Probiotic microcapsule as well as preparation method and application thereof

Abstract

The invention discloses a probiotic microcapsule and a preparation method and application thereof, the probiotic microcapsule has a dual protection structure, and comprises an inner core and a wall material layer coated outside the inner core; the inner core is composed of porous starch, and probiotics are embedded in an internal pore structure of the porous starch; the wall material layer is composed of cellulose derivatives. Dissolving starch in water to prepare a starch solution; dissolving a cellulose derivative in water to prepare a cellulose solution; mixing the starch solution with the probiotic suspension, and stirring for 0.5-1 hour at the temperature of 37 DEG C at the rotating speed of 150-200 rpm; then adding a cellulose solution, and continuously stirring for 0.5-1 hour; and centrifuging the mixed solution at 8000-10000 rpm for 10-30 minutes, collecting a precipitate, and freeze-drying the precipitate to obtain the probiotic microcapsule. A dual protection structure is constructed through the porous starch and the cellulose derivative, the high survival rate of the probiotics in multiple environments of processing, digestion, storage and the like is achieved, and the method has the advantage of being simple in process.

Description

Technical Field

[0001] This invention relates to the field of food and pharmaceutical preparation technology, and in particular to a probiotic microcapsule, its preparation method and application. Background Technology

[0002] With the increasing awareness of health consumption, the global probiotic product market has experienced explosive growth. Probiotics, due to their regulatory effect on the gut microbiota, have shown significant potential in improving digestive function, enhancing immunity, and intervening in metabolic diseases. Among them, *Lactobacillus plantarum*, as a strain with strong acid resistance and excellent colonization ability, is widely used in fermented foods, dietary supplements, and pharmaceuticals. However, current oral probiotic preparations still face serious challenges: on the one hand, the gastric acid and bile salt environment significantly reduce the survival rate of live bacteria; on the other hand, conventional freeze-dried powders, liquid dosage forms, and tablets are easily affected by temperature and humidity during storage and transportation, leading to bacterial inactivation. Studies show that the live bacteria loss rate of commercially available probiotic products generally exceeds 50% within their shelf life, severely restricting their functionality. Therefore, developing probiotic protection technologies that combine high stability and high survival rate has become an urgent need for the industry.

[0003] To improve the tolerance of probiotics during processing and digestion, microencapsulation technology has gradually become a research hotspot. This technology encapsulates bacteria in protective materials using physical or chemical methods, effectively blocking adverse external factors. Existing microcapsules mostly use traditional wall materials such as sodium alginate and chitosan, but these suffer from low encapsulation efficiency, insufficient mechanical strength, or poor biocompatibility. In contrast, cellulose and its derivatives (such as hydroxypropyl methylcellulose) are considered ideal novel encapsulation materials due to their excellent film-forming properties, pH responsiveness, and biodegradability. Studies have shown that cellulose-based microcapsules can maintain structural integrity in gastric juice and target the release of live bacteria in the intestine, thereby improving bioavailability. However, single microcapsule technology still cannot meet the industrialization needs of probiotic preparations, especially during subsequent tableting, where microcapsules are prone to structural damage due to mechanical pressure or frictional heat, leading to secondary inactivation. Achieving synergistic optimization of microcapsule and tablet processes has become a key technological breakthrough.

[0004] In traditional tableting processes, probiotics are often directly mixed with excipients such as fillers and disintegrants before compression, but this method significantly damages bacterial viability. Existing research mainly focuses on low-temperature tableting or the addition of protective agents, but problems such as high equipment costs, slow tablet disintegration, or excipient interference with bacterial activity still exist. Therefore, there is an urgent need for an integrated solution that balances process feasibility and bacterial protection. Among the currently published literature and patents, for example, Chinese Patent Publication No. CN117378764A discloses a probiotic microcapsule and its preparation method and application. It adopts a traditional "core material-wall material" single-layer gel encapsulation structure, in which a gel network is formed by materials such as sodium alginate and chitosan under the action of a cross-linking agent to encapsulate the probiotics. Its protective effect mainly relies on a single physical barrier, and the wall material depends on the compounding of natural polysaccharides such as sodium alginate and chitosan, and calcium carbonate is required as a cross-linking agent, Span-type emulsifiers, and acetic acid-containing vegetable oils as inducers, making the raw material system relatively complex. Summary of the Invention

