A microcapsule composition with slow-release function and a preparation method and application thereof

Abstract

The application relates to the technical field of microcapsule preparation, and particularly discloses a microcapsule composition with a slow-release function, a preparation method thereof and application. Small molecule peptides and amino acids generated by three-stage hydrolysis of soybean protein can be interwoven with malt dextrin in a network structure, so that a dense and coherent composite wall material network structure is jointly constructed, which can effectively resist the erosion of gastric acid and provide physical isolation protection for probiotics. When the microcapsule enters the neutral to weak alkaline environment in the intestinal tract, the pH value in the intestinal tract is increased, the original hydrogen bond network structure between molecules is destroyed, so that the originally dense gel network structure becomes loose and greatly swells. At the same time, the hydrolyzed soybean protein is further hydrolyzed under the action of trypsin in the intestinal tract, the microcapsule wall material structure is gradually loosened, the bacteria gradually release in the swelling-disintegration process, the intestinal tract targeted slow release is realized, and the probiotic function is exerted.

Description

Technical Field

[0001] This invention relates to the field of microcapsule preparation technology, specifically to a microcapsule composition with sustained-release function, its preparation method, and its application. Background Technology

[0002] Probiotics, as live microorganisms, can benefit the host's health by improving the balance of the gut microbiota when ingested in sufficient quantities. Probiotics have shown great potential in promoting digestive health, enhancing immune function, and regulating metabolism, and are widely used in functional foods, dietary supplements, and pharmaceuticals. However, most probiotics are extremely sensitive to external environmental conditions. High temperatures and mechanical shear forces during food processing, as well as oxygen and temperature fluctuations during storage, can easily lead to a rapid decline in their activity. Even more challenging is the human gastrointestinal tract, a challenging environment where the strong acid in the stomach can cause severe damage, resulting in the inactivation of many live bacteria before they even reach the colonization site in the intestines. This significantly limits their practical application and the commercialization of these products.

[0003] To address the core challenges of probiotics' environmental intolerance and poor stability, researchers are dedicated to developing effective encapsulation and protection technologies. Microencapsulation technology is considered a promising strategy, aiming to encapsulate individual or colony probiotics within a semi-permeable or hermetically sealed protective layer. This layer acts as a physical barrier as the probiotics traverse the highly acidic environment of the stomach, effectively isolating them from direct attacks by harmful factors and significantly improving bacterial survival rates. Simultaneously, the microcapsule wall material provides a relatively stable internal microenvironment for the strains, mitigating the long-term effects of adverse factors such as moisture and oxygen on bacterial vitality. Microencapsulated probiotics not only better tolerate processing and storage stress but, more importantly, can achieve targeted release into the gut, ensuring a sufficient quantity of live bacteria reaches the site of action to fully realize their intended probiotic effects.

[0004] Therefore, developing a probiotic microcapsule with a simple preparation process, widely available raw materials, low cost, and stable performance has good application prospects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a microcapsule composition with sustained-release function, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing a microcapsule composition with sustained-release function, comprising the following steps: S1. Preparation of hydrolyzed soybean protein powder Soy protein is added to water and stirred evenly. Then, it undergoes three-stage hydrolysis. In the first stage of hydrolysis, alkaline protease is added. In the second stage, flavor protease and bromelain are added. In the third stage, carboxypeptidase B is added. After hydrolysis, the enzymes are inactivated by high temperature. After cooling, the mixture is centrifuged, filtered, concentrated, and dried to obtain hydrolyzed soy protein powder.

[0007] In this step, the mass ratio of soybean protein, water, alkaline protease, flavor protease, bromelain, and carboxypeptidase B is 10-20:100-150:0.5-1:0.5-1:0.5-1:0.3-0.6.

[0008] In this step, the pH of the first stage of hydrolysis is 9.5-10.0, for example, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0 can be selected; the hydrolysis temperature is 50-60℃, for example, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, or 60℃ can be selected; the hydrolysis time is 60-90 minutes, for example, 60 minutes, 70 minutes, 80 minutes, or 90 minutes can be selected; however, it is not limited to the listed values, and other unlisted values ​​within the range are also applicable.

