High-activity probiotic microcapsule suitable for dairy product fermentation and preparation method thereof

Abstract

The invention relates to the technical field of dairy product fermentation, and discloses a high-activity probiotic microcapsule suitable for dairy product fermentation and a preparation method thereof.The microcapsule is prepared from, by weight, 6.7-13.3 parts of bifidobacterium lactis, 3.3-6.7 parts of lactobacillus plantarum, 9.1-15.2 parts of Arabic gum, 13.6-22.7 parts of maltodextrin, 7.3-12.1 parts of chitosan, 15.1-30.2 parts of trehalose, 3.8-7.5 parts of glycerinum and 1.1-2.3 parts of ascorbic acid. The molecular weight of the chitosan is 80kDa, and the weight part ratio of the bifidobacterium lactis to the lactobacillus plantarum is 2: 1. In the invention, an inner core is formed through a eutectic structure of bifidobacterium lactis, lactobacillus plantarum and trehalose to protect the probiotics, so that the problems that the existing probiotics are easily influenced by the external environment in the storage and fermentation processes, the activity is reduced, and the fermentation effect of dairy products is poor are solved.

Description

Technical Field

[0001] This invention relates to the field of dairy fermentation technology, and in particular to highly active probiotic microcapsules suitable for dairy fermentation and their preparation method. Background Technology

[0002] Probiotics in dairy fermentation are a class of live microorganisms that play a crucial role in the fermentation process of dairy products, offering numerous health benefits. Common probiotics in dairy fermentation include lactic acid bacteria and bifidobacteria. Lactic acid bacteria convert lactose into lactic acid, giving dairy products their unique sour taste and texture, while simultaneously inhibiting the growth of harmful bacteria and maintaining the balance of the intestinal microecology. Bifidobacteria can improve intestinal function, promote nutrient absorption, and enhance the body's immunity. These probiotics play a key role in dairy fermentation. On the one hand, they produce various beneficial substances through metabolic activities, such as vitamins and short-chain fatty acids, enhancing the nutritional value of dairy products. On the other hand, the fermentation process alters the structure of dairy products, making them easier for the body to digest and absorb, suitable for different groups, especially those with lactose intolerance. Appropriate intake of dairy products containing fermented probiotics helps regulate the intestinal flora, promote digestion, and strengthen the body. However, the activity of probiotics is affected by factors such as storage conditions and processing techniques. It is important to choose fresh, properly stored dairy products to ensure the intake of a sufficient number of live probiotics and to fully realize their health benefits.

[0003] Existing probiotics are easily affected by the external environment during storage and fermentation, leading to reduced activity and thus causing poor fermentation results in dairy products. Summary of the Invention

[0004] To overcome the above shortcomings, this invention provides highly active probiotic microcapsules suitable for dairy product fermentation and their preparation method, aiming to improve the problem that existing probiotics are easily affected by the external environment during storage and fermentation, resulting in reduced activity and thus poor fermentation effect in dairy products.

[0005] To achieve the above objectives, the present invention provides the following technical solution: Highly active probiotic microcapsules suitable for dairy product fermentation comprise the following raw materials in parts by weight: 6.7-13.3 parts of *Bifidobacterium lactis*, 3.3-6.7 parts of *Lactobacillus plantarum*, 9.1-15.2 parts of gum arabic, 13.6-22.7 parts of maltodextrin, 7.3-12.1 parts of chitosan, 15.1-30.2 parts of trehalose, 3.8-7.5 parts of glycerol, and 1.1-2.3 parts of ascorbic acid. The chitosan has a molecular weight of 80 kDa. The weight ratio of *Bifidobacterium lactis* to *Lactobacillus plantarum* is 2:1, and the total weight of *Bifidobacterium lactis* and *Lactobacillus plantarum* is 10-20 parts. The total viable count of *Bifidobacterium lactis* and *Lactobacillus plantarum* is ≥1.5 × 10⁻⁶. 12 CFU / g.

[0006] By adopting the above technical solution, the core of the microcapsule is formed by the eutectic structure of Bifidobacterium lactis, Lactobacillus plantarum and trehalose to protect the probiotics. The shell is formed by the composite matrix of gum arabic and maltodextrin to provide physical support. The membrane formed by the cross-linked layer of chitosan and ascorbic acid resists adverse external factors. This allows the microcapsule to effectively protect the activity of probiotics during storage and fermentation, thus solving the problem that existing probiotics are easily affected by the external environment during storage and fermentation, resulting in reduced activity and poor fermentation effect of dairy products.

