 maintained a higher level of 71.74%. The water holding capacity of the examples was about 1.13 times that of the comparative examples at the end point, which directly proved the excellent ability of the examples to maintain product texture stability over a long period. The water holding capacity of Example 1 remained at a high level throughout (84.13% on the 1st day → 70.63% on the 21st day), with the best overall stability. Example 2 showed a unique late recovery ability, with the water holding capacity increasing from 65.59% on the 7th day to 71.74% on the 21st day, a trend that the comparative examples did not have, proving that the microencapsulated bacteria could still play a role in the late fermentation period and strengthen the gel network. The results showed that the microencapsulated Bifidobacterium breve in the examples effectively slowed down the rate of decline in the water holding capacity of the yogurt during the shelf life, significantly inhibited the separation of whey, and improved the appearance and texture stability of the product. As Ma (2020) et al. found, the addition of denatured whey protein isolate and inulin microcapsules increased the water holding capacity of the yogurt by about 2%. However, there is still a significant gap compared with the probiotic microcapsules prepared by the method of the present application. At the same time, by comparing Example 1, Comparative Example 1 and Comparative Example 2, it can be seen that there is a synergistic effect between the starter and the double-layer microcapsules in the method of the present application, which can synergistically improve the relevant properties of the fermented milk rich in probiotic microcapsules prepared, especially when the mass ratio of the starter to the double-layer microcapsules is 0.004:1, there is a significant synergistic effect between the two, which can significantly synergistically improve the relevant properties of the fermented milk rich in probiotic microcapsules prepared.

[0067] (3) Texture and apparent viscosity determination The test method is as follows: the hardness, consistency, cohesiveness and gumminess of the yogurt are determined by a texture analyzer. The probe type is A / BE, the trigger type is automatic-10g, the pre-test speed is 1.0 mm / s, the test speed is 1.0 mm / s, the post-test speed is 10.0 mm / s, the measurement distance is 80% of the height of the yogurt, and the data acquisition rate is 400 pps; the apparent viscosity is detected by No. 3 rotor in NDJ-99 digital viscometer at 60 r / min.

[0068] Table 1 Change in yogurt texture during storage period

[0069] The changes of yogurt texture during storage are shown in Table 1. During the 21-day storage period, the yogurt of Example 1 and Example 2 showed significant technical effects superior to the comparative examples in maintaining the stability of yogurt texture and delaying the deterioration during storage. By the 21st day, the hardness (106.76 gf) and chewiness (24.28 gf) of Comparative Example 1 decreased sharply, and the springiness (0.60) was severely lost, indicating that the gel network had been destroyed and the mouthfeel had significantly deteriorated. In contrast, the decrease in the examples was very moderate. In particular, the hardness (124.26 gf) of Example 2 was significantly higher than that of all other samples, and the chewiness (34.31 gf) was also superior to that of Comparative Example 1 (24.28 gf). This proves that the examples can most effectively inhibit the destruction of the gel structure, maintain the fullness of the product form and the toughness of the mouthfeel. The springiness reflects the recovery ability of the product after being pressed, and the gumminess simulates the force required to chew a semi-solid sample into a swallowable state. By the 21st day, the springiness (0.60) of Comparative Example 1 had deteriorated severely, and the springiness (0.65) of Comparative Example 2 was also significantly lower than that of Example 1 (0.71) and Example 2 (0.72). This indicates that the examples can better maintain the three-dimensional structure of the gel network, so that the product can still maintain better form after chewing. By the 21st day, the gumminess (47.56 gf) of Example 2 was comparable to that of Comparative Example 2 (46.59 gf), but much higher than that of Comparative Example 1 (40.43 gf) whose structure had been severely destroyed. This indicates that the product of the present application can provide more consistent mouthfeel strength throughout the shelf life, avoiding the soft and weak mouthfeel in the later period. The results show that the microencapsulated Bifidobacterium breve yogurt of the examples is significantly superior to the comparative examples in terms of hardness, springiness and chewiness, which are core indicators of mouthfeel, at the end of the 21-day storage period, substantially extending the optimal eating texture period of the product. At the same time, by comparing Example 1, Comparative Example 1 and Comparative Example 2, it can be seen that there is a synergistic effect between the starter and the double-layer microcapsules in the method of the present application, which can synergistically improve the relevant properties of the fermented milk rich in probiotic microcapsules prepared, especially when the mass ratio of the starter to the double-layer microcapsules is 0.004:1, the synergistic effect is significant, which can significantly synergistically improve the relevant properties of the fermented milk rich in probiotic microcapsules prepared.