[0009] In this step, the pH of the second stage of hydrolysis is 6.5-7.0, for example, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0 can be selected; the hydrolysis temperature is 50-55℃, for example, 50℃, 51℃, 52℃, 53℃, 54℃, or 55℃ can be selected; and the hydrolysis time is 30-60 minutes, for example, 30 minutes, 40 minutes, 50 minutes, or 60 minutes can be selected, but it is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0010] In this step, the pH of the third stage of hydrolysis is 8.0-8.5, for example, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5 can be selected; the hydrolysis temperature is 48-52℃, for example, 48℃, 49℃, 50℃, 51℃, or 52℃ can be selected; and the hydrolysis time is 30-60 minutes, for example, 30 minutes, 40 minutes, 50 minutes, or 60 minutes can be selected, but it is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0011] In this step, the soy protein is first subjected to a three-stage enzymatic hydrolysis: the first stage uses alkaline protease to dissociate the large soy protein molecules into smaller peptides, significantly reducing the protein molecular weight and exposing more hydrophilic and hydrophobic groups; the second stage uses flavor protease and bromelain to further hydrolyze the large protein molecules into medium and small peptides, improving their solubility and functionality; the third stage adds specific carboxypeptidase B, which can effectively remove the exposed hydrophobic amino acids from the ends of the peptide chains, preventing the hydrophobic peptides from damaging the lipid bilayer structure of the probiotic cell membrane, thus avoiding cell membrane damage, leakage of contents, and death; through graded hydrolysis, the spherical soy protein is opened up, generating a large number of small peptides that are both hydrophilic and lipophilic, which can more effectively adsorb onto the surface of probiotics and form a stable interfacial film.

[0012] S2, Preparation of wall material solution The wall material solution is obtained by dissolving hydrolyzed soybean protein powder and maltodextrin in PBS buffer.

[0013] In this step, the ratio of hydrolyzed soy protein powder, maltodextrin, and PBS buffer is 10-15g:5-10g:150mL. For example, you can choose 10g:5g:150mL, 10g:8g:150mL, 10g:10g:150mL, 12g:5g:150mL, 12g:10g:150mL, 15g:5g:150mL, 15g:8g:150mL, or 15g:10g:150mL, but it is not limited to the values ​​listed. Other unlisted values ​​within the range are also applicable.

[0014] In this step, the small peptides and amino acids produced by the three-stage hydrolysis of soy protein, due to their smaller size and more reaction sites, can interact more extensively with maltodextrin molecules (such as electrostatic attraction and hydrogen bonding), and intertwine with maltodextrin in the network structure, thus jointly constructing a dense and coherent composite wall material network structure. This effectively resists the erosion of gastric acid and provides physical isolation protection for probiotics. When the microcapsules enter the neutral to weakly alkaline environment of the intestine, the pH value in the intestine increases, causing the original hydrogen bond network structure between molecules to be destroyed, thereby making the originally dense gel network structure loose and swollen. At the same time, the hydrolyzed soy protein is further hydrolyzed under the action of intestinal trypsin and other enzymes, and the microcapsule wall structure gradually loosens. The bacteria are gradually released during the swelling-disintegration process, achieving targeted and sustained release in the intestine, thereby exerting its probiotic function.

[0015] S3. Preparation of microcapsule composition The probiotic liquid is added to the wall material solution, mixed evenly, and then freeze-dried to obtain the microcapsule composition.

[0016] In this step, the volume ratio of probiotic solution to wall material solution is 3-6:100. For example, you can choose 3:100, 4:100, 5:100, or 6:100, but it is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0017] In this step, the number of live bacteria in the probiotic solution must be ≥1.0×10⁻⁶. 6 CFU / mL.

[0018] In this step, the probiotics are selected from at least one of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium longum, and Lactobacillus bulgaricus.

[0019] Secondly, the present invention provides a microcapsule composition with sustained-release function prepared by the above-described preparation method.

[0020] Thirdly, the present invention also provides the application of the above-mentioned microcapsule composition with sustained-release function in the preparation of food, health products or pharmaceuticals.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention firstly performs a three-stage enzymatic hydrolysis of soybean protein: the first stage uses alkaline protease to dissociate the large-molecule soybean protein into smaller peptides, which greatly reduces the molecular weight of the protein and exposes more hydrophilic and hydrophobic groups; the second stage uses flavor protease and bromelain to further hydrolyze the large-molecule protein into medium and small-molecule peptides, improving its solubility and functionality; the third stage adds specific carboxypeptidase B, which can effectively remove the hydrophobic amino acids exposed at the ends of the peptide chains, avoiding damage to the lipid bilayer structure of the probiotic cell membrane by the hydrophobic peptides, which would lead to cell membrane damage, leakage of contents and death; through graded hydrolysis, the spherical soybean protein is opened up to generate a large number of small-molecule peptides that are both hydrophilic and lipophilic, which can be more effectively adsorbed on the surface of probiotics and form a stable interfacial membrane.

