A method for the preparation of dha oil-in-water emulsions with enhanced probiotic processing and delivery

Abstract

This invention provides a method for preparing a DHA oil-in-water emulsion for probiotic processing and delivery, relating to the field of bioengineering technology. The method includes the following steps: dissolving Tween 80 and glycerol in a buffer solution, then adding DHA and mixing thoroughly to obtain a suspension; repeatedly extruding the suspension 15 times using a liposome extruder; and diluting the product obtained in the previous step with a buffer solution to obtain the DHA oil-in-water emulsion. By preparing DHA into an oil-in-water emulsion, the problem of DHA's immiscibility with water is solved, while simultaneously reducing or even eliminating the unique taste of DHA's unsaturated fatty acids, thus broadening the application range of fatty acids in the pretreatment and protection of probiotics. Furthermore, this preparation method is simple, saves steps, and facilitates industrial production.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method for preparing a DHA oil-in-water emulsion processed and delivered by beneficial probiotics. Background Technology

[0002] Many probiotics offer multiple health benefits to the host, including immune regulation, improvement of metabolic syndrome, treatment of inflammatory bowel disease, and regulation of gut microbiota. Based on these benefits, probiotics are widely used in food, dietary supplements, pharmaceuticals, and animal feed. However, probiotics face several significant challenges in these applications. Firstly, before being added to commercially available products, probiotics typically undergo pretreatment processes such as freeze-drying and spray-drying, which can easily affect their activity. Secondly, during delivery through the gastrointestinal tract, probiotics are easily killed by the highly acidic environment of gastric juice and various intestinal enzymes, significantly reducing the number and activity of viable bacteria reaching the colonization site. These two challenges present unavoidable obstacles to the application of probiotics.

[0003] Current protective strategies for pretreatment typically involve adding preservatives such as sucrose, glycerol, and skim milk powder. Traditional preservatives usually do not involve fatty acids, largely because fatty acids are insoluble in water and difficult to disperse evenly in water-based bacterial suspensions. This leads to uneven protection, with some cells not being protected, and high concentrations of fatty acids may even damage cell membranes. To address the issue of gastrointestinal stressors during delivery, common strategies include enteric-coated capsules, microencapsulation, hydrogel encapsulation, and genetic engineering. Hydrogels are high-molecular-weight compounds that can form three-dimensional network structures through physical cross-linking (hydrogen bonds, van der Waals forces, hydrophobic interactions, etc.) or chemical cross-linking (enzymatic cross-linking, etc.). Encapsulating probiotics within this three-dimensional network structure provides both a barrier against external stressors and a habitat for the encapsulated probiotics. However, this three-dimensional network structure is not without its drawbacks. Due to its submicron or even micron-sized pores, small molecules in gastric and intestinal fluids, H+, etc., can cause damage. + These substances can enter the gel through the mesh, significantly reducing the hydrogel's protective effect on probiotics.

[0004] Docosahexaenoic acid (DHA), as an omega-3 fatty acid, offers numerous health benefits, such as anti-inflammatory properties, promoting brain development in infants and young children, protecting vision, and safeguarding brain health. However, its application also faces some significant challenges. Firstly, as an unsaturated fatty acid, DHA has an unpleasant flavor and is not suitable for direct consumption; adding it directly to food can easily affect its flavor. Secondly, DHA is also difficult to dissolve in water, which poses a significant challenge to its application.

[0005] Therefore, this invention is proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for preparing a DHA oil-in-water emulsion for enhancing probiotic processing and delivery. By preparing DHA into an oil-in-water emulsion, this method offers a novel approach to solving existing problems in DHA applications. It not only resolves the issue of DHA's immiscibility with water but also reduces or even eliminates the unique flavor of DHA's unsaturated fatty acids after the emulsion is formed. Secondly, with the water immiscibility issue resolved, the DHA oil-in-water emulsion holds promise for use as a pretreatment protectant for probiotics. Thirdly, in the delivery of probiotics protected by gel, the introduction of the DHA oil-in-water emulsion blocks the gel pores, thereby reducing their size and enhancing the protective effect of the gel on the gastrointestinal delivery of probiotics.

