Preparation method of dialdehyde sodium alginate microcapsule loaded with probiotics and plant polyphenol

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a preparation method of dialdehyde sodium alginate microcapsules loaded with probiotics and plant polyphenols, the preparation method comprises the following steps: (1) dissolving the plant polyphenols and probiotic bacterial suspension in water, and uniformly mixing to obtain a core layer solution; (2) mixing and reacting the core layer solution, the dialdehyde sodium alginate solution and the glycosylated pea protein to obtain a reaction solution; and (3) mixing the reaction solution with a calcium ion solution, and freeze-drying to obtain the probiotic capsule. The raw materials adopted by the probiotic capsule are all natural biomacromolecules, so that the probiotic capsule has high biocompatibility and safety and does not have food safety risks. In addition, the two raw materials are cheap and easy to obtain, have good degradability, and are economical and environment-friendly.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and specifically relates to a preparation method of a dialdehyde sodium alginate microcapsule loaded with probiotics and plant polyphenols. BACKGROUND

[0002] Probiotics are a class of active microorganisms that are beneficial to the health of the host. The Food and Agriculture Organization of the United Nations and the World Health Organization define them as "live microorganisms that, when ingested in sufficient quantities, can bring one or more health benefits to the host". They mainly include Lactobacillus and Bifidobacterium, etc. This kind of microorganism shows significant potential in the treatment of gastrointestinal diseases, especially in the replacement of high-dose antibiotic treatment of antibiotic-associated diarrhea, viral gastrointestinal infection, etc. However, probiotics are easily affected by high temperature, pH change and gastrointestinal environment during food processing and human intake, and are inactivated. In order to play a function, it needs to meet the prerequisite of "maintaining cell integrity, intestinal adhesion colonization and the number reaching at least 10 6 CFU / g", which greatly limits its actual application effect.

[0003] In order to solve the problem of easy inactivation of probiotics, various delivery systems (such as emulsion, gel, microcapsule, etc.) and auxiliary ingredients such as plant polyphenols have been widely concerned. Among them, microcapsules can build a physical barrier to isolate the external environment by physical and chemical or mechanical methods to encapsulate probiotics in the wall material, and have the advantages of high mechanical strength, good stability, high encapsulation efficiency, etc. Natural biological macromolecules (such as proteins, polysaccharides) have become an ideal choice for microcapsule wall materials due to their low cost, excellent storage stability, good safety and environmental protection. For example, pea protein isolate, as a plant protein, has the characteristics of low cost, low allergenicity and high nutritional value, but it has the defect of poor water solubility and emulsification; glycosylation modification can improve its functional properties and enhance intestinal adhesion, and dialdehyde sodium alginate can cross-link with glycosylated proteins under the mediation of calcium ions to form a stable gel network structure, further improving the strength of the microcapsule. Although the existing technology has explored many microcapsule formulas, some of the schemes still have shortcomings: for example, the composite dialdehyde sodium alginate probiotic microcapsule disclosed in Chinese invention patent 202410830214.5 contains modified attapulgite, nano silicon dioxide and other ingredients, and its safety needs to be verified; the commonly used chitosan and gelatin wall materials may cause probiotics to be released prematurely under acidic or body temperature conditions, affecting the efficacy.

[0004] Therefore, it is of great significance to develop a green, safe and healthy probiotic microcapsule delivery system to overcome the defects of single material and improve the protection effect of probiotics through synergistic effect, which promotes the development and production of probiotic related products. SUMMARY

[0005] Based on this, the application provides a preparation method of a bifunctional dialdehyde sodium alginate microcapsule loaded with probiotics and plant polyphenols.

[0006] In order to achieve the above-mentioned purpose, the application can adopt the following technical scheme: The application provides a preparation method of a bifunctional dialdehyde sodium alginate microcapsule loaded with probiotics and plant polyphenols. (1) dissolving plant polyphenols and probiotic bacterial suspension in water and mixing to obtain a core layer solution; (2) mixing and reacting the core layer solution, a bifunctional dialdehyde sodium alginate solution and sugar-based pea protein to obtain a reaction solution; (3) mixing the reaction solution with a calcium ion solution and freeze-drying to obtain probiotic bacterial capsules.