[0005] The purpose of this invention is to provide a probiotic microcapsule, its preparation method, and its application. This invention constructs a dual protective structure using porous starch and cellulose derivatives, and utilizes microencapsulation technology combining physical adsorption and encapsulation to form a protective barrier that possesses high encapsulation efficiency, heat resistance, pressure resistance, and resistance to gastrointestinal digestion. This achieves a high survival rate of probiotics under multiple environments including processing, digestion, and storage, and has the advantages of simple process and suitability for industrial production.

[0006] The technical solution of the present invention is as follows: a probiotic microcapsule, the probiotic microcapsule having a dual protective structure, including a core and a wall material layer covering the core; the core is composed of porous starch, and the probiotics are embedded in the internal pore structure of the porous starch; the wall material layer is composed of cellulose derivatives.

[0007] In the above-mentioned probiotic microcapsules, the cellulose derivative is selected from at least one of microcrystalline cellulose, hydroxypropyl cellulose, and hydroxypropyl methylcellulose.

[0008] The aforementioned probiotic microcapsules contain one or more of the following probiotics: Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Streptococcus thermophilus, and Saccharomyces boulardii.

[0009] The aforementioned method for preparing probiotic microcapsules includes the following steps:

[0010] S1. Dissolve starch in water to prepare a starch solution with a mass concentration of 0.5-2.5%; dissolve cellulose derivatives in water to prepare a cellulose solution with a mass concentration of 0.1-0.5%.

[0011] S2. Mix the starch solution obtained in step S1 with a concentration of 10... 7 -10 9 The probiotic suspension of CFU / ml was mixed at a volume ratio of 2:1 to 1:5 and stirred at 150-200 rpm for 0.5-1 hour at 37°C; then the cellulose solution obtained in step S1 was added and stirring was continued for 0.5-1 hour.

[0012] S3. Centrifuge the mixture obtained in step S2 at 8000-10000 rpm for 10-30 minutes, collect the precipitate, and freeze-dry it to obtain the probiotic microcapsules.

[0013] In the aforementioned method for preparing probiotic microcapsules, in step S1, the mass concentration of the starch solution is 1.5%, and the mass concentration of the cellulose solution is 0.3%.

[0014] In the aforementioned method for preparing probiotic microcapsules, in step S2, the concentration of the probiotic suspension is 10. 8 The bacterial suspension and starch solution were mixed in a volume ratio of 1:1 (CFU / ml).

[0015] An application of a probiotic microcapsule, the probiotic microcapsule being used to prepare a probiotic microcapsule tablet, the probiotic microcapsule tablet comprising the probiotic microcapsule and pharmaceutically or food-acceptable excipients; the weight ratio of the probiotic microcapsule to the excipient is 1:4 to 2:3.

[0016] In the aforementioned applications, the excipients include the following raw materials in parts by weight: 20-30 parts maltodextrin, 2-5 parts microcrystalline cellulose, 15-35 parts sorbitol, 0.1-0.2 parts citric acid, and 0.5-2 parts magnesium stearate.

[0017] The aforementioned application involves the preparation of the probiotic microcapsule tablets as follows: maltodextrin, microcrystalline cellulose, sorbitol, citric acid, and magnesium stearate are mixed in a specific ratio for 10-30 minutes, followed by wet granulation, drying, sieving, and grinding to obtain a mixed excipient; the probiotic microcapsules are then mixed uniformly with the mixed excipients at a ratio of 1:4 to 2:3 to obtain a tableting mixture; 0.5-1.0 g of the tableting mixture is taken and compressed under a pressure of 5-15 kPa for 1-10 minutes to obtain the probiotic microcapsule tablets.

[0018] The application of a probiotic microcapsule tablet as described above in the preparation of food or pharmaceuticals with the function of regulating intestinal flora.