[0070] The changes of yogurt apparent viscosity during storage are shown in Table 2. Figure 7During the 21-day storage period, the Examples 1 and 2 exhibited significant technical effects over the Comparative Examples in establishing and maintaining high apparent viscosity of the yogurt system. The apparent viscosity values of the Examples of the present application were consistently significantly higher than all the Comparative Examples during the 21-day storage period. At the initial stage of storage (Day 1), the initial viscosity of Example 1 (3.05 Pa-s) and Example 2 (2.98 Pa-s) were about 17% and 34% higher than Comparative Example 1 (2.60 Pa-s) respectively, which indicated that the present Examples helped to form a more viscous initial gel structure. This advantage was maintained throughout the shelf life. By Day 21, the viscosity values of Example 1 (3.07 Pa-s) and Example 2 (3.15 Pa-s) were still steadily maintained above 3.0 Pa-s, while Comparative Example 1 and Comparative Example 2 were only 2.62 Pa-s and 2.62 Pa-s respectively. The viscosity values of the Examples were about 20% higher than the Comparative Examples. The viscosities of the Comparative Examples exhibited varying degrees of fluctuation over time (e.g. Comparative Example 2: 2.23 → 2.49 → 2.74 → 2.62), which indicated that the gel network structure was unstable. The apparent viscosity of Example 1 steadily increased from 3.05 Pa-s on Day 1 to a peak of 3.22 Pa-s on Day 14, and was maintained at 3.07 Pa-s on Day 21, exhibiting very high stability. Example 2 also exhibited a smooth change trend (2.98 → 3.14 → 3.18 → 3.15 Pa-s). This small fluctuation range proved that the present Examples effectively maintained the stability of the yogurt system, avoiding severe viscosity deterioration during the storage period. The results showed that the present Examples of microencapsulated B. breve yogurt helped to form a yogurt with higher initial viscosity and a more thick mouthfeel, directly improving the initial quality perception of the product; and the yogurt product could consistently maintain excellent fluid properties throughout the shelf life. At the same time, by comparing Example 1, Comparative Example 1 and Comparative Example 2, it can be seen that there is a synergistic effect between the starter and the double-layer microcapsules in the method of the present application, which can synergistically improve the relevant properties of the fermented milk rich in probiotic microcapsules prepared, especially when the mass ratio of the starter to the double-layer microcapsules is 0.004:1, there is a significant synergistic effect between the two, which can significantly synergistically improve the relevant properties of the fermented milk rich in probiotic microcapsules prepared.

[0071] 4. Determination of B. breve content of the yogurt prepared in Examples 1 and 2 The test method was as follows: 25 g of microencapsulated yogurt was placed in 225 mL of a preheated 37°C deencapsulation liquid, vortexed to mix uniformly, and incubated in a 37°C constant temperature incubator at a speed of 150 r / min for 4 h. The culture was terminated in an ice water bath, and the corresponding sample was taken out, gradiently diluted, and then plated for counting to determine the survival rate of the probiotics.