[0007] Preferably, the degree of esterification of the gum arabic is ≥75%, and the DE value of the maltodextrin is 10-15.

[0008] Preferably, the ascorbic acid and chitosan form a coating layer through electrostatic adsorption, and the coating layer accounts for 3%-5% of the total raw material mass.

[0009] Preferably, the microcapsule has a core-shell-membrane three-layer structure: the core is a eutectic structure of Bifidobacterium lactis, Lactobacillus plantarum and trehalose, the shell is a composite matrix of gum arabic and maltodextrin, and the membrane is a cross-linked layer of chitosan and ascorbic acid.

[0010] Preferably, the thickness ratio of the core-shell-membrane three-layer structure is 2:3:1, and the membrane surface is distributed with micropores with a diameter of 200-500 nm.

[0011] The preparation method of highly active probiotic microcapsules suitable for dairy product fermentation includes the following steps:

[0012] S1. Raw material preparation: Weigh each component according to the formula;

[0013] S2. Pre-crosslinking treatment of wall material: Add 50℃ deionized water, gum arabic, maltodextrin and chitosan to a constant temperature stirring tank, stir with an anchor stirrer at 200-300r / min until completely dissolved, adjust the pH to 5.5, add calcium chloride solution accounting for 0.2% of the total mass of the system, control the temperature in the constant temperature stirring tank at 50±2℃, and perform the crosslinking reaction for 30min to form a pre-crosslinked colloid;

[0014] S3. Activation of the protective agent: Trehalose, glycerol, and ascorbic acid are mixed in a vacuum mixing vessel. The temperature inside the vacuum mixing vessel is controlled at 58-62℃ and the vacuum degree is controlled at -0.07 to 0.09MPa. The mixture is stirred for 0.9-1.1 hours to form an activated protective agent.

[0015] S4. Composite Emulsification: Bifidobacterium lactis, Lactobacillus plantarum, pre-crosslinked colloid, and activating protectant are mixed in a weight ratio of 1:1:3:2 and then placed in an emulsifier. The speed of the emulsifier is controlled at 3000±200 r / min for homogenization, and the homogenization time is controlled at 20±2 min. After homogenization, it is transferred to a high-pressure homogenizer. The pressure of the high-pressure homogenizer is controlled at 150±5 MPa. The high-pressure homogenization cycle is repeated 3 times, with an interval of 5 min between each cycle, to form an oil-in-water emulsion.

[0016] S5. Spray-fluidized bed combined drying: The emulsion is dried using a spray-fluidized bed combined equipment. The spray-fluidized bed combined equipment uses a dual-fluid nozzle with a nozzle orifice diameter controlled at 1.0-1.5 mm, an atomization pressure controlled at 0.25-0.35 MPa, and a feed rate controlled at 10-15 mL / min. In the spray-fluidized bed combined equipment, the fluidized bed inlet air temperature is controlled at 185-195℃, the fluidized bed temperature is controlled at 58-62℃, and the fluidized bed air velocity is controlled at 1.3-1.7 m / s. The dried emulsion has a moisture content of ≤3% and forms microcapsules.

[0017] S6. Surface Modification: The dried microcapsules were added to a constant-temperature shaking reactor at a ratio of 1% (g / mL) of 1% β-cyclodextrin solution for shaking modification. The pH of the solution was adjusted to 7.0±0.1 using sodium hydroxide or hydrochloric acid solution. The temperature of the shaking reactor was controlled at 28-32℃, the shaking speed at 90-110 r / min, and the shaking time at 1.8-2.2 h. The shaking modification was carried out in the dark. After shaking modification, the microcapsules were separated by centrifugation and filtration. The centrifugation speed was controlled at 3000 r / min, and the centrifugation time was controlled at 5 min. After centrifugation, the microcapsules were placed in a vacuum drying oven with a vacuum degree controlled at -0.09 MPa and the temperature inside the vacuum drying oven controlled at 40℃ until constant weight was achieved.

[0018] Preferably, in step S4, nitrogen gas is introduced for protection during the homogenization process, with the nitrogen flow rate controlled at 5-10 L / min, and monitored using an oxygen concentration sensor to maintain the oxygen content ≤0.1%.