[0019] Compared with the prior art, the present invention has the following significant advantages:

[0020] 1. This invention innovatively constructs a dual protection system using porous starch and cellulose derivatives. Porous starch serves as a carrier, firmly immobilizing probiotics through physical adsorption, reducing bacterial loss during subsequent processing. Cellulose derivatives (microcrystalline cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose) are used as encapsulation materials, utilizing their excellent film-forming properties and hydrogen bonds with porous starch to form a stable protective barrier around the bacteria. This dual structure effectively blocks damage to probiotics from extreme environments in the digestive tract, such as gastric acid and bile salts, while also resisting local environmental fluctuations during processing. This significantly enhances the survival rate of probiotics during digestion, ensuring more live bacteria reach the intestines and colonize, fully leveraging their function in regulating the intestinal flora. Furthermore, this dual structure eliminates the need for additional protective agents such as skim milk powder, whey protein, and trehalose, effectively blocking damage to probiotics from extreme digestive environments like stomach acid and bile salts. It also resists local environmental fluctuations during processing. Incubation in a 1% bile salt environment for 5 hours only resulted in a decrease of 1.97 log CFU / g in the number of live probiotics, significantly enhancing the survival ability of probiotics during digestion. This ensures that more live bacteria can reach the intestines and colonize, fully leveraging their function of regulating the intestinal flora.

[0021] 2. This invention optimizes the selection of microcapsule wall materials (especially cellulose derivatives) and tablet forming parameters, enabling the microcapsules to maintain structural integrity during tableting. The survival rate of probiotics after tableting exceeds 40%, solving the industry pain point of easy breakage and secondary inactivation of bacteria caused by traditional microcapsule tableting. At the same time, the resulting tablets have good physical stability, meeting the conventional physical performance requirements of food and pharmaceutical preparations, and ensuring rapid release of probiotics in the intestine, avoiding the impact of slow tablet disintegration on bacterial activity.

[0022] 3. The probiotic microcapsules and tablets of the present invention can effectively resist the influence of temperature and humidity changes on probiotic activity during storage and transportation. Even after being stored at 37°C for 30 days, the probiotics protected by double encapsulation can still maintain a high survival rate of live bacteria. Compared with probiotic products that are not encapsulated or are encapsulated only, the products of the present invention can significantly slow down the rate of bacterial inactivation. Even under normal storage conditions, they can maintain a high level of live bacteria for a long time, greatly extend the shelf life of the product, reduce the risk of product failure due to improper storage, and reduce cost losses in the production and distribution process.

[0023] 4. The raw materials used in this invention (porous starch, cellulose derivatives, conventional excipients, etc.) are all commonly used materials in the food and pharmaceutical fields, with wide availability and controllable costs, eliminating the need to rely on scarce or expensive raw materials. The preparation process, including bacterial suspension preparation, wall material dissolution, centrifugation, freeze-drying, and tableting, are all mature and conventional processes in the industry, requiring no special equipment investment. The operation process is simple and easy to control, and can be directly connected to existing food or pharmaceutical production lines, reducing the technical threshold and equipment costs for large-scale production, and possessing good prospects for industrial application.

[0024] 5. The probiotic microcapsules prepared by this invention have good compatibility. They can be used alone in foods such as yogurt and beverages, or as a core ingredient in the preparation of intestinal flora regulation drugs. Probiotic microcapsule tablets (such as compressed candies) further optimize the product form, achieve precise and controllable dosage, eliminate the need for additional operations such as reconstitution, and are easy to carry and store. They effectively solve the problems of inconvenience and high storage requirements of traditional freeze-dried powders and liquids, meet the consumption needs of different scenarios, and improve the user experience. Attached Figure Description

[0025] Figure 1 This demonstrates the effect of different wall materials on the encapsulation efficiency of Lactobacillus plantarum;

[0026] Figure 2 The changes in microcapsule survival rate before and after freeze-drying were shown;

[0027] Figure 3 The survival of encapsulated probiotics under acidic conditions was demonstrated;

[0028] Figure 4 The survival of encapsulated probiotics under bile salt conditions was shown;

[0029] Figure 5 The tablet survival rate of each group was shown;

[0030] Figure 6 The crumb density of each group was displayed;

[0031] Figure 7 The hardness of each group was displayed;

[0032] Figure 8 The breakdown time for each group was displayed;

[0033] Figure 9 The changes in tablet survival rate after 30 days of storage at 4°C were shown.

