Probiotic microcapsule as well as preparation method and application thereof

Abstract

The invention provides a probiotic microcapsule as well as a preparation method and application thereof, and belongs to the technical field of microbial microcapsules. The preparation method of the probiotic microcapsule provided by the invention comprises the following steps: providing an embedding material which comprises porous starch and probiotics embedded in pores of the porous starch and adhered to the surface of the porous starch; mixing the embedded material, a cross-linking agent and a dispersing agent, and performing cross-linking treatment to obtain a cross-linked material; and carrying out freeze drying on the cross-linked material to obtain the probiotic micro-capsule. The probiotic microcapsule prepared by the method disclosed by the invention is relatively high in survival rate of probiotics.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial microcapsules, and in particular to a probiotic microcapsule and a preparation method and application thereof. Background Art

[0002] Probiotics are live microorganisms that are beneficial to the host's health when given in sufficient quantities. They are mainly colonized in the colon of the human intestine. A large number of studies have found that intestinal diseases, sub-health, aging, etc. are all related to the types and abundance of intestinal probiotics. Therefore, the effective supplementation of probiotics has become a key issue of concern in academia and industry.

[0003] Oral administration has become the main way to supplement probiotics because of its simplicity and convenience, but oral probiotics are easily inactivated by the adverse effects of gastric acid and bile salts in the body. Encapsulation can isolate probiotics from the external environment and effectively avoid the impact of the gastrointestinal environment. However, most of the established probiotic encapsulation methods, such as emulsification and complex coagulation, have problems of complex processes and high costs. The development of simple and feasible new probiotic encapsulation methods is an urgent need for industrial upgrading and development. At present, there is a related technology that uses porous starch as an encapsulation material to encapsulate probiotics, but the product obtained by this method has poor ability to resist gastric acid and bile salts from entering the colon, and the survival rate of probiotics is low. Summary of the invention

[0004] The purpose of the present invention is to provide a probiotic microcapsule and a preparation method and application thereof. The probiotic microcapsule prepared by the method of the present invention has a high survival rate of probiotics.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for preparing probiotic microcapsules, comprising the following steps: Providing an embedding material, the embedding material comprising porous starch and probiotics embedded in the pores of the porous starch and adhered to the surface of the porous starch; The embedding material, the crosslinking agent and the dispersing agent are mixed and crosslinked to obtain a crosslinked material; The cross-linked material is freeze-dried to obtain the probiotic microcapsule.

[0006] Preferably, the cross-linking agent comprises phosphate; and the mass ratio of the cross-linking agent to the porous starch is 0.05~0.5:1.

[0007] Preferably, the phosphate includes one or more of trimetaphosphate, tripolyphosphate and hexametaphosphate.

[0008] Preferably, the dispersant comprises physiological saline, and the pH value of the dispersant is 8-10.

[0009] Preferably, the cross-linking treatment is carried out at a temperature of 35-55° C. and for a time of 0.5-5 h.

[0010] Preferably, the probiotics include rod-shaped probiotics, and the cross-sectional diameter of the rod-shaped probiotics is 0.5-2.0 μm; the pore size of the porous starch is 1.0-3.0 μm.

[0011] Preferably, the probiotics include one or more of the genera Lactobacillus, Bifidobacterium, Escherichia coli, Bacillus, Clostridium butyricum and Saccharomyces.

[0012] Preferably, the porous starch is prepared by enzymatic hydrolysis of starch, and the enzymes used in the enzymatic hydrolysis include amylase and / or saccharifying enzyme; the temperature of the enzymatic hydrolysis is 30-60°C, and the time is 8-24h.

[0013] The present invention provides probiotic microcapsules prepared by the preparation method described in the above technical solution.

[0014] The present invention provides the use of the probiotic microcapsules described in the above technical solution as additives for food, health products, feed or medicine.

[0015] Beneficial effects: The present invention provides an embedding material, which includes porous starch and probiotics embedded in the pores of the porous starch and adhered to the surface of the porous starch; the embedding material, a cross-linking agent and a dispersant are mixed and cross-linked to obtain a cross-linked material; the cross-linked material is freeze-dried to obtain the probiotic microcapsule. The present invention adopts porous starch as embedding material to realize probiotic embedding, and on this basis adopts cross-linking agent to carry out cross-linking treatment, and combines physical embedding technology with in-situ cross-linking technology to prepare probiotic microcapsules, which can cover the in-situ pores of part of the porous starch embedding probiotic system, improve the embedding rate of probiotics, and can also enhance the thermal stability, acid resistance and enzymolysis resistance of probiotic microcapsules, so as to better resist the adverse environment of the gastrointestinal tract, and the survival rate of probiotics is higher; at the same time, the resistant starch formed by in-situ cross-linking can respond to the flora and amylase in the colon intestine, promote the dissociation of the system in the colon part, and target release in the colon mucus layer; the resistant starch formed by in-situ cross-linking can also be used as the fermentation carbon source of probiotics, so as to more effectively realize the targeted release and colonization of probiotics in the colon part, and further enhance the probiotic function of probiotic microcapsules. Therefore, the probiotic microcapsules prepared by the method of the present invention can realize the controlled release and colonization of the colon region in vivo. In addition, the method of the present invention also has the advantages of simple production process, low energy consumption, small investment, and easy large-scale production.

