Probiotic microcapsules and methods for their preparation

Abstract

The present application provides a probiotic microcapsule and a preparation method thereof. The probiotic microcapsule comprises a core material made of probiotics, a freeze-drying protective layer made of a freeze-drying protective agent and a wall material made of polysaccharides, wherein the freeze-drying protective layer is interposed between the core material and the wall material. The freeze-drying protective agent used in the probiotic microcapsule of the present application can reduce the damage to the bacterial body in the freezing and drying process through synergistic effect; and the wall material can form an acid-stable, dense and hard shell outside the bacterial body, greatly improving the stress resistance and stability of the probiotic in the production, storage and digestion processes. The probiotic microcapsule of the present application can be used for preparing food, cosmetics, medicines and feed.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a probiotic microcapsule and a preparation method thereof. BACKGROUND

[0002] Probiotics are a kind of active microorganisms that can produce beneficial effects on the health of the host. They can adhere to the intestinal tract of the host and inhibit the growth of pathogenic bacteria by competing for nutrients or producing organic acids, bacteriocins, hydrogen peroxide and other substances, thereby maintaining the balance of intestinal microecology and regulating intestinal function. With the enhancement of people's health awareness, probiotics have gradually entered a stage of rapid development in China and have been widely used in the fields of health food, functional food, cosmetics, medicine and the like. However, probiotics generally have the problems of easy inactivation under high temperature and high humidity, poor tolerance to acid, bile salts and free radicals, and short storage period.

[0003] In the industrial production of probiotic powder, in order to maximize the survival rate of bacterial cells, freeze-drying method is usually used for dehydration treatment of wet bacterial cells, and an appropriate amount of protective agent is generally added during freeze-drying process to reduce the death of bacterial cells. However, during transportation, sales, storage and other processes, the naked bacterial cells are still vulnerable to the influence of environment, temperature and humidity and are difficult to survive for a long time. Microcapsule technology is to use embedding materials that can form a film to wrap the core material in a semi-permeable or sealed capsule film with a diameter ranging from 1 to 5000 microns. The microcapsule wall material can act as a barrier to protect the core material from the influence of external environment, gastric acid, bile salts and other adverse factors, thereby improving the commercial value and economic value of the core material. Chinese Patent Application No. 202011579685.1 discloses a probiotic microcapsule and its preparation method and application, which comprises probiotic bacteria, natural fibers and a hydrophobic carrier, wherein the mass ratio of probiotic bacteria to natural fibers is 0.4:1 to 1.2:1, and the preparation method comprises the following steps: (1) dispersing probiotic bacteria in natural fibers; (2) dispersing the mixture obtained in step (1) in a hydrophobic carrier to form a microcapsule; (3) drying the microcapsule obtained in step (2). Defects: insoluble dietary fiber has no good gelation and film formation, the mechanical strength of the wall material is weak, the stability is poor, and the safety of the probiotic microcapsule obtained by this method needs to be confirmed. Chinese Patent Application No. 201410652096.X discloses a probiotic microcapsule added with prebiotics and a preparation method, wherein the core material comprises probiotic agents, prebiotics, traditional Chinese medicine extracts and skimmed milk powder, the wall material is a mixture of sodium alginate and protein wall material with a ratio of 2:1 and a final concentration of 3%-5%, and the protein wall material is soy protein or whey protein. The above-mentioned patent has the following defects: the raw material preparation process is strict and difficult to control, and it is difficult to apply to mass production; the core material formula is complex and easy to cause multiple damage to probiotic bacteria; in addition, the capsule shell made of sodium alginate has a large pore size, and the protein is difficult to compensate for its defects to form a hard shell. At present, the microcapsule technology in the industry generally has complex process and is not easy to scale up production, and during the preparation and freeze-drying of wet capsules, the naked probiotic bacteria directly contact with the wall material, which easily causes the inactivation of part of the probiotic bacteria, reduces the product value and increases the cost. Therefore, the above problems need to be solved. SUMMARY

[0004] The present application provides a probiotic microcapsule and a preparation method thereof, and the specific implementation manner is as follows:

