Preparation method of probiotic microcapsule with high stress resistance

Abstract

The invention provides a preparation method of a probiotic microcapsule with high stress resistance, and belongs to the field of foods, pharmaceutical preparations or feeds. The microcapsule prepared by the preparation method has excellent technical effects of excellent high temperature resistance and digestive juice resistance, capability of protecting a core material from being inactivated in a spray drying process, high embedding rate, difficulty in disintegration in simulated gastric acid, good slow-release effect in simulated intestinal juice and the like; and the stability of the probiotics which have heat sensitivity and are not resistant to digestive juice is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food (including general food or health food), pharmaceutical preparation or feed, and in particular to a preparation method of high-stress-resistance probiotic microcapsules. BACKGROUND

[0002] Microcapsule technology can provide a physical barrier for the core material, effectively masking the unpleasant taste and odor of the core material, enhancing its tolerance to external adverse environment, and improving stability. For heat-sensitive and unstable core materials such as probiotics, active drugs, etc., microcapsule technology is an important way to improve their stability and bioavailability. Especially for probiotics, in the actual processing, storage and digestion process, they are easily affected by external adverse environment, resulting in a significant decrease in the number and activity of the final colonization in the human intestinal tract, and even complete inactivation, making it difficult to exert the probiotic effect. However, the existing microcapsule preparation methods have problems such as high material cost, complex processing, low embedding efficiency, and unsuitability for large-scale production, etc., and cannot exhibit high stress resistance in the processing, storage and digestion processes. Patent CN114287632A proposes a method for preparing sodium alginate-chitosan-inulin probiotic microcapsules by extrusion method, which can improve the stability of probiotics in gastric juice, but its preparation efficiency is low, not suitable for industrial production, and the average particle size of the microcapsules reaches 4.5 mm, which is not conducive to the sensory evaluation when ingested.

[0003] Spray drying is a commonly used microcapsule preparation technology, which has the advantages of low cost, simple operation, high preparation efficiency and continuous production, and is often used in industrial production. However, the high temperature in the spray drying process can cause great damage to the core material, which needs further improvement.

[0004] In summary, there is currently no microcapsule with good stability that can simply and effectively improve the tolerance of the core material to digestion and heat treatment, and is suitable for large-scale preparation. SUMMARY

[0005] To solve the above technical problems, the present application provides the following technical solutions.

[0006] In a first aspect, the present application provides a preparation method of high-stress-resistance microcapsules.

[0007] A preparation method of high-stress-resistance microcapsules, comprising the following steps: (1) dissolving wall material A in water to obtain a wall material A solution, wherein the wall material A is at least one of whey protein, soy protein and sodium caseinate; (2) dissolving wall material B in water to obtain a wall material B solution, wherein the wall material B is at least one of collagen and gelatin; (3) Dissolve wall material C in water to obtain wall material C solution, wherein wall material C is seaweed polysaccharide; then mix the wall material C solution with the wall material B solution evenly to obtain mixed solution 1; (4) Mix the core material with the wall material A solution obtained in step (1) to obtain mixed solution 2. Then add the mixed solution 1 obtained in step (3) to mixed solution 2 and mix thoroughly to obtain a suspension. (5) The suspension obtained in step (4) is spray-dried to obtain the high stress-resistant microcapsules.

[0008] In some embodiments, the molecular weight of the seaweed polysaccharide is ≥100 kDa. In some preferred embodiments, the molecular weight of the seaweed polysaccharide is ≥1000 kDa.

[0009] In some embodiments, the seaweed polysaccharide is at least one selected from the following: *Ulva prolifera* polysaccharide, *Sargassum fusiforme* polysaccharide, *Asparagus cochinchinensis* polysaccharide, *Ulva lactuca* polysaccharide, and kelp polysaccharide. In some preferred embodiments, the seaweed polysaccharide is *Ulva prolifera* polysaccharide with a molecular weight ≥1000 kDa.

[0010] In some embodiments, the method for preparing the seaweed polysaccharide includes: extracting the seaweed polysaccharide by water extraction using seaweed as raw material, then separating and purifying it, and drying it to obtain the seaweed polysaccharide; wherein the seaweed includes at least one of Ulva prolifera, Sargassum fusiforme, Asparagus cochinchinensis, Ulva lactuca, and kelp.