[0007] Preferably, the preparation method satisfies one or more of the following conditions: (a1) the plant polyphenols are selected from one or more combinations of oridonin, catechin or tea polyphenols; (b1) the probiotics are selected from one or more combinations of Lactobacillus plantarum, Bifidobacterium longum or Lactobacillus rhamnosus; (c1) the calcium ion solution is a calcium chloride solution.

[0008] Preferably, the preparation method satisfies one or more of the following conditions: (a2) the preparation method of the bifunctional dialdehyde sodium alginate solution comprises: oxidizing sodium alginate by sodium periodate and dialyzing, then freeze-drying to obtain bifunctional dialdehyde sodium alginate, and dissolving the bifunctional dialdehyde sodium alginate in water to obtain a bifunctional dialdehyde sodium alginate solution; (b2) the preparation method of the sugar-based pea protein comprises: preparing the sugar-based pea protein by a hydrothermal method.

[0009] Preferably, the preparation method satisfies one or more of the following conditions: (a3) the addition amount of the plant polyphenols is (2 mg-4 mg) / 100 mL of the probiotic bacterial suspension; (b3) the volume concentration of the bifunctional dialdehyde sodium alginate solution is 1%-3%; (c3) the volume ratio of the core layer solution to the bifunctional dialdehyde sodium alginate solution is 1:(0.5-1.5); (d3) the mass concentration of the sugar-based pea protein is 0.5%-1.5%; (e3) the volume concentration of the calcium ion solution is 0.5%-1.5%, and the pH is 3-5.

[0010] Preferably, the above preparation method meets one or more of the following conditions: (a4) in step (2), the reaction conditions include: 400 rpm stirring for 1 h; (b4) in step (3), the freeze-drying conditions include: feed flow rate 8 mL / min, air flow 350 L / h, air pressure 0.1 MPa, and temperature -60℃.

[0011] Another aspect of the present application provides a bifunctional dialdehyde sodium alginate microcapsule loaded with probiotics and plant polyphenols prepared by the above preparation method.

[0012] The beneficial effects of the present application include: (1) The probiotic capsules provided by the present application use natural biological macromolecules as raw materials, which have high biocompatibility and safety, and do not pose a food safety risk. In addition, both raw materials are inexpensive and easy to obtain, and have good biodegradability, economic and environmental protection.

[0013] (2) The probiotic capsules provided by the present application have pH sensitivity, i.e., colon targeting function. In the stomach, the carboxyl groups of ALG are in a non-ionized state, forming an insoluble structure. When entering the intestine, the carboxyl groups of ALG become ionized, causing the microcapsule structure to disintegrate. Therefore, the microcapsule has a higher release amount in the intestine, which can significantly improve the survival rate of probiotics after passing through the stomach environment.

[0014] (3) The preparation method of the probiotic capsules provided by the present application has mild conditions, simple operation, and is green and environmentally friendly. The prepared microcapsules have the advantages of high encapsulation efficiency, high survival rate of probiotics, and strong stability. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a schematic diagram of sodium alginate oxidation reaction; Figure 2 is a schematic diagram of the preparation process of probiotic microcapsules; Figure 3 is the survival rate of different probiotic microcapsules after simulated gastrointestinal digestion. DETAILED DESCRIPTION

[0016] The examples are provided to better illustrate the present application, but are not the only embodiments of the present application. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above description of the application still fall within the scope of the present application.

[0017] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless otherwise defined, all terms of art used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of the disclosure. As used herein, it is also to be understood that the use of the singular includes the plural unless specifically stated otherwise. As used herein, it is also to be understood that the use of the term "and / or," as well as the use of "comprising," "including," "containing," "consisting of," "consisting essentially of,” means "one or more of the listed items," unless specifically stated otherwise. As used herein, "may" means "one or more of the listed items," unless specifically stated otherwise.