[0005] A probiotic microcapsule, the microcapsule comprises a core material made of probiotic bacteria, a freeze-drying protective layer made of a freeze-drying protective agent and a wall material made of polysaccharide, wherein the freeze-drying protective layer is between the core material and the wall material;

[0006] Further, the diameter of the microcapsule is 4-300 microns;

[0007] Further, the freeze-drying protective layer is made of three freeze-drying protective agents of chlorophyllin, inulin and trehalose, and the wall material is composed of three polysaccharides of sodium alginate, pectin and chitosan;

[0008] Further, the mass ratio of chlorophyllin, inulin and trehalose is (2-6):2:1.

[0009] Further, the mass ratio of sodium alginate and pectin is (9-30):1.

[0010] Further, the probiotic bacteria are selected from one or more of Lactobacillus plantarum, Lactobacillus fermentum, Bifidobacterium, Lactobacillus casei, Lactobacillus acidophilus and Lactobacillus rhamnosus.

[0011] In addition, the application also provides a preparation method of the probiotic bacteria microcapsule. 8 The probiotic bacteria microcapsule is prepared by mixing a probiotic bacteria suspension with a live bacteria concentration of 10

[0012] Further, the preparation process of the freeze-drying protective agent solution comprises the following steps: mixing chlorophyllin, inulin and trehalose in a mass ratio of (2-6):2:1, adding 9-10 times of deionized water, and stirring uniformly to prepare a freeze-drying protective agent solution.

[0013] Further, the preparation process of the sodium alginate-pectin mixed solution comprises the following steps: mixing sodium alginate and pectin in a mass ratio of (9-30):1, adding 50-100 times of deionized water, and stirring at 35-45 DEG C for 20-40 min to obtain a sodium alginate-pectin mixed solution.

[0014] Further, the preparation process of the chitosan solution comprises the following steps: adding chitosan in 1% glacial acetic acid as a solvent to prepare a chitosan solution with a mass percentage of 0.8-1.2%, and stirring at 18-25 DEG C for 20-40 min.

[0015] Due to the above technical scheme, the application has the following beneficial technical effects:

[0016] 1. The present application provides a probiotic microcapsule, on the one hand, the microcapsule prepared by synergistic effect of orobanchin as a natural active substance and probiotics has the effect of reducing uric acid, on the other hand, orobanchin, inulin and trehalose as freeze-drying protectants can improve the viable cell count during the freeze-drying process of the microcapsule, significantly improve the cell death caused by the rupture of cell membrane and excessive water loss of the bacteria due to freeze-drying;

[0017] 2. In the present application, sodium alginate, pectin and chitosan synergistically form a dense polyelectrolyte complex microcapsule wall to protect the bacteria;

[0018] 3. The present application also considers the weak alkaline nature of orobanchin, which acts as a freeze-drying protective agent between the core material and the wall material, and can neutralize part of the gastric acid when the wall material of the probiotic microcapsule is decomposed by acid, thereby improving the tolerance of the bacteria to gastric acid and achieving a "multi-layer" protection effect on the bacteria. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0020] A probiotic microcapsule, the microcapsule comprising a core material made of probiotics, a freeze-drying protective layer made of freeze-drying protectants and a wall material made of polysaccharides, wherein the freeze-drying protective layer is between the core material and the wall material. The diameter of the microcapsule is 4-300 μm. The probiotic microcapsule adds a freeze-drying protective layer between the core material and the wall material, which enhances the stress resistance and survival rate of probiotics during production, storage and digestion, etc.

[0021] Specifically, the freeze-drying protective layer is made of orobanchin, inulin and trehalose, and the wall material is composed of sodium alginate, pectin and chitosan. Orobanchin has multiple effects in the microcapsule, on the one hand, orobanchin as a natural active substance has the effect of reducing uric acid in synergistic effect with probiotics, on the other hand, orobanchin, inulin and trehalose as freeze-drying protectants can improve the viable cell count during the freeze-drying process of the microcapsule, significantly improve the cell death caused by the rupture of cell membrane and excessive water loss of the bacteria due to freeze-drying. The present application makes full use of the properties of orobanchin to make the prepared microcapsule have an important effect of reducing uric acid, and the freeze-drying survival rate, viable cell count and storage stability of the prepared microcapsule are higher.