[0007] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides a method for preparing a DHA oil-in-water emulsion processed and delivered by probiotics, comprising the following steps: S1. Dissolve Tween 80 and glycerol in buffer solution, then add DHA and mix well to obtain a suspension; S2. The suspension is repeatedly squeezed 15 times using a liposome extruder; S3. Dilute the product obtained in S2 with buffer solution to obtain DHA oil-in-water emulsion.

[0008] Furthermore, in step S1, during the step of adding DHA and mixing it evenly, the mixture is stirred at a speed of 600 rad / min for 10 min.

[0009] Furthermore, the buffer solution is 0.5×PBS.

[0010] Furthermore, in S1, the final concentration of Tween 80 in the suspension is 1 w / v%-3 w / v, the final concentration of glycerol is 2 w / v%-4 w / v, and the final concentration of DHA is 10 w / v%-18 w / v.

[0011] Furthermore, the final concentrations of Tween 80 in the suspension were 2 w / v, glycerol 3 w / v, and DHA 15 w / v.

[0012] Furthermore, in S2, the filter membrane used in the liposome extruder is a polycarbonate filter membrane with a pore size of 5 μm.

[0013] Furthermore, in step S3, the final concentration of DHA in the DHA oil-in-water emulsion is obtained to be 0.0075 w / v.

[0014] The present invention also provides a DHA oil-in-water emulsion prepared by the above preparation method.

[0015] The present invention also provides a freeze-drying protectant, comprising: DHA oil-in-water emulsion, glycerol, sucrose, skim milk powder and buffer solution; The composition, by mass percentage, consists of 0.0075 w / v DHA oil-in-water emulsion, 2 w / v glycerol, 6 w / v sucrose, 6 w / v skim milk powder, with the remainder being water or buffer solution.

[0016] Furthermore, the buffer solution was 0.5×PBS.

[0017] The present invention also provides a synergistic gel comprising: gelatin, gellan gum, and an oil-in-water emulsion of DHA.

[0018] The present invention also provides a probiotic-loaded synergistic protective gel, comprising: gelatin, gellan gum, probiotic suspension and DHA oil-in-water emulsion.

[0019] The present invention also provides a method for preparing the above-mentioned probiotic-loaded synergistic protective gel, comprising the following steps: dissolving gelatin and gellan gum in DHA oil-in-water emulsion at 60°C until completely dissolved, and when the temperature drops to 45°C, taking gelatin, gellan gum, DHA oil-in-water emulsion and probiotic suspension.

[0020] Furthermore, in the step of taking gelatin, gellan gum, DHA oil-in-water emulsion and probiotic suspension, the volume ratio of gelatin, gellan gum, DHA oil-in-water emulsion and probiotic suspension is 6:6:3:2.

[0021] This invention also provides the application of the above-mentioned DHA oil-in-water emulsion in the preparation of a protective agent to improve the gastrointestinal delivery tolerance of probiotics.

[0022] This invention also provides the application of the above-mentioned DHA oil-in-water emulsion in the preparation of probiotic activity protectants.

[0023] This invention also provides the application of the above-mentioned DHA oil-in-water emulsion, or freeze-drying protectant, or synergistic gel in the preparation of products that protect the activity of probiotics.

[0024] Furthermore, the probiotic is a strain of Bifidobacterium.

[0025] The present invention has the following technical effects: (1) Micron-sized DHA oil-in-water emulsions were prepared by liposome extruders, which solved the application bottleneck of poor water solubility and unpleasant flavor of DHA. A controllable particle size and stable properties emulsion system was obtained, making it possible to uniformly disperse fatty acids in the aqueous phase.

[0026] (2) When the DHA emulsion was used in combination with traditional freeze-drying protectants, the survival rate and number of live bacteria of probiotics were significantly improved after freeze-drying, which proved the effectiveness of DHA emulsion as an auxiliary freeze-drying protectant and broadened the application scope of fatty acids in the pretreatment protection of probiotics.