[0034] Figure 10 The changes in tablet survival rate after 30 days of storage at 25°C were shown.

[0035] Figure 11The study showed the changes in tablet survival rate after 30 days of storage at 37°C. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0037] Example 1: A probiotic microcapsule, the probiotic microcapsule having a dual protective structure, including a core and a wall material layer covering the core; the core is composed of porous starch, and the probiotics are embedded in the internal pore structure of the porous starch; the wall material layer is composed of a cellulose derivative. In this example, the preparation and formulation application of the probiotic microcapsule are as follows:

[0038] (1) Preparation of bacterial suspension: Lactobacillus plantarum powder was inoculated at a concentration of 1% into MRS broth medium (37℃, 24 hours) for activation. Subsequently, the activated culture was transferred to MRS broth medium at a concentration of 3% and passaged twice (37℃, 24 hours). Bacteria were harvested by centrifugation at 5000 rpm for 10 minutes at 4℃, and then washed three times in NaCl solution (0.85% w / v). The sediment was resuspended in saline solution to obtain a suspension with approximately 10... 9 A suspension of *Lactobacillus plantarum* at CFU / mL. This embodiment uses *Lactobacillus plantarum* as the experimental bacteria. In other embodiments, the probiotics may be one or more combinations of *Lactobacillus plantarum*, *Lactobacillus acidophilus*, *Lactobacillus rhamnosus*, *Lactobacillus casei*, *Bifidobacterium infantis*, *Bifidobacterium longum*, *Bifidobacterium breve*, *Streptococcus thermophilus*, and *Saccharomyces boulardii*.

[0039] (2) Preparation of probiotic microcapsules: Before encapsulation, 1 g of starch and 0.1 g of microcrystalline cellulose were dissolved in 100 mL of water to obtain 1% starch solution and 0.1% microcrystalline cellulose solution, respectively. The probiotic suspension was mixed with the starch solution (volume ratio 1:1) and stirred at 180 rpm for 0.5 h at 37 °C. Then, 0.1% microcrystalline cellulose solution was added and stirring was continued for 0.5 h. The probiotic microcapsules were collected by centrifugation at 8000 rpm for 15 min and frozen at -20 °C for 25 min. Finally, the samples were freeze-dried.

[0040] (3) Preparation of probiotic tablets: Mix the following excipients by weight, wherein maltodextrin accounts for 25 parts, microcrystalline cellulose accounts for 2 parts, sorbitol accounts for 20 parts, citric acid accounts for 0.1 parts, and magnesium stearate accounts for 1 part. Mix and stir the various materials of the excipients for 15 minutes. Obtain the excipients by wet granulation, drying, sieving, and grinding. Mix the probiotic microcapsules and excipients in a ratio of 1:4 to obtain a powder mixture. Take 0.8 g of the powder mixture and press it at 8 kPa for 5 minutes to obtain probiotic microcapsule tablets.

[0041] Example 2: A probiotic microcapsule, wherein the probiotic microcapsule has a dual protective structure, including a core and a wall material layer covering the core; the core is composed of porous starch, and the probiotics are embedded in the internal pore structure of the porous starch; the wall material layer is composed of a cellulose derivative. In this example, the preparation and formulation application of the probiotic microcapsule are as follows:

[0042] (1) Preparation of bacterial suspension: Lactobacillus plantarum powder was inoculated at a concentration of 1% into MRS broth medium (37℃, 24 hours) for activation. Subsequently, the activated culture was transferred to MRS broth medium at a concentration of 3% and passaged twice (37℃, 24 hours). Bacteria were harvested by centrifugation at 5000 rpm for 10 minutes at 4℃, and then washed three times in NaCl solution (0.85% w / v). The sediment was resuspended in saline solution to obtain a suspension with approximately 10... 9 A suspension of Lactobacillus plantarum at CFU / mL.