[0016] Furthermore, the present invention adopts phosphate as a cross-linking agent, which has mild cross-linking conditions and is conducive to the survival of probiotics. The present invention adopts in-situ cross-linking technology to allow the encapsulated material (i.e., porous starch encapsulating probiotics) to interact through phosphate bonds and ionic bonds to form probiotic microcapsules with a three-dimensional network structure. This structure can effectively cover up part of the in-situ pores of the porous starch encapsulated probiotic system and improve the encapsulation rate of probiotics.

[0017] Furthermore, the present invention utilizes enzymatic hydrolysis to prepare porous starch having a pore size matching the size of probiotics, which facilitates the probiotics to enter the interior of the porous starch and improves the embedding rate; and the present invention uses porous starch as an embedding material, without the need to use other reagents, has high embedding efficiency, low cost, and is green and safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The following is a preparation flow chart and action mechanism diagram of probiotic microcapsules in an embodiment of the present invention; Figure 2 This is a SEM image of the E. coli microcapsules prepared in Example 1; Figure 3 This is a SEM image of the Lactobacillus acidophilus microcapsules prepared in Example 2; Figure 4 This is a SEM image of Clostridium butyricum microcapsules prepared in Example 3; Figure 5 This is a graph showing the changes in storage survival rates of E. coli microcapsules and E. coli encapsulation systems; Figure 6 This is a graph showing the changes in storage survival rates of Lactobacillus acidophilus microcapsules, Lactobacillus acidophilus embedding systems, a certain Junkang Lactobacillus acidophilus tablet and a certain peptide home Lactobacillus acidophilus powder; Figure 7 This is a graph showing the changes in the storage survival rate of Clostridium butyricum microcapsules, Clostridium butyricum embedding system and a certain peptide house. Figure 8 This is the SEM image of the E. coli microcapsules that simulate the enzymatic hydrolysis of colon amylase; Fig. 9 This is the SEM image of the microcapsules of Lactobacillus acidophilus enzymatically hydrolyzed by simulating colon amylase; Fig.10 This is the SEM image of Clostridium butyricum microcapsules that simulates the enzymatic hydrolysis of colon amylase; Fig.11 This is a graph showing the results of the in vivo adhesion and colonization test of Escherichia coli microcapsules; Fig.12 This is a graph showing the results of the in vivo adhesion and colonization test of Lactobacillus acidophilus microcapsules; Fig.13 This is the in vivo adhesion and colonization test result of Clostridium butyricum microcapsules. DETAILED DESCRIPTION

[0019] The present invention provides a method for preparing probiotic microcapsules, comprising the following steps: Providing an embedding material, the embedding material comprising porous starch and probiotics embedded in the pores of the porous starch and adhered to the surface of the porous starch; The embedding material, the crosslinking agent and the dispersing agent are mixed and crosslinked to obtain a crosslinked material; The cross-linked material is freeze-dried to obtain the probiotic microcapsule.

[0020] In the present invention, unless otherwise specified, the raw materials used are commercially available products well known to those skilled in the art or are prepared by methods well known to those skilled in the art.

[0021] The present invention provides an embedding material, the embedding material includes porous starch and probiotics embedded in the pores of the porous starch and adhered to the surface of the porous starch. As an embodiment of the present invention, the probiotics may include rod-shaped probiotics; the probiotics may include one or more of Lactobacillus, Bifidobacterium, Escherichia coli, Bacillus, Clostridium butyricum and Saccharomyces, specifically Lactobacillus, Bifidobacterium, Escherichia coli, Bacillus, Clostridium butyricum or Saccharomyces. As an embodiment of the present invention, the cross-sectional diameter of the rod-shaped probiotics can be 0.5-2.0 μm, specifically 0.5-0.8 μm, 0.5-1.2 μm or 1.0-2.0 μm; the pore size of the porous starch can be 1.0-3.0 μm, specifically 1.0-1.5 μm, 1.5-2.5 μm or 2.0-3.0 μm; wherein the pore size of the porous starch is not less than the cross-sectional diameter of the rod-shaped probiotics to ensure that the rod-shaped probiotics can be fully embedded in the pores of the porous starch.