[0022] As a green plant nutrient, wheat green pigment is rich in protein and active substances such as polysaccharides, plant flavones and polyphenols. Studies have shown that protein can form a protective film outside the bacterial cell, thereby wrapping the bacterial cell; the hydrogen bond of polysaccharides can replace water to combine with the protein on the bacterial cell membrane, forming a more stable spatial structure; in addition, the plant flavones and polyphenols in wheat green pigment have antioxidant effects, which can block the attack of oxygen and free radicals on the bacterial cell, increase the stress resistance of probiotics, and prolong the survival rate of probiotics during the shelf life. Inulin is a water-soluble dietary fiber and a commonly used prebiotic, which can form a glassy protective shell around the cell membrane. Fuculose has strong water-holding capacity and can enter the cell to prevent the excessive loss of intracellular bound water, thereby protecting the structure and function of biological macromolecules.

[0023] Sodium alginate, pectin and chitosan synergistically form a dense shell to protect the bacterial cell. Sodium alginate is a natural polysaccharide and has become one of the most commonly used materials for microcapsule wall materials due to its good gelation and thermal stability. However, sodium alginate is sensitive to acid and has porosity, and when passing through the stomach, gastric acid can easily diffuse into the capsule interior to attack the core material. Therefore, the stress resistance of the microcapsule is reduced under gastric juice conditions, which limits its wide application in probiotic microcapsules. Pectin widely exists in plant cell walls of fruits and vegetables and has good emulsifying and film-forming properties and is stable to gastric acid. The use of pectin and sodium alginate as wall materials can significantly improve the gastric acid resistance of the microcapsule. Chitosan can form a polyelectrolyte complex with sodium alginate and pectin through electrostatic interaction, further coating sodium alginate and pectin to form a hard and dense microcapsule shell.

[0024] Example 1

[0025] Preparation method of probiotic microcapsules:

[0026] (1) Preparation of core material: under sterile conditions, the low-temperature preserved Lactobacillus plantarum and Lactobacillus casei strains were inoculated and activated, then inoculated in MRS liquid medium and cultured for 48 h, centrifuged at 4℃ and 5000 rmp for 15 min, and the bacterial cells were resuspended with sterilized physiological saline to obtain a bacterial suspension;

[0027] (2) Preparation of freeze-drying protective agent solution: wheat green pigment, inulin and fuculose were mixed in a mass ratio of 4:2:1, 9 times of deionized water was added, and the mixture was stirred uniformly to prepare a freeze-drying protective agent solution, which was prepared; specifically, the wheat green pigment was prepared by water extraction from barley seedlings and highland barley seedlings;

[0028] (3) Preparation of wall material: preparation of sodium alginate-pectin mixed solution: sodium alginate and pectin were mixed in a mass ratio of 20:1, 70 times of deionized water was added, and the mixture was stirred at 40℃ and 300 r / min for 30 min to obtain a sodium alginate-pectin mixed solution.

[0029] Chitosan solution preparation: 1% glacial acetic acid as solvent, add chitosan, prepared into a chitosan solution with a mass percentage of 1%, under the condition of 20℃, 250r / min, stirring for 30min;

[0030] (4) Microcapsule preparation: mix the probiotic bacteria suspension and the freeze-drying protective agent mixture in a mass ratio of 1:1 to obtain mixture A, then mix mixture A with the sodium alginate pectin mixture in a mass ratio of 1:2 to obtain mixture B, stir at 10℃ for 40min; use a 1mL syringe to add mixture B dropwise into the chitosan solution, stir at room temperature for 40min, place at -4℃ for 1h, centrifuge, and collect the wet capsules; pre-freeze the wet capsules at -20℃ for 24h, and use vacuum freeze-drying to prepare probiotic microcapsules.