[0019] Preferably, in S5, in the spray-fluidized bed combined equipment, the vertical distance between the bottom of the spray drying tower and the top of the fluidized bed is 75-85cm, and they are connected by a guide pipe.

[0020] The present invention has the following beneficial effects:

[0021] 1. In this invention, the core of the microcapsule is formed by the eutectic structure of Bifidobacterium lactis, Lactobacillus plantarum and trehalose to protect the probiotics. The shell is formed by the composite matrix of gum arabic and maltodextrin to provide physical support. The membrane formed by the cross-linked layer of chitosan and ascorbic acid resists adverse external factors. This allows the microcapsule to effectively protect the activity of the probiotics during storage and fermentation, thereby solving the problem that existing probiotics are easily affected by the external environment during storage and fermentation, resulting in reduced activity and poor fermentation effect of dairy products.

[0022] 2. In this invention, Bifidobacterium lactis and Lactobacillus plantarum are combined in a 2:1 weight ratio to form a metabolic synergy. This is combined with the slow swelling characteristics of the gum arabic-maltodextrin-chitosan gradient cross-linked wall material, the trehalose-glycerol-ascorbic acid antioxidant protection system, the size sieving effect of the 200-500nm micropores in the membrane layer, and the pH-responsive release mechanism of chitosan. This enables a dynamic process in which probiotics colonize and produce acid in the early stage of fermentation, stably metabolize and produce aroma in the middle stage, and release live bacteria on demand in the later stage. This simultaneously optimizes the efficiency of substrate diffusion and product discharge, avoids the problems of sudden release inactivation and metabolic inhibition of traditional microcapsules, significantly shortens the fermentation cycle, and increases the amount of flavor substances generated.

[0023] 3. In this invention, the temperature, stirring speed, and cross-linking time of the pre-cross-linking of the wall material are precisely controlled by a constant-temperature reactor, enabling gum arabic and chitosan to form a uniform network structure under the action of calcium chloride, thus improving the consistency of the wall material. A two-stage process using a high-shear emulsifier and a high-pressure homogenizer refines the emulsion particle size to ≤5μm, and nitrogen protection inhibits oxidation, ensuring the stability of the protective agent's effective components. A spray-fluidized bed combined with dual-fluid nozzle parameters and fluidized bed temperature and rate control, combined with precise 75-85cm spacing for flow guidance, achieves rapid surface curing of microcapsules and low-temperature drying of the core, avoiding thermal damage and particle breakage. Standardized control of process parameters throughout the entire process ensures uniform microcapsule particle size and a viable bacteria retention rate of ≥92% at each stage from wall material preparation to drying and modification, significantly improving the quality uniformity and stability of industrial production. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the preparation steps of the highly active probiotic microcapsules suitable for dairy product fermentation proposed in this invention. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] See appendix Figure 1 This invention provides highly active probiotic microcapsules suitable for dairy product fermentation, comprising the following raw materials in parts by weight: 6.7-13.3 parts of Bifidobacterium lactis, 3.3-6.7 parts of Lactobacillus plantarum, 9.1-15.2 parts of gum arabic, 13.6-22.7 parts of maltodextrin, 7.3-12.1 parts of chitosan, 15.1-30.2 parts of trehalose, 3.8-7.5 parts of glycerol, and 1.1-2.3 parts of ascorbic acid. The chitosan has a molecular weight of 80 kDa. The weight ratio of Bifidobacterium lactis to Lactobacillus plantarum is 2:1, the total weight of Bifidobacterium lactis and Lactobacillus plantarum is 10-20 parts, and the total viable count of Bifidobacterium lactis and Lactobacillus plantarum is ≥1.5 × 10⁻⁶. 12 CFU / g.

[0027] Specifically, Bifidobacterium lactis and Lactobacillus plantarum were combined at a weight ratio of 2:1, resulting in a total viable count ≥1.5 × 10⁻⁶. 12 The controlled concentration of CFU / g creates a metabolic synergistic effect, improving the fermentation efficiency of dairy products. Gum arabic, maltodextrin, and chitosan are combined in a weight ratio of 1:1.5:0.8 to form a gradient cross-linked wall material. Among them, 80kDa chitosan forms an acid-resistant film layer through electrostatic adsorption, enabling the microcapsules to achieve an encapsulation rate of ≥90% in a pH 4.0 environment. Trehalose provides skeletal protection, glycerol provides moisturizing, and ascorbic acid provides antioxidant effects. These components are combined in a weight ratio of 4:1:0.3 to form a ternary protection system. Combined with the chitosan coating layer, this increases the survival rate of live bacteria stored at 4℃ for 6 months to over 85%.

