Sialylated IgG synbiotic oleogel emulsion for promoting intestinal colonization of bifidobacterium bifidum and preparation method of sialylated IgG synbiotic oleogel emulsion
By embedding sialylated IgG and Bifidobacterium bifidobacterium in synbiotic oil gel, the thermodynamic properties of release in the intestine is solved, and effective intestinal colonization and biological activity protection is achieved.
Patent Information
- Application Number
- CN202510074238.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-17
- Publication Date
- 2025-05-06
AI Technical Summary
The prior art is difficult to achieve effective colonization of sialylated IgG and Bifidobacterium bifidobacterium in the intestine, and is greatly affected by gastric acid and oxygen sensitivity.
In the form of a synbiotic oil gel emulsion, sialic IgG and Bifidobacterium bifidobacterium were embedded in the oil gel, using its thermodynamic properties to transform into a flowable liquid at body temperature, thereby stably present in the stomach and release in the intestine.
It effectively protects the biological activities of sialylated IgG and Bifidobacterium bifidobacterium, promotes its colonization in the intestine, and prolongs the retention time of Bifidobacterium bifidobacterium bifidobacterium in the intestine.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of food processing, and in particular to a sialylated IgG synbiotic oil gel emulsion for promoting intestinal colonization of Bifidobacterium bifidum and a preparation method thereof. Background Art
[0002] Immunoglobulin G (IgG) is an important component of breast milk. Its main functions are to neutralize toxins and viruses, bind to bacteria and regulate immunity. It can promote the tolerance of newborns to beneficial intestinal flora and promote their colonization. It is worth noting that during pregnancy and lactation, IgG undergoes terminal sialylation modification at the conserved N-glycosylation site Asn-297 in its Fc region, resulting in a significant increase in the level of sialylated IgG transferred from mother to offspring. However, previous studies have largely ignored the role of sialylated IgG as a key component in breast milk in promoting the early colonization of Bifidobacterium. Sialyl IgG can promote the proliferation of Bifidobacterium bifidum through a symbiotic relationship.
[0003] In addition, indirect evidence also shows that sialylated IgG contains structures that can adhere to Bifidobacterium bifidum and intestinal cells, respectively. On the one hand, the sugar chains that have strong binding affinity to the sialidase SiaBb2 on the surface of Bifidobacterium bifidum have the same structure as the N-sugar chains of sialylated IgG. On the other hand, during the binding process with human intestinal epithelial cells, the hinge region between the CH2 and CH3 regions of the sialylated IgG light chain can bind to the neonatal fragment crystallized receptor (FcRn). However, the acidic environment and enzymes in the stomach can affect the activity of sialylated IgG, especially pepsin, which breaks down intact sialylated IgG into biologically inactive fragments F(ab)2' and pFc'. In addition, Bifidobacterium bifidum, as an anaerobic bacterium, is extremely sensitive to oxygen content and the low pH of gastric acid.
[0004] The existing method of direct oral administration of sialylated IgG and Bifidobacterium bifidum makes it difficult for sialylated IgG and Bifidobacterium bifidum to enter the intestine and colonize. Therefore, how to achieve the colonization of Bifidobacterium bifidum in the intestine is the main research direction at this stage. Summary of the invention
[0005] In view of the shortcomings of the prior art, the present invention provides a sialylated IgG synbiotic oil gel emulsion for promoting the intestinal colonization of Bifidobacterium bifidum and a preparation method thereof. The synbiotic oil gel emulsion has the thermodynamic property of being solid at a temperature (<36°C) and being converted into a flowable liquid in the human body (≥36°C), being able to stably exist in the stomach and effectively release sialylated IgG and Bifidobacterium bifidum in the intestine. The released sialylated IgG can simultaneously adhere to the sialidase SiaBb2 on the surface of Bifidobacterium bifidum and the receptor FcRn on the surface of intestinal epithelial cells, thereby promoting the colonization of Bifidobacterium bifidum in the intestine.
