Delivery method for improving survival rate of probiotics based on double emulsions and complex coacervation

Abstract

The invention discloses a targeted delivery method for improving the survival rate of probiotics based on double emulsions and complex coacervation, and belongs to the technical field of biological delivery and slow release. The method comprises the following steps: (1) preparing an internal water phase, namely dissolving mannitol, dwarf violet polysaccharide and gelatin in water, and mixing with probiotics to prepare a bacterial suspension as the internal water phase; (2) preparing an oil phase: adding PGPR into vitamin A oil to obtain the oil phase; (3) preparing an outer water phase: adding gelatin and sucrose ester into water to obtain the outer water phase; (4) preparing a probiotic carrier based on double emulsions: mixing the oil phase with the inner water phase, and then mixing with the outer water phase to obtain the probiotic double-emulsion carrier; and (5) preparing the probiotic microcapsules based on complex coacervation: adding a curing agent into the probiotic double-emulsion carrier, stirring, centrifuging, washing and freeze-drying to obtain the probiotic microcapsules. According to the double-layer emulsion microcapsule, the water environment is isolated through the oil phase, the structural stability is enhanced, gastric acid erosion can be delayed, and targeted slow release of the intestinal tract can be achieved.

Description

Technical Field

[0001] The invention relates to a probiotic targeted delivery method for improving the survival rate of probiotics based on double emulsion and complex coacervation, and belongs to the technical field of biological delivery and sustained release. Background Art

[0002] Over the past two decades, consumers have increasingly prioritized healthier foods and those with greater nutritional value. In light of this new reality, functional foods have gained prominence, representing one of the most dynamic and innovative categories in the food industry. Among these, probiotic products have seen increasing market acceptance year after year, becoming a booming industry with significant implications for promoting human health and improving quality of life. As researchers delve deeper into the gut microbiome and the importance of imbalance in various diseases, we've discovered that probiotic supplementation can help restore balance in the gut ecosystem and enhance immunity.

[0003] The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) define probiotics as living microorganisms that, when consumed in sufficient quantities, confer health benefits on the host. However, there is currently no consensus on what constitutes a "sufficient quantity." It is generally accepted that a minimum of 6 log CFU / g or 6 log CFU / ml of probiotics is required to exert their beneficial effects in the host's intestines. However, during processing, storage, and digestion, probiotics are affected by their own viability and adverse external environments, often resulting in a decrease in the number and activity of probiotics that ultimately colonize the human intestine, or even their disappearance, making it difficult to achieve the desired effect. To ensure that probiotics maintain their numbers and viability before reaching the human intestine to regulate the body's microecological balance, enhance their inhibitory effects on harmful microorganisms, promote nutrient digestion and absorption, and improve the body's metabolic capacity, it is crucial to select appropriate carrier materials to encapsulate the probiotics and enhance their survival rate.

[0004] The encapsulation rate and survival rate directly reflect the effectiveness of probiotic encapsulation. The survival rate of probiotics in the stomach and their release in the intestines are directly related to their survival and colonization in the intestine. Furthermore, the slow release of probiotic microcapsules in the intestine also facilitates their colonization. The adhesion and colonization of probiotics in the intestines form a protective layer that prevents harmful bacteria and toxins from invading the intestinal mucosa, thereby protecting intestinal health. Furthermore, the colonization of probiotics in the intestines can promote the development and function of the immune system, enhance the barrier function of the intestinal mucosa, and improve the body's resistance. Probiotics adhered to the intestines help break down nutrients in food, promote their absorption and utilization, and increase nutrient utilization. Therefore, improving the survival rate of probiotics transported to the intestine during digestion is crucial for maintaining intestinal health and promoting overall health.

[0005] The unique water solubility of probiotics and common wall materials makes single-layer emulsion encapsulation difficult, while the survival and activity of probiotics are susceptible to environmental factors such as pH, temperature, and oxygen concentration. Currently, mainstream matrix-loaded microcapsules are typically composed of polymers or gels, but their inner walls offer limited barrier capabilities against the external environment. Rapid degradation of the matrix material in the presence of gastric acid can lead to premature release of probiotics, while temperature fluctuations can also cause the polymer structure to deform or dissolve. The pore size and material properties of the matrix microcapsules also make release rate control difficult. Excessively small pores can trap probiotics, while excessively large pores can lead to premature release. Notably, traditional microcapsules lack targeted release design and rely on passive diffusion or physical excretion for distribution, making them difficult to adapt to the pH and enzymatic environments of different intestinal regions. Microcapsules made of a single material cannot withstand intestinal mechanical movement and the action of digestive enzymes, easily rupturing and resulting in uneven release of the strain. Simple matrix dissolution release mechanisms, however, prevent precise control of release rate and dosage, shortening the duration of viable bacteria. Although current research has improved targeting through nano-coatings and pH-responsive materials, it still faces technical bottlenecks such as insufficient verification of material biocompatibility and poor process stability of multi-layer structures.