[0072] Further, the unpacking liquid is prepared by dissolving 6.8 g of potassium dihydrogen phosphate in 250 mL of distilled water, then adding 77 mL of 0.2 mol / L sodium hydroxide and 500 mL of distilled water. Then add 10 g of trypsin, mix and adjust the pH to 6.8±0.1 with 0.2 mol / L sodium hydroxide or 0.2 mol / L hydrochloric acid, and then dilute to 1000 mL with distilled water, and filter sterilized with a 0.22 μm filter.

[0073] The Bifidobacterium breve in the yogurts prepared in Examples 1-2 and Comparative Examples 1-2 was detected according to GB 4789.35-2023, and the results are shown in Table 2.

[0074] Table 2 Change of Bifidobacterium breve in fermented milk during storage period

[0075] As can be seen from Table 2, Examples 1 and 2 exhibit significant advantages in improving the storage stability of probiotic yogurt. The specific results are as follows: after fermentation, the initial viable cell count of Bifidobacterium breve in Examples 1 and 2 reached 1.4×10 8 , 3.4×10 7 CFU / mL, respectively, indicating that the preparation method of the present application can effectively guarantee the final cell count. On the 5th day of storage, the viable cell count of Examples 1 and 2 still maintained at 1.58×10 8 , 1.9×10 7 CFU / mL, respectively, which was significantly higher than that of Comparative Example 2 (1.28×10 6 CFU / mL), indicating that the present application can effectively improve the survival ability of probiotics in the early stage of storage. During the entire 25-day storage period, Examples 1 and 2 always maintained a high viable cell count. Even at the end of storage (25th day), the viable cell count of Examples 1 and 2 was still 1.3×10 7 , 6.2×10 6CFU / mL, while the control sample 2 was unable to detect viable bacteria from the 15th day. After 21 days of storage, the survival rate of unencapsulated B. breve decreased by 100%. In contrast, B. breve encapsulated in microcapsules exhibited excellent survival rate, with only about 1 order of magnitude decrease. This result fully demonstrates that the present application can significantly delay the decline of bacterial bodies and effectively maintain the number of viable bacteria in the product during long-term storage through the protection of microcapsules. In summary, the microcapsule scheme provided by the present application can significantly enhance the survival stability during storage, prolong the shelf life of the product, and improve the functionality and added value of the product. At the same time, as can be seen from the comparison of example 1, comparative example 1 and comparative example 2, the present application has a synergistic effect between the fermenting agent and the double-layer microcapsule, which can synergistically improve the related properties of the fermented milk rich in probiotic microcapsules prepared, especially when the mass ratio of the fermenting agent to the double-layer microcapsule is 0.004:1, the two have a significant synergistic effect, which can significantly synergistically improve the related properties of the fermented milk rich in probiotic microcapsules prepared.

[0076] Although the embodiments of the present application are disclosed for illustrative purposes, those skilled in the art can understand that various alternatives, changes and modifications are possible without departing from the spirit and scope of the present application and the appended claims, therefore, the scope of the present application is not limited to the disclosed content of the embodiments.

Claims

1. A method for preparing a highly active probiotic yogurt protected by double-layer microcapsules, characterized in that: Includes the following steps: (1) Preparation of monolayer microcapsules: After mixing probiotic suspension and protein solution, cross-linking is carried out in vegetable oil by transglutaminase (TG) to form gel particles encapsulating probiotics. The particles are washed with Ringer's solution to obtain monolayer microcapsules. (2) Preparation of bilayer microcapsules: After pretreatment of the single-layer microcapsules prepared in step (1) in sodium alginate solution, they are transferred to a cross-linking environment containing calcium ions to allow sodium alginate to react with calcium ions and prepare sodium alginate calcium-encapsulated bilayer microcapsules. (3) Preparation of yogurt: Mix raw milk and white sugar, homogenize, sterilize and cool, then add double-layer microcapsules and starter culture, and ferment to prepare yogurt.