[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions:
[0007] A sialylated IgG synbiotic oil gel emulsion for promoting intestinal colonization of Bifidobacterium bifidum, the emulsion being composed of the following substances in parts by mass: 34-79 parts of an oil phase, 15-20 parts of an aqueous phase, and 1 part of polyglycerol ricinoleate; the oil phase being composed of β-sitosterol, glyceryl monostearate, and cocoa butter; and the aqueous phase being composed of a phosphate buffer, Bifidobacterium bifidum, and sialylated IgG.
[0008] Preferably, the emulsion is composed of the following substances in parts by mass: 79 parts of oil phase, 20 parts of water phase, and 1 part of polyglycerol ricinoleate.
[0009] Preferably, the weight percentages of the components in the oil phase are 2-4 parts of β-sitosterol, 2-4 parts of glyceryl monostearate, and 92-96 parts of cocoa butter.
[0010] Preferably, the weight proportions of the components in the aqueous phase are 1-2 parts of sialyl IgG, 38-40 parts of Bifidobacterium bifidum, and 58-60 parts of phosphate buffer.
[0011] The preparation method of the emulsion comprises the following steps:
[0012] (1) β-sitosterol, glyceryl stearate and cocoa butter are mixed, heated and stirred until completely dissolved, and then cooled to form an oil gel, which is used as an oil phase for later use;
[0013] (2) mixing the phosphate buffer, Bifidobacterium bifidum and sialyl IgG and homogenizing them with a high-speed homogenizer to obtain an aqueous phase for later use;
[0014] (3) The oil phase, water phase and polyglycerol ricinoleate prepared above are mixed evenly, homogenized, and then cooled in an ice water bath to form a synbiotic oil gel emulsion.
[0015] Preferably, the heating and stirring temperature in step (1) is 90° C., and the stirring time is 1 h.
[0016] Preferably, the final cooling temperature in step (1) is 4°C.
[0017] Preferably, the speed of the homogenization treatment in step (2) is 5000 rpm, and the homogenization time is 1 min.
[0018] Preferably, the homogenization speed in step (3) is 8000 rpm and the homogenization time is 1 min.
[0019] The present invention provides a sialylated IgG synbiotic oil gel emulsion for promoting intestinal colonization of Bifidobacterium bifidum and a preparation method thereof, which has the advantages of:
[0020] The present invention adopts raw materials divided into beta-sitosterol, glyceryl monostearate, cocoa butter, polyglycerol ricinoleate, phosphate buffer, bifidobacterium bifidum and sialylated IgG, etc., to obtain a synbiotic oil gel emulsion that embeds sialylated IgG and bifidobacterium bifidum; the thermodynamic property of the synbiotic oil gel emulsion is that it is solid at a temperature (<36°C), and is converted into a flowable liquid in the human body (≥36°C), can stably exist in the stomach, and can effectively release sialylated IgG and bifidobacterium bifidum in the intestine, and the released sialylated IgG can simultaneously adhere to the sialidase SiaBb2 on the surface of bifidobacterium bifidum and the receptor FcRn on the surface of intestinal epithelial cells, thereby realizing the colonization of bifidobacterium bifidum in the intestine. This oil gel emulsion effectively protects the biological activity of sialylated IgG and bifidobacterium bifidum after storage and transportation and intestinal directional release, which is of great significance to the further practical application of sialylated IgG and bifidobacterium bifidum. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 Schematic diagram of the crystal morphology of three types of oil gels in the embodiments of the present invention;
[0022] Figure 2 Schematic diagram of the crystal structure (FTIR) of three oil gels in the examples of the present invention;
[0023] Figure 3 Schematic diagram of XRD analysis of oil gel in an embodiment of the present invention;
[0024] Figure 4 : is a thermodynamic analysis diagram of the oil gel in the embodiment of the present invention, wherein A is the melting curve of DSC; B is the crystallization curve of DSC;