[0006] Some bacterial strains have specific requirements for delivery systems. Negatively charged bacteria (such as Lactobacillus plantarum) easily bind to positively charged wall materials, and acid-producing bacteria (such as Lactobacillus plantarum) may change the local pH, requiring the pH buffering capacity of the wall material or the addition of cell protectants to maintain stability. At the same time, some strains (such as Bacillus subtilis) secrete multiple extracellular enzymes that can degrade the polysaccharide materials they directly contact. It is worth considering whether the encapsulating wall material can simultaneously achieve high encapsulation efficiency of multiple probiotics after embedding, high survival rate after storage and exposure to gastric fluid, sustained release effect in the intestine, and high adhesion and colonization rates. At the same time, achieving good encapsulation effect for multiple probiotic strains is very difficult. Summary of the Invention

[0007] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a targeted delivery method for improving the survival rate of probiotics based on double emulsions and complex coacervation. Another purpose of the present invention is to provide a method for improving the survival rate of core material strains by using double emulsions and microcapsules prepared by the above preparation method. Yet another purpose of the present invention is to provide the application of the above-mentioned probiotic emulsions and microcapsules based on double emulsions and complex coacervation. The present invention isolates the water environment through the oil phase through double-layer emulsion microcapsules, enhances structural stability, delays gastric acid erosion and achieves intestinal targeted sustained release. The oil phase in the double-layer emulsion structure provided by the present invention can directly serve as a physical barrier to limit the migration of strains in the inner water phase to the outside world, avoid such destabilizing factors, and help prevent external pollution factors from contaminating and poisoning the inner water phase strains.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] The present invention provides a delivery method for improving the survival rate of probiotics based on double emulsion and complex coagulation, comprising the following steps:

[0010] (1) preparing the inner aqueous phase: dissolving mannitol, dwarf yew polysaccharide, and gelatin in water as a composite freeze-drying protective agent; mixing the probiotics with the composite freeze-drying protective agent to prepare a bacterial suspension as the inner aqueous phase;

[0011] (2) Preparation of oil phase: Add polyglycerol ricinoleate (PGPR) to vitamin A oil and dissolve for 20-40 min to obtain the oil phase;

[0012] (3) Preparing the external aqueous phase: hydrating gelatin and sucrose ester at 40-50°C for 20-40 min, centrifuging while hot to remove impurities, and adjusting the pH to 3-4 to obtain the external aqueous phase;

[0013] (4) Preparation of a probiotic carrier based on a double emulsion: at room temperature, uniformly mixing the oil phase and the inner aqueous phase to obtain a primary emulsion, and uniformly mixing the primary emulsion and the outer aqueous phase to obtain a secondary emulsion, i.e., a probiotic double emulsion carrier;

[0014] (5) Preparation of probiotic microcapsules based on complex coacervation: adding a curing agent to a probiotic double emulsion carrier at 50-60°C, stirring, centrifuging, and washing to obtain wet probiotic microcapsules;

[0015] (6) pre-freezing the wet microcapsules and then freeze-drying them to obtain probiotic microcapsule powder.

[0016] In one embodiment, in step (1), the probiotics are one or more of Bacillus megaterium, Bacillus velezensis, Bacillus coagulans, Lactobacillus rhamnosus, Bacillus amyloliquefaciens, and Lactobacillus plantarum, and the concentration of the probiotics in the bacterial suspension is 8 to 10 log CFU / 100 ml, preferably 8.74 log CFU / 100 ml.

[0017] In one embodiment, the probiotics are obtained by centrifuging a probiotic culture, or by centrifuging and washing a probiotic culture; preferably, the probiotic culture is continuously subcultured two or more times, and then the probiotic culture is centrifuged at 4-20°C and 5000-8000 rpm, and the supernatant is removed, or the probiotic sludge is obtained by washing three times with a physiological saline solution (0.9%) after centrifugation and removing the supernatant. The obtained probiotic sludge is suspended in a composite freeze-drying protective agent to prepare a bacterial suspension as the internal aqueous phase.

[0018] In one embodiment, in step (1), the concentration of mannitol in the composite lyoprotectant is 3-4 wt%, the concentration of dwarf taxane polysaccharide is 4-5 wt%, and the concentration of gelatin is 2-3 wt%.

[0019] Preferably, in step (1), the concentration of mannitol in the composite lyophilization protectant is 3.0wt%, the concentration of dwarf taxane polysaccharide is 4.4wt%, and the concentration of gelatin is 2.0wt%, so that the survival rate of probiotics during the freeze-drying process reaches more than 80%, which is more than 60% higher than that of naked bacteria.

[0020] In one embodiment, in step (2), the oil phase is a mixed oil phase of PGPR and vitamin A oil, the purity of the vitamin A oil is 99%, and the ratio of PGPR to vitamin A oil is 4-4.5 g:100 ml.

[0021] Preferably, in step (2), the ratio of PGPR to vitamin A oil in the mixed oil phase is 4g:100ml.

[0022] In one embodiment, in step (3), the mass concentration of gelatin in the external aqueous phase is 1-2 wt%, and the mass concentration of the sucrose ester is 3-5 wt%.

[0023] In one embodiment, in step (4), the rotation speed for forming the primary emulsion is 900-1200 rpm, the emulsification time is 40-80 min, and the volume ratio of the oil phase to the internal water phase is 6:1-8:1.

[0024] Preferably, in step (4), the rotation speed for forming the primary emulsion is 1227.96 rpm, the emulsification time is 61.94 min, and the volume ratio of the oil phase to the internal water phase is 7.12:1.

[0025] In one embodiment, in step (4), the rotation speed for forming the secondary emulsion is 600-1200 rpm, the emulsification time is 100-140 min, and the volume ratio of the primary emulsion to the external aqueous phase is 1:3-1:6, preferably 1:4-1:5.

[0026] Preferably, in step (4), the rotation speed for forming the secondary emulsion is 900.04 rpm, the emulsification time is 121.06 min, and the volume ratio of the primary emulsion to the external aqueous phase is 4.62:1.

[0027] In one embodiment, in step (5), the curing agent is a 1-2 wt% carboxymethyl cellulose (CMC) dispersion, and the addition amount of the CMC dispersion is 20% to 25% of the total mass of the external aqueous phase and the carboxymethyl cellulose-water dispersion.

[0028] Preferably, in step (5), the curing agent is a 1 wt% CMC solution, and the amount of the CMC dispersion added is 22.2% of the external aqueous phase; the stirring speed is 600-1200 rpm, and the time is 60-80 min.

[0029] In one embodiment, in step (5), before adding the curing agent, the pH of the system is maintained at 3-4.