[0011] In some embodiments, the preparation method includes: extracting seaweed plants in water at 80℃-100℃ (e.g., 80℃, 85℃, 90℃, 95℃ or 100℃) for 2 h-6 h, wherein 20 g to 500 g (e.g., 20 g, 30 g, 40 g, 50 g, 60 g, 70 g, 80 g, 90 g, 100 g, 150 g, 200 g, 250 g, 300 g, 350 g, 400 g, 450 g or 500 g) of the seaweed plants are added per liter of water; centrifuging, taking the supernatant, separating and purifying it using a separation membrane, collecting the retentate, drying it, and obtaining the seaweed polysaccharide; In some embodiments, the separation membrane has a molecular weight cutoff of 100 kDa. In some preferred embodiments, the separation membrane has a molecular weight cutoff of 1000 kDa.

[0012] In some embodiments, the content of wall material A in the suspension is 5 g / L to 20 g / L, calculated based on the total volume of the suspension. In some embodiments, the content of wall material A in the suspension is 5 g / L, 10 g / L, 15 g / L, or 20 g / L, calculated based on the total volume of the suspension.

[0013] In some embodiments, the content of wall material B in the suspension is 10 g / L to 40 g / L, calculated based on the total volume of the suspension. In some embodiments, the content of wall material B in the suspension is 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, or 40 g / L, calculated based on the total volume of the suspension.

[0014] In some embodiments, the content of seaweed polysaccharides in the suspension is 1 g / L to 20 g / L, calculated based on the total volume of the suspension. In some embodiments, the content of seaweed polysaccharides in the suspension is 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, or 20 g / L, calculated based on the total volume of the suspension.

[0015] In some embodiments, the core material is probiotics, and the ratio of the number of viable bacteria in the core material to the total mass of wall material A, wall material B and seaweed polysaccharide in the suspension in step (4) is (10) 6 ~10 11 CFU: 1 g. In some embodiments, the core material is probiotics, and the ratio of the number of viable bacteria in the core material to the total mass of wall material A, wall material B and seaweed polysaccharide in the suspension in step (4) is 10. 6 CFU: 1 g, 10 7 CFU: 1 g, 10 8 CFU: 1 g, 10 9 CFU: 1 g, 10 10 CFU: 1 g or 10 g 11 CFU: 1 g.

[0016] In some embodiments, the core material is a drug, and the mass ratio of the core material in step (4) to the total mass of wall material A, wall material B and seaweed polysaccharide in the suspension is (0.1~1.0):1.0. In some embodiments, the core material is a drug, and the mass ratio of the core material in step (4) to the total mass of wall material A, wall material B and seaweed polysaccharide in the suspension is 0.1:1.0, 0.2:1.0, 0.3:1.0, 0.4:1.0, 0.5:1.0, 0.6:1.0, 0.7:1.0, 0.8:1.0, 0.9:1.0 or 1.0:1.0.

[0017] In some embodiments, dissolving wall material B in water in step (2) is performed at a temperature of 50°C to 80°C. In some embodiments, dissolving wall material B in water in step (2) is performed at a temperature of 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C.

[0018] In some embodiments, the dissolution of seaweed polysaccharides in step (3) is carried out at a temperature of 50°C to 80°C. In some embodiments, the dissolution of seaweed polysaccharides in step (3) is carried out at a temperature of 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C.

[0019] In some embodiments, the parameters for spray drying in step (5) are: inlet air temperature of 100℃~180℃, outlet air temperature of 60℃~100℃, and feed flow rate of 5 mL / min~20 mL / min. In some embodiments, the parameters for spray drying in step (5) are: inlet air temperature of 100℃, 110℃, 120℃, 130℃, 140℃, 150℃, 160℃, 170℃, or 180℃, outlet air temperature of 60℃, 70℃, 80℃, 90℃, or 100℃, and feed flow rate of 5 mL / min, 6 mL / min, 7 mL / min, 8 mL / min, 9 mL / min, 10 mL / min, 11 mL / min, 12 mL / min, 13 mL / min, 14 mL / min, 15 mL / min, 16 mL / min, 17 mL / min, 18 mL / min, 19 mL / min, or 20 mL / min.

[0020] In some embodiments, the probiotics include at least one of Lactobacillus plantarum, Bifidobacterium, Lactobacillus casei, and Bacillus subtilis.

[0021] In some embodiments, the drug includes at least one of ibuprofen, norfloxacin, avermectin, and enrofloxacin.

[0022] In a second aspect, the present invention provides a highly stress-resistant microcapsule prepared according to the preparation method described in the first aspect.

[0023] A highly stress-resistant microcapsule prepared according to the preparation method described in the first aspect.