[0028] The degree of esterification of gum arabic is ≥75%, and the DE value of maltodextrin is 10-15.

[0029] Specifically, a gum arabic esterification degree of ≥75% can improve the cross-linking efficiency between wall material molecules and enhance the mechanical strength of the microcapsule shell; a maltodextrin DE value of 10-15 gives it suitable viscosity and permeability, promotes the bonding between the wall material and probiotics interface, and synergistically optimizes the integrity and anti-swelling properties of microcapsule encapsulation.

[0030] Ascorbic acid and chitosan form a coating layer through electrostatic adsorption, which accounts for 3%-5% of the total raw material mass.

[0031] Specifically, the coating layer formed by ascorbic acid and chitosan through electrostatic adsorption (accounting for 3%-5% of the total raw material mass) can build an antioxidant barrier and impart a negative charge to the surface of the microcapsules, inhibiting the oxidation reaction between probiotics and the external environment, while enhancing the dispersion stability of the microcapsules in the milk system.

[0032] The microcapsules have a core-shell-membrane three-layer structure: the core is a eutectic structure of Bifidobacterium lactis, Lactobacillus plantarum and trehalose, the shell is a composite matrix of gum arabic and maltodextrin, and the membrane is a cross-linked layer of chitosan and ascorbic acid.

[0033] Specifically, the core eutectic structure enables physical encapsulation and vitrification protection of live bacteria, inhibiting activity decay during storage; the shell composite matrix provides mechanical support and a slow-release channel, adapting to the fermentation environment of dairy products; the membrane cross-linking layer forms a chemical barrier, enhancing acid resistance and delaying oxidation reactions, synergistically improving the stability of microcapsules and the controllability of live bacteria release.

[0034] The thickness ratio of the core-shell-membrane three-layer structure is 2:3:1, and the membrane surface has micropores with a diameter of 200-500 nm.

[0035] Specifically, the functional allocation of each layer is optimized by using a three-layer structure with a thickness ratio of 2:3:1. The core provides sufficient space for live bacteria, the shell layer ensures structural strength, and the membrane layer achieves a precise barrier function. The 200-500nm micropores on the surface of the membrane layer can regulate the substrate diffusion rate, allowing probiotics to be released on demand during the fermentation of dairy products, while blocking the intrusion of large molecular impurities and maintaining stable activity.

[0036] The preparation method of highly active probiotic microcapsules suitable for dairy product fermentation includes the following steps:

[0037] S1. Raw material preparation: Weigh each component according to the formula;

[0038] S2. Pre-crosslinking treatment of wall material: Add 50℃ deionized water, gum arabic, maltodextrin and chitosan to a constant temperature stirring tank, stir with an anchor stirrer at 200-300r / min until completely dissolved, adjust the pH to 5.5, add calcium chloride solution accounting for 0.2% of the total mass of the system, control the temperature in the constant temperature stirring tank at 50±2℃, and perform the crosslinking reaction for 30min to form a pre-crosslinked colloid;

[0039] S3. Activation of the protective agent: Trehalose, glycerol, and ascorbic acid are mixed in a vacuum mixing vessel. The temperature inside the vacuum mixing vessel is controlled at 58-62℃ and the vacuum degree is controlled at -0.07 to 0.09MPa. The mixture is stirred for 0.9-1.1 hours to form an activated protective agent.

[0040] S4. Composite Emulsification: Bifidobacterium lactis, Lactobacillus plantarum, pre-crosslinked colloid, and activating protectant are mixed in a weight ratio of 1:1:3:2 and then placed in an emulsifier. The speed of the emulsifier is controlled at 3000±200 r / min for homogenization, and the homogenization time is controlled at 20±2 min. After homogenization, it is transferred to a high-pressure homogenizer. The pressure of the high-pressure homogenizer is controlled at 150±5 MPa. The high-pressure homogenization cycle is repeated 3 times, with an interval of 5 min between each cycle, to form an oil-in-water emulsion.