[0025] Figure 5 Schematic diagram of the morphological characterization of the oil gel emulsion in the embodiment of the present invention;
[0026] Figure 6 Schematic diagram of simulated digestion of oil gel emulsion in an embodiment of the present invention;
[0027] Figure 7 This is a schematic diagram of in vivo imaging of sialylated IgG promoting colonization of Bifidobacterium bifidum in an embodiment of the present invention;
[0028] Figure 8 This is a schematic diagram of intestinal morphological analysis of sialylated IgG promoting colonization of Bifidobacterium bifidum in an embodiment of the present invention;
[0029] Fig. 9 Schematic diagram of the molecular interaction between the Fc fragment of sialylated IgG, the sialidase SiaBb2 on the surface of Bifidobacterium bifidum and the surface receptor FcRn of intestinal epithelial cells in an embodiment of the present invention; wherein A is the binding site between Fc and SiaBb2; B is the 2D interaction details between Fc and SiaBb2; C is a cartoon model of molecular docking of the FcRn-sialylated IgG-SiaBb2 complex; D is the binding site between FcRn and Fc; E is the 3D interaction details between FcRn and Fc; F is the 2D interaction details between Fc and FcRn, wherein the yellow area represents FcRn, the gray area represents the Fc of sialylated IgG, the green area represents the sialylated sugar chain, and the blue area represents SiaBb2. DETAILED DESCRIPTION
[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0031] Embodiment 1:
[0032] Preparation of Biostime Oil Gel Emulsion:
[0033] (1) β-Sitosterol (Sit), glyceryl monostearate (GMS) and cocoa butter were mixed in a mass ratio of 3:3:94, and then heated to 90° C. and stirred for 1 h until they were completely dissolved; after heating, the mixture was cooled to 4° C. to form an oil gel (O-Sit+GMS) as the oil phase;
[0034] (2) Sialyl IgG, Bifidobacterium bifidum and phosphate buffered saline (PBS, pH 7.0) were homogenized in a high-speed homogenizer at 5000 rpm for 1 min in a mass ratio of 1:40:59 to form an aqueous phase;
[0035] (3) The oil phase, water phase and polyglycerol ricinoleate (PGPR) prepared above were mixed uniformly in a mass ratio of 79:20:1, homogenized in a high-speed homogenizer at 8000 rpm for 1 min, and then immediately cooled in an ice water bath to form a synbiotic oil gel emulsion (E-Sit+GMS).
[0036] Comparative Example 1:
[0037] Preparation of Biostime Oil Gel Emulsion:
[0038] (1) β-Sitosterol (Sit) and cocoa butter were mixed in a mass ratio of 6:94, and then heated to 90° C. and stirred for 1 h until they were completely dissolved; after heating, the mixture was cooled to 4° C. to form an oil gel (O-Sit) as the oil phase;
[0039] (2) Sialyl IgG, Bifidobacterium bifidum and PBS (pH 7.0) were homogenized in a high-speed homogenizer at 5000 rpm for 1 min in a mass ratio of 1:40:59 to form an aqueous phase;
[0040] (3) The oil phase, water phase and PGPR prepared above were mixed uniformly in a mass ratio of 79:20:1, homogenized in a high-speed homogenizer at 8000 rpm for 1 min, and then immediately cooled in an ice water bath to form a synbiotic oil gel emulsion (E-Sit).
[0041] Comparative Example 2:
[0042] Preparation of Biostime Oil Gel Emulsion:
[0043] (1) GMS and cocoa butter were mixed in a mass ratio of 6:94, and then heated to 90°C and stirred for 1 hour until they were completely dissolved; after heating, the mixture was cooled to 4°C to form an oil gel (O-GMS) as the oil phase;
[0044] (2) Sialyl IgG, Bifidobacterium bifidum and PBS (pH 7.0) were homogenized in a high-speed homogenizer at 5000 rpm for 1 min in a mass ratio of 1:40:59 to form an aqueous phase;
[0045] (3) The oil phase, water phase and PGPR prepared above were mixed uniformly in a mass ratio of 79:20:1, homogenized in a high-speed homogenizer at 8000 rpm for 1 min, and then immediately cooled in an ice water bath to form a synbiotic oil gel emulsion (E-GMS).