[0024] Thirdly, the present invention provides a pharmaceutical preparation.

[0025] A pharmaceutical formulation comprising a high-resistance microcapsule prepared according to the preparation method described in the first aspect or the high-resistance microcapsule described in the second aspect.

[0026] Fourthly, the present invention provides a food product.

[0027] A food product comprising high stress-resistant microcapsules prepared by the preparation method described in the first aspect or high stress-resistant microcapsules described in the second aspect, wherein the core material of the high stress-resistant microcapsules is probiotics.

[0028] In some embodiments, the food is a regular food or a health food.

[0029] Fifthly, the present invention provides a feed.

[0030] A feed comprising high stress-resistant microcapsules prepared by the preparation method described in the first aspect or high stress-resistant microcapsules described in the second aspect, wherein the core material of the high stress-resistant microcapsules is probiotics.

[0031] Beneficial effects Compared with the prior art, the technical solution provided by the present invention has at least one of the following beneficial technical effects: (1) The preparation method provided by the present invention is simple. The seaweed polysaccharide-based wall material provided by the present invention can reduce the influence of high temperature and dehydration on the activity of the core material during spray drying. The average diameter of the obtained high stress-resistant microcapsules is <50 μm and the encapsulation rate is ≥90%. The obtained microcapsules have a high encapsulation rate, and the survival rate of the probiotic core material after spray drying is high. The loss rate of the drug core material that is unstable at high temperature is low after spray drying.

[0032] (2) The high-resistance microcapsules prepared by the preparation method provided by the present invention can improve the stability of the core material during digestion. The high-resistance microcapsules do not disintegrate in simulated gastric juice and have a good sustained-release effect in simulated intestinal juice.

[0033] (3) Compared with not adding any of the wall materials A, B and C described in this invention, the microcapsules obtained by adding the wall materials A, B and C provided in this invention at the same time have unexpected technical effects of gastric acid resistance, which helps to avoid the destruction of probiotics or drugs in the microcapsules in gastric or intestinal fluid.

[0034] (4) Compared with the microcapsules obtained by adding any one of the wall materials A, B and C of the present invention without adding any one of them, the microcapsules obtained by adding the wall materials A, B and C of the present invention have unexpected technical effects of resistance to intestinal fluid (containing bile salts).

[0035] (5) Compared with other molecular weight seaweed polysaccharides, the microcapsules obtained by using seaweed polysaccharides with a molecular weight (preferably ≥1000KDa) provided by the present invention have unexpected technical effects of resistance to gastric juice and intestinal juice (containing bile salts), which helps to avoid the destruction of probiotics or drugs in the microcapsules in gastric juice or intestinal juice.

[0036] (6) Compared with other seaweed polysaccharides, the microcapsules obtained by using the seaweed polysaccharide type provided by the present invention have unexpected technical effects of resistance to gastric juice and intestinal juice (containing bile salts), which helps to avoid the destruction of probiotics or drugs in the microcapsules in gastric juice or intestinal juice.

[0037] (7) The high stress-resistant microcapsules prepared by the preparation method provided by the present invention can significantly improve the heat resistance of the core material (e.g., when using probiotics as the core material, the survival rate of probiotics is ≥90% after heat treatment at 60°C for 30 minutes; and the survival rate of probiotics is ≥6% after heat treatment at 90°C for 10 minutes), which has unexpected technical effects and is beneficial to the processing stability and storage stability of microcapsules.

[0038] (8) Compared with other molecular weight seaweed polysaccharides, the microcapsules obtained by using seaweed polysaccharides with a molecular weight (preferably ≥1000KDa) provided by the present invention have unexpected high temperature resistance, which is beneficial to the processing stability and storage stability of the microcapsules.

[0039] (9 Compared with other seaweed polysaccharides, the microcapsules obtained by using the seaweed polysaccharide type provided by the present invention have unexpected high temperature resistance, which is beneficial to the processing stability and storage stability of the microcapsules.

[0040] Terminology Explanation In the foregoing description of this invention, all figures disclosed herein, whether or not the words “approximately” or “about” are used, are approximate values. Based on the disclosed figures, the value of each figure may vary by less than ±10% or by a difference that is considered reasonable by those skilled in the art, such as ±1%, ±2%, ±3%, ±4%, or ±5%.

[0041] The terms “optional,” “optional,” or “optionally” refer to events or situations that may, but are not necessarily, occur as described below.