[0022] As an embodiment of the present invention, the porous starch can be prepared by enzymatic hydrolysis of starch, and the enzyme used in the enzymatic hydrolysis may include amylase and / or saccharifying enzyme, specifically amylase or saccharifying enzyme; the amylase may include α-amylase and / or β-amylase, specifically α-amylase or β-amylase; in an embodiment of the present invention, the enzyme used in the enzymatic hydrolysis may be saccharifying enzyme used alone, or may be a combination of α-amylase, β-amylase and saccharifying enzyme. As an embodiment of the present invention, based on the mass of the porous starch, the enzyme activity of the alpha amylase can be 400~2000U / g, and further can be 400~800U / g; the enzyme activity of the beta amylase can be 400~10000U / g, and further can be 400~2000U / g, and further can be 600~1000U / g; the enzyme activity of the saccharifying enzyme can be 400~10000U / g, and further can be 600~2000U / g, and further can be 800~1000U / g. The present invention does not specifically limit the type of the starch, and any type of starch known to those skilled in the art can be used. In the embodiment of the present invention, corn starch is specifically used. As an embodiment of the present invention, the temperature of the enzymatic hydrolysis treatment can be 30-60°C, specifically 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C; the time can be 8-24h, specifically 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h or 24h. In the embodiment of the present invention, starch is specifically mixed with the enzyme used for the enzymatic hydrolysis treatment, and enzymatic hydrolysis treatment is performed to obtain the porous starch. In the embodiment of the present invention, enzymatic hydrolysis treatment is performed under the above conditions, so that the obtained porous starch has a pore size that matches the size of the probiotics (such as the cross-sectional diameter size of the rod-shaped probiotics), which is convenient for the probiotics to be fully embedded in the pores of the porous starch through embedding treatment.

[0023] As an embodiment of the present invention, the method for preparing the embedding material may include the following steps: mixing porous starch with a fermentation liquid containing probiotics, and performing an embedding treatment to obtain the embedding material.

[0024] The present invention does not specifically limit the method for obtaining the fermentation broth containing probiotics, and a method well known to those skilled in the art can be used. Specifically, the probiotics can be cultured at a high density to obtain the fermentation broth containing probiotics. The number of probiotics in the fermentation broth containing probiotics can be 1×10 9 ~1×10 11 CFU / mL, specifically 1×10 9 CFU / mL, 1×10 10 CFU / mL or 1×10 11CFU / mL. As an embodiment of the present invention, the dosage ratio of the porous starch to the fermentation broth containing probiotics can be 0.5~1.5g:10mL, specifically 0.5g:10mL, 0.8g:10mL, 1.2g:10mL or 1.5g:10mL. As an embodiment of the present invention, the temperature of the embedding treatment can be 4~20℃, specifically 4℃, 6℃, 8℃, 10℃, 12℃, 15℃, 18℃ or 20℃; the time can be 4~72h, specifically 4h, 8h, 16h, 24h, 36h, 48h or 72h; the embedding treatment can be carried out under stirring conditions, and the stirring speed can be 100~150r / min, specifically 100r / min, 120r / min or 150r / min. As an embodiment of the present invention, the embedding treatment may further include: performing solid-liquid separation on the liquid obtained after the embedding treatment, and collecting the solid material as the embedding material; the solid-liquid separation method may be centrifugation; the centrifugal speed may be 4000~6000×g / min, specifically 4000×g / min, 5000×g / min or 6000×g / min; the time may be 5~15min, specifically 5min, 10min or 15min.