[0043] (2) Preparation of probiotic microcapsules: Before encapsulation, 1.5 g of starch and 0.3 g of hydroxypropyl cellulose were dissolved in 100 mL of water to obtain a starch solution with a mass concentration of 1.5% and a hydroxypropyl cellulose solution with a mass concentration of 0.1%, respectively. The *Lactobacillus plantarum* suspension was mixed with the starch solution (1:2) and stirred at 200 rpm for 1 h at 37 °C. Then, 0.1% hydroxypropyl cellulose solution was added and stirring was continued for 1 h. The *Lactobacillus plantarum* microcapsules were collected by centrifugation at 8000 rpm for 25 min and frozen at -20 °C for 25 min. Finally, the samples were freeze-dried.

[0044] (3) Preparation of probiotic tablets: Mix the following excipients by weight, wherein maltodextrin accounts for 30 parts, microcrystalline cellulose accounts for 5 parts, sorbitol accounts for 30 parts, citric acid accounts for 0.2 parts, and magnesium stearate accounts for 2 parts. Mix and stir the various materials of the excipients for 20 minutes. Obtain the excipients by wet granulation, drying, sieving, and grinding. Mix the probiotic microcapsules and excipients in a ratio of 1:3 to obtain a uniform powder mixture. Take 1g of the powder mixture and press it at 10kPa for 5 minutes to obtain probiotic microcapsule tablets.

[0045] Example 3: A probiotic microcapsule, wherein the probiotic microcapsule has a dual protective structure, including a core and a wall material layer covering the core; the core is composed of porous starch, and the probiotics are embedded in the internal pore structure of the porous starch; the wall material layer is composed of a cellulose derivative. In this example, the preparation and formulation application of the probiotic microcapsule are as follows:

[0046] (1) Preparation of bacterial suspension: Lactobacillus plantarum powder was inoculated at a concentration of 1% into MRS broth medium (37°C, 24 hours) for activation. Subsequently, the activated culture was transferred to MRS broth medium at a concentration of 3% and passaged twice (37°C, 24 hours). Bacteria were harvested by centrifugation at 5000 rpm for 10 minutes at 4°C, and then washed three times in NaCl solution (0.85% w / v). The sediment was resuspended in saline solution to obtain a suspension with approximately 10... 9 A suspension of Lactobacillus plantarum at CFU / mL.

[0047] (2) Preparation of probiotic microcapsules: Before encapsulation, 1.5 g of starch and 0.3 g of hydroxypropyl methylcellulose were dissolved in 100 mL of water to obtain a starch solution with a mass concentration of 1.5% and a hydroxypropyl methylcellulose solution with a mass concentration of 0.3%, respectively. The *Lactobacillus plantarum* suspension was mixed with the starch solution (volume ratio 1:1) and stirred at 180 rpm for 1 h at 37 °C. Then, the hydroxypropyl methylcellulose solution was added and stirring was continued for 1 h. The *Lactobacillus plantarum* microcapsules were collected by centrifugation at 10,000 rpm for 30 min and frozen at -20 °C for 30 min. Finally, the samples were freeze-dried.

[0048] (3) Preparation of probiotic tablets: Mix the following excipients by weight, wherein maltodextrin accounts for 25 parts, microcrystalline cellulose accounts for 3 parts, sorbitol accounts for 25 parts, citric acid accounts for 0.15 parts, and magnesium stearate accounts for 1 part. Mix and stir the various materials of the excipients for 20 minutes. Obtain the excipients by wet granulation, drying, sieving, and grinding. Mix the probiotic microcapsules and excipients in a ratio of 1:2 to obtain a powder mixture. Take 1g of the powder mixture and press it at 10kPa for 10 minutes to obtain probiotic microcapsule tablets.

[0049] Example 4: A probiotic microcapsule, wherein the probiotic microcapsule has a dual protective structure, including a core and a wall material layer covering the core; the core is composed of porous starch, and the probiotics are embedded in the internal pore structure of the porous starch; the wall material layer is composed of a cellulose derivative. In this example, the preparation and formulation application of the probiotic microcapsule are as follows:

[0050] (1) Preparation of bacterial suspension: Lactobacillus plantarum powder was inoculated at a concentration of 1% into MRS broth medium (37°C, 24 hours) for activation. Subsequently, the activated culture was transferred to MRS broth medium at a concentration of 3% and passaged twice (37°C, 24 hours). Bacteria were harvested by centrifugation at 5000 rpm for 10 minutes at 4°C, and then washed three times in NaCl solution (0.85% w / v). The sediment was resuspended in saline solution to obtain a suspension with approximately 10... 9 A suspension of Lactobacillus plantarum at CFU / mL.