[0018] In a first aspect, the present application provides a preparation method of a dialdehyde sodium alginate microcapsule loaded with probiotics and plant polyphenols, the preparation method comprising: (1) dissolving the plant polyphenols and the probiotic bacterial suspension in water and mixing to obtain a core layer solution; (2) mixing and reacting the core layer solution, a dialdehyde sodium alginate solution, and a sugar-based pea protein to obtain a reaction solution; (3) mixing the reaction solution with a calcium ion solution and freeze-drying to obtain the probiotic bacterial microcapsule.

[0019] It should be noted that the present application uses probiotics as the main raw material, supplemented by other additives to prepare probiotic microcapsules. The use of oxidized dialdehyde sodium alginate and sugar-based pea protein to encapsulate probiotics and plant polyphenols improves the survival rate and intestinal adhesion of probiotics. Plant polyphenols can enhance the intestinal adhesion of probiotics. Pea protein (PPI) is a plant protein derived from peas, which has the unique advantages of low allergenicity, low cost, and renewability. By glycosylating the sugar-based pea protein, it can combine the initial properties of polysaccharides and proteins, and enhance its water solubility and surface charge quantity, achieving the encapsulation of a single substance for probiotics. Sodium alginate is a natural polysaccharide extracted from seaweed, which is considered to have high biological safety as a natural hydrophilic colloid, and is the first choice for materials in microcapsule preparation. Oxidized dialdehyde sodium alginate can further enhance the gel properties, allowing the microcapsules to stay in the intestine for a longer period of time, promoting the colonization of probiotics. By cross-linking these two materials with calcium ions, a network structure can be formed spontaneously, allowing probiotics to be retained within it, achieving the encapsulation and protection of probiotics. In addition, the present application uses sugar-based pea protein and dialdehyde sodium alginate cross-linking as the wall material of the probiotic microcapsule. Sugar-based pea protein has the unique advantage of low allergenicity compared to other plant proteins. Furthermore, the addition of plant polyphenols promotes the colonization of probiotics in the intestine, thereby exerting their efficacy. The present application innovatively adds polyphenols to the microcapsule, which will further improve the adhesion of probiotics.

[0020] Specifically, plant polyphenols are also a kind of prebiotics, which can reduce the damage of oxidative stress to probiotics during storage and provide certain nutrition for probiotics. In addition, plant polyphenols have been proved to enhance the intestinal adhesion of probiotics, the delivery system based on encapsulation technology can effectively protect probiotics from the influence of the external environment, and the delivery system with intestinal targeting delivery characteristics can promote the colonization of probiotics in the intestine, so that probiotics can better exert their effects. Ordinary prebiotics lack the adhesion effect of polyphenols, and synthetic antioxidants have the risk of disrupting the balance of flora. In addition, pea protein isolate, as a kind of plant protein, has the great advantages of low cost, low allergenicity and high nutritional value. Although it has been used in many delivery systems, it still has the defects of poor functionality such as water solubility and emulsification. The functional properties can be significantly improved by glycosylation modification, and the defects can be overcome. Notably, the adhesion can be enhanced after glycosylation, which further promotes the colonization of probiotics in the intestine. Glycosylation combines the adhesion, water solubility of polysaccharides and the low allergenicity of pea protein isolate, which is not possessed by single polysaccharide or protein. The modified dialdehyde sodium alginate can be cross-linked with glycosylated proteins through hydrogen bonds and electrostatic interactions. Under the mediation of calcium ions, the alpha-L-guluronate of dialdehyde sodium alginate can be further cross-linked with glycosylated proteins or itself to form a stable and dense gel network structure, which enhances the strength of the microcapsule and effectively protects the probiotics from inactivation under the influence of the external environment. The commonly used wall materials such as chitosan and gelatin may cause the probiotics to be released in advance under acidic or body temperature, thereby failing to play a role. Therefore, by using plant polyphenols, glycosylated pea protein isolate and dialdehyde sodium alginate, the defects of single material can be overcome, and a synergistic effect can be formed to effectively protect the probiotics from the influence of the external environment and promote their effects in the intestine.

[0021] In some specific examples, the above preparation method meets one or more of the following conditions: (a1) the plant polyphenols are selected from one or more combinations of resveratrol, catechin or tea polyphenols; (b1) the probiotics are selected from one or more combinations of Lactobacillus plantarum, Bifidobacterium longum or Lactobacillus rhamnosus; (c1) the calcium ion solution is a calcium chloride solution.