[0022] (2) The small molecule peptides and amino acids produced by the three-stage hydrolysis of soybean protein provided by the present invention have smaller molecules and more reaction sites, which can interact more extensively with maltodextrin molecules (such as electrostatic attraction, hydrogen bonding, etc.) and intertwine with maltodextrin in the network structure, thereby jointly constructing a dense and coherent composite wall material network structure, which can effectively resist the erosion of gastric acid and provide physical isolation protection for probiotics. When the microcapsules enter the neutral to weakly alkaline environment of the intestine, the pH value in the intestine increases, which leads to the destruction of the original hydrogen bond network structure between molecules, thereby making the originally dense gel network structure loose and swollen. At the same time, the hydrolyzed soybean protein will also be further hydrolyzed under the action of intestinal trypsin, etc., and the microcapsule wall material structure will gradually loosen. The bacteria will be gradually released during the swelling-disintegration process, realizing targeted sustained release in the intestine, thereby exerting its probiotic function. Attached Figure Description

[0023] Figure 1 The graph shows the gastric acid resistance test results of the microcapsule compositions obtained in different groups; Figure 2 The graph shows the intestinal fluid release test results of the microcapsule compositions obtained in different groups. Detailed Implementation

[0024] The present invention will be further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0025] It should be noted that, unless otherwise specified, all chemical reagents involved in this invention were purchased through commercial channels.

[0026] The alkaline protease, flavor protease, and bromelain used in the embodiments of this invention are from Jiangsu Jiujia Biotechnology Co., Ltd.; the CAS number of carboxypeptidase B, 9025-24-5, is from Hubei Jiahuixingcheng Biotechnology Co., Ltd.; and the probiotics are selected from Lactobacillus plantarum PS128.

[0027] Example 1 A method for preparing a microcapsule composition with sustained-release function includes the following steps: S1. Add 10g of soy protein to 100mL of water, stir well, and then perform three-stage hydrolysis. In the first stage of hydrolysis, add 0.5g of alkaline protease, the hydrolysis pH is 10.0, the hydrolysis temperature is 55℃, and the hydrolysis time is 60min. In the second stage of hydrolysis, add 0.5g of alkaline protease and 0.5g of bromelain, the hydrolysis pH is 7.0, the hydrolysis temperature is 55℃, and the hydrolysis time is 60min. In the third stage of hydrolysis, add 0.3g of carboxypeptidase B, the hydrolysis pH is 8.5, the hydrolysis temperature is 50℃, and the hydrolysis time is 60min. After hydrolysis, perform high-temperature enzyme inactivation, cool, centrifuge, filter, concentrate, and dry to obtain hydrolyzed soy protein powder. S2. Dissolve 10g of hydrolyzed soybean protein powder and 5g of maltodextrin in 150mL of PBS buffer to obtain the wall material solution. S3. Add 5 mL of Lactobacillus plantarum PS128 bacterial suspension to 100 mL of wall material solution, wherein the viable count in the bacterial suspension is 1.0 × 10⁻⁶. 10 After mixing CFU / mL evenly and freeze-drying, the microcapsule composition is obtained.

[0028] Example 2 A method for preparing a microcapsule composition with sustained-release function includes the following steps: S1. Add 15g of soy protein to 100mL of water, stir well, and then perform three-stage hydrolysis. In the first stage of hydrolysis, add 0.8g of alkaline protease, the hydrolysis pH is 10.0, the hydrolysis temperature is 55℃, and the hydrolysis time is 60min. In the second stage of hydrolysis, add 0.8g of alkaline protease and 0.6g of bromelain, the hydrolysis pH is 7.0, the hydrolysis temperature is 55℃, and the hydrolysis time is 60min. In the third stage of hydrolysis, add 0.5g of carboxypeptidase B, the hydrolysis pH is 8.5, the hydrolysis temperature is 50℃, and the hydrolysis time is 60min. After hydrolysis, perform high-temperature enzyme inactivation, cool, centrifuge, filter, concentrate, and dry to obtain hydrolyzed soy protein powder. S2. Dissolve 10g of hydrolyzed soybean protein powder and 8g of maltodextrin in 150mL of PBS buffer to obtain the wall material solution. S3. Add 3 mL of Lactobacillus plantarum PS128 bacterial suspension to 100 mL of wall material solution, wherein the viable bacteria count in the bacterial suspension is 1.0 × 10⁻⁶. 10 After mixing CFU / mL evenly and freeze-drying, the microcapsule composition is obtained.