[0027] (3) A modified gel with enhanced efficacy was prepared by introducing DHA emulsion into the gelatin-gellan gel composite gel. The emulsion droplets physically filled the micropores of the gel network, effectively blocking the penetration of small molecule stress components such as gastric acid, bile salts, and digestive enzymes. Simulated gastrointestinal treatment experiments showed that, compared with free bacteria and traditional gels, the viable counts of Bifidobacterium longum and Bifidobacterium breve encapsulated in the modified gel were better maintained or even significantly increased after 2 hours of simulated gastric juice treatment and 8 hours of simulated intestinal juice treatment, significantly improving the tolerance and survival rate of probiotics during gastrointestinal delivery. Attached Figure Description

[0028] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 Flowchart for the preparation of DHA oil-in-water emulsion; Figure 2 Particle size of DHA oil-in-water emulsion; Figure 3 Comparison of live bacteria count (CFU) of probiotics before and after freeze-drying; Figure 4 Comparison of the survival rates of probiotics before and after freeze-drying; Figure 5 : The number of viable Bifidobacterium longum BL21 bacteria before and after 2 hours of treatment with simulated gastric juice under different conditions; Figure 6 : The number of viable Bifidobacterium breve BBr60 before and after 2 hours of treatment with simulated gastric juice under different conditions; Figure 7: The number of viable Bifidobacterium longum BL21 bacteria before and after 8 hours of treatment with simulated intestinal fluid under different conditions; Figure 8 The viable count of Bifidobacterium breve BBr60 before and after 8 hours of treatment with simulated intestinal fluid under different conditions. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0031] In a first aspect, the present invention provides a method for preparing a DHA oil-in-water emulsion processed and delivered by synergistic probiotics, comprising the following steps: S1. Dissolve Tween 80 and glycerol in buffer solution, then add DHA and mix well to obtain a suspension; S2. The suspension is repeatedly squeezed 15 times using a liposome extruder; S3. Dilute the product obtained in S2 with buffer solution to obtain DHA oil-in-water emulsion.

[0032] In the preparation of DHA oil-in-water emulsions using a high-energy mechanical input method, the magnitude of the energy input is crucial for controlling the emulsion particle size. Insufficient energy input makes emulsion formation difficult, and even if successful, the emulsion is often unstable. Excessive energy input results in extremely small emulsion particle sizes, even reaching the nanometer scale. This invention uses a liposome extruder as the emulsion preparation device, which can control the droplet size by changing the pore size of the filter membrane. Furthermore, the emulsion prepared using this method is stable and less prone to water-oil separation.

[0033] In some embodiments, during step S1, when adding DHA and mixing it evenly, the mixture is stirred at a speed of 600 rad / min for 10 min.

[0034] In some embodiments, the buffer solution is 0.5×PBS.

[0035] In some embodiments, in S1, the final concentration of Tween 80 in the suspension is 1 w / v%-3 w / v, the final concentration of glycerol is 2 w / v%-4 w / v, and the final concentration of DHA is 10 w / v%-18 w / v.

[0036] In some embodiments, the final concentration of Tween 80 in the suspension is 2 w / v, the final concentration of glycerol is 3 w / v, and the final concentration of DHA is 15 w / v.

[0037] The dosage of each substance was limited. A Tween 80 concentration that was too low (<1%) was insufficient to completely emulsify the 15% DHA oil phase, while a concentration that was too high (>3%) might introduce too much surfactant, affecting probiotic activity. The practically workable concentration range for DHA was 10%-18%, with 15% being the optimal value in the examples. Too low a concentration would result in poor efficiency, while too high a concentration would exceed the emulsification capacity and lead to demulsification. This concentration range ensured the success rate and stability of emulsion formation.

[0038] In some embodiments, in S2, the filter membrane used by the liposome extruder is a polycarbonate filter membrane with a pore size of 5 μm.

[0039] Polycarbonate membranes have uniform pore size and smooth surfaces, making them suitable for preparing liposome-based emulsions with controllable particle size using liposome extrusion. After repeated extrusion, the droplet size is approximately 2 μm, demonstrating that this pore size can effectively shear large oil droplets and form stable micron-sized emulsions. Pores that are too small (e.g., submicron-sized) require higher pressure and may damage the DHA structure or form unwanted nanoemulsions. Precise control of emulsion particle size is achieved through repeated extrusion and selection of the filter membrane. Unlike conventional high-pressure homogenization methods, this approach is simple to operate and convenient to use.

[0040] In some embodiments, in step S3, the final concentration of DHA in the DHA oil-in-water emulsion is obtained to be 0.0075 w / v.

[0041] This concentration can be directly applied to the working concentration of lyophilization protectants and synergistic gels, while also being easy to store and precisely add.