[0022] It should be noted that the plant polyphenols, probiotics and calcium ion solution in the present application are well known in the art, and preferably the above listed.

[0023] In some specific examples, the above preparation method meets one or more of the following conditions: (a2) the preparation method of the dialdehyde sodium alginate solution comprises: oxidizing sodium alginate by sodium periodate and dialyzing, then freeze-drying to obtain dialdehyde sodium alginate, dissolving the dialdehyde sodium alginate in water to obtain the dialdehyde sodium alginate solution; The preparation method of the glycosylated pea protein comprises: preparing the glycosylated pea protein by hydrothermal method with the pea protein and carboxymethyl chitosan.

[0024] It should be noted that the preparation method of the glycosylated pea protein and the dialdehyde sodium alginate solution in the present application are known in the art, for example, the above-mentioned methods. In addition, the process of oxidizing sodium alginate by sodium periodate can refer to the synthesis route shown in Figure 1

[0025] In some specific examples, the above preparation method meets one or more of the following conditions: (a3) The plant polyphenol addition amount is (2 mg-4 mg) / 100 mL of probiotic bacterial suspension; (b3) The volume concentration of the dialdehyde sodium alginate solution is 1%-3%; (c3) The volume ratio of the core layer solution to the dialdehyde sodium alginate solution is 1:(0.5-1.5); (d3) The mass concentration of the glycosylated pea protein is 0.5%-1.5%; (e3) The volume concentration of the calcium ion solution is 0.5%-1.5%, and the pH is 3-5.

[0026] It should be noted that the plant polyphenol addition amount in the present application can be (2 mg-4 mg) / 100 mL of probiotic bacterial suspension, for example, 2.5 mg / 100 mL, 3 mg / 100 mL or 3.5 mg / 100 mL, etc.; in addition, the volume concentration of the dialdehyde sodium alginate solution can be 1.5%, 2% or 2.5%, etc.; in addition, the mass concentration of the glycosylated pea protein is 0.5%-1.5%, for example, 0.7%, 1% or 1.2%, etc.; in addition, the volume concentration of the calcium ion solution is 0.5%-1.5%, for example, 0.7%, 1% or 1.3%, etc.

[0027] In some specific examples, the above preparation method meets one or more of the following conditions: (a4) In step (2), the reaction conditions include: 400 rpm stirring for 1 h; (b4) In step (3), the freeze-drying conditions include: feed flow rate 8 mL / min, air flow rate 350 L / h, air pressure 0.1 MPa, and temperature-60℃.

[0028] In a second aspect, the present application provides a dialdehyde sodium alginate microcapsule loaded with probiotics and plant polyphenols prepared by the above preparation method.

[0029] ​It should be noted that the probiotic capsule provided by the present application has pH sensitivity, i.e. colon targeting function. When in the stomach, the carboxyl group of ALG is in a non-ionized state, forming an insoluble structure. When entering the intestine, the carboxyl group of ALG becomes ionized, thereby causing the microcapsule structure to disintegrate. Therefore, the microcapsule has a higher release amount in the intestine, which can significantly improve the survival rate of probiotics after passing through the stomach environment.

[0030] In order to better understand the present application, the content of the present application will be further illustrated below in combination with specific examples, but the content of the present application is not limited to the following examples only.

[0031] Preparation examples In the following examples, the MRS liquid medium is composed of: peptone (10 g / L), beef powder (10 g / L), yeast powder (5 g / L), glucose (20 g / L), potassium phosphate dibasic (2 g / L), ammonium citrate dibasic (2 g / L), sodium acetate (3 g / L), magnesium sulfate (0.2 g / L), manganese sulfate (0.04 g / L), Tween 80 (1 g / L), and water is added to 1 L.

[0032] In the following examples, sodium alginate is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. and is of analytical grade; probiotics are purchased from Shanghai Balang Biotechnology Co., Ltd.; pea protein is purchased from Shanghai Balang Biotechnology Co., Ltd. and has a protein content of 80% and is of food grade.

[0033] In the following examples, the preparation steps of the microcapsule can be carried out with reference to the flowchart shown in Figure 2 .