[0042] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0043] The term "wt%" indicates a percentage by mass.

[0044] The term "KDa" stands for kilodalton.

[0045] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples. Attached Figure Description

[0046] Figure 1 The graphs show the release of samples from Examples 3, 4, 5 and Comparative Example 2 in simulated gastric and intestinal fluid environments, respectively. Detailed Implementation

[0047] To enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to provide a more detailed description of the present invention.

[0048] The reagents or consumables used in this invention can be purchased from the market or prepared by the methods described in this invention.

[0049] The sources of *Lactobacillus plantarum* used in the following examples or comparative examples are as follows: Lactobacillus plantarum: Lactiplantibacillus plantarum The strain number is DSM 20174.

[0050] Example 1: Preparation of seaweed polysaccharides Seaweed was extracted in water at 90℃ for 3 h, with 50 g of the seaweed added per liter of water; after centrifugation, the supernatant was collected and purified using a separation membrane with a molecular weight cutoff of 100 kDa. The retentate was collected, dried, and the seaweed polysaccharide was obtained, with a molecular weight ≥100 kDa. The seaweed was selected from *Ulva prolifera*, *Sargassum fusiforme*, *Asparagus cochinchinensis*, *Ulva oleracea*, or kelp to obtain *Ulva prolifera* polysaccharide, *Sargassum fusiforme* polysaccharide, *Asparagus cochinchinensis* polysaccharide, *Ulva oleracea* polysaccharide, or kelp polysaccharide, respectively.

[0051] Example 2: Preparation of seaweed polysaccharides Seaweed was extracted in water at 90℃ for 3 hours, with 50 g of the seaweed added per liter of water. After centrifugation, the supernatant was collected and purified using a separation membrane with a molecular weight cutoff of 1000 kDa. The retentate was collected, dried, and the seaweed polysaccharide was obtained, with a molecular weight ≥1000 kDa. The seaweed selected included *Ulva prolifera*, *Sargassum fusiforme*, *Gynostemma pentaphyllum*, *Ulva lactuca*, kelp, or coral algae, respectively, to obtain *Ulva prolifera* polysaccharide, *Sargassum fusiforme* polysaccharide, *Gynostemma pentaphyllum* polysaccharide, *Ulva lactuca* polysaccharide, kelp polysaccharide, or coral algae polysaccharide.

[0052] Examples 3-6: Preparation of Highly Resistant Probiotic Microcapsules Formula: See Table 1.

[0053] Table 1: High-resistance probiotic microcapsule formulation, average microcapsule diameter, spray-dried survival rate, and encapsulation efficiency.

[0054] Preparation method: 1. Preparation of core material The *Lactobacillus plantarum* strain was inoculated into MRS medium and cultured at a constant temperature of 37°C and a rotation speed of 120 rpm for 14 h for activation. The activated *Lactobacillus plantarum* culture was then cultured again using the same procedure to obtain a *Lactobacillus plantarum* strain culture that had been activated twice. One L of the twice-activated *Lactobacillus plantarum* culture yielded a viable cell concentration of approximately 10⁻⁶. 8 The core material was obtained by centrifuging at 8000 rpm for 10 min with CFU / mL and collecting the wet bacterial cells.

[0055] 2. Preparation method of highly stress-resistant probiotic microcapsules (1) Dissolve wall material A in water to obtain wall material A solution; (2) Dissolve wall material B in water at 70°C to obtain wall material B solution; (3) Dissolve wall material C in water at 70°C to obtain wall material C solution; then mix the wall material C solution with the wall material B solution to obtain mixed solution 1; (4) Mix the core material with the wall material A solution obtained in step (1) to obtain mixed solution 2. Then add mixed solution 1 obtained in step (3) to mixed solution 2 and mix evenly to obtain a suspension. The content of wall material A in the suspension is 10 g / L, the content of wall material B is 20 g / L, and the content of wall material C is 5 g / L. 10 g of total wall material (i.e., wall material A + wall material B + wall material C) is added. 8 The core material described in CFU; (5) The suspension obtained in step (4) is spray-dried to obtain the high stress-resistant microcapsules; the parameters of the spray drying are: inlet air temperature of 180℃, outlet air temperature of 80℃, and feed flow rate of 10 mL / min.

[0056] The average diameter of the microcapsules, the survival rate after spray drying, and the encapsulation rate are shown in Table 1.