[0025] After obtaining the embedding material, the present invention mixes the embedding material, the crosslinking agent and the dispersant, and performs a crosslinking treatment to obtain a crosslinked material. As an embodiment of the present invention, the crosslinking agent may include a phosphate (specifically a food-grade phosphate), the phosphate may include one or more of trimetaphosphate, tripolyphosphate and hexametaphosphate, and the phosphate may include a sodium salt. As an embodiment of the present invention, the phosphate may include one or more of sodium trimetaphosphate, sodium tripolyphosphate and sodium hexametaphosphate, specifically sodium trimetaphosphate, or a compound of sodium trimetaphosphate and sodium tripolyphosphate (at this time, the mass ratio of sodium trimetaphosphate to sodium tripolyphosphate can be 90~99:1~10, specifically 99:1), and can also be a compound of sodium trimetaphosphate, sodium tripolyphosphate and sodium hexametaphosphate (at this time, the mass ratio of sodium trimetaphosphate, sodium tripolyphosphate and sodium hexametaphosphate can be 80~90:5~10:5~10, specifically 90:5:5). As an embodiment of the present invention, the mass ratio of the cross-linking agent to the porous starch can be 0.05-0.5:1, specifically 0.05:1, 0.1:1, 0.15:1, 0.2:1, 0.25:1, 0.3:1, 0.35:1, 0.4:1, 0.45:1 or 0.5:1. As an embodiment of the present invention, the dispersant can include physiological saline, and the pH value of the dispersant can be 8-10, specifically 8, 8.5, 9, 9.5 or 10; the amount ratio of the embedding material to the dispersant can be 0.01-1g:10mL, specifically 0.01g:10mL, 0.05g:10mL, 0.1g:10mL, 0.5g:10mL, 0.8g:10mL or 1g:10mL. As an embodiment of the present invention, the embedding material can be resuspended in the dispersant, and then the resulting suspension is mixed with a crosslinking agent for crosslinking. As an embodiment of the present invention, the temperature of the crosslinking treatment can be 35-55°C, specifically 35°C, 40°C, 45°C, 50°C or 55°C; the time can be 0.5-5h, specifically 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h or 5h; the crosslinking treatment can be carried out under vortexing conditions, and the vortexing speed can be 100-150r / min, specifically 100r / min, 120r / min or 150r / min.As an embodiment of the present invention, the cross-linking treatment may further include: performing solid-liquid separation on the liquid obtained after the cross-linking treatment, and collecting the solid material as the cross-linked material; the solid-liquid separation method may be centrifugation; the centrifugal rate may be 4000~6000×g / min, specifically 4000×g / min, 5000×g / min or 6000×g / min; the time may be 5~15min, specifically 5min, 10min or 15min.

[0026] After obtaining the cross-linked material, the present invention freeze-dries the cross-linked material to obtain the probiotic microcapsules. As an embodiment of the present invention, the freeze-drying temperature can be -60 to -40°C, specifically -60°C, -55°C, -50°C, -45°C or -40°C; the time can be 24 to 72 hours, specifically 24 hours, 36 hours, 48 ​​hours, 60 hours or 72 hours; the freeze-drying can be specifically carried out in a freeze dryer.

[0027] The present invention provides probiotic microcapsules prepared by the preparation method described in the above technical solution.

[0028] The present invention provides the use of the probiotic microcapsules described in the above technical solution as additives for food, health products, feed or medicine. The probiotic microcapsules described in the present invention are used as additives for food, health products, feed or medicine to regulate intestinal flora, improve sub-health status, etc.

[0029] Figure 1 The following is a flow chart of the preparation of probiotic microcapsules and a diagram of the mechanism of action in the embodiments of the present invention. The technical scheme in the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1 (1) Use a fermentation tank to culture E. coli at a high density (cross-section diameter of 0.5-0.8 μm) so that the total number of E. coli in the fermentation broth is 1×10 9 CFU / mL; 1g starch (specifically corn starch) was mixed with 800U saccharifying enzyme, and enzymatic hydrolysis was performed at 50°C for 8h to obtain porous starch (pore size of 1.0-1.5μm); the porous starch was mixed with fermentation liquid at a ratio of 0.5g:10mL, and embedding treatment was performed at a temperature of 20°C and a stirring rate of 150r / min for 8h, and then centrifuged at 4000×g / min for 15min, and the precipitate was collected as the embedding material; (2) resuspending the embedding material in physiological saline with a pH value of 9 to obtain a suspension, wherein the amount ratio of the embedding material to the physiological saline is 1 g:10 mL; adding a crosslinking agent (specifically sodium trimetaphosphate, and the mass ratio of the porous starch to the crosslinking agent is 1:0.05) to the suspension, performing a crosslinking treatment at a temperature of 35° C. and a vortexing rate of 150 r / min for 1.5 h, and then centrifuging at 4000×g / min for 15 min, and collecting the precipitate as the crosslinked material; (3) The cross-linked material is placed in a freeze dryer and freeze-dried at -60°C for 48 hours to obtain Escherichia coli microcapsules.