[0051] (2) Preparation of probiotic microcapsules: Before encapsulation, 2.5 g of starch and 0.5 g of microcrystalline cellulose were dissolved in 100 mL of water to obtain a starch solution with a mass concentration of 2.5% and a microcrystalline cellulose solution with a mass concentration of 0.5%, respectively. The *Lactobacillus plantarum* suspension was mixed with the starch solution (volume ratio 1:1) and stirred at 200 rpm for 1 h at 37 °C. Then, the hydroxymicrocrystalline cellulose solution was added and stirring was continued for 1 h. The *Lactobacillus plantarum* microcapsules were collected by centrifugation at 10,000 rpm for 30 min and frozen at -20 °C for 30 min. Finally, the samples were freeze-dried.

[0052] (3) Preparation of probiotic tablets: Mix the following excipients by weight: maltodextrin 22 parts, microcrystalline cellulose 3 parts, sorbitol 35 parts, citric acid 0.15 parts, and magnesium stearate 1.5 parts. Mix the various excipients for 30 minutes. Obtain the excipients by wet granulation, drying, sieving, and grinding. Mix the probiotic microcapsules and excipients in a 2:3 ratio to obtain a powder mixture. Take 1g of the powder mixture and press it at 10kPa for 10 minutes to obtain probiotic microcapsule tablets.

[0053] Experimental Example: The steps and processes in this experimental example are the same as in Example 3, except that an orthogonal experiment was used to study the effect of different wall materials on microcapsules. In this experimental example, tablets made directly from bacterial powder without starch and cellulose encapsulation were used as the control group. Experimental group 1 (also known as the PS group) was named using porous starch as the encapsulation material only; experimental group 2 (also known as the PS-MCC group) was named using porous starch and microcrystalline cellulose as the encapsulation material; experimental group 3 (also known as the PS-HPC group) was named using porous starch and hydroxypropyl cellulose as the encapsulation material; and experimental group 4 (also known as the PS-HPMC group) was named using porous starch and hydroxypropyl methylcellulose as the encapsulation material. The testing methods in this experimental example are as follows:

[0054] (1) Determination of microencapsulated bacterial activity and encapsulation efficiency

[0055] First, 1.0 g of microcapsules was weighed and added to 9.0 mL of α-amylase (125 mg, 8 U / mg). The mixture was hydrolyzed in PBS at 60 Hz for 15 min. A series of diluted samples were then applied to MRS agar plates. After incubation at 37°C for 48 hours, colony-forming units (CFU) were counted.

[0056] ;

[0057] In the formula, It is the number of live Lactobacillus plantarum in the microcapsules (logCFU / g). It represents the number of live Lactobacillus plantarum before encapsulation (log CFU / g).

[0058] Figure 1 The effects of different wall materials on the encapsulation efficiency of *Lactobacillus plantarum* were demonstrated. For example... Figure 1 As shown, the encapsulation efficiency using porous starch alone was approximately 65%. The encapsulation efficiency significantly improved after introducing cellulose derivatives, with experimental group 2 reaching approximately 75%, experimental group 3 approximately 85%, and experimental group 4 approaching 90%. This indicates that the dual encapsulation structure of porous starch and cellulose derivatives can effectively improve the encapsulation efficiency of probiotics, and the effects of different cellulose derivatives varied: hydroxypropyl methylcellulose (HPMC) > hydroxypropyl cellulose (HPC) > microcrystalline cellulose (MCC).

[0059] (2) Thermal stability, acid resistance and bile salt resistance of microcapsules

[0060] Heat resistance stability: To evaluate the protective effect of encapsulation on the viability of Lactobacillus plantarum obtained from different temperature treatments, 100 mg of sample was immersed in 1 mL of PBS, heated in water for 15 min (30℃, 45℃), then cooled to room temperature in an ice-water bath, and the viability of Lactobacillus plantarum was measured.