[0030] Preferably, before adding the curing agent, the pH of the system is maintained at 3.5.

[0031] In one embodiment, in step (5), the stirring speed is 600 to 1200 rpm, and the stirring time is 50 to 80 min.

[0032] Preferably, in step (5), the stirring speed is 900 rpm and the stirring time is 60 min.

[0033] In one embodiment, in step (6), the wet microcapsules are treated by pre-freezing the wet microcapsules at -80°C for 12 to 36 hours (preferably 24 hours) and then drying them using a freeze-drying method to obtain a probiotic microcapsule powder. The freeze-drying method has the following drying conditions: a temperature of -70°C and a drying time of 24 to 48 hours.

[0034] The present invention also provides probiotic microcapsules prepared by the above method.

[0035] The present invention also provides the use of the above-mentioned probiotic microcapsules in the preparation of gastric acid-resistant oral products, wherein the products include foods, medicines, and health products; and the health products include probiotic preparations.

[0036] The present invention uses a gelatin-carboxymethyl cellulose copolymer as the wall material. Gelatin, as a common collagen hydrolyzed protein, has good biocompatibility, and neither the main body nor the hydrolyzed product will cause human rejection reactions. Carboxymethyl cellulose is a cellulose derivative obtained through chemical modification, with a high carboxyl content, which can form a copolymer with the amino group in gelatin. This type of copolymer has good controlled release ability. The acidic environment of the stomach will promote cross-linking and protect the core material from gastric acid erosion. When the carrier enters the duodenum, the alkaline environment will cause both gelatin and carboxymethyl cellulose to be negatively charged, repelling each other and promoting disintegration. The primary emulsion will promote the intestinal wall cilia to capture the carried probiotics, achieving targeted delivery.

[0037] Double-layer emulsion-loaded bacterial microcapsules provide a physical barrier by forming an outer layer and an inner layer structure, which can effectively isolate probiotics from direct contact with adverse external environments (such as acids and enzymes). This barrier can mitigate the impact of external stimuli on probiotics, thereby improving their survival rate. Compared with traditional single-layer emulsions, this structure effectively isolates the inner aqueous phase from direct contact with the external environment, preventing both contamination of the bacterial suspension in the inner aqueous phase by the external environment and the escape of the bacterial suspension. It has advantages in structural stability and resistance to environmental pressure. At the same time, we use sugars, alcohols and proteins as inner aqueous phase carriers instead of physiological saline, which can form a protective film for the strains, mitigate the negative impact of the external environment, enhance the fluidity of the cell membrane, improve the ability to adapt to environmental changes, and protect the cell walls and cell membranes of microorganisms. At the same time, it can absorb water and form hydrogen bonds, increasing the moisture content of the inner aqueous phase, providing energy for microorganisms, keeping the microorganisms hydrated, and helping to maintain their metabolic activities.

[0038] The present invention demonstrates that the probiotic emulsions and microcapsules based on double emulsions and complex coagulation have superior encapsulation efficiency and survival rate compared to delivery systems using only complex coagulation and without protective agent screening, demonstrating the superiority of the double emulsion system and composite protective agent. Furthermore, the bacteria-loaded microcapsules produced by combining the double emulsion system with complex coagulation technology not only have superior intestinal targeting and storage stability compared to single complex coagulation bacteria-loaded microcapsules, but also possess superior storage stability, which is beneficial for the storage and application of probiotic preparations. The composite protective agent, double emulsion system, and complex coagulation method are combined to enhance the survival rate and targeted delivery characteristics of the core material strain.

[0039] Beneficial effects

[0040] (1) The present invention optimizes the core-shell structure of the probiotic-carrying microcapsules by using a double emulsion system mediated by PGPR and sucrose esters and a copolymer of gelatin and CMC at low pH as the wall material of the probiotic-carrying microcapsules. The encapsulation efficiency is increased by about 20% compared with that of single complex coacervate microcapsules. The intestinal-targeted controlled release characteristics of the microcapsules containing Bacillus velezensis prepared by the present invention are improved by about 20% compared with single complex coacervate microcapsules and by about 60% compared with naked bacteria. The storage stability is also improved by about 10% compared with that of single complex coacervate microcapsules and by about 60% compared with naked bacteria.

[0041] (2) The present invention optimizes the composite lyophilization protectant through RSM. The optimized composite lyophilization protectant is about 20% higher than the single lyophilization protectant and about 60% higher than the naked bacteria.

[0042] (3) In simulated in vitro digestion, the release curve can be fitted with the Hixson-Crowell model. The survival rate in artificial gastric fluid is always greater than 85%, which is about 200% higher than that of single complex coacervate microcapsules and about 60% higher than that of naked bacteria. Sustained release is achieved within 6 hours in artificial intestinal fluid, which helps the probiotics to colonize in the intestine.

[0043] (4) The composite freeze-drying protective agent and wall material used in the present invention are both natural products derived from plants or microorganisms, and have good biocompatibility. The conditions during the microgelation process are mild and do not involve high temperature, high-speed stirring, high-pressure homogenization and other process flows. They can effectively protect the activity of probiotics. Compared with high-pressure homogenization emulsification and gelation, the preparation method of the bacteria-loaded microcapsules of the present invention increases the activity of probiotics by about 50%. In addition, the process steps of the present invention are relatively simple, and the raw materials are cheap and easily available, which is beneficial for production enterprises to control production costs and improve production efficiency.