[0031] Example 2 (1) Use a fermentation tank to culture Lactobacillus acidophilus at a high density (cross-section diameter of 0.5-1.2 μm) so that the total number of Lactobacillus acidophilus in the resulting fermentation broth is 1×10 10 CFU / mL; 1g starch (specifically corn starch) was mixed with 800U saccharifying enzyme, and enzymatic hydrolysis was performed at 50°C for 24h to obtain porous starch (pore size of 1.5-2.5μm); the porous starch was mixed with fermentation liquid at a ratio of 1g:10mL, and embedding treatment was performed at a temperature of 4°C and a stirring rate of 100r / min for 16h, and then centrifuged at 5000×g / min for 15min, and the precipitate was collected as the embedding material; (2) resuspending the embedding material in physiological saline with a pH value of 8 to obtain a suspension, wherein the amount ratio of the embedding material to the physiological saline is 1 g:10 mL; adding a crosslinking agent (specifically a compound of sodium trimetaphosphate and sodium tripolyphosphate in a mass ratio of 99:1, and a mass ratio of the porous starch to the crosslinking agent of 1:0.15) to the suspension, performing a crosslinking treatment at a temperature of 55° C. and a mixing rate of 50 r / min for 2 h, and then centrifuging at 6000×g / min for 15 min, and collecting the precipitate as the crosslinked material; (3) placing the cross-linked material in a freeze dryer and freeze-drying it at -50°C for 48 hours to obtain Lactobacillus acidophilus microcapsules.

[0032] Example 3 (1) Use a fermentation tank to culture Clostridium butyricum at a high density (cross-section diameter of 1.0-2.0 μm) so that the total number of Clostridium butyricum in the resulting fermentation broth is 1×10 11CFU / mL; 1g starch (specifically corn starch), 400U α-amylase, 600U β-amylase and 800U saccharifying enzyme were mixed, and enzymatic hydrolysis was performed at 55°C for 16h to obtain porous starch (pore size of 2.0-3.0μm); the porous starch was mixed with fermentation liquid at a ratio of 1.5g:10mL, and embedding treatment was performed at a temperature of 10°C and a stirring rate of 120r / min for 24h, and then centrifuged at 6000×g / min for 15min, and the precipitate was collected as the embedding material; (2) resuspending the embedding material in physiological saline with a pH value of 10 to obtain a suspension, wherein the amount ratio of the embedding material to the physiological saline is 1 g:10 mL; adding a crosslinking agent (specifically a compound of sodium trimetaphosphate, sodium tripolyphosphate and sodium hexametaphosphate in a mass ratio of 90:5:5, and the mass ratio of the porous starch to the crosslinking agent is 1:0.25) to the suspension, performing a crosslinking treatment at a temperature of 40° C. and a vortexing rate of 100 r / min for 1 h, and then centrifuging at 4000×g / min for 15 min, and collecting the precipitate as the crosslinked material; (3) placing the cross-linked material in a freeze dryer and freeze-drying it at -40°C for 48 hours to obtain Clostridium butyricum microcapsules.

[0033] Test Example 1 1 g of each of the Escherichia coli microcapsules, Lactobacillus acidophilus microcapsules and Clostridium butyricum microcapsules prepared in Example 1, Example 2 and Example 3 were taken and their morphology and structure were observed using a scanning electron microscope.

[0034] Figure 2 This is the SEM image of the E. coli microcapsules prepared in Example 1. Figure 3 This is the SEM image of the Lactobacillus acidophilus microcapsules prepared in Example 2. Figure 4 This is a SEM image of the Clostridium butyricum microcapsules prepared in Example 3. The results show that Escherichia coli, Lactobacillus acidophilus, and Clostridium butyricum are dispersed inside and on the surface of porous starch granules, adjacent porous starches are aggregated, and some in-situ pores are shielded, which indicates that after the cross-linking treatment, starch aggregates with a porous structure are formed, thereby effectively encapsulating the target probiotics in the microcapsules and significantly enhancing their structural stability.

[0035] Test Example 2 Take 1g each of the Escherichia coli microcapsules, Lactobacillus acidophilus microcapsules, and Clostridium butyricum microcapsules prepared in Example 1, Example 2, and Example 3, and 1g each of the Escherichia coli embedding system, Lactobacillus acidophilus embedding system, and Clostridium butyricum embedding system obtained after freeze-drying the embedding materials prepared in step (1) of Example 1, step (1) of Example 2, and step (1) of Example 3, add 10mL of physiological saline, vortex oscillation, and use the PBS gradient dilution method to coat and count, and determine the embedding amount of each probiotic microcapsule and embedding system. The results are shown in Table 1. As shown in Table 1, the embedding amount of the Escherichia coli microcapsules is 1.56±0.08×10 9 CFU / g, which is significantly higher than the embedding capacity of the uncrosslinked system (1.33±0.16×10 9 CFU / g; the embedding capacity of Lactobacillus acidophilus microcapsules was 20.00±0.29×10 9 CFU / g, which is significantly higher than the embedding capacity of the uncrosslinked system 4.00±0.82 ×10 9 CFU / g; the embedding capacity of Clostridium butyricum microcapsules was 4.35±0.29×10 9 CFU / g, which is significantly higher than the embedding capacity of the uncross-linked system (2.88±0.33×10 9 CFU / g.