[0042] Secondly, the present invention also provides a DHA oil-in-water emulsion prepared by the above preparation method.

[0043] Thirdly, the present invention also provides a freeze-drying protectant, comprising: DHA oil-in-water emulsion, glycerol, sucrose, skim milk powder and buffer solution; The composition, by mass percentage, consists of 0.0075 w / v DHA oil-in-water emulsion, 2 w / v glycerol, 6 w / v sucrose, 6 w / v skim milk powder, with the remainder being water or buffer solution.

[0044] In some embodiments, the buffer solution is 0.5×PBS.

[0045] Glycerol and sucrose replace water molecules during freeze-drying to form hydrogen bonds with cell membrane phospholipids, maintaining membrane integrity; skim milk powder provides physical encapsulation of protein matrix; DHA droplets adhere to the surface of bacteria, replenishing lipid losses in the cell membrane during freeze-drying and dehydration, enhancing membrane repair capacity, and effectively realizing the application of fatty acids as freeze-drying auxiliary protective agents.

[0046] Fourthly, the present invention also provides a synergistic gel comprising: gelatin, gellan gum, and an oil-in-water emulsion of DHA.

[0047] Gelatin-gellan gum forms a three-dimensional network gel framework, during which DHA droplets are trapped in the network pores, physically filling and blocking submicron to micron-sized pores. This gel effectively blocks small molecules such as gastric acid, bile salts, and digestive enzymes from penetrating through the gel network, significantly enhancing the survival and proliferation of probiotics in a simulated gastrointestinal environment compared to traditional gels.

[0048] Fifthly, the present invention also provides a probiotic-loaded synergistic protective gel, comprising: gelatin, gellan gum, probiotic suspension and DHA oil-in-water emulsion.

[0049] In a sixth aspect, the present invention also provides a method for preparing the above-mentioned probiotic-loaded synergistic protective gel, comprising the following steps: dissolving gelatin and gellan gum in a DHA oil-in-water emulsion at 60°C until completely dissolved, and when the temperature drops to 45°C, taking out the gelatin, gellan gum, DHA oil-in-water emulsion and probiotic suspension.

[0050] In some embodiments, in the steps of taking gelatin, gellan gum, DHA oil-in-water emulsion and probiotic suspension, the volume ratio of gelatin, gellan gum, DHA oil-in-water emulsion and probiotic suspension is 6:6:3:2.

[0051] In a seventh aspect, the present invention also provides the application of the above-mentioned DHA oil-in-water emulsion in the preparation of a protective agent that improves the gastrointestinal delivery tolerance of probiotics.

[0052] Eighthly, the present invention also provides the application of the above-mentioned DHA oil-in-water emulsion, or freeze-drying protectant, or synergistic gel in the preparation of products that protect the activity of probiotics.

[0053] In some embodiments, the probiotic is a Bifidobacterium strain.

[0054] The following is a detailed explanation using specific embodiments: Example 1: Preparation of DHA oil-in-water emulsion Dissolve 0.2 g Tween 80 and 0.3 g glycerol in 10 mL of 0.5×PBS, then add 3 g DHA (50% purity) to achieve a final concentration of 2% (w / v) for Tween 80, 3% (w / v) for glycerol, and 15% (w / v) for DHA. Stir at 600 rad / min for 10 min until homogeneous. Pass the suspension through a liposome extractor 15 times (using a polycarbonate membrane with a pore size of 5 μm) to obtain a DHA emulsion (preparation process as follows). Figure 1As shown), the resulting emulsion was then diluted 2000 times with 0.5×PBS to achieve a final DHA concentration of 0.0075%. The particle size of the emulsion was then analyzed using a Baxter particle size potentiometer, and the obtained particle size was 2 μm. Figure 2 ).