[0044] In summary, the present invention uses mannitol, dwarf paclitaxel polysaccharide, and gelatin as composite freeze-drying protective agents, uses gelatin and CMC as microcapsule wall materials, and uses a composite coacervation method to prepare probiotic-loaded microcapsules based on double-layer emulsions. Probiotic-loaded microcapsules with high protection ability can be prepared at a lower cost and with a relatively simple process method. DETAILED DESCRIPTION

[0045] The present invention provides a targeted delivery method for improving the survival rate of probiotics based on double emulsions and complex coagulation, comprising the following steps:

[0046] (1) Preparation of the inner aqueous phase: Mannitol, dwarf yew polysaccharide, and gelatin were dissolved in water as a composite lyophilization protectant. 8-10 log CFU / 100 ml of probiotics were centrifuged, washed, and then mixed with the composite lyophilization protectant to prepare a bacterial suspension as the inner aqueous phase;

[0047] (2) Preparation of oil phase: Add 4-4.5 g / 100 ml PGPR to 9% vitamin A oil and dissolve for 30 min to obtain the oil phase;

[0048] (3) Preparation of the external aqueous phase: Add 1 g / ml gelatin and 3 g / 100 ml sucrose ester to deionized water, hydrate at 40-50°C for 30 min, centrifuge while hot, and adjust the pH to 3-4 to obtain the external aqueous phase;

[0049] (4) Preparation of a probiotic carrier based on a double emulsion: The oil phase and the inner aqueous phase were uniformly mixed at a ratio of 7:1 to obtain a primary emulsion. The primary emulsion and the outer aqueous phase were uniformly mixed at a ratio of 1:5 to obtain a probiotic double emulsion carrier;

[0050] (5) Preparation of probiotic microcapsules based on complex coacervation: CMC was added to the probiotic double emulsion carrier at a gelatin-CMC ratio of 4:1 to 5:1, and wet probiotic microcapsules were obtained after stirring, centrifugation, and washing;

[0051] (6) pre-freezing the wet microcapsules and then freeze-drying them to obtain probiotic microcapsule powder.

[0052] In the present invention, a double emulsion system, a complex coacervation method, and a composite lyophilization protective agent are used in combination to obtain bacterial-loaded microcapsules with improved core material strain survival rate and intestinal targeted sustained release. In the present invention, the oil phase is 4-4.5g / 100ml PGPR added to 9% vitamin A oil, and more preferably 4g / 100ml PGPR. The composite lyophilization protective agent is 3-4wt% mannitol, 4-5wt% dwarf yew polysaccharide, and 2-3wt% gelatin. Preferably, mannitol 3.07wt%, dwarf yew polysaccharide 4.42wt%, and gelatin 2.01wt%.

[0053] After obtaining the composite freeze-dried protective agent, the present invention mixes the composite freeze-dried protective agent with probiotics to obtain a composite freeze-dried protective agent-probiotic mixed bacterial suspension. In the present invention, the probiotics include one or more of Bacillus megaterium, Bacillus velez, Bacillus coagulans, Lactobacillus rhamnosus, Bacillus amyloliquefaciens, and Lactobacillus plantarum. The frozen and preserved probiotic culture is centrifuged at 4°C after subculture, and the supernatant is removed and washed three times with a sterilized physiological saline (0.9%) solution and then centrifuged to obtain a probiotic mud. After the composite freeze-dried protective agent is mixed with the probiotics, the solution needs to be mixed evenly using a vortex oscillator.

[0054] The primary emulsion of the present invention has a rotation speed of 900-1200 rpm, an emulsification time of 40-80 min, and a phase ratio of 6:1-8:1. Preferably, the rotation speed is 1227.96 rpm, the emulsification time is 61.94 min, and the phase ratio is 7.12:1. The secondary emulsion has a rotation speed of 600-1200 rpm, an emulsification time of 100-140 min, and a phase ratio of 4:1-5:1. Preferably, the secondary emulsion has a rotation speed of 900.04 rpm, an emulsification time of 121.06 min, and a phase ratio of 4.62:1.

[0055] After obtaining the secondary emulsion of the double emulsion system, the present invention adds CMC to the probiotic double emulsion carrier at a gelatin-CMC ratio of 4:1 to 5:1. After stirring, centrifugation, and washing, wet probiotic microcapsules are obtained. Preferably, the CMC dispersion is added in an amount of 22.2% of the gelatin dispersion in the external aqueous phase. The CMC dispersion should be added 60 minutes after the primary emulsion is added to the external aqueous phase, and mixing is continued for 60 minutes. Before adding the CMC, the system pH is maintained at 3.5, the stirring speed is 900 rpm, and the stirring time is 60 minutes.

[0056] After obtaining the wet bacteria-loaded microcapsules, the present invention freeze-dries the wet bacteria-loaded microcapsules to obtain bacteria-loaded microcapsule powder. In the present invention, the freeze-drying treatment method for the wet bacteria-loaded microcapsules is to pre-freeze the wet bacteria-loaded microcapsules at -80°C for 12 to 36 hours, preferably 24 hours.

[0057] The present invention also provides the use of the probiotic-loaded microcapsules in preparing a gastric acid-resistant oral probiotic preparation. The present invention has no particular limitation on the dosage form and excipients of the gastric acid-resistant oral probiotic microcapsule preparation, and conventional probiotic dosage forms and excipients in the art can be used.

[0058] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1: A targeted delivery method for improving the survival rate of probiotics based on double emulsions and complex coacervation

[0060] The specific preparation method is as follows:

[0061] (1) Preparation of bacterial suspension: After thawing, 100 μl of frozen Bacillus velezensis (Bacillus velezensis NSZ-YBGJ0001, CGMCC: No. 14384) was added to 100 ml of PDB liquid culture medium and cultured continuously at 37°C and 150 rpm in a shaker for 48 h. After three consecutive subcultures, the strain culture was centrifuged at 4°C and 5000 rpm, washed three times with sterile saline solution (0.9%), and the supernatant was removed. The suspension was resuspended in 10 ml of a composite lyoprotectant containing 3.07 wt% mannitol, 4.42 wt% dwarf yew polysaccharide, and 2.01 wt% gelatin by vortexing to obtain a Bacillus velezensis suspension with a concentration of 8.7 log CFU / ml.