[0036] Table 1 Encapsulation amount of each probiotic microcapsule and encapsulation system in Examples 1 to 3

[0037] Test Example 3 Take 1 g each of the Escherichia coli microcapsules, Lactobacillus acidophilus microcapsules and Clostridium butyricum microcapsules prepared in Example 1, Example 2 and Example 3, and 1 g each of the Escherichia coli embedding system, Lactobacillus acidophilus embedding system and Clostridium butyricum embedding system obtained after freeze-drying the embedding materials prepared in step (1) of Example 1, step (1) of Example 2 and step (1) of Example 3, 1 g of a certain bacteria-contained Lactobacillus acidophilus tablet, and 1 g each of a certain peptide-home Lactobacillus acidophilus powder and Clostridium butyricum powder, and place them in a constant temperature incubator at 4°C (RH=25±5%). Take samples on the 0th, 7th, 14th, 28th, 60th and 90th day of storage, respectively, perform gradient dilution plate counts, detect the number of viable bacteria and calculate the survival rate, as shown in detail. Figure 5~Figure 7 As shown, different letters at the same time point represent significant differences, and the same letters represent no significant differences.

[0038] Figure 5 This is a graph showing the changes in storage survival rates of E. coli microcapsules and E. coli encapsulation systems. The results show that when stored at 4°C for 90 days, the survival rate of E. coli in the E. coli microcapsules prepared by cross-linking treatment was higher than 80%, which was significantly higher than the survival rate of the E. coli encapsulation system.

[0039] Figure 6 This is a storage survival rate change chart of Lactobacillus acidophilus microcapsules, Lactobacillus acidophilus embedding system, a certain bacteria-contained Lactobacillus acidophilus tablets and a certain peptide-home Lactobacillus acidophilus powder. The results show that when stored at 4°C for 90 days, the survival rate of Lactobacillus acidophilus in the Lactobacillus acidophilus microcapsules prepared by cross-linking treatment was higher than 80%, which was higher than the survival rate of Lactobacillus acidophilus in a certain peptide-home, higher than the survival rate of Lactobacillus acidophilus embedding system, and higher than the survival rate of Lactobacillus acidophilus in a certain bacteria-contained Lactobacillus acidophilus.

[0040] Figure 7 This is a graph showing the changes in the storage survival rate of Clostridium butyricum microcapsules, Clostridium butyricum encapsulation system and a certain peptide house. The results show that after storage at 4°C for 90 days, the survival rate of Clostridium butyricum in the Clostridium butyricum microcapsules prepared by cross-linking treatment was higher than 80%, which was higher than the survival rate of Clostridium butyricum in a certain peptide house, and significantly higher than the survival rate of Clostridium butyricum encapsulation system.

[0041] Test Example 4 Take 1g each of the Escherichia coli microcapsules, Lactobacillus acidophilus microcapsules and Clostridium butyricum microcapsules prepared in Example 1, Example 2 and Example 3, and 1g each of the Escherichia coli embedding system, Lactobacillus acidophilus embedding system and Clostridium butyricum embedding system obtained by freeze-drying the embedding materials prepared in step (1) of Example 1, step (1) of Example 2 and step (1) of Example 3, 1g each of a certain bacteria-contained Lactobacillus acidophilus tablet, and 1g each of a certain peptide-contained Lactobacillus acidophilus powder and Clostridium butyricum powder, and add 10mL of a simulated gastric flask preheated to 37°C. The cells were centrifuged at 6000 × g / min for 15 min, the precipitate was collected, and 10 mL of simulated intestinal fluid preheated to 37 °C was added for resuspending. The cells were then digested for 4 h. The cells were centrifuged at 6000 × g / min for 15 min, the precipitate was collected, and 10 mL of normal saline was added for resuspending. 1 mL of sample solution was drawn for gradient dilution. The number of viable bacteria was measured according to the plate count method, and the survival rate was calculated. The results are shown in Table 2.