[0057] Comparative Example 1: Preparation of Probiotic Powder The *Lactobacillus plantarum* strain was inoculated into MRS medium and cultured at a constant temperature of 37°C and a rotation speed of 120 rpm for 14 h for activation. The activated *Lactobacillus plantarum* culture was then cultured again using the same procedure to obtain a *Lactobacillus plantarum* strain culture that had been activated twice. One L of the twice-activated *Lactobacillus plantarum* culture yielded a viable cell concentration of approximately 10⁻⁶. 8 After centrifuging at 8000 rpm for 10 min, the bacterial precipitate was collected and evenly dispersed in 1 L of sterile water to obtain a bacterial suspension.

[0058] The bacterial suspension was spray-dried while being stirred, with an inlet air temperature of 180℃, an outlet air temperature of 80℃, and a feed flow rate of 10 mL / min. A white powder of *Lactobacillus plantarum* was collected, with a spray-dried survival rate of only 0.01%.

[0059] Comparative Examples 2-4: Investigation of Wall Materials The wall material was investigated based on the formulation of Example 3, and the specific formulation is shown in Table 2.

[0060] Table 2: Probiotic microcapsule formulation, average microcapsule diameter, spray-dried survival rate, and encapsulation efficiency

[0061] Preparation method: 1. Preparation of core material The preparation of the core material is the same as in Examples 3-6.

[0062] 2. Preparation method of highly stress-resistant probiotic microcapsules The preparation method of highly resistant probiotic microcapsules in Example 3 is as follows: the components not added in each comparative example are replaced with water of the corresponding weight, and the rest of the operation is the same as in Example 3.

[0063] Comparative Examples 7-9: Investigation of wall materials with different molecular weights C The wall materials C with different molecular weights were investigated based on the formulation of Example 3. The specific formulation is shown in Table 3.

[0064] Table 3: Investigation of wall materials with different molecular weights (C)

[0065] I. Preparation method of seaweed polysaccharides (1) Preparation method of Ulva polysaccharide (molecular weight 50 KDa ~ 100 KDa): Extract Ulva in water at 90℃ for 3h, wherein 50 g of Ulva is added for every 1 liter of water; centrifuge, take the supernatant, separate and purify it using a separation membrane with a molecular weight cutoff of 100 KDa, collect the permeate, and then separate and purify it again using a separation membrane with a molecular weight cutoff of 50 KDa, collect the retentate, dry it, and obtain the Ulva polysaccharide (molecular weight 50 KDa ~ 100 KDa).

[0066] (2) Preparation method of Ulva polysaccharide (molecular weight 10 KDa ~ 50 KDa): Extract Ulva in water at 90℃ for 3 h, wherein 50 g of Ulva is added for every 1 liter of water; centrifuge, take the supernatant, separate and purify it using a separation membrane with a molecular weight cutoff of 50 KDa, collect the permeate, and then separate and purify it again using a separation membrane with a molecular weight cutoff of 10 KDa, collect the retentate, dry it, and obtain the Ulva polysaccharide (molecular weight 10 KDa ~ 50 KDa).

[0067] (3) Preparation method of Ulva polysaccharide (molecular weight <10 KDa): Ulva was extracted in water at 90℃ for 3 h, with 50 g of Ulva added per 1 liter of water; centrifuged, the supernatant was collected, and the supernatant was separated and purified using a separation membrane with a molecular weight cutoff of 10 KDa. The permeate was collected and dried to obtain the Ulva polysaccharide (molecular weight <10 KDa).

[0068] II. Microcapsule preparation methods: 1. Preparation of core material The preparation of the core material is the same as in Examples 3-6.

[0069] 2. Preparation method of highly stress-resistant probiotic microcapsules The preparation method of the highly stress-resistant probiotic microcapsules in Example 3 is the same as in Example 3, except that the wall material C is different.

[0070] Comparative Examples 10-12: Investigation of Different Seaweed Polysaccharides Different seaweed polysaccharides were investigated based on the formulation of Example 3. The specific formulations are shown in Table 4.

[0071] Table 4: Investigation of different seaweed polysaccharides

[0072] Preparation method: 1. Preparation of core material The preparation of the core material is the same as in Examples 3-6.

[0073] 2. Preparation method of highly stress-resistant probiotic microcapsules The preparation method of the highly stress-resistant probiotic microcapsules in Example 3 is the same as in Example 3, except that the wall material C is different.