[0062] (2) Preparation of oil phase: Add 4 g of PGPR to 100 ml of 9% vitamin A oil and dissolve at 600 rpm for 30 min to obtain the oil phase;

[0063] (3) Preparation of the external aqueous phase: 1 g of gelatin was dissolved in 100 ml of sterile water and 3 g of sucrose ester was added. The mixture was hydrated at 600 rpm and 50°C for 30 min until completely dissolved. The mixture was centrifuged while hot to remove impurities. The pH was adjusted to 3-4 using acetic acid (1%) and sodium hydroxide (1%) to obtain a 1 wt% gelatin dispersion.

[0064] (4) Preparation of primary emulsion: Bacillus velezensis suspension was added dropwise to the oil phase at a ratio of 1:7.12, at a rotation speed of 1227.96 rpm and an emulsification time of 61.94 min, and mixed uniformly to obtain a primary emulsion;

[0065] (5) Preparation of secondary emulsion: The primary emulsion was added dropwise to the external aqueous phase at a ratio of 1:4.62 at a rotation speed of 900.04 rpm and an emulsification time of 121.06 min, and mixed uniformly to obtain a secondary emulsion;

[0066] (6) Preparation of bacteria-loaded microcapsules: The pH of the secondary emulsion system was adjusted to 3.5 using acetic acid (1%) and sodium hydroxide (1%). 1 wt% carboxymethyl cellulose-water dispersion was added to the secondary emulsion. The amount of carboxymethyl cellulose-water dispersion added was 22.2% of the total mass of the external aqueous phase and the carboxymethyl cellulose-water dispersion. The stirring speed was 900 rpm and the stirring time was 60 min. Wet bacteria-loaded microcapsules were obtained after washing three times with sterile physiological saline (0.9%). The wet bacteria-loaded microcapsules were pre-frozen at -80°C for 24 h and then dried using vacuum freeze drying (-70°C for 24 h) to obtain Bacillus velezensis-loaded microcapsules.

[0067] Example 2: A targeted delivery method for improving the survival rate of probiotics based on double emulsions and complex coacervation

[0068] The Bacillus Velez subtilis in Example 1 was replaced with Bacillus megaterium (Priesteria megaterium 2203LBZ02, CGMCC: No. 24901) to prepare Bacillus megaterium-loaded microcapsules. The remaining steps were the same as in Example 1.

[0069] Example 3: A targeted delivery method for improving the survival rate of probiotics based on double emulsions and complex coacervation

[0070] The Bacillus Velez in Example 1 was replaced with Bacillus coagulans (Bacillus coagulans JSSW-LA-07, CGMCC: No. 2602) to prepare Bacillus coagulans-loaded microcapsules. The remaining steps were the same as in Example 1.

[0071] Example 4: A targeted delivery method for improving the survival rate of probiotics based on double emulsions and complex coacervation

[0072] The Bacillus velezensis in Example 1 was replaced by Lactobacillus rhamnosus (CGMCC No. 13310) to prepare Lactobacillus rhamnosus-loaded microcapsules. The remaining steps were the same as in Example 1.

[0073] Example 5: A targeted delivery method for improving the survival rate of probiotics based on double emulsions and complex coacervation

[0074] The Bacillus Velez subtilis in Example 1 was replaced with Bacillus amyloliquefaciens (CGMCC: No. 12593) to prepare Bacillus amyloliquefaciens-loaded microcapsules. The remaining steps were the same as in Example 1.

[0075] Example 6: A targeted delivery method for improving the survival rate of probiotics based on double emulsions and complex coacervation

[0076] The Bacillus Velez subtilis in Example 1 was replaced with Lactobacillus plantarum (Lactobacillus plantarum LP-Only, CGMCC: No. 1258) to prepare Lactobacillus plantarum-loaded microcapsules. The remaining steps were the same as in Example 1.

[0077] Comparative Example 1: Omitting the double-layer emulsion system

[0078] The preparation method of single complex coacervate bacteria-loaded microcapsules is as follows:

[0079] (1) Preparation of bacterial suspension: After thawing, 100 μl of frozen Bacillus velezensis (Bacillus velezensis NSZ-YBGJ0001, CGMCC: No. 14384) was added to 100 ml of PDB liquid culture medium and cultured continuously at 37°C and 150 rpm in a shaker for 48 h. After three consecutive subcultures, the strain culture was centrifuged at 4°C, washed three times with sterile saline solution (0.9%), and the supernatant was removed. 10 ml was resuspended in a composite lyoprotectant containing 3.07 wt% mannitol, 4.42 wt% dwarf yew polysaccharide, and 2.01 wt% gelatin to obtain a Bacillus velezensis suspension with a concentration of 8.7 log CFU / ml.

[0080] (2) Complex coagulation behavior: 1 wt% gelatin dispersion was added to the above bacterial suspension (1 g of gelatin was dissolved in 100 ml of sterile water, hydrated at 600 rpm and 50°C for 30 min until completely dissolved, and impurities were removed by centrifugation while hot to obtain a 1 wt% gelatin dispersion). The pH of the system was adjusted to 3.5 using acetic acid (1%) and sodium hydroxide (1%). 1 wt% CMC dispersion was added to the above solution. The amount of CMC dispersion added was 22.2% of the total mass of the 1 wt% gelatin dispersion and the carboxymethyl cellulose-water dispersion. The stirring speed was 900 rpm and the stirring time was 60 min.