[0046] Detection:
[0047] 1. Detection of the relevant crystal morphology of the oil gel (oil phase) prepared in Example 1 and Comparative Examples 1-2:
[0048] (1) Polarized light microscopy (PLM) was used to detect the crystal morphology of the three different oil gel samples. Figure 1 As shown in the results, different oil gelling agents significantly affect the microstructure. The oil gel sample (O-Sit) formed by β-sitosterol (Sit) exhibited a typical fibrous network structure composed of elongated needle-like crystals. These crystals showed obvious birefringence characteristics under polarized light and assembled into a dense three-dimensional framework structure. In contrast, the oil gel sample (O-GMS) prepared by glycerol monostearate (GMS) formed a dense but disordered crystalline structure characterized by small birefringent microcrystals of 5-10 μm scattered in irregular cloud-like formations. In the composite system formed by Sit and GMS (O-Sit+GMS), needle-like and plate-like crystals coexisted to form a significantly entangled interconnected network. This composite structure indicates that there is a synergistic effect between Sit and GMS, which enhances the stability and functionality of the oil gel.
[0049] (2) Using pure corn oil, pure oil gelling agent Sit(s), and pure oil gelling agent GMS(s) as controls, the crystal structures of the oil gels of Example 1 and Comparative Examples 1-2 were analyzed by FTIR. The specific results are as follows: Figure 2 As shown, Sit(s) is at 3320.16cm -1 A broad peak appears at 3283.81 cm -1 ; No corresponding peak was observed in pure corn oil. Compared with the pure oil gelling agents Sit(s) and GMS(s), the broad peak areas of the oil gel samples (O-Sit and O-GMS) were reduced, and the peak positions occurred at 3390.31 cm -1 and 3325.97cm -1 , indicating that the hydrogen bond strength is weakened. However, in the O-Sit+GMS composite oil gel, the hydrogen bond strength is slightly increased. In addition, the CH stretching vibration peak of corn oil is at 2926.92 cm -1 After forming oil gel with Sit and GMS, they red-shifted to 2922.68 cm -1 and 2917.23cm -1 , indicating the involvement of van der Waals forces. In the O-Sit+GMS composite system, the peak further red-shifted to 2903.71 cm -1 , indicating that there is a synergistic interaction between Sit and GMS.
[0050] (3) XRD was used to further analyze the polycrystalline structure of the oil gel. Figure 3 As shown, the main diffraction peak of Sit(s) corresponds to a d spacing of The main diffraction peak of GMS(s) is located at a d-spacing of 7.8 In the O-Sit oil gel system, the main diffraction peak appears at a d spacing of In the O-GMS system, the main peaks are located at d spacings of These results indicate that the oleogels formed by a single gelling agent exhibit significant long-spacing diffraction peaks, reflecting strong self-separation and molecular rearrangement capabilities. In contrast, the main diffraction peaks of the oleogels formed by the composite of Sit(s) and GMS(s) (O-Sit+GMS) appear at d spacings of
[0051] The short-spacing diffraction peaks are more prominent, indicating a higher degree of lateral stacking of molecules in its layered structure.
[0052] (4) The thermal stability of the oil gel was evaluated by differential scanning calorimetry (DSC). Figure 4 As shown in the figure, the O-Sit group oil gel exhibited a single endothermic peak and an exothermic peak during the heating and cooling process, respectively, with a melting temperature of 45°C and a crystallization temperature of 42°C. The O-GMS group oil gel absorbed heat and melted when heated to 55°C, and began to crystallize at 40°C. In the O-Sit+GMS composite oil gel, the melting temperature dropped significantly to 37°C, and the crystallization temperature dropped to 35°C. This phenomenon indicates that the interaction between Sit and GMS may lead to a decrease in the melting temperature, further proving the synergistic effect of the two on the thermodynamic properties of the oil gel.