[0074] Experimental Example 1: Survival rate determination in simulated gastric and intestinal fluids I. Solution Preparation (1) Preparation of simulated gastric juice (pH 1.8): Measure 80 mL of ultrapure water, add 1 g of pepsin and disperse evenly, adjust the pH to 1.8 with 10% hydrochloric acid, transfer to a 100 mL volumetric flask and make up to volume, filter with a 0.22 μm sterile filter membrane for later use.

[0075] (2) Preparation of simulated intestinal fluid (pH 6.8): Prepare 0.2 mol / L pH 6.8 phosphate buffer, add trypsin at a final concentration of 1 mg / mL and ox bile salt at a final concentration of 3 mg / mL, and after complete dissolution, filter with a 0.22 μm sterile filter membrane for later use.

[0076] II. Operation (1) Probiotic microcapsules or probiotic powder prepared in the above examples or comparative examples were added to simulated gastric juice (pH 1.8). The mixture was incubated at a constant temperature of 37℃ and 100 rpm for 2 h with shaking. The microcapsules or powder were collected by centrifugation at 8000 rpm for 5 min. After cell wall disruption, the mixture was diluted with sterile water. The total bacterial count was determined using the plate count method of GB4789.2-2016. The results are shown in Table 1.

[0077] (2) Probiotic microcapsules or probiotic powder prepared in the above examples or comparative examples were added to simulated intestinal fluid (pH 6.8). The mixture was incubated at a constant temperature of 37℃ with shaking at 100 rpm for 2 h. The microcapsules or powder were collected by centrifugation at 8000 rpm for 5 min. After cell wall disruption, the mixture was diluted with sterile water, and the total bacterial count was determined using the plate count method of GB4789.2-2016. The results are shown in Table 5.

[0078] Simulated gastric fluid survival rate = (Number of colonies after treatment with simulated gastric fluid / Initial total number of colonies) × 100% Simulated intestinal fluid survival rate = (Number of colonies after treatment with simulated intestinal fluid / Initial total number of colonies) × 100% Table 5: Survival rates of different probiotic microcapsules or probiotic powders in simulated gastric and intestinal fluids.

[0079] in conclusion: (1) Comparative Example 1 contained naked probiotic bacteria, which showed poor tolerance in simulated gastric juice. After 2 hours of incubation in simulated gastric juice, the survival rate of live bacteria was 0%. Comparative Example 2 contained no seaweed polysaccharide in its microcapsule wall material, resulting in relatively poor protection for probiotics. After 2 hours of incubation in simulated gastric juice, the survival rate of live bacteria was 12%. Comparative Examples 3 and 4 contained seaweed polysaccharide but no sodium caseinate or gelatin. After 2 hours of incubation in simulated gastric juice, the survival rate of live bacteria was 0%. Comparative Examples 5 and 6 contained only sodium caseinate or gelatin microcapsules. After 2 hours of incubation in simulated gastric juice, the survival rates of live bacteria were 0.2% and 1.3%, respectively. Compared to not adding any of the wall materials A, B, and C described in this invention, the microcapsules obtained by adding all of the wall materials A, B, and C provided in this invention (Examples 3-6) showed unexpected technical effects in resisting simulated gastric juice.

[0080] (2) Similarly, compared with not adding any one of the wall materials A, B and C described in this invention, the microcapsules obtained by adding the wall materials A, B and C provided in this invention at the same time (Examples 3-6) have unexpected technical effects of being resistant to simulated intestinal fluid (containing bile salts).

[0081] (3) Although the microcapsules of Comparative Examples 7 to 9 contained Ulva prolifera polysaccharide, the molecular weight of Ulva prolifera polysaccharide was <10 kDa. The smaller the molecular weight, the worse the protective effect on probiotics in simulated gastric juice or simulated intestinal juice. Therefore, it is shown that compared with seaweed polysaccharides of other molecular weights, the microcapsules obtained by using seaweed polysaccharides with a molecular weight (preferably ≥1000 kDa) provided by the present invention have unexpected technical effects of resistance to gastric juice and intestinal juice (containing bile salts).

[0082] (4) The microcapsules of Comparative Examples 10 to 12 contained coral polysaccharide, sodium alginate and carrageenan, respectively, but the survival rate of probiotics in simulated gastrointestinal fluid was significantly lower than that of Examples 3 to 6; indicating that compared with other seaweed polysaccharides, the microcapsules obtained by using the seaweed polysaccharide type provided by the present invention have unexpected technical effects of resistance to gastric juice and intestinal juice (containing bile salts).