[0081] (3) Preparation of bacteria-loaded microcapsules: Wet bacteria-loaded microcapsules were obtained after washing with physiological saline three times. The wet bacteria-loaded microcapsules were pre-frozen at -80°C for 24 h and then dried using vacuum freeze drying to obtain Bacillus velezensis-loaded microcapsules.

[0082] Comparative Example 2: Omitting the composite freeze-drying protective agent

[0083] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to Example 1, the difference is that the composite lyophilization protectant in Example 1 is replaced with sterile physiological saline (0.9%), and the remaining steps are the same as those in Example 1.

[0084] Comparative Example 3: Preferably, the composite freeze-drying protective agent is replaced by a single freeze-drying protective agent

[0085] Specific implementation method: Refer to Example 1, except that the composite lyoprotectant in Example 1 is replaced by a single lyoprotectant (mannitol 3.07wt%, trehalose 4.42wt%, dwarf taxan polysaccharide 4.42wt%, or gelatin 2.01wt%), and the remaining steps are the same as Example 1.

[0086] Comparative Example 4: Unembedded naked bacteria

[0087] After thawing the frozen Bacillus Velezii, 100 μl was added to 100 ml of PDB liquid culture medium, activated at 37°C for 36 hours, and serially subcultured three times. The strain culture was centrifuged at 4°C, washed three times with sterile saline solution (0.9%), and the supernatant was removed. The culture was resuspended in sterile saline solution (0.9%) and lyophilized to obtain bacterial powder.

[0088] Example 7: Performance Characterization

[0089] The microcapsules obtained in Examples 1 to 6 of the present invention have a double-layer emulsion embedding structure, an average particle size of the microcapsules is about 230 μm, and the wall material is formed by the electrostatic bonding of gelatin and CMC.

[0090] 1. Embedding Efficiency: 1 g of the probiotic microcapsules prepared in Examples 1-6 and Comparative Examples 1-3 were dissolved in 20 ml of normal saline (0.9%, pH 7.0) and counted. For cell counting, the bacterial suspension was serially diluted, plated, and incubated at 37°C for 24 h. Plates with 20-200 colonies were selected for counting, and the results were expressed as log CFU / ml. The embedding efficiency was calculated using the following formula, and the results are shown in Table 1.

[0091]

[0092] Where W1 is the number of viable bacteria in the bacterial suspension (log CFU / ml), and W2 is the number of viable bacteria after release (log CFU / ml)

[0093] Table 1 Embedding rate of embodiment and comparative example

[0094]

[0095]

[0096] The encapsulation efficiency of Examples 1-6 was all above 90%, demonstrating that the encapsulation method of the present invention has broad applicability and is suitable for one or more of Bacillus megaterium, Bacillus velezensis, Lactobacillus plantarum, Bacillus amyloliquefaciens, Bacillus coagulans, and Lactobacillus rhamnosus. Compared to Comparative Examples 1-3, the double emulsion system in Example 1 increased the encapsulation efficiency by 1.36 times, the composite lyoprotectant increased the encapsulation efficiency by 2.13 times, and the composite lyoprotectant increased the encapsulation efficiency by 1.37-1.71 times compared to a single lyoprotectant. The double emulsion and complex coacervation-based encapsulation method prepared by the present invention exhibits excellent encapsulation efficiency.

[0097] 2. Storage Stability: 2 g of the probiotic microcapsules or powders prepared in Examples 1-6 and Comparative Examples 1-4 were stored at 25°C for 5 weeks. The survival rate was measured every 7 days. The survival rate was calculated according to the following formula, and the results are shown in Table 2.

[0098]

[0099] Where, E1 is the number of viable bacteria in the microcapsules (log CFU / ml), and W2 is the number of viable bacteria in the microcapsules after storage (log CFU / ml).

[0100] Table 2 Storage survival rate of the embodiments and comparative examples

[0101]

[0102] The probiotic microcapsules prepared according to Examples 1 to 6 had a survival rate of more than 80% at 5 weeks. Compared with Comparative Example 1, the double emulsion system increased the survival rate at week 5 by about 3.41 times. Compared with Comparative Example 2, the composite freeze-drying protective agent increased the survival rate at week 5 by about 3.01 times. Compared with Comparative Example 3, the composite freeze-drying protective agent increased the survival rate at week 5 by about 1.25 to 1.39 times. Compared with naked bacteria, the storage stability after encapsulation was improved by about 60%. This shows that the double emulsion system and the composite freeze-drying protective agent have a significant protective effect on the core material strain. This is because in the double-layer emulsion system, the presence of the oil phase reduces the contamination of the core material by the external environment and limits the diffusion of cells in the inner water phase to the outside world. The composite lyophilization protectant can serve as a prebiotic for the core material strain, thereby prolonging the survival time of the probiotics. Therefore, the bacteria-loaded microcapsules prepared by the present invention based on the double emulsion system and the composite lyophilization protectant can ensure the survival rate of the probiotics under room temperature conditions and effectively improve the protection ability of the probiotics.

[0103] 3. Frozen recovery rate:

[0104] The Bacillus velezensis suspension obtained in step (1) of Example 1 is recorded as a composite freeze-drying protective agent bacterial suspension, that is, Bacillus velezensis is resuspended in a composite freeze-drying protective agent containing 3.07 wt% mannitol, 4.42 wt% dwarf yew polysaccharide, and 2.01 wt% gelatin to obtain a Bacillus velezensis composite freeze-drying protective agent bacterial suspension.