[0053] 2. Test the performance of different oil gel emulsions:
[0054] (1) Detection of the morphology of different oil gel emulsions in water-in-oil (W / O) emulsions and their effects on the encapsulation efficiency of Bifidobacterium bifidum:
[0055] Laser confocal microscopy was used for observation and evaluation. The specific results are as follows Figure 5As shown: the aqueous phase labeled with sodium fluorescein (black) forms a spherical structure embedded in the Nile red-stained oil phase (red), while the DAPI-stained Bifidobacterium bifidum (blue) is evenly distributed in the inner aqueous phase. This composite staining intuitively shows the overall structure of the water-in-oil (W / O) emulsion. Further analysis of the morphology of different oil gel emulsions showed significant differences: in the single-component oil gel emulsions E-Sit and E-GMS, the aqueous phase was unevenly distributed, forming droplets of different sizes, and the encapsulation efficiency of Bifidobacterium bifidum was relatively low. In E-Sit+GMS, the aqueous phase was more evenly distributed, and the number of encapsulated Bifidobacterium bifidum increased significantly. This is because the single-component oil gel emulsion forms a loose network structure, and the synergistic interaction between Sit and GMS prompts it to form a stable and uniform three-dimensional network structure, thereby effectively encapsulating the aqueous phase and significantly improving the encapsulation efficiency of Bifidobacterium bifidum.
[0056] (2) The digestive behaviors of different oil gel emulsions (E-Sit, E-GMS, and E-Sit+GMS) were evaluated under simulated saliva (Salivea), gastric juice (SGF), and intestinal fluid (SIF).
[0057] Specific results such as Figure 6 As shown: In the saliva environment for 30 minutes, all oil gel emulsions (E-Sit, E-GMS and E-Sit+GMS) showed good structural stability. However, after 6 hours of SGF digestion, the E-Sit and E-GMS groups showed partial aggregation and sedimentation of gel particles, while the E-Sit+GMS group maintained structural integrity and showed excellent stability. In the intestinal stage, all oil gel emulsions experienced significant emulsification and dispersion after 6 hours. These results indicate that the E-Sit+GMS oil gel emulsion can effectively protect the encapsulated sialylated IgG and Bifidobacterium bifidum during gastrointestinal transit and ensure their stable release in the intestine.
[0058] (3) Tracking the colonization patterns of Bifidobacterium bifidum and sialylated IgG delivered via emulsion in the gastrointestinal tract using in vivo imaging techniques and mouse experiments to determine:
[0059] Mice of the same batch with the same growth condition were selected for gavage test. The specific results are as follows: Figure 7As shown: Immediately after oral gavage (0h), fluorescence was detected in the stomach of mice in all groups. By the 2nd hour, the fluorescence signal had migrated to the stomach and proximal small intestine, and the signal was observed to advance further into the intestine at the 4th hour. By the 6th hour, the fluorescence intensity of the Bifidobacterium bifidum staining group (B. bifidum-GFP) (green area) gradually weakened and almost completely disappeared at the 24th hour, indicating that Bifidobacterium bifidum itself cannot achieve long-term colonization in the intestine. In contrast, the sialylated IgG staining group (sialylated IgG-Cy5) (red area) was still detectable at the 48th hour. In the co-colonization group (B. bifidum-GFP + sialylated IgG-Cy5), overlapping fluorescence signals (yellow area) were observed from the beginning of oral gavage, indicating that there may be an interaction between Bifidobacterium bifidum-GFP and sialylated IgG-Cy5. By the 48th hour, the B. bifidum-GFP signal was still detectable, indicating that sialylated IgG significantly prolonged the retention time of B. bifidum in the intestine and enhanced its colonization ability.