[0042] As shown in Table 2, the survival rate of the E. coli microcapsules constructed by the in situ cross-linking method in the gastrointestinal environment was 12.19%, and the number of viable bacteria was 1.30±0.06×10 7 CFU / g, exceeding the threshold for effective action of probiotics in the intestine (≥10 6 CFU / g), which is much higher than the survival rate of E. coli embedding system (3.04%).

[0043] As shown in Table 2, the survival rate of Lactobacillus acidophilus microcapsules constructed by in-situ cross-linking of porous starch in the gastrointestinal environment was 5.63%, and the number of viable bacteria was 4.50±0.03×10 7CFU / g, exceeding the threshold for effective action of probiotics in the intestine (≥10 6 CFU / g), while the survival rates of Lactobacillus acidophilus embedding system, Junkang Lactobacillus acidophilus tablets and Peptide Home Lactobacillus acidophilus powder were all lower than 0.1%.

[0044] As shown in Table 2, the survival rate of Clostridium butyricum microcapsules constructed by in situ cross-linking of porous starch in the gastrointestinal environment was 4.80%, and the number of viable bacteria was 1.75±0.25×10 6 CFU / g, exceeding the threshold for effective action of probiotics in the intestine (≥10 6 CFU / g), while the survival rates of Clostridium butyricum embedding system and Clostridium butyricum powder of a certain peptide house were both lower than 0.1%.

[0045] Table 2 Simulated gastrointestinal digestion test results of each sample

[0046] Note: ND means not detected Test Example 5 This test example simulates the response of the colon amylase of the probiotic microcapsules. Specifically, 1 g of each of the Escherichia coli microcapsules, Lactobacillus acidophilus microcapsules and Clostridium butyricum microcapsules prepared in Example 1, Example 2 and Example 3 were taken, 10 mL of 5000 U / mL pullulanase 1×PBS solution preheated to 37°C was added, and enzymolysis was performed for 4 hours. Then, the mixture was centrifuged at 6000×g / min for 15 minutes, and the precipitate was collected. After freeze-drying, the morphology and structure were observed using a scanning electron microscope.

[0047] Figure 8 This is the SEM image of the E. coli microcapsule being hydrolyzed by colon amylase. Fig. 9 This is the SEM image of the microcapsules of Lactobacillus acidophilus enzymatically hydrolyzed by simulating colon amylase. Fig.10 The SEM image of the Clostridium butyricum microcapsule simulated by colon amylase enzymatic hydrolysis. The results show that the starch granules of Escherichia coli microcapsules, Lactobacillus acidophilus microcapsules and Clostridium butyricum microcapsules are depolymerized, dispersed and broken. This shows that the probiotic microcapsules prepared by the present invention can respond to amylase in the colon area, thereby achieving the colon-targeted delivery effect of probiotics.

[0048] Test Example 6 This test case simulates in vivo adhesion and colonization, as follows: Take the E. coli microcapsules prepared in Example 1 and prepare them into 1.25×10 6 CFU / mL of microcapsule solution, mice in the experimental group 1 were gavaged with 0.4 mL of microcapsule solution, mice in the control group 1 were gavaged with 0.4 mL of normal saline, and mice in the control group 2 were gavaged with 0.4 mL of E. coli concentration of 1.25×10 6After continuous gavage of 7 days with normal saline containing CFU, the mice were fasted for 24 hours, and the feces were collected. The abundance of Escherichia coli in the feces was detected by fluorescent quantitative PCR.

[0049] The Lactobacillus acidophilus microcapsules prepared in Example 2 were prepared with physiological saline to a concentration of 1.25×10 6 CFU / mL of microcapsule solution, mice in the experimental group 2 were gavaged with 0.4 mL of microcapsule solution, mice in the control group 3 were gavaged with 0.4 mL of normal saline, and mice in the control group 4 were gavaged with 0.4 mL of Lactobacillus acidophilus with a concentration of 1.25×10 6 CFU of physiological saline, and 5 mice in the control group were gavaged with 0.4 mL of a certain Junkang Lactobacillus acidophilus tablet with a Lactobacillus acidophilus concentration of 1.25×10 6 CFU of physiological saline, and 6 mice in the control group were gavaged with 0.4 mL of a peptide-made Lactobacillus acidophilus powder with a Lactobacillus acidophilus concentration of 1.25×10 6 After continuous gavage of 7 days with normal saline containing CFU, the mice were fasted for 24 hours, and the feces were collected. The abundance of Lactobacillus acidophilus in the feces was detected by fluorescent quantitative PCR.