[0105] Similarly, in step (1) of Comparative Example 3, mannitol was used alone, and the resulting Bacillus velez suspension was recorded as a mannitol bacterial suspension, i.e., Bacillus velez was resuspended in a single freeze-drying protective agent of 3.07 wt% mannitol to obtain a mannitol bacterial suspension of Bacillus velez. In step (1) of Comparative Example 3, dwarf yew polysaccharide was used alone, and the resulting Bacillus velez suspension was recorded as a dwarf yew polysaccharide bacterial suspension, i.e., Bacillus velez was resuspended in a single freeze-drying protective agent of 4.42 wt% dwarf yew polysaccharide to obtain a dwarf yew polysaccharide bacterial suspension of Bacillus velez. In step (1) of Comparative Example 3, gelatin was used alone, and the resulting Bacillus velez suspension was recorded as a gelatin bacterial suspension, i.e., Bacillus velez was resuspended in a single freeze-drying protective agent of 2.01 wt% gelatin to obtain a gelatin bacterial suspension of Bacillus velez. In step (1) of Comparative Example 3, no lyoprotectant was used but physiological saline was used, and the obtained Bacillus Velez suspension was recorded as physiological saline bacterial suspension, that is, Bacillus Velez was resuspended in 0.9% sterile physiological saline to obtain Bacillus Velez physiological saline bacterial suspension.

[0106] The bacterial suspension obtained above was pre-cooled at -30°C for 24 hours and then transferred to a vacuum freeze dryer for 48 hours. After drying, the bacterial powder was reconstituted in 100 ml of sterile saline for 1 hour (37°C, 150 rpm) and then the cells were counted. The recovery rate was used as an indicator to evaluate the effectiveness of the lyoprotectant. For cell counting, the bacterial suspension was diluted exponentially and spread on plates. After incubation at 37°C for 24 hours, culture dishes with 20-200 colonies were selected for counting. The results were calculated as CFU / mL and are shown in Table 3.

[0107] Table 3 Effect of lyophilization protective agents

[0108]

[0109] The present invention optimizes the composite lyophilization protectant through RSM. The optimized composite lyophilization protectant is about 20% higher than that of a single lyophilization protectant and about 60% higher than that of naked bacteria.

[0110] 4. Acid Resistance: The encapsulated probiotic cells or bacterial powders prepared in Examples 1-6 and Comparative Examples 1-4 were exposed to artificial gastric digestive fluid, and their survival rates were assessed using an in vitro release model. Artificial gastric fluid: 0.2 g of NaCl was accurately weighed and dissolved in 100 ml of sterile water. The solution was sterilized by autoclaving at 121°C for 20 min. Under aseptic conditions, 3 g / L of porcine gastric mucosal pepsin was added, and the pH was adjusted to 2.5 with 1 N HCl and 1 N NaOH. The solution was then sterilized by filtration through a 0.22 μm aqueous sterile syringe. 1 g of the encapsulated probiotic cells prepared in Examples 1-6 and Comparative Examples 1-3 was placed in 10 ml of artificial gastric fluid for 2 hours of simulated digestion. The simulated digestion conditions were all at 100 rpm and 37°C. During the simulated digestion, 200 μl of the digestive fluid was collected every 30 minutes for cell counting to track the digestion progress. The results are shown in Table 3.

[0111] Table 4 Acid resistance of Examples and Comparative Examples

[0112]

[0113] The probiotic microcapsules prepared according to Examples 1 to 6 had survival rates higher than 85% in simulated gastric juice digestion within 2 hours. Compared with Comparative Example 1, the double emulsion system increased the survival rate in simulated gastric juice digestion within 2 hours by about 2.02 times. Compared with Comparative Example 2, the composite freeze-dried protective agent increased the survival rate in simulated gastric juice digestion within 2 hours by about 1.03 times. Compared with Comparative Example 3, the composite freeze-dried protective agent increased the survival rate in the 5th week by about 1.03 to 1.06 times, and the gastric tolerance after encapsulation was increased by about 60% compared to naked bacteria. This shows that the double emulsion system has a significant acid-resistant effect on the core material strain. After encapsulation, excess protons may not have a substantial effect on the cells because they interact with the acidic and alkaline biopolymer groups around the encapsulated cells and participate in the protonation equilibrium. Therefore, the bacteria-loaded microcapsules prepared in the present invention based on the double emulsion system and the composite freeze-drying protectant can ensure the survival rate of probiotics under gastric digestion conditions. The double-layer emulsion delivery system enables the core material strain to pass through the highly acidic gastric environment with high activity and be targeted to the intestine for slow release.

[0114] 5. Intestinal Targeting: The encapsulated probiotic cells or bacterial powders prepared in Examples 1-6 and Comparative Examples 1-4 were exposed to artificial intestinal digestive fluid, and their survival rates were assessed using an in vitro release model. Artificial intestinal fluid: 0.2 g of NaCl and 0.45 g of bile salts were accurately weighed and dissolved in 100 ml of sterile water. The solution was sterilized by autoclaving at 121°C for 20 min. Under sterile conditions, 2 g / L of trypsin was added, and the pH was adjusted to 7.5 with 1 N NaOH. The solution was then sterilized by filtration through a 0.22 μm aqueous sterile syringe. 1 g of the encapsulated probiotic cells prepared in Examples 1-6 and Comparative Examples 1-3 was placed in 10 ml of artificial intestinal fluid for 4 hours of simulated digestion. The simulated digestion environment was 100 rpm and 37°C. During the simulated digestion, 200 μl of the upper digestive fluid was collected every 60 minutes for cell counting to track the digestion progress. The results are shown in Table 4.

[0115] Table 5 Enteric release properties of Examples and Comparative Examples

[0116]

[0117] The probiotic microcapsules prepared according to Examples 1 to 6 had a release rate higher than 90% in simulated intestinal fluid digestion within 4 hours. Compared with Comparative Example 1, the double emulsion system increased the release rate in simulated intestinal fluid digestion within 4 hours by about 1.36 times. Compared with Comparative Example 2, the composite lyophilization protectant increased the release rate in simulated intestinal fluid digestion within 4 hours by about 1.03 times. Compared with Comparative Example 3, the composite lyophilization protectant increased the release rate in simulated intestinal fluid digestion within 4 hours by about 1.01 to 1.04 times. Compared with Comparative Example 4, it was increased by about 60% compared with naked bacteria. The pH-mediated gelatin and CMC complexes have negative charges at the high pH of the intestinal environment, which is conducive to repulsion between them, resulting in the rupture of the microcapsule structure and the release of the probiotic content, which helps to facilitate the colonization of probiotics in the intestine.