[0031] Example 2

[0032] Preparation method of probiotic microcapsules:

[0033] (1) Preparation of core material: under sterile operation, inoculate the low-temperature preserved Lactobacillus fermentum strain, then inoculate in MRS liquid medium and culture for 48h, centrifuge at 4℃, 5000rmp for 15min, obtain the bacterial body, resuspend the bacterial body with sterilized physiological saline to obtain a bacterial suspension;

[0034] (2) Preparation of freeze-drying protective agent: mix wheat green pigment, inulin and trehalose in a mass ratio of 2:2:1, add 10 times deionized water, stir uniformly to prepare a freeze-drying protective agent solution, and reserve; specifically, the wheat green pigment is prepared by water extraction from buckwheat seedlings;

[0035] (3) Preparation of wall material: preparation of sodium alginate pectin mixture: mix sodium alginate and pectin in a mass ratio of 9:1, add 80 times deionized water, stir at 45℃, 250r / min for 20min, and obtain the sodium alginate pectin mixture;

[0036] Chitosan solution preparation: 1% glacial acetic acid as solvent, add chitosan, prepared into a chitosan solution with a mass percentage of 0.8%, under the condition of 25℃, 250r / min, stirring for 20min;

[0037] (4) Microcapsule preparation: the probiotic bacteria suspension and the freeze-drying protectant mixture were mixed at a mass ratio of 1:2 to obtain mixture A, mixture A was mixed with the sodium alginate pectin mixture at a mass ratio of 1:2 to obtain mixture B, and stirring was performed at 15°C for 30 min; mixture B was added dropwise into the chitosan solution using a 1 mL syringe, stirring was performed at room temperature for 30 min, and solidification was performed at -6°C for 1 h, centrifugation was performed, and wet capsules were collected; the wet capsules were pre-frozen at -20°C for 24 h, and vacuum freeze-drying was performed to obtain the probiotic bacteria microcapsules.

[0038] Example 3

[0039] Preparation method of probiotic bacteria microcapsules:

[0040] (1) Preparation of core material: under sterile operation, the low-temperature preserved Bifidobacterium strain was inoculated and activated, then was inoculated in MRS liquid medium and cultured for 48 h, centrifugation was performed at 4°C and 5000 rpm for 15 min, the bacterial body was obtained, and the bacterial body was resuspended with sterilized normal saline to obtain a bacterial suspension;

[0041] (2) Preparation of freeze-drying protectant: deionized water was used as a solvent, and malt green, inulin and trehalose were mixed at a mass ratio of 6:2:1, 9 times of deionized water was added, and stirring was performed to obtain a freeze-drying protectant solution, which was prepared for use; specifically, the malt green was prepared from barley seedlings by water extraction;

[0042] (3) Preparation of wall material: preparation of sodium alginate pectin mixture: sodium alginate and pectin were mixed at a mass ratio of 30:1, 60 times of deionized water was added, and stirring was performed at 35°C and 300 r / min for 40 min to obtain the sodium alginate pectin mixture;

[0043] Preparation of chitosan solution: 1% glacial acetic acid was used as a solvent, chitosan was added, and a chitosan solution with a mass percentage of 1.2% was prepared, stirring was performed at 18°C and 250 r / min for 40 min;

[0044] (4) Microcapsule preparation: the probiotic bacteria suspension and the freeze-drying protectant mixture were mixed at a mass ratio of 1:2 to obtain mixture A, mixture A was mixed with the sodium alginate pectin mixture at a mass ratio of 1:2 to obtain mixture B, and stirring was performed at 10°C for 40 min; mixture B was added dropwise into the chitosan solution using a 1 mL syringe, stirring was performed at room temperature for 50 min, and solidification was performed at -5°C for 1 h, centrifugation was performed, and wet capsules were collected; the wet capsules were pre-frozen at -20°C for 24 h, and vacuum freeze-drying was performed to obtain the probiotic bacteria microcapsules.