[0034] Example 1 (1) Preparation of bacterial suspension (1-1) Lactobacillus plantarum (ACCC 11095) was inoculated in MRS liquid medium and cultured at 37±1℃ for 24 h, and then centrifuged at 4℃ and 4000 g for 15 min to obtain Lactobacillus plantarum precipitate; (1-2) The Lactobacillus plantarum precipitate was washed twice with phosphate buffer solution (PBS solution, 0.1 M, pH=7.4); (1-3) The washed precipitate was dissolved in sterile normal saline to obtain Lactobacillus plantarum suspension; the concentration of the bacterial suspension was 1×10 8 ~1×10 9 CFU / mL.

[0035] (2) Preparation of glycosylated pea protein powder (2-1) Accurately weigh 2 g of commercial pea protein powder (Shanghai Beilang Biotechnology Co., Ltd.) and dissolve it in 100 mL of deionized water. Stir thoroughly to prepare a 2% glycosylated pea protein solution. (2-2) Adjust the pH of the glycosylated pea protein solution to 12 using 1M NaOH and stir magnetically (400 rpm, 2 h) to obtain a mixed solution. (2-3) After sonicating the mixed solution (80% power, 6 min), heat it at 85℃ for 15 min, adjust the pH to 7 with 1M HCl, and centrifuge (7000 rpm, 15 min) to obtain pea protein solution; (2-5) Pea protein solution was freeze-dried under vacuum (-80℃, 24h) to obtain pea protein powder; (2-6) Accurately weigh equal amounts of pea protein powder and carboxymethyl chitosan, add a certain amount of deionized water and adjust the pH to 9 using 1M NaOH to obtain a solution with a concentration of 2 mg / mL, place it in a water bath at 90℃ and heat for 90 min; after heating, quickly place it in an ice-water bath to cool and terminate the reaction, and freeze dry under vacuum (-80℃, 24 h) to obtain glycosylated pea protein powder.

[0036] (3) Preparation of dialdehyde sodium alginate solution (3-1) Weigh 6g of sodium alginate and dissolve it in 120mL of 50% ethanol solution, and dissolve 2g of sodium periodate in 20mL of deionized water. Mix the two solutions and stir at 40℃ in the dark for 6h. Then add ethylene glycol to stop the reaction and obtain the reaction solution. (3-2) The reaction solution was transferred to a dialysis bag (8-14 kDa) and dialyzed with ultrapure water for 72 h, followed by vacuum freeze drying (-80℃, 24 h) to obtain sodium dialdehyde aldehyde; (3-3) Take 2g, 4g and 6g of sodium dialdehyde alkanoate respectively and dissolve them in 100mL of deionized water to obtain 2%, 4% and 6% sodium dialdehyde alkanoate solutions.

[0037] (4) Preparation of core layer solution Under magnetic stirring (400 rpm, 1 h), 3 mg of plant polyphenol (resveratrol, molecular weight 228.24, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to the bacterial suspension prepared in (1) above and dissolved completely to obtain a core layer solution; the concentration of plant polyphenol in the core layer solution was 0.03 mg / mL. (5) Preparation of microcapsules (5-1) To 100 mL of the core solution prepared in (4) above, 2 g of the glycosylated pea protein powder prepared in (2) above and 100 mL of the dialdehyde sodium alginate solution prepared in (3) above were added, and the pH was adjusted to 7.0, and stirring was performed at 400 rpm for 1 h to ensure sufficient reaction to obtain a reaction solution; (5-2) Microcapsules were prepared by freeze spray drying, and the reaction solution in (5-1) above was mixed with a CaCl2 solution (1% w / v, pH = 4) through a nozzle and sprayed to obtain Lactobacillus plantarum microcapsules; the parameters for freeze spray drying were set as follows: feed flow rate 8 mL / min, air flow rate 350 L / h, air pressure 0.1 MPa, and temperature -60°C.

[0038] Example 2 Example 2 was substantially the same as Example 1, except that Bifidobacterium longum (ATCC 15707) was used in Example 2 instead of Lactobacillus plantarum in Example 1, and other operations were the same as in Example 1 to obtain Bifidobacterium longum microcapsules.