[0118] 6. Intestinal Adhesion: The probiotic microcapsules or powders described in Examples 1 to 6 and Comparative Examples 1 to 4 were subjected to an in vitro intestinal adhesion test. The specific method was as follows: on a sterile operating table, the intestines of male rats were cut open, and four portions of intestinal tissue (1.5 cm × 1.5 cm × 0.2 cm) were placed on sterile glass slides. 50 μl of Bacillus Velez suspension (blank group), 0.01 g each of the probiotic microcapsules of Examples 1 to 6, and the probiotic microcapsules of Comparative Examples 1 to 3 were added dropwise and dissolved in 50 μl of normal saline. The tubes were placed in sterile centrifuge tubes, shaken in a 37°C water bath shaker for 2 h, and then removed and rinsed with 300 μl of normal saline. The rinse fluid was collected for gradient dilution, and the number of viable bacteria was counted. The adhesion rate was calculated according to the following formula. The results are shown in Table 5.

[0119] Adhesion rate (%) = (total number of bacteria - number of non-adherent bacteria) / total number of bacteria × 100%

[0120] The results show that the adhesion rate of the Bacillus velezensis microcapsules (Example 1) prepared by the present invention in the intestine reaches 83.80%, which is about 70% higher than that of naked bacteria. The adhesion rates of the probiotic microcapsules prepared according to Examples 1 to 6 are all higher than 80%. Compared with Comparative Example 1, the double emulsion system increases the adhesion rate by about 2.06 times. Compared with Comparative Example 2, the adhesion rate is increased by about 1.03 times. Compared with Comparative Example 3, the adhesion rate is increased by about 1.03 to 1.05 times. When the carrier enters the duodenum, the alkaline environment will cause gelatin and CMC to be negatively charged, repel each other and promote disintegration. The double-layer emulsion system will promote the intestinal wall cilia to capture the probiotics carried, thereby achieving targeted delivery.

[0121] Table 6 Intestinal Adhesion of Examples and Comparative Examples

[0122]

[0123] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A targeted delivery method for improving the survival rate of probiotics based on double emulsion and complex coacervation, characterized in that: The following steps are involved: (1) preparing the inner aqueous phase: dissolving mannitol, dwarf yew polysaccharide, and gelatin in water as a composite freeze-drying protective agent; mixing the probiotics with the composite freeze-drying protective agent to prepare a bacterial suspension as the inner aqueous phase; (2) Preparation of oil phase: Add polyglycerol ricinoleate to vitamin A oil and dissolve for 20-40 minutes to obtain the oil phase; (3) Preparation of external aqueous phase: gelatin and sucrose ester were added to deionized water, hydrated at 40-50°C for 20-40 min, and centrifuged while hot to obtain the external aqueous phase; (4) Preparation of a probiotic carrier based on a double emulsion: uniformly mixing the oil phase with the inner aqueous phase to obtain a primary emulsion; uniformly mixing the primary emulsion with the outer aqueous phase to obtain a probiotic double emulsion carrier; (5) Preparation of probiotic microcapsules based on complex coacervation: Add a curing agent to the probiotic double emulsion carrier, stir, centrifuge, wash, and freeze-dry to obtain probiotic microcapsules.

2. The method according to claim 1, wherein In step (1), the probiotics are one or more of Bacillus megaterium, Bacillus velezensis, Bacillus coagulans, Lactobacillus rhamnosus, Bacillus amyloliquefaciens, and Lactobacillus plantarum, and the concentration of the probiotics in the suspension is 8-10 log CFU / 100 ml.

3. The method according to claim 1, wherein In step (1), the concentration of mannitol in the composite freeze-drying protectant is 3-4 wt%, the concentration of dwarf yew polysaccharide is 4-5 wt%, and the concentration of gelatin is 2-3 wt%.

4. The method according to claim 1, wherein In step (2), the purity of the vitamin A oil is 99%, and the ratio of PGPR to vitamin A oil is 4-4.5 g:100 ml.

5. The method according to claim 1, wherein In step (3), the mass concentration of gelatin in the external aqueous phase is 1-2%, and the concentration of the sucrose ester is 3-5%.

6. The method according to claim 1, wherein In step (4), the volume ratio of the oil phase to the inner water phase in the primary emulsion is 6:1 to 8:1, and the volume ratio of the primary emulsion to the outer water phase in the secondary emulsion is 1:3 to 1:

6.

7. The method according to claim 1, wherein In step (4), the rotation speed for forming the primary emulsion is 1227.96 rpm and the emulsification time is 61.94 min; the rotation speed for forming the secondary emulsion is 600-1200 rpm and the emulsification time is 100-140 min.

8. The method according to claim 1, wherein In step (5), the curing agent is a carboxymethyl cellulose dispersion, and the amount of carboxymethyl cellulose dispersion added is 20% to 25% of the total mass of the external aqueous phase and the carboxymethyl cellulose-water dispersion; before adding the curing agent, the pH of the system is maintained at 3 to 4; the stirring speed is 600 to 1200 rpm, and the stirring time is 50 to 80 minutes.

9. Probiotic microcapsules prepared by the method according to any one of claims 1 to 8.

10. Use of the probiotic microcapsule according to claim 9 in preparing a gastric acid-resistant oral product, characterized in that: The products include food, medicine, and health care products; the health care products include probiotic preparations.

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