[0050] The Clostridium butyricum microcapsules prepared in Example 3 were prepared with physiological saline to a concentration of 1.25×10 6 CFU / mL of microcapsule solution, 3 mice in the experimental group were gavaged with 0.4 mL of microcapsule solution, 7 mice in the control group were gavaged with 0.4 mL of normal saline, and 8 mice in the control group were gavaged with 0.4 mL of Clostridium butyricum. The concentration of the microcapsule solution was 1.25×10 6 CFU of physiological saline, and 9 mice in the control group were gavaged with 0.4 mL of a peptide-based Clostridium butyricum powder with a Clostridium butyricum concentration of 1.25×10 6 After continuous gavage of normal saline containing CFU for 7 days, the mice were fasted for 24 hours, and the feces were collected. The abundance of Clostridium butyricum in the feces was detected by fluorescent quantitative PCR.

[0051] The primer sequences involved in the above experiments are as follows: The forward primer for E. coli is: 5'-TGTTGCTTTGTTTAATTCYGATAAGC-3', SEQ ID NO.1; the reverse primer is: 5'-GGAATCGGAGTATAGTTTACACCAA-3', SEQ ID NO.2; The forward primer for Lactobacillus acidophilus is: 5'-TGCTGACGAATGGGAAAC-3', SEQ ID NO.3; the reverse primer is: 5'-TGCCAGGACTTGGGTAGAT-3', SEQ ID NO.4; The forward primer of Clostridium butyricum is: 5'-GTGCCGCCGCTAACGCATTAAGTAT-3', SEQ ID NO.5; the reverse primer is: 5'-ACCATGCACCACCTGTCTTCCTGCC-3', SEQ ID NO.6.

[0052] Fig.11 This is the in vivo adhesion and colonization test result of Escherichia coli microcapsules. It can be seen that the relative expression of Escherichia coli in the feces of mice in the experimental group 1 is significantly higher than that in the control group 1 and the control group 2, indicating that the Escherichia coli microcapsules can effectively protect and promote the colonization of Escherichia coli in the intestine.

[0053] Fig.12 This is the in vivo adhesion and colonization test result of Lactobacillus acidophilus microcapsules. It can be seen that the relative expression of Lactobacillus acidophilus in the feces of mice in the experimental group 2 was significantly higher than that in the control group 3, control group 4, control group 5 and control group 6, indicating that Lactobacillus acidophilus microcapsules can effectively protect and promote the colonization of Lactobacillus acidophilus in the intestine.

[0054] Fig.13 This is the in vivo adhesion and colonization test result of Clostridium butyricum microcapsules. It can be seen that the relative expression of Clostridium butyricum in the feces of mice in the experimental group 3 was significantly higher than that in the control group 7, control group 8 and control group 9, indicating that Clostridium butyricum microcapsules can effectively protect and promote the colonization of Clostridium butyricum in the intestine.

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing probiotic microcapsules, comprising the following steps: Providing an embedding material, the embedding material comprising porous starch and probiotics embedded in the pores of the porous starch and adhered to the surface of the porous starch; The embedding material, the crosslinking agent and the dispersing agent are mixed and crosslinked to obtain a crosslinked material; The cross-linked material is freeze-dried to obtain the probiotic microcapsule.

2. The preparation method according to claim 1, characterized in that: The cross-linking agent includes phosphate; the mass ratio of the cross-linking agent to the porous starch is 0.05~0.5:

1.

3. The preparation method according to claim 2, characterized in that: The phosphate includes one or more of trimetaphosphate, tripolyphosphate and hexametaphosphate.

4. The preparation method according to claim 1, characterized in that: The dispersant includes physiological saline, and the pH value of the dispersant is 8-10.

5. The preparation method according to any one of claims 1 to 4, characterized in that The temperature of the cross-linking treatment is 35-55° C., and the time is 0.5-5 h.

6. The preparation method according to claim 1, characterized in that: The probiotics include rod-shaped probiotics, and the cross-sectional diameter of the rod-shaped probiotics is 0.5-2.0 μm; the pore size of the porous starch is 1.0-3.0 μm.

7. The preparation method according to claim 1 or 6, characterized in that: The probiotics include one or more of the genera Lactobacillus, Bifidobacterium, Escherichia coli, Bacillus, Clostridium butyricum and Saccharomyces.

8. The preparation method according to claim 1 or 6, characterized in that: The porous starch is prepared by enzymatic hydrolysis of starch, wherein the enzymes used in the enzymatic hydrolysis include amylase and / or saccharifying enzyme; the temperature of the enzymatic hydrolysis is 30-60° C., and the time is 8-24 hours.

9. The probiotic microcapsule prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the probiotic microcapsule according to claim 9 as an additive for food, health products, feed or medicine.

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