[0039] Example 3 Example 3 was substantially the same as Example 1, except that Lactobacillus rhamnosus (ATCC 5310) was used in Example 3 instead of Lactobacillus plantarum in Example 1, and other operations were the same as in Example 1 to obtain Lactobacillus rhamnosus microcapsules.

[0040] Comparative Example 1 Characterization tests In the following tests, the MRS solid medium was composed of: peptone (10 g / L), beef powder (10 g / L), yeast powder (5 g / L), glucose (20 g / L), potassium phosphate dibasic (2 g / L), ammonium citrate dibasic (2 g / L), sodium acetate (3 g / L), magnesium sulfate (0.2 g / L), manganese sulfate (0.04 g / L), Tween 80 (1 g / L), agar (15 g / L), and water was added to 1 L.

[0041] (I) encapsulation efficiency test (1) 0.1 g of the microcapsules prepared in different examples were respectively suspended in 10 mL of phosphate buffer to obtain a microcapsule suspension; (2) The microcapsule suspension was treated by a high-speed dispersion homogenizer (5000 rpm for 30 s) to release the probiotics from the microcapsules, and then the solution was diluted with sterile deionized water to obtain a bacterial suspension; (3) Then, the number of viable cells of the probiotics in the bacterial suspension was determined by plate counting method, specifically: 100 μL of the bacterial suspension was smeared on the MRS solid medium, and incubated in a 37°C constant temperature incubator for 48 h; (4) The encapsulation efficiency was calculated by the following formula: Encapsulation efficiency = (number of viable bacteria in microcapsules / number of viable bacteria in bacterial suspension in Example 1(1)) x 100%.

[0042] The encapsulation efficiency of the microcapsules prepared in different examples is shown in Table 1.

[0043] Table 1. Encapsulation efficiency of the microcapsules prepared in examples and comparative examples

[0044] (B) Particle size 0.1 g of the microcapsules prepared in different examples were suspended in 10 mL of phosphate buffer to obtain a microcapsule suspension, and the particle size of the microcapsules was measured at room temperature using a Malvern particle size analyzer. The results are shown in Table 2.

[0045] Table 2. Particle size of the microcapsules prepared in examples and comparative examples

[0046] Performance test In the following tests, the MRS solid medium was composed of: peptone (10 g / L), beef powder (10 g / L), yeast powder (5 g / L), glucose (20 g / L), potassium phosphate dibasic (2 g / L), ammonium citrate dibasic (2 g / L), sodium acetate (3 g / L), magnesium sulfate (0.2 g / L), manganese sulfate (0.04 g / L), Tween 80 (1 g / L), agar (15 g / L), and water to 1 L.

[0047] (A) Test of simulated digestion of probiotic colonization (1) 1 g of the microcapsules prepared in different examples using 4% dialdehyde sodium alginate was suspended in 9 mL of simulated gastric juice preheated to 37°C and mixed thoroughly to obtain a mixture. The simulated gastric juice was prepared by dissolving NaCl (2.0 g / L) and pepsin (3.2 g / L) in sterile deionized water and adjusting the pH to 2 with 1 M HCl; (2) The mixture was placed in a constant temperature shaker and placed in a 37°C water bath at a speed of 100 rpm for 2 hours to simulate gastric digestion; (3) Every 30 min, 1 mL of the mixture after simulated gastric digestion was taken, then 9 mL of phosphate buffer was added, vortexed for 5 min, and 1 mL of the solution was diluted to an appropriate concentration before being determined for viable bacteria according to plate counting to calculate the number of viable cells; (4) After the end of the simulated gastric digestion, the pH of the system was adjusted to 7.0, 9 mL of simulated intestinal fluid was added and mixed thoroughly, and then the mixture was placed in a 37°C constant temperature shaker for continuous digestion for 4 h, and the viable cell count was determined every 30 min according to the method described in the gastric stage; the simulated intestinal fluid was obtained by dissolving pancreatin (1.0 g / L) and porcine bile salt (0.8 g / L) in sterile PBS (0.2 M).