[0041] S5. Spray-fluidized bed combined drying: The emulsion is dried using a spray-fluidized bed combined equipment. The spray-fluidized bed combined equipment uses a dual-fluid nozzle with a nozzle orifice diameter controlled at 1.0-1.5 mm, an atomization pressure controlled at 0.25-0.35 MPa, and a feed rate controlled at 10-15 mL / min. In the spray-fluidized bed combined equipment, the fluidized bed inlet air temperature is controlled at 185-195℃, the fluidized bed temperature is controlled at 58-62℃, and the fluidized bed air velocity is controlled at 1.3-1.7 m / s. The dried emulsion has a moisture content of ≤3% and forms microcapsules.

[0042] S6. Surface Modification: The dried microcapsules were added to a constant-temperature shaking reactor at a ratio of 1% (g / mL) of 1% β-cyclodextrin solution for shaking modification. The pH of the solution was adjusted to 7.0±0.1 using sodium hydroxide or hydrochloric acid solution. The temperature of the shaking reactor was controlled at 28-32℃, the shaking speed at 90-110 r / min, and the shaking time at 1.8-2.2 h. The shaking modification was carried out in the dark. After shaking modification, the microcapsules were separated by centrifugation and filtration. The centrifugation speed was controlled at 3000 r / min, and the centrifugation time was controlled at 5 min. After centrifugation, the microcapsules were placed in a vacuum drying oven with a vacuum degree controlled at -0.09 MPa and the temperature inside the vacuum drying oven controlled at 40℃ until constant weight was achieved.

[0043] Specifically, S2 pre-crosslinking of wall materials: Through constant temperature stirring and pH control, gum arabic and chitosan are directionally crosslinked under the action of calcium chloride to form a pre-crosslinked colloid with a network structure, improving the mechanical strength and encapsulation efficiency of the wall material; S3 activation of protective agents: Vacuum environment and temperature control promote the formation of intermolecular hydrogen bonds of trehalose, ascorbic acid, etc., enhancing the compatibility between protective agents and probiotics, while reducing the oxidation loss of ascorbic acid; S4 composite emulsification: Two-stage homogenization process (high shear + high pressure) ensures that probiotics are uniformly dispersed in the wall material. The material-protectant system forms a stable emulsion with a particle size ≤5μm, laying the foundation for the uniformity of microcapsules; S5 combined drying: spray drying rapidly solidifies the shell, fluidized bed low temperature maintains the activity of live bacteria, dual-fluid nozzle and wind speed control achieves controllable microcapsule particle size of 5-20μm, and water content ≤3% ensures storage stability; S6 surface modification: β-cyclodextrin inclusion complexation gives the microcapsule surface lipophilicity, light-proof vibration avoids degradation of active ingredients, centrifugation and vacuum drying remove excess modifiers, ensuring product purity and dispersibility.

[0044] In S4, nitrogen gas is introduced for protection during homogenization, with the nitrogen flow rate controlled at 5-10 L / min. An oxygen concentration sensor is used to monitor the oxygen content and maintain it at ≤0.1%.

[0045] Specifically, by introducing nitrogen gas during the homogenization process and controlling the oxygen content to ≤0.1%, oxygen in the system can be eliminated, the oxidation reaction between probiotics and protectants (such as ascorbic acid) can be inhibited, the activity of live bacteria can be maintained, and the biostability of microcapsules during the emulsification stage can be ensured.

[0046] In S5, in the spray-fluidized bed combined equipment, the vertical distance between the bottom of the spray drying tower and the top of the fluidized bed is 75-85cm, and they are connected by a guide pipe.

[0047] Specifically, the vertical distance between the bottom of the spray drying tower and the top of the fluidized bed is 75-85cm and they are connected by a guide pipe. This ensures that after the atomized droplets have completed their initial solidification in the drying tower, they can smoothly transition to the fluidized bed. This avoids the particles falling and breaking due to excessive spacing or the airflow being disturbed due to insufficient spacing, thus ensuring the continuity and structural integrity of the microcapsule drying process.

[0048] The following is a further description with reference to specific embodiments:

[0049] Example 1:

[0050] Highly active probiotic microcapsules suitable for dairy product fermentation comprise the following raw materials in parts by weight: 13.3 parts Bifidobacterium lactis, 6.7 parts Lactobacillus plantarum, 15.2 parts gum arabic, 22.7 parts maltodextrin, 12.1 parts chitosan, 30.2 parts trehalose, 7.5 parts glycerol, and 2.3 parts ascorbic acid. The chitosan has a molecular weight of 80 kDa. The weight ratio of Bifidobacterium lactis to Lactobacillus plantarum is 2:1, with a total weight of 20 parts. The total viable count of Bifidobacterium lactis and Lactobacillus plantarum is ≥1.5 × 10⁻⁶. 12 CFU / g.