[0045] Comparative Example 1

[0046] Compared with Example 1, the freeze-drying protective agent was not added to the malt green pigment, and the sodium alginate pectin solution was prepared by mixing sodium alginate and pectin at a mass ratio of 5:1, adding 40 times water and stirring, and the other parts were the same.

[0047] Comparative Example 2

[0048] Compared with Example 1, the malt green pigment was prepared from highland barley seedlings, the mass ratio of malt green pigment, inulin and trehalose in the freeze-drying protective agent was 2:1:1, the wall material did not add pectin, and the other parts were the same.

[0049] Comparative Example 3

[0050] Compared with Example 1, the freeze-drying protective agent was not added to the probiotic microcapsule, and the other parts were the same.

[0051] The probiotic microcapsules prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to embedding rate and survival rate test, in-vitro gastric acid resistance test, bile salt resistance test and storage resistance test, respectively.

[0052] 1. Embedding rate and survival rate test method:

[0053] Embedding rate and survival rate test: wet (dry) probiotic microcapsules were added to 3% sodium citrate solution, mixed uniformly and placed in a constant temperature shaker, the speed and temperature were set at 180 r / min and 37±1℃, and shaken for 60 min to make the probiotic microcapsules crack. After cracking, viable bacteria count was performed, and the calculation formula was as follows:

[0054] Embedding rate / % = P1 / P0 x 100

[0055] Freeze-drying survival rate / % = P2 / P1 x 100

[0056] Viable bacteria rate / % = P2 / P0 x 100

[0057] In the formula: P0 is the total number of viable bacteria (CFU) of probiotic bacterial suspension; P1 is the total number of viable bacteria (CFU) of wet capsules; P2 is the total number of viable bacteria (CFU) of dry capsules.

[0058] 2. In-vitro gastric acid resistance test:

[0059] Preparation of artificial gastric juice: first prepare 50 mL of 0.1 mlo / L hydrochloric acid solution, take 5 g of pepsin and add to 8.2 mL of 0.1 mlo / L hydrochloric acid solution, add appropriate amount of deionized water and stir uniformly, and adjust the volume to 500 mL in a volumetric flask, adjust the pH to 2.0, and finally filter sterilize with a 0.22 μm filter membrane, store in a 4℃ refrigerator, and use.

[0060] Gastric acid resistance test: 1.0 g of microcapsules prepared in Examples 1-3 and Comparative Examples 1-3 were weighed into 100 mL conical flasks, 50 mL of artificial gastric juice was added, and after mixing, the mixture was placed in a constant temperature shaker, with a speed and temperature setting of 180 r / min, 37±1℃, and shaken for 100 min. The viable bacteria count in each sample was determined at 0, 20, 30, 35, 40, 45, 50, 55, 60 min, to determine the complete release time and gastric acid resistance of the probiotic bacteria.

[0061] 3. Determination method of bile salt resistance test:

[0062] Preparation of bile salt solution: 0.5% bile salt solution was prepared using sterilized physiological saline. Bile salt resistance test: A certain amount of microcapsules prepared in Examples 1-3 and Comparative Examples 1-3 were weighed and dissolved in 0.5% bile salt solution to prepare a 10% microcapsule solution, which was then placed in a 37℃ constant temperature incubator for 4h. The culture solution was then removed and the viable bacteria count was determined to determine the bile salt resistance.

[0063] 4. Storage resistance test:

[0064] The microcapsules prepared in Examples 1-3 and Comparative Examples 1-3 were placed in a 37℃ constant temperature incubator for 30 days. At the 30th day, a certain amount of sample was removed and the viable bacteria count was determined, and the survival rate of the probiotic bacteria under 37℃ storage conditions was calculated. The total number of lactic acid bacteria was determined according to the National Food Safety Standard GB 4789.35-2016.