[0055] Example 2: Protection of DHA oil-in-water emulsion in probiotic freeze-drying Traditional freeze-drying preservatives: 2% (w / v) glycerin, 6% (w / v) sucrose, 6% (w / v) skim milk powder; Improved freeze-drying protectant: 0.0075% DHA oil-in-water emulsion, 2% (w / v) glycerin, 6% (w / v) sucrose, 6% (w / v) skim milk powder; Before lyophilizing Bifidobacterium longum and Bifidobacterium breve (both purchased from Microcon Probiotics (Suzhou) Co., Ltd.), 100 μL of bacterial solution was diluted and spread to obtain the viable count before lyophilization. Then, Bifidobacterium longum and Bifidobacterium breve were centrifuged to obtain bacterial precipitates. The two strains were then resuspended in the two lyophilization protectants and lyophilized. The lyophilized bacterial powder was resuspended in an equal volume of PBS as before lyophilization, vortexed, and then 100 μL of bacterial solution was diluted and spread to obtain the viable count after lyophilization. By comparing the viable count and survival rate of probiotics under the protection of the two lyophilization protectants, the auxiliary protective effect of DHA emulsion during the lyophilization process can be characterized. The experiment was conducted in triplicate.

[0056] Experimental Results Analysis: The number of viable bacteria after freeze-drying of DHA emulsion combined with a traditional freeze-drying protectant (…) Figure 3 ) and survival rate ( Figure 4 All were higher than the single protective agent experimental group without emulsion.

[0057] Example 3: Enhanced protective effect of DHA water-in-oil emulsion gel Preparation of two types of bacterial-carrying gels: Add 0.8g of gelatin to 12.5mL of sterile water at 60℃, and then place it in a 60℃ constant temperature water bath until completely dissolved to obtain a gelatin solution. Add 0.2g of gellan gum to 12.5mL of sterile water at 60℃, and then place it in a 60℃ constant temperature water bath until completely dissolved to obtain a gellan gum solution. After the temperature of the gelatin solution and the gellan gum solution drops to about 45℃, mix 0.6mL of gelatin, 0.6mL of gellan gum, 0.3mL of sterile water and 0.2mL of Bifidobacterium longum or Bifidobacterium breve respectively, stir well, and then place in a 4℃ refrigerator to cool for 2 hours to prepare the traditional gelatin-gellan gum composite gel loaded with bacteria (divided into GG@BL21 gel loaded with Bifidobacterium longum BL21 and GG@BBr60 gel loaded with Bifidobacterium breve BBr60).

[0058] The preparation methods for gelatin and gellan gum remain unchanged. Instead of 0.3 mL of sterile water, an equal volume of the prepared DHA oil-in-water emulsion is replaced. Under the same conditions, gelatin-DHA-gellan gum synergistic modified gels loaded with bacteria are obtained (divided into GDG@BL21 gel loaded with Bifidobacterium longum BL21 and GDG@BBr60 gel loaded with Bifidobacterium breve BBr60).

[0059] Preparation of simulated gastric and intestinal fluids: Dissolve 0.3g of pepsin in 25mL of sterile water, then bring the volume to 30mL. Adjust the pH to 3.0 to simulate the pH of gastric fluid after eating to obtain simulated gastric fluid. Dissolve 0.3g of trypsin, 0.3g of pancreatic enzyme, 0.255g of NaCl, and 0.09g of bile salts in 25mL of sterile water, then bring the volume to 30mL. Adjust the pH to 6.8 to obtain simulated intestinal fluid.

[0060] Two 200 μL samples of free Bifidobacterium longum and Bifidobacterium breve, along with two cross-linked conventional and modified gels, were taken. 4.9 mL of 6 mol / L urea was added to the conventional gel, and 4.9 mL of 6 mol / L urea was added to the modified gel to induce gel lysis and release the bacteria completely. PBS was then added to all three experimental groups to ensure a final volume of 12 mL. After vortexing, 100 μL of each sample was diluted appropriately and plated. The samples were incubated at 37°C in an anaerobic workstation for 48 h, and viable cell counts were performed to obtain the CFU before simulated intestinal fluid treatment. Another sample was placed in 4.9 mL of simulated gastric fluid and anaerobically incubated at 37°C for 2 hours to simulate the intestinal environment. After 2 hours, 4.9 mL of 6 mol / L urea was added to the sample to induce lysis and release the bacteria. PBS was then added to all three experimental groups to ensure a final volume of 12 mL. After vortexing, 100 μL of the sample was diluted to an appropriate concentration and plated. The samples were then anaerobically incubated at 37°C for 48 hours, followed by viable cell counting to obtain the CFU of the strain treated with simulated gastric fluid. By comparing the CFU before and after simulated gastric fluid treatment, the protective effects of the two gels on Bifidobacterium longum and Bifidobacterium breve can be directly reflected. Triple copies of the plated samples were used.