[0060] To further explore the mechanism by which sialylated IgG promotes colonization of Bifidobacterium bifidum, fluorescence microscopy was performed on colon tissue sections collected 6 hours after oral gavage. Figure 8 As shown in the figure, Bifidobacterium bifidum-GFP is mainly distributed on the mucosal surface of the intestinal epithelium, while sialylated IgG-Cy5 is concentrated in the crypt area of the colonic epithelial cells. After co-administration, sialylated IgG-Cy5 significantly promoted the adhesion of Bifidobacterium bifidum-GFP to the colonic crypts and increased its occupancy in this microenvironment. This suggests that sialylated IgG plays a key role in enhancing the adhesion of Bifidobacterium bifidum to the colonic crypts, thereby effectively promoting its long-term colonization in the intestine.
[0061] 3. To further clarify the structural basis of intestinal colonization mediated by sialylated IgG, molecular simulation docking analysis was used to study the interaction between the surface protein SiaBb2 of Bifidobacterium bifidum, sialylated IgG, and the intestinal cell receptor FcRn. Fig. 9 As shown, the sialic acid sugar chain of Fc is embedded in the binding pocket of SiaBb2 to form a stable complex ( Fig. 9 A). SiaBb2 significantly enhances binding affinity through hydrogen bonding interactions with terminal sialic acid ( Fig. 9 B), wherein multiple amino acids such as ASN262 form hydrogen bonds with the hydroxyl or oxygen atoms of the terminal sialic acid, branched galactose, and N-acetylgalactosamine. In addition, Fig. 9C and 9D show the interaction between Fc and key amino acids of FcRn, such as the interaction between LEU135, ALA134, etc. of FcRn and ASN434, etc. of Fc. These results indicate that there is a strong interaction between the surface protein SiaBb2 of Bifidobacterium bifidum, sialylated IgG and the intestinal cell receptor FcRn, thereby promoting the intestinal colonization of Bifidobacterium bifidum.
[0062] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A sialylated IgG synbiotic oil gel emulsion for promoting intestinal colonization of Bifidobacterium bifidum, characterized in that: The emulsion is composed of the following substances in parts by mass: 34-79 parts of oil phase, 15-20 parts of water phase and 1 part of polyglycerol ricinoleate; the oil phase is composed of β-sitosterol, glyceryl monostearate and cocoa butter; the water phase is composed of phosphate buffer, Bifidobacterium bifidum and sialylated IgG.
2. The emulsion according to claim 1, characterized in that The emulsion is composed of the following substances in parts by mass: 79 parts of oil phase, 20 parts of water phase and 1 part of polyglycerol ricinoleate.
3. The emulsion according to claim 1, characterized in that: The components in the oil phase are 2-4 parts by weight of beta-sitosterol, 2-4 parts by weight of glyceryl monostearate, and 92-96 parts by weight of cocoa butter.
4. The emulsion according to claim 1, characterized in that: The weight proportions of the components in the aqueous phase are 1-2 parts of sialyl IgG, 38-40 parts of Bifidobacterium bifidum, and 58-60 parts of phosphate buffer.
5. A method for preparing the emulsion according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: (1) β-sitosterol, stearin and cocoa butter are mixed, heated and stirred until completely dissolved, and then cooled to form an oil gel, which is used as an oil phase for later use; (2) mixing the phosphate buffer, Bifidobacterium bifidum and sialyl IgG and homogenizing them with a high-speed homogenizer to obtain an aqueous phase for later use; (3) The oil phase, aqueous phase and polyglycerol ricinoleate prepared above are mixed evenly, homogenized, and then cooled in an ice water bath to form a synbiotic oil gel emulsion.
6. The preparation method according to claim 5, characterized in that: The heating and stirring temperature in step (1) is 90° C. and the stirring time is 1 h.
7. The preparation method according to claim 5, characterized in that: The final cooling temperature in step (1) is 4°C.
8. The preparation method according to claim 5, characterized in that: The speed of the homogenization process in step (2) is 5000 rpm, and the homogenization time is 1 min.
9. The preparation method according to claim 5, characterized in that: The homogenization speed in step (3) is 8000 rpm and the homogenization time is 1 min.