[0083] In summary, the experimental results (Table 5) show that after the probiotics were encapsulated in seaweed polysaccharide / sodium caseinate / gelatin microcapsules (Examples 3-6), their tolerance to simulated gastrointestinal fluid was significantly improved, thus enhancing the stability of the probiotics in digestive fluids.

[0084] Experimental Example 2: Survival Rate Determination During Heat Treatment The probiotic microcapsules or probiotic powders prepared in the above examples or comparative examples were subjected to heat treatment at 60℃ for 30 min and at 90℃ for 10 min, respectively, and then serially diluted with sterile water. The total bacterial count was determined using the plate count method of GB4789.2-2016. The results are shown in Table 6.

[0085] Heat treatment survival rate = (Number of colonies after heat treatment / Initial total number of colonies) × 100% Table 6: Survival rate of different probiotic microcapsules or probiotic powders during heat treatment

[0086] in conclusion: (1) Comparative Example 1 uses naked probiotic bacteria, which have poor tolerance to high temperatures and a survival rate of 0%. Comparative Example 2's microcapsule wall material does not contain seaweed polysaccharides, so its protective effect on probiotics is limited under high temperatures. However, the microcapsules provided by this invention (Examples 3-6) have unexpected high-temperature resistance.

[0087] (2) Although the microcapsules of Comparative Examples 7 to 9 contain the addition of seaweed polysaccharide, the molecular weight of seaweed polysaccharide is <10 KDa. The smaller the molecular weight, the worse the protective effect on probiotics under high temperature. This indicates that compared with seaweed polysaccharides of other molecular weights, the microcapsules obtained by using seaweed polysaccharides with a molecular weight (preferably ≥1000 KDa) provided by this invention have unexpected high temperature resistance.

[0088] (3) Coral algae polysaccharide, sodium alginate and carrageenan were used to replace the algae polysaccharide in Example 3 in the microcapsules of Comparative Examples 10 to 12, respectively. However, the microcapsules obtained after the replacement had limited protective effect on probiotics in a high-temperature environment. This shows that compared with other algae polysaccharides, the microcapsules obtained by using the algae polysaccharide type provided by the present invention have unexpected high-temperature resistance.

[0089] In summary, the experimental results (Table 5) show that probiotics encapsulated in seaweed polysaccharide / sodium caseinate / gelatin microcapsules (Examples 3-6) have good heat resistance, which greatly improves the probiotics' tolerance to high-temperature environments.

[0090] Experimental Example 3: Release in the gastrointestinal fluid environment The probiotic microcapsules prepared in the above examples or comparative examples were added to simulated gastric juice and subjected to constant temperature shaking at 37°C and 100 rpm. Odulosity (OD) was measured after 0, 30, 60, 90, and 120 min. 600After the simulated gastric juice treatment was completed, the simulated gastric juice was discarded by centrifugation, and then added to the simulated intestinal juice. The mixture was kept at a constant temperature of 37℃ and oscillated at 100 rpm. The OD values ​​were measured at 60 min, 120 min, 180 min, and 240 min after the addition of the simulated intestinal juice. 600 The results are shown Figure 1 .

[0091] Conclusion: The highly resilient microcapsules of the embodiment were stable in simulated gastric fluid for 2 hours, showing little disintegration and leakage of probiotics. However, they gradually disintegrated and released probiotics in simulated intestinal fluid, demonstrating a significant sustained-release / controlled-release effect. Comparative Example 2 was relatively stable in simulated gastric fluid for 2 hours, showing little leakage of probiotics. It disintegrated and released probiotics within 1 hour in simulated intestinal fluid, but showed no significant sustained-release effect.

[0092] Example 7: Preparation of Enrofloxacin Microcapsules Formula: See Table 7.

[0093] Table 7: Enrofloxacin microcapsule formulation, average microcapsule diameter, enrofloxacin content and encapsulation efficiency after spray drying

[0094] Preparation method: (1) Dissolve wall material A in water to obtain wall material A solution; (2) Dissolve wall material B in water at 70°C to obtain wall material B solution; (3) Dissolve wall material C in water at 70°C to obtain wall material C solution; then mix the wall material C solution with the wall material B solution to obtain mixed solution 1; (4) Mix the core material with the wall material A solution obtained in step (1) to obtain mixed solution 2. Then add the mixed solution 1 obtained in step (3) to mixed solution 2 and mix evenly to obtain a suspension. The content of wall material A in the suspension is 10 g / L, the content of wall material B in the suspension is 20 g / L, and the content of wall material C in the wall material C is 5 g / L. 0.5 g of the core material is added for every 1 g of total wall material (i.e., wall material A + wall material B + wall material C). (5) The suspension obtained in step (4) is spray-dried to obtain the high stress-resistant microcapsules; the parameters of the spray drying are: inlet air temperature of 180℃, outlet air temperature of 80℃, and feed flow rate of 10 mL / min.