[0061] The viable cell counts of *Lactobacillus plantarum* embedded under different temperature treatments are shown in Table 1. Table 1

[0062] Table 1 shows that at 30℃: the unencapsulated control group had the lowest viable bacterial count, experimental group 1 using only porous starch showed a slight increase, while the introduction of cellulose derivatives resulted in significantly higher viable bacterial counts in experimental groups 2-4. At 45℃: the unencapsulated control group showed a significant decrease in viable bacterial count, experimental group 1 using only porous starch showed some protection, while experimental groups 2-4 showed more significant protection, with experimental group 3 maintaining the highest viable bacterial count and exhibiting the best thermal stability.

[0063] Figure 2 The changes in microcapsule survival rate before and after freeze-drying were shown. Figure 2It can be seen that the number of viable bacteria in the unencapsulated control group was higher before freeze-drying, but decreased significantly after drying, indicating that freeze-drying alone causes great damage to probiotics. Experimental group 1, which used only porous starch, retained some viable bacteria after drying, but it was still significantly lower than the combination of the present invention. Experimental groups 2-4 of the present invention showed a significantly reduced decrease in viable bacteria after freeze-drying, demonstrating excellent protective effects, with very little difference between the number of viable bacteria after drying and before drying.

[0064] Acid stability: The microcapsules were added to MRS liquid medium with pH 3 and an inoculum of 5% and treated at 37°C for 4 hours. Samples were taken at 0, 1, 2 and 4 hours for serial dilution and colony counting was performed at three appropriate dilution gradients.

[0065] Bile salt stability: 1 g of microcapsules were transferred to test tubes containing no bile salts and those containing 1% bile salts, and incubated at 37°C for 5 h. Samples were serially diluted at 0, 1, 3, and 5 h. 1 mL of the diluted solution was decanted and added to MRS culture on sterile petri dishes. After incubation at 37°C for 48 h, colonies were counted.

[0066] Figure 3 The study showed the survival of encapsulated probiotics under acidic conditions. Figure 4 The survival of encapsulated probiotics under bile salt conditions was shown; among them Figure 3 and Figure 4 The bars in the chart, from left to right, represent the control group and experimental groups 1-4, respectively. Figure 3 and Figure 4 The results show that the survival rate of probiotics in untreated microcapsules decreased by 0.19 log CFU / g and 2.92 log CFU / g in acidic and bile salt environments, respectively. In contrast, the microbiota encapsulated in cellulose exhibited better protection against environmental factors, with experimental group 4 showing the best performance, showing a decrease of only 0.07 log CFU / g and 1.97 log CFU / g in acidic and bile salt environments, respectively.

[0067] (3) Tablet survival rate, brittleness, hardness, disintegration time and storage stability

[0068] Tablet survival rate: The prepared Lactobacillus plantarum probiotic tablets were stored at 4℃ and 25℃ for 4 weeks, and then taken out weekly and their bacterial activity was determined by plate counting method.

[0069] Crumbliness, hardness, and disintegration time: The crumbliness, hardness, and disintegration time of probiotic tablets are determined in accordance with the relevant provisions of the Pharmacopoeia of the People's Republic of China (National Pharmacopoeia Commission, 2020).

[0070] Storage stability: The shelf stability of probiotic microencapsulated tablets with different encapsulation materials was evaluated at 4°C, 25°C and 37°C for 0, 7, 14, 21 and 30 days. Figure 5 The survival rate of tablets in each group was shown. Figure 6 The crispness of each group was displayed. Figure 7 The hardness of each group was displayed. Figure 8 The breakdown time for each group is displayed. From Figures 5-8 It can be seen that the microencapsulated tablets formed from probiotics encapsulated in microcapsules can improve bacterial activity, and the friability of all samples is less than 1%. The hardness of the probiotic tablets is improved, which is attributed to the excellent compressibility of the microcapsules of this invention. After microencapsulation, the tablet disintegration time is significantly shortened compared with the control group. Rapid disintegration is beneficial for maintaining probiotic activity and enhancing colonization ability.