[0051] The degree of esterification of gum arabic is ≥75%, and the DE value of maltodextrin is 10-15.

[0052] Ascorbic acid and chitosan form a coating layer through electrostatic adsorption, which accounts for 3%-5% of the total raw material mass.

[0053] The microcapsules have a core-shell-membrane three-layer structure: the core is a eutectic structure of Bifidobacterium lactis, Lactobacillus plantarum and trehalose, the shell is a composite matrix of gum arabic and maltodextrin, and the membrane is a cross-linked layer of chitosan and ascorbic acid.

[0054] The thickness ratio of the core-shell-membrane three-layer structure is 2:3:1, and the membrane surface has micropores with a diameter of 200-500 nm.

[0055] The preparation method of highly active probiotic microcapsules suitable for dairy product fermentation includes the following steps:

[0056] S1. Raw material preparation: Weigh each component according to the formula;

[0057] S2. Pre-crosslinking treatment of wall material: Add 50℃ deionized water, gum arabic, maltodextrin and chitosan to a constant temperature stirring tank, stir with an anchor stirrer at 200-300r / min until completely dissolved, adjust the pH to 5.5, add calcium chloride solution accounting for 0.2% of the total mass of the system, control the temperature in the constant temperature stirring tank at 50±2℃, and perform the crosslinking reaction for 30min to form a pre-crosslinked colloid;

[0058] S3. Activation of the protective agent: Trehalose, glycerol, and ascorbic acid are mixed in a vacuum mixing vessel. The temperature inside the vacuum mixing vessel is controlled at 58-62℃ and the vacuum degree is controlled at -0.07 to 0.09MPa. The mixture is stirred for 0.9-1.1 hours to form an activated protective agent.

[0059] S4. Composite Emulsification: Bifidobacterium lactis, Lactobacillus plantarum, pre-crosslinked colloid, and activating protectant are mixed in a weight ratio of 1:1:3:2 and then placed in an emulsifier. The speed of the emulsifier is controlled at 3000±200 r / min for homogenization, and the homogenization time is controlled at 20±2 min. After homogenization, it is transferred to a high-pressure homogenizer. The pressure of the high-pressure homogenizer is controlled at 150±5 MPa. The high-pressure homogenization cycle is repeated 3 times, with an interval of 5 min between each cycle, to form an oil-in-water emulsion.

[0060] S5. Spray-fluidized bed combined drying: The emulsion is dried using a spray-fluidized bed combined equipment. The spray-fluidized bed combined equipment uses a dual-fluid nozzle with a nozzle orifice diameter controlled at 1.0-1.5 mm, an atomization pressure controlled at 0.25-0.35 MPa, and a feed rate controlled at 10-15 mL / min. In the spray-fluidized bed combined equipment, the fluidized bed inlet air temperature is controlled at 185-195℃, the fluidized bed temperature is controlled at 58-62℃, and the fluidized bed air velocity is controlled at 1.3-1.7 m / s. The dried emulsion has a moisture content of ≤3% and forms microcapsules.

[0061] S6. Surface Modification: The dried microcapsules were added to a constant-temperature shaking reactor at a ratio of 1% (g / mL) of 1% β-cyclodextrin solution for shaking modification. The pH of the solution was adjusted to 7.0±0.1 using sodium hydroxide or hydrochloric acid solution. The temperature of the shaking reactor was controlled at 28-32℃, the shaking speed at 90-110 r / min, and the shaking time at 1.8-2.2 h. The shaking modification was carried out in the dark. After shaking modification, the microcapsules were separated by centrifugation and filtration. The centrifugation speed was controlled at 3000 r / min, and the centrifugation time was controlled at 5 min. After centrifugation, the microcapsules were placed in a vacuum drying oven with a vacuum degree controlled at -0.09 MPa and the temperature inside the vacuum drying oven controlled at 40℃ until constant weight was achieved.

[0062] In S4, nitrogen gas is introduced for protection during homogenization, with the nitrogen flow rate controlled at 5-10 L / min. An oxygen concentration sensor is used to monitor the oxygen content and maintain it at ≤0.1%.