[0029] Example 3 A method for preparing a microcapsule composition with sustained-release function includes the following steps: S1. Add 20g of soy protein to 100mL of water, stir well, and then perform three-stage hydrolysis. In the first stage of hydrolysis, add 1g of alkaline protease, the hydrolysis pH is 10.0, the hydrolysis temperature is 55℃, and the hydrolysis time is 60min. In the second stage of hydrolysis, add 1g of alkaline protease and 1g of bromelain, the hydrolysis pH is 7.0, the hydrolysis temperature is 55℃, and the hydrolysis time is 60min. In the third stage of hydrolysis, add 0.6g of carboxypeptidase B, the hydrolysis pH is 8.5, the hydrolysis temperature is 50℃, and the hydrolysis time is 60min. After hydrolysis, perform high-temperature enzyme inactivation. After cooling, centrifuge, filter, concentrate, and dry to obtain hydrolyzed soy protein powder. S2. Dissolve 10g of hydrolyzed soybean protein powder and 10g of maltodextrin in 150mL of PBS buffer to obtain the wall material solution. S3. Add 6 mL of Lactobacillus plantarum PS128 bacterial suspension to 100 mL of wall material solution, wherein the viable bacteria count in the bacterial suspension is 1.0 × 10⁻⁶. 10 After mixing CFU / mL evenly and freeze-drying, the microcapsule composition is obtained.

[0030] Comparative Example 1 A method for preparing a microcapsule composition with sustained-release function includes the following steps: S1. Add 10g of soy protein and 5g of maltodextrin to 150mL of PBS buffer and mix well to obtain the wall material solution. S2. Add 5 mL of Lactobacillus plantarum PS128 bacterial suspension to 100 mL of wall material solution, wherein the viable bacteria count in the bacterial suspension is 1.0 × 10⁻⁶. 10 After mixing CFU / mL evenly and freeze-drying, the microcapsule composition is obtained.

[0031] Compared to Comparative Example 1 and Example 1, no hydrolysis treatment was performed on the soybean protein.

[0032] Comparative Example 2 A method for preparing a microcapsule composition with sustained-release function includes the following steps: S1. Add 10g of soy protein to 100mL of water, stir well, and then perform two-stage hydrolysis. In the first stage of hydrolysis, add 0.5g of alkaline protease, the hydrolysis pH is 10.0, the hydrolysis temperature is 55℃, and the hydrolysis time is 60min. In the second stage of hydrolysis, add 0.5g of alkaline protease and 0.5g of bromelain, the hydrolysis pH is 7.0, the hydrolysis temperature is 55℃, and the hydrolysis time is 60min. After hydrolysis, perform high-temperature enzyme inactivation, cool, centrifuge, filter, concentrate, and dry to obtain hydrolyzed soy protein powder. S2. Dissolve 10g of hydrolyzed soybean protein powder and 5g of maltodextrin in 150mL of PBS buffer to obtain the wall material solution. S3. Add 5 mL of Lactobacillus plantarum PS128 bacterial suspension to 100 mL of wall material solution, wherein the viable count in the bacterial suspension is 1.0 × 10⁻⁶. 10 After mixing CFU / mL evenly and freeze-drying, the microcapsule composition is obtained.

[0033] Compared with Example 1, Comparative Example 2 did not include the addition of carboxypeptidase B for further hydrolysis.

[0034] Comparative Example 3 A method for preparing a microcapsule composition with sustained-release function includes the following steps: S1. Add 10g of soy protein to 100mL of water, stir well, and then perform three-stage hydrolysis. In the first stage of hydrolysis, add 0.5g of alkaline protease, the hydrolysis pH is 10.0, the hydrolysis temperature is 55℃, and the hydrolysis time is 60min. In the second stage of hydrolysis, add 0.5g of alkaline protease and 0.5g of bromelain, the hydrolysis pH is 7.0, the hydrolysis temperature is 55℃, and the hydrolysis time is 60min. In the third stage of hydrolysis, add 0.3g of neutral protease, the hydrolysis pH is 7.0, the hydrolysis temperature is 50℃, and the hydrolysis time is 60min. After hydrolysis, perform high-temperature enzyme inactivation, cool, centrifuge, filter, concentrate, and dry to obtain hydrolyzed soy protein powder. S2. Dissolve 10g of hydrolyzed soybean protein powder and 5g of maltodextrin in 150mL of PBS buffer to obtain the wall material solution. S3. Add 5 mL of Lactobacillus plantarum PS128 bacterial suspension to 100 mL of wall material solution, wherein the viable count in the bacterial suspension is 1.0 × 10⁻⁶. 10 After mixing CFU / mL evenly and freeze-drying, the microcapsule composition is obtained.