[0061] The simulated gastric fluid was replaced with simulated intestinal fluid, the incubation time was changed to 8 hours, and the other conditions remained unchanged, which verified the gel protection effect under simulated intestinal fluid treatment.

[0062] Results analysis: Simulated gastric juice treatment: After 2 hours of simulated gastric juice treatment, the CFU of free Bifidobacterium longum (BL21) decreased, as did the CFU of the conventional gel-encapsulated Bifidobacterium longum (GG@BL21). However, the CFU of the modified gel-encapsulated Bifidobacterium longum (GDG@BL21) with DHA incorporation did not show a significant change. Figure 5 In all three experimental groups of *Bifidobacterium breve*, the CFU level increased after 2 hours of treatment, which is related to the proliferation of the bacteria themselves. However, the CFU increase was more significant in the modified gel-encapsulated *Bifidobacterium breve* group with DHA (GDG@BBr60). Figure 6 In other words, during the 2 hours of simulated gastric juice treatment, the bacteria in GDG@BBr60 experienced the least inhibitory effect.

[0063] Simulated intestinal fluid treatment: After 8 hours of simulated intestinal fluid treatment, the CFU of free Bifidobacterium longum (BL21) decreased significantly, while the CFU of Bifidobacterium longum encapsulated in the conventional gel (GG@BL21) increased. This indicates that the conventional gel also has a certain protective effect on BL21. However, the increase in CFU of Bifidobacterium longum encapsulated in the modified gel with added DHA (GDG@BL21) was much greater than that of GG@BL21. Figure 7 This indicates that the addition of DHA emulsion greatly enhances the protective effect of the gel against BL21. The trends shown by the three experimental groups of Bifidobacterium breve are consistent with those of the three experimental groups of Bifidobacterium longum, which further demonstrates that DHA emulsion can enhance the protective effect of the gel against probiotics.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a DHA oil-in-water emulsion processed and delivered by beneficial probiotics, characterized in that, Includes the following steps: S1. Dissolve Tween 80 and glycerol in buffer solution, then add DHA and mix well to obtain a suspension; S2. The suspension is repeatedly squeezed 15 times using a liposome extruder; S3. Dilute the product obtained in S2 with buffer solution to obtain DHA oil-in-water emulsion.

2. The method for preparing DHA oil-in-water emulsion by probiotic processing and delivery according to claim 1, characterized in that, In S1, the final concentration of Tween 80 in the suspension is 1 w / v%-3 w / v, the final concentration of glycerol is 2 w / v%-4 w / v, and the final concentration of DHA is 10 w / v%-18 w / v.

3. The method for preparing DHA oil-in-water emulsion by probiotic processing and delivery according to claim 1, characterized in that, In S2, the filter membrane used in the liposome extruder is a polycarbonate filter membrane with a pore size of 5 μm.

4. The method for preparing DHA oil-in-water emulsion by probiotic processing and delivery according to claim 1, characterized in that, In step S3, the final concentration of DHA in the DHA oil-in-water emulsion is obtained to be 0.0075 w / v.

5. A DHA oil-in-water emulsion prepared by the method for preparing DHA oil-in-water emulsion processed and delivered by probiotics as described in any one of claims 1-4.

6. A freeze-drying protectant, characterized in that, include: The DHA oil-in-water emulsion, glycerol, sucrose, skim milk powder, and buffer solution as described in claim 5; The composition, by mass percentage, consists of 0.0075 w / v DHA oil-in-water emulsion, 2 w / v glycerol, 6 w / v sucrose, 6 w / v skim milk powder, with the remainder being water or buffer solution.

7. A synergistic gel, characterized in that, include: Gelatin, gellan gum, and the DHA oil-in-water emulsion as described in claim 5.

8. The use of the DHA oil-in-water emulsion as described in claim 5 in the preparation of a protective agent to improve the gastrointestinal delivery tolerance of probiotics.

9. The use of the DHA oil-in-water emulsion as described in claim 5, the freeze-drying protectant as described in claim 6, or the synergistic gel as described in claim 7 in the preparation of products that protect the activity of probiotics.

10. The application according to claim 9, characterized in that, The probiotics are strains of the genus Bifidobacterium.

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