[0071] Figure 9 , Figure 10 and Figure 11 The changes in tablet survival rates after 30 days of storage at 4℃, 25℃, and 37℃ are shown in the figure. The bars in the graph, from left to right, represent the control group and experimental groups 1-4, respectively. The results indicate that the survival rate of probiotics gradually decreases with increasing temperature and storage time. Higher temperatures (such as 37℃) significantly reduce microbial survival rates, while storage conditions near 0℃ slow down the rate of harmful chemical reactions. However, microencapsulation further improves the storage stability of probiotic tablets.

[0072] In summary, this invention utilizes porous starch as a carrier and cellulose derivatives as encapsulation materials to prepare microcapsules. By optimizing the microcapsule wall material ratio and tablet forming parameters, the loss of probiotics during processing and storage is reduced, providing a new approach for the development of highly active probiotic solid preparations.

Claims

1. A probiotic microcapsule, characterized in that, The probiotic microcapsule has a dual protective structure, including a core and a wall material layer covering the core; the core is made of porous starch, and the probiotics are embedded in the internal pore structure of the porous starch; the wall material layer is made of cellulose derivative.

2. The probiotic microcapsule according to claim 1, characterized in that, The cellulose derivative is selected from at least one of microcrystalline cellulose, hydroxypropyl cellulose, and hydroxypropyl methylcellulose.

3. The probiotic microcapsule according to claim 1, characterized in that, The probiotics include one or more combinations of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Streptococcus thermophilus, and Saccharomyces boulardii.

4. The method for preparing probiotic microcapsules according to any one of claims 1-3, characterized in that: Includes the following steps: S1. Dissolve starch in water to prepare a starch solution with a mass concentration of 0.5-2.5%; dissolve cellulose derivatives in water to prepare a cellulose solution with a mass concentration of 0.1-0.5%. S2. Mix the starch solution obtained in step S1 with a concentration of 10... 7 -10 9 The probiotic suspension of CFU / ml was mixed at a volume ratio of 2:1 to 1:5 and stirred at 150-200 rpm for 0.5-1 hour at 37°C; then the cellulose solution obtained in step S1 was added and stirring was continued for 0.5-1 hour. S3. Centrifuge the mixture obtained in step S2 at 8000-10000 rpm for 10-30 minutes, collect the precipitate, and freeze-dry it to obtain the probiotic microcapsules.

5. The method for preparing probiotic microcapsules according to claim 4, characterized in that: In step S1, the mass concentration of the starch solution is 1.5%, and the mass concentration of the cellulose solution is 0.3%.

6. The method for preparing probiotic microcapsules according to claim 4, characterized in that: In step S2, the concentration of the probiotic suspension is 10. 8 The bacterial suspension and starch solution were mixed in a volume ratio of 1:1 (CFU / ml).

7. An application of a probiotic microcapsule, characterized in that: The probiotic microcapsules are used to prepare probiotic microcapsule tablets, which contain the probiotic microcapsules as described in claims 1-3 and pharmaceutically or food-grade excipients.

8. The application according to claim 7, characterized in that: The excipients include the following raw materials in parts by weight: 20-30 parts maltodextrin, 2-5 parts microcrystalline cellulose, 15-35 parts sorbitol, 0.1-0.2 parts citric acid, and 0.5-2 parts magnesium stearate.

9. The application according to claim 8, characterized in that: The probiotic microcapsule tablets are prepared as follows: maltodextrin, microcrystalline cellulose, sorbitol, citric acid, and magnesium stearate are mixed in a certain proportion for 10-30 minutes, followed by wet granulation, drying, sieving, and grinding to obtain a mixed excipient; the probiotic microcapsules and the mixed excipients are mixed evenly in a ratio of 1:4 to 2:3 to obtain a tableting mixture; 0.5-1.0g of the tableting mixture is taken and compressed under a pressure of 5-15 kPa for 1-10 minutes to obtain the probiotic microcapsule tablets.

10. The use of a probiotic microcapsule tablet as described in any one of claims 7-9 in the preparation of food or pharmaceuticals with the function of regulating intestinal flora.

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