[0065] Table 1 Summary of test results of probiotic microcapsules prepared in Examples 1-3 and Comparative Examples 1-3

[0066]

[0067]

[0068] As can be seen from the data in Table 1 above, the probiotic microcapsules with added freeze-drying protectants have higher embedding rate, freeze-drying survival rate, viable bacteria rate, acid and bile salt resistance, and storage stability. First, after 30 days of storage at 37℃, the viable bacteria count of the probiotic microcapsules with added freeze-drying protectants increased, which was due to the fact that the barley green and inulin in the freeze-drying protectants could promote the proliferation of probiotic bacteria. Second, the synergistic effect of sodium alginate, pectin and chitosan better protected the bacterial cells, and the barley green, inulin and trehalose as freeze-drying protectants could improve the viable bacteria load and significantly improve the death of bacterial cells caused by freeze-drying. Finally, the shell wall made of sodium alginate, pectin and chitosan could better delay the release of the microcapsules in gastric acid, and the complex freeze-drying protectants could improve the acid and bile salt resistance of the probiotic bacteria, effectively protecting the viable bacteria to reach the intestinal tract.

[0069] It is worth noting that the malt chlorophyll plays a very important role in the freeze-drying protectant, compared with the microcapsules without malt chlorophyll such as Comparative Example 1 and Comparative Example 3, the freeze-drying survival rate and the viable bacteria rate of the probiotic bacteria are the highest, and the difference is 30% and 35% respectively, which shows that the probiotic bacteria microcapsules with malt chlorophyll have the effect of reducing uric acid, in addition, the freeze-drying survival rate, viable bacteria rate, acid and bile salt resistance and storage stability of the probiotic bacteria microcapsules are higher.

[0070] In addition, it should be understood that, although the present specification is described in terms of embodiments, not every embodiment contains only one independent technical solution, and the description of the specification is only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can be combined appropriately to form other embodiments that those skilled in the art can understand.

Claims

1. A probiotic microcapsule, characterized in that, The microcapsule comprises a core material made of probiotics, a freeze-drying protective layer made of a freeze-drying protectant, and a wall material made of polysaccharides, wherein the freeze-drying protective layer is located between the core material and the wall material; the freeze-drying protective layer is made of three freeze-drying protectants: wheatgrass, inulin, and trehalose, and the wall material is composed of three polysaccharides: sodium alginate, pectin, and chitosan; the mass ratio of wheatgrass, inulin, and trehalose is (2-6):2:

1.

2. The probiotic microcapsule according to claim 1, characterized in that, The diameter of the microcapsules is 4-300 μm.

3. The probiotic microcapsule according to claim 1, characterized in that, The mass ratio of sodium alginate to pectin is (9-30):

1.

4. The probiotic microcapsule according to claim 1, characterized in that, The probiotics are one or more of the following: Lactobacillus plantarum, Lactobacillus fermentum, Bifidobacterium, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus rhamnosus.

5. A method for preparing probiotic microcapsules, characterized in that, The concentration of live bacteria reached 10. 8 A mixture of CFU / g probiotic suspension and lyophilization protectant solution was prepared by mixing in a mass ratio of 1:(1-2) to obtain mixture A. Mixture A was then mixed with sodium alginate pectin solution in a mass ratio of 1:2 to obtain mixture B. Mixture B was added dropwise to chitosan solution and stirred at room temperature for 30-50 minutes. After standing for 1-2 hours, the mixture was centrifuged to obtain wet capsules. The wet capsules were then dried to obtain probiotic microcapsules. The preparation process of the freeze-drying protectant solution includes: mixing wheatgrass, inulin and trehalose in a mass ratio of (2-6):2:1, adding 9-10 times the amount of deionized water, and stirring evenly to prepare the freeze-drying protectant solution.

6. The method for preparing probiotic microcapsules according to claim 5, characterized in that, The preparation process of the sodium alginate and pectin mixture includes: mixing sodium alginate and pectin at a mass ratio of (9-30):1, adding 50-100 times the amount of deionized water, and stirring at 35-45℃ for 20-40 min to obtain the sodium alginate and pectin mixture solution.

7. The method for preparing probiotic microcapsules according to claim 5, characterized in that, The chitosan solution preparation process includes: adding chitosan to 1% glacial acetic acid as solvent to prepare a chitosan solution with a mass percentage of 0.8-1.2%, and stirring for 20-40 minutes at 18-25℃.

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