[0095] The average diameter of the microcapsules, the survival rate after spray drying, and the encapsulation rate are shown in Table 7.

[0096] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A method for preparing highly stress-resistant microcapsules, characterized in that, Includes the following steps: (1) Dissolve wall material A in water to obtain wall material A solution, wherein wall material A is at least one of whey protein, soy protein and sodium caseinate; (2) Dissolve wall material B in water to obtain wall material B solution, wherein wall material B is at least one of collagen and gelatin; (3) Dissolve wall material C in water to obtain wall material C solution, wherein wall material C is seaweed polysaccharide; then mix the wall material C solution with the wall material B solution evenly to obtain mixed solution 1; (4) Mix the core material with the wall material A solution obtained in step (1) to obtain mixed solution 2. Then add mixed solution 1 obtained in step (3) to mixed solution 2 and mix thoroughly to obtain a suspension. The core material is probiotics. The ratio of the number of viable bacteria in the core material in step (4) to the total mass of wall material A, wall material B and seaweed polysaccharide in the suspension is (10). 6 ~10 11 CFU: 1 g; (5) The suspension obtained in step (4) is spray-dried to obtain the high stress-resistant microcapsules.

2. According to the preparation method of claim 1, the molecular weight of the seaweed polysaccharide is ≥100 kDa, preferably ≥1000 kDa; Optionally, the seaweed polysaccharide is at least one of the following: sea lettuce polysaccharide, Sargassum fusiforme polysaccharide, Asparagus cochinchinensis polysaccharide, Ulva lactuca polysaccharide, and kelp polysaccharide; Preferably, the seaweed polysaccharide is *Ulva prolifera* polysaccharide; More preferably, the seaweed polysaccharide is a seaweed polysaccharide with a molecular weight of ≥1000 kDa.

3. The preparation method according to any one of claims 1-2, wherein the preparation method of the seaweed polysaccharide comprises: The seaweed polysaccharide is obtained by water extraction using seaweed as raw material, followed by separation, purification, and drying; the seaweed includes at least one of Ulva prolifera, Sargassum fusiforme, Asparagus cochinchinensis, Ulva lactuca, and kelp.

4. The preparation method according to claim 3, wherein the preparation method comprises: Seaweed was extracted in water at 80℃-100℃ for 2 h-6 h, with 20 g to 500 g of the seaweed added per liter of water; the seaweed was centrifuged, the supernatant was collected, and the supernatant was purified by separation membrane. The retentate was collected and dried to obtain the seaweed polysaccharide. Optionally, the molecular weight cutoff of the separation membrane is 100 kDa; preferably, the molecular weight cutoff of the separation membrane is 1000 kDa.

5. The preparation method according to any one of claims 1-4, wherein the content of wall material A in the suspension is 5 g / L to 20 g / L, calculated based on the total volume of the suspension; and / or Based on the total volume of the suspension, the content of wall material B in the suspension is 10 g / L to 40 g / L; and / or Based on the total volume of the suspension, the content of seaweed polysaccharides in the suspension is 1 g / L to 20 g / L.

6. The preparation method according to any one of claims 1-5, wherein dissolving the wall material B in water in step (2) is performed at 50°C to 80°C; and / or In step (3), dissolving the seaweed polysaccharide in water is carried out at a temperature of 50°C to 80°C; and / or The parameters for spray drying in step (5) are: inlet air temperature of 100℃~180℃, outlet air temperature of 60℃~100℃, and feed flow rate of 5 mL / min~20 mL / min.

7. The preparation method according to claim 1, wherein the probiotics include at least one of Lactobacillus plantarum, Bifidobacterium, Lactobacillus casei, and Bacillus subtilis.

8. A highly stress-resistant microcapsule prepared by the preparation method according to any one of claims 1-7.

9. A pharmaceutical formulation comprising a high-resistance microcapsule prepared by any one of claims 1-7 or the high-resistance microcapsule of claim 8.

10. A food or feed comprising a high-resistance microcapsule prepared by the preparation method according to any one of claims 1-7 or the high-resistance microcapsule according to claim 8, wherein the core material of the high-resistance microcapsule is probiotics.

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