[0035] Compared with Example 1, Comparative Example 3 uses neutral protease instead of carboxypeptidase B.

[0036] Gastric acid resistance test: The microcapsule compositions obtained in Examples 1-3 and Comparative Examples 1-3 were suspended in sterilized simulated gastric fluid and incubated in a 37°C incubator in the dark for 2 hours. Then, 0.5 mL was transferred to a sterile centrifuge tube and centrifuged at 3000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was resuspended in sterile PBS buffer. The precipitate was then plated onto Mucin medium and anaerobically cultured at 37°C for 48 hours. The colony count on each plate was then calculated, and the number of surviving bacteria (CFU / mL) in the samples after incubation in simulated gastric fluid was calculated based on the dilution factor. The survival rate was calculated, and the results are as follows: Figure 1 As shown, from Figure 1 As can be seen from the above, compared with comparative examples 1-3, the present invention can improve the survival rate of probiotics in gastric juice by performing a three-stage enzymatic hydrolysis of soybean protein.

[0037] Intestinal fluid release test: 1g of the microcapsule composition obtained in Example 1 and Comparative Example 1 was weighed into 50mL of simulated intestinal fluid and placed on a shaker at 150r / m and 37℃ for 2h. One tube was taken out at 30min, 60min, 90min, and 120min respectively, and the intestinal fluid was directly aspirated to determine the viable bacterial count. The results are as follows: Figure 2 As shown, from Figure 2 As can be seen, the microcapsules prepared in Comparative Example 1 were almost completely disintegrated in simulated intestinal fluid at 60 min. The present invention, through three-stage hydrolysis of soybean protein, can produce a large number of small-molecule oligopeptides and free amino acids, which greatly increases the cross-linking sites with maltodextrin molecules, helps to form a dense and stable composite gel network structure, and can delay the disintegration time of microcapsules to 90 min, thus achieving a sustained-release effect.

[0038] Finally, it should be noted that the above embodiments do not limit the present invention in any way. Those skilled in the art can make modifications and improvements based on the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a microcapsule composition with sustained-release function, characterized in that, Includes the following steps: S1. Preparation of hydrolyzed soy protein powder: Soy protein is added to water and stirred evenly. Then, three-stage hydrolysis is carried out. In the first stage of hydrolysis, alkaline protease is added. In the second stage of hydrolysis, flavor protease and bromelain are added. In the third stage of hydrolysis, carboxypeptidase B is added. After hydrolysis, the enzymes are inactivated by high temperature. After cooling, the mixture is centrifuged, filtered, concentrated and dried to obtain hydrolyzed soy protein powder. S2. Preparation of wall material solution: Dissolve hydrolyzed soybean protein powder and maltodextrin in PBS buffer to obtain the wall material solution; S3. Preparation of microcapsule composition: Add probiotic bacterial solution to wall material solution, mix evenly and freeze dry to obtain microcapsule composition; In step S1, the mass ratio of soybean protein, water, alkaline protease, flavor protease, bromelain, and carboxypeptidase B is 10-20:100-150:0.5-1:0.5-1:0.5-1:0.3-0.

6. In step S1, the first stage of hydrolysis has a pH of 9.5-10.0, a hydrolysis temperature of 50-60℃, and a hydrolysis time of 60-90 min; the second stage of hydrolysis has a pH of 6.5-7.0, a hydrolysis temperature of 50-55℃, and a hydrolysis time of 30-60 min; the third stage of hydrolysis has a pH of 8.0-8.5, a hydrolysis temperature of 48-52℃, and a hydrolysis time of 30-60 min. In step S2, the ratio of hydrolyzed soybean protein powder, maltodextrin, and PBS buffer is 10-15g:5-10g:150mL.

2. The preparation method according to claim 1, characterized in that, In step S3, the volume ratio of probiotic solution to wall material solution is 3-6:

100.

3. The preparation method according to claim 1, characterized in that, In step S3, the number of live bacteria in the probiotic solution is ≥1.0×10⁻⁶. 6 CFU / mL.

4. The preparation method according to claim 1, characterized in that, In step S3, the probiotics are selected from at least one of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium longum, and Lactobacillus bulgaricus.

5. The microcapsule composition with sustained-release function prepared by the preparation method according to any one of claims 1-4.

6. The use of the microcapsule composition with sustained-release function as described in claim 5 in the preparation of food, health products or pharmaceuticals.

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