[0063] In S5, in the spray-fluidized bed combined equipment, the vertical distance between the bottom of the spray drying tower and the top of the fluidized bed is 75-85cm, and they are connected by a guide pipe.

[0064] Example 2:

[0065] The difference between this embodiment and Embodiment 1 above is that:

[0066] The sample contained 10 parts Bifidobacterium lactis, 5 parts Lactobacillus plantarum, 12.15 parts gum arabic, 18.15 parts maltodextrin, 9.7 parts chitosan, 22.65 parts trehalose, 5.65 parts glycerol, and 1.7 parts ascorbic acid. The chitosan had a molecular weight of 80 kDa. The weight ratio of Bifidobacterium lactis to Lactobacillus plantarum was 2:1, and the total weight of Bifidobacterium lactis and Lactobacillus plantarum was 15 parts. The total viable count of Bifidobacterium lactis and Lactobacillus plantarum was ≥1.5 × 10⁻⁶. 12 CFU / g.

[0067] Example 3:

[0068] The difference between this embodiment and Embodiment 1 above is that:

[0069] The sample contained 6.7 parts Bifidobacterium lactis, 3.3 parts Lactobacillus plantarum, 9.1 parts gum arabic, 13.6 parts maltodextrin, 7.3 parts chitosan, 15.1 parts trehalose, 3.8 parts glycerol, and 1.1 parts ascorbic acid. The chitosan had a molecular weight of 80 kDa. The weight ratio of Bifidobacterium lactis to Lactobacillus plantarum was 2:1, and the total weight of Bifidobacterium lactis and Lactobacillus plantarum was 10 parts. The total viable count of Bifidobacterium lactis and Lactobacillus plantarum was ≥1.5 × 10⁻⁶. 12 CFU / g.

[0070] Table 1:

[0071]

[0072]

[0073] The table above compares the probiotic survival rates of each group after storage at 4℃ in the dark, at 0, 3, and 6 months. The viable count was determined using the plate count method (GB4789.35-2016), and the survival rate was calculated as (viable count after storage / initial viable count × 100%). Comparative Example 1 uses a single Bifidobacterium lactis microcapsule with a core-shell two-layer structure (no membrane layer) and identical manufacturing processes. Comparative Example 2 uses microcapsules with a 1:1 bacterial ratio and no ascorbic acid protectant. As shown in Comparative Example 1 in Table 1, this invention utilizes the eutectic structure of Bifidobacterium lactis, Lactobacillus plantarum, and trehalose. The microcapsules form a core to protect probiotics, utilize a composite matrix of gum arabic and maltodextrin to form a shell for physical support, and employ a cross-linked layer of chitosan and ascorbic acid to form a membrane to resist adverse external factors. This allows the microcapsules to effectively protect the activity of probiotics during storage and fermentation. By utilizing a three-layer wall material, the problem of existing probiotics being easily affected by the external environment during storage and fermentation, leading to reduced activity and poor fermentation results in dairy products, is solved. As shown in Comparative Example 2 in Table 1, the ascorbic acid protective agent system of this invention can play an antioxidant role, thereby ensuring the long-term activity of probiotics.

[0074] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A highly active probiotic microcapsule suitable for dairy product fermentation, characterized in that, The raw materials include the following parts by weight: 6.7-13.3 parts of Bifidobacterium lactis, 3.3-6.7 parts of Lactobacillus plantarum, 9.1-15.2 parts of gum arabic, 13.6-22.7 parts of maltodextrin, 7.3-12.1 parts of chitosan, 15.1-30.2 parts of trehalose, 3.8-7.5 parts of glycerol, and 1.1-2.3 parts of ascorbic acid. The chitosan has a molecular weight of 80 kDa. The weight ratio of Bifidobacterium lactis to Lactobacillus plantarum is 2:1, and the total weight of Bifidobacterium lactis and Lactobacillus plantarum is 10-20 parts. The total viable count of Bifidobacterium lactis and Lactobacillus plantarum is ≥1.5 × 10⁻⁶. 12 CFU / g.

2. The highly active probiotic microcapsules suitable for dairy product fermentation according to claim 1, characterized in that: The gum arabic has an esterification degree of ≥75%, and the maltodextrin has a DE value of 10-15.

3. The highly active probiotic microcapsules suitable for dairy product fermentation according to claim 1, characterized in that: The ascorbic acid and chitosan form a coating layer through electrostatic adsorption, and this coating layer accounts for 3%-5% of the total raw material mass.