[0048] The detection results are shown in Table 2. Figure 3 As shown in Table 2, the first 120 min is the gastric environment, and 120 min to 360 min is the intestinal environment.

[0049] (II) Stability test The microcapsules prepared in Example 1 to Example 3 with 4% dialdehyde sodium alginate were placed at 4°C, and the viable cell count of probiotics was determined on the 0th day, 15th day and 30th day, respectively. The cell count measurement method is as follows: the probiotics were released from the microcapsules by treating the solution with a high-speed dispersion homogenizer, and then the solution was diluted by a certain multiple; the viable cell count of probiotics was determined by plate counting method; 100 μL of sample was smeared on MRS solid culture medium and incubated in a 37°C constant temperature incubator for 48 h; wherein the high-speed dispersion homogenizer treatment conditions are: rotation speed 5000 rpm, time 30 s. The test results are shown in Table 3.

[0050] Table 3 Stability test of microcapsules prepared in different examples

[0051] From the above Table 3, it can be seen that the viable number of the microcapsules prepared in Example 1 to Example 3 does not change significantly after being placed for 30 days, indicating good stability.

[0052] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A method for preparing a dialdehyde sodium alginate microcapsule loaded with probiotics and plant polyphenols, characterized by, The preparation method comprises: (1) dissolving plant polyphenols and probiotic bacterial suspension in water and mixing to obtain a core layer solution; (2) mixing and reacting the core layer solution, dialdehyde sodium alginate solution and sugar-based pea protein to obtain a reaction solution; (3) mixing the reaction solution with a calcium ion solution and freeze-drying to obtain a probiotic bacterial capsule.

2. The production method according to claim 1, characterized by, The preparation method meets one or more of the following conditions: (a1) the plant polyphenols are selected from one or more combinations of resveratrol, catechin or tea polyphenols; (b1) the probiotics are selected from one or more combinations of Lactobacillus plantarum, Bifidobacterium longum or Lactobacillus rhamnosus; (c1) the calcium ion solution is a calcium chloride solution.

3. The production method according to claim 1 or 2, characterized by, The preparation method meets one or more of the following conditions: (a2) the preparation method of the dialdehyde sodium alginate solution comprises: oxidizing sodium alginate with sodium periodate and dialyzing, then freeze-drying to obtain dialdehyde sodium alginate, which is dissolved in water to obtain a dialdehyde sodium alginate solution; (b2) the preparation method of the sugar-based pea protein comprises: preparing by hydrothermal method with pea protein and carboxymethyl chitosan.

4. The production method according to claim 1 or 2, characterized by, The preparation method meets one or more of the following conditions: (a3) the plant polyphenol addition amount is (2 mg-4 mg) / 100 mL of probiotic bacterial suspension; (b3) the volume concentration of the dialdehyde sodium alginate solution is 1%-3%; (c3) the volume ratio of the core layer solution to the dialdehyde sodium alginate solution is 1:(0.5-1.5); (d3) the mass concentration of the sugar-based pea protein is 0.5%-1.5%; (e3) the volume concentration of the calcium ion solution is 0.5%-1.5%, and the pH is 3-5.

5. The preparation method according to claim 3, characterized in that, The preparation method meets one or more of the following conditions: (a3) the plant polyphenol addition amount is (2 mg-4 mg) / 100 mL of probiotic bacterial suspension; (b3) the volume concentration of the dialdehyde sodium alginate solution is 1%-3%; (c3) the volume ratio of the core layer solution to the dialdehyde sodium alginate solution is 1:(0.5-1.5); (d3) the mass concentration of the sugar-based pea protein is 0.5%-1.5%; (e3) the volume concentration of the calcium ion solution is 0.5%-1.5%, and the pH is 3-5.

6. The production method according to claim 1, 2 or 5, characterized by, The preparation method meets one or more of the following conditions: (a4) in step (2), the reaction conditions include 400 rpm stirring for 1 h; (b4) in step (3), the freeze-drying conditions include: feed flow rate 8 mL / min, air flow 350 L / h, air pressure 0.1 MPa, and temperature -60℃.

7. The dialdehyde sodium alginate microcapsule loaded with probiotics and plant polyphenols prepared by the preparation method of any one of claims 1-6.

Images