2. The preparation method according to claim 1, characterized in that: The probiotic suspension mentioned in step (1) is prepared by inoculating probiotics into MRS liquid culture medium, allowing them to stand, collecting the bacterial sludge by centrifugation, and resuspending them in 0.85% physiological saline to obtain a bacterial count of 10. 9 -10 10 CFU / mL bacterial suspension.

3. The preparation method according to claim 1, characterized in that: In step (1), the probiotics are functional probiotics, including one or more of the following listed in the "List of Strains that can be Used in Food".

4. The preparation method according to claim 1, characterized in that: The protein mentioned in step (1) is a protein that can be cross-linked by TG enzyme, including sodium caseinate, whey protein, and soy protein isolate, and the mass fraction of protein in the protein solution is 10%; Alternatively, the vegetable oil mentioned in step (1) may be one or more of soybean oil, sunflower seed oil, corn oil, and flaxseed oil; Alternatively, the specific preparation method of step (1) is as follows: the bacterial suspension and the 10% protein solution are mixed at a volume ratio of 1:8-10, glutamine transaminase is added to make its enzyme activity 15-25 U / g protein, the above mixture is mixed with vegetable oil at a volume ratio of 1:4-6, and stirred at 42℃ and 600 rpm / min for 4-5 hours to form gel particles. After separation, the particles are washed three times with Ringer's reagent to obtain monolayer microcapsules. Alternatively, the preparation method of the Ringer's solution in step (1) is as follows: dissolve 8.6g of sodium chloride, 0.3g of potassium chloride and 0.28g of calcium chloride in 1000mL of distilled water to obtain the Ringer's solution.

5. The preparation method according to claim 1, characterized in that: In step (2), the mass fraction of sodium alginate in the sodium alginate solution is 1%; Alternatively, the calcium salt used in step (2) may be selected from one or more of calcium chloride, calcium lactate, calcium phosphate, and calcium carbonate, with a calcium ion concentration of 0.1 mol / L; Alternatively, the bilayer microcapsules in step (2) have a complete spherical structure with an average particle size of 383 μm and an encapsulation rate of 56.21%. Alternatively, the specific preparation method of step (2) is as follows: add monolayer microcapsules to a 1% sodium alginate solution at a mass percentage of 15%-18% (w / v), stir at 600 rpm / min for 30 min, and collect the gel particles by centrifugation; resuspend the gel particles in soybean oil containing 0.05% Tween 80 and 0.1 mol / L calcium ions, stir at an initial speed of 1000 rpm / min for 5 min to make the microcapsules uniformly dispersed, then reduce the speed to 700 rpm / min and maintain it for 30 min. Subsequently, add a solution containing 0.1 mol / L calcium ions to break up the emulsion, and collect the bilayer microcapsules by centrifugation.

6. The preparation method according to claim 1, characterized in that: The raw milk used in step (3) must comply with Chinese standards. The raw milk purchase standard includes skimmed or partially skimmed milk; Alternatively, in step (3), the amount of white sugar added is 5-7% by mass, the amount of fermenting agent added is 0.004%-0.01% by mass, and the amount of microcapsules added is 0.1%-0.2% by mass. In step (3), the starter culture is a conventional yogurt starter culture, including Streptococcus thermophilus and Lactobacillus bulgaricus.

7. The preparation method according to claim 1, characterized in that: The specific preparation method of step (3) is as follows: add white sugar to the raw milk and stir and mix for 45 minutes at 55℃ and 700rpm / min to obtain raw milk mixture; after homogenizing, sterilizing and cooling the raw milk mixture, inoculate the starter culture and microcapsules for co-fermentation to prepare high-activity probiotic yogurt protected by double-layer microcapsules.

8. The preparation method according to claim 1, characterized in that: The fermentation temperature in step (3) is 40-45℃, the fermentation time is 6-8h, and fermentation is stopped when the pH reaches about 4.

6.

9. The highly active probiotic yogurt protected by double-layer microcapsules prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the preparation method according to any one of claims 1 to 8 in the preparation of yogurt.

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