4. The highly active probiotic microcapsules suitable for dairy product fermentation according to claim 1, characterized in that: The microcapsule has a core-shell-membrane three-layer structure: the core is a eutectic structure of Bifidobacterium lactis, Lactobacillus plantarum and trehalose, the shell is a composite matrix of gum arabic and maltodextrin, and the membrane is a cross-linked layer of chitosan and ascorbic acid.

5. The highly active probiotic microcapsules suitable for dairy product fermentation according to claim 4, characterized in that: The thickness ratio of the core-shell-membrane three-layer structure is 2:3:1, and the membrane surface is distributed with micropores with a diameter of 200-500nm.

6. The method for preparing highly active probiotic microcapsules suitable for dairy product fermentation according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Raw material preparation: Weigh each component according to the formula; S2. Pre-crosslinking treatment of wall material: Add 50℃ deionized water, gum arabic, maltodextrin and chitosan to a constant temperature stirring tank, stir with an anchor stirrer at 200-300r / min until completely dissolved, adjust the pH to 5.5, add calcium chloride solution accounting for 0.2% of the total mass of the system, control the temperature in the constant temperature stirring tank at 50±2℃, and perform the crosslinking reaction for 30min to form a pre-crosslinked colloid; S3. Activation of the protective agent: Trehalose, glycerol, and ascorbic acid are mixed in a vacuum mixing vessel. The temperature inside the vacuum mixing vessel is controlled at 58-62℃ and the vacuum degree is controlled at -0.07 to 0.09MPa. The mixture is stirred for 0.9-1.1 hours to form an activated protective agent. S4. Composite Emulsification: Bifidobacterium lactis, Lactobacillus plantarum, pre-crosslinked colloid, and activating protectant are mixed in a weight ratio of 1:1:3:2 and then placed in an emulsifier. The speed of the emulsifier is controlled at 3000±200 r / min for homogenization, and the homogenization time is controlled at 20±2 min. After homogenization, it is transferred to a high-pressure homogenizer. The pressure of the high-pressure homogenizer is controlled at 150±5 MPa. The high-pressure homogenization cycle is repeated 3 times, with an interval of 5 min between each cycle, to form an oil-in-water emulsion. S5. Spray-fluidized bed combined drying: The emulsion is dried using a spray-fluidized bed combined equipment. The spray-fluidized bed combined equipment uses a dual-fluid nozzle with a nozzle orifice diameter controlled at 1.0-1.5 mm, an atomization pressure controlled at 0.25-0.35 MPa, and a feed rate controlled at 10-15 mL / min. In the spray-fluidized bed combined equipment, the fluidized bed inlet air temperature is controlled at 185-195℃, the fluidized bed temperature is controlled at 58-62℃, and the fluidized bed air velocity is controlled at 1.3-1.7 m / s. The dried emulsion has a moisture content of ≤3% and forms microcapsules. S6. Surface Modification: The dried microcapsules were added to a constant-temperature shaking reactor at a ratio of 1% (g / mL) of 1% β-cyclodextrin solution for shaking modification. The pH of the solution was adjusted to 7.0±0.1 using sodium hydroxide or hydrochloric acid solution. The temperature of the shaking reactor was controlled at 28-32℃, the shaking speed at 90-110 r / min, and the shaking time at 1.8-2.2 h. The shaking modification was carried out in the dark. After shaking modification, the microcapsules were separated by centrifugation and filtration. The centrifugation speed was controlled at 3000 r / min, and the centrifugation time was controlled at 5 min. After centrifugation, the microcapsules were placed in a vacuum drying oven with a vacuum degree controlled at -0.09 MPa and the temperature inside the vacuum drying oven controlled at 40℃ until constant weight was achieved.

7. The method for preparing highly active probiotic microcapsules suitable for dairy product fermentation according to claim 6, characterized in that: In step S4, nitrogen gas is introduced for protection during the homogenization process, with the nitrogen flow rate controlled at 5-10 L / min. An oxygen concentration sensor is used to monitor the oxygen content and maintain it at ≤0.1%.

8. The method for preparing highly active probiotic microcapsules suitable for dairy product fermentation according to claim 6, characterized in that: In S5, in the spray-fluidized bed combined equipment, the vertical distance between the bottom of the spray drying tower and the top of the fluidized bed is 75-85cm, and they are connected by a guide pipe.

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