Multi-component synergistic emulsion gel with function of regulating intestinal flora and preparation method of multi-component synergistic emulsion gel

Abstract

The invention relates to the technical field of food processing, in particular to multi-component synergistic emulsion gel with an intestinal flora regulating function and a preparation method of the multi-component synergistic emulsion gel. The emulsion gel is prepared from the following raw materials in parts by weight: 1.0 to 3.0 parts of EGCG-collagen peptide conjugate; 3.0 to 5.0 parts of fructo-oligosaccharide; 2.0 to 3.0 parts of resistant starch; 1.0 to 3.0 parts of polyunsaturated fatty acid; 0.5 to 2.5 parts of a gel matrix; 0.1 to 1.0 part of a natural flavoring agent; 0.1 to 0.5 part of an emulsifier; and 0.1 to 0.5 part of calcium chloride. The beneficial effects comprise that functional components are designed cooperatively, fructo-oligosaccharide, resistant starch and polyunsaturated fatty acids are combined, and generation of short-chain fatty acids is promoted through bifidobacterium proliferation and metabolism complementation of butyric acid producing bacteria; the EGCG-fish collagen peptide conjugate partially replaces a chemical emulsifier, has the functions of oxidation resistance and interface stabilization, and solves the problem of DHA / EPA oxidation; a pH-sensitive gel network structure is formed by crosslinking calcium ions with sodium alginate, so that damage of high temperature to prebiotics and DHA / EPA is avoided, and colon-targeted release is realized at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and in particular to a multi-component synergistic emulsion gel with the function of regulating intestinal flora and a preparation method thereof. Background Art

[0002] Imbalances in the homeostasis of the intestinal flora are closely related to metabolic syndrome, neurological diseases, and immune dysfunction. The use of substances such as prebiotics, probiotics, synbiotics, and postbiotics is an important means of regulating intestinal microecological imbalances. Known prebiotics include oligofructose, inulin, and resistant starch, but single prebiotics (such as oligosaccharides) are insufficiently targeted to specific bacterial communities. For example, oligofructose can promote the proliferation of bifidobacteria, while resistant starch can induce the metabolism of butyrate-producing bacteria. If the two are combined, the production of short-chain fatty acids can be increased by 2.3 times. Therefore, the synergistic application of multiple prebiotics is currently an important means to enhance the effectiveness of prebiotics.

[0003] For example, the authorized invention patent "A milk companion for infant formula milk powder and its preparation method" (application number: CN2011103594385) uses fat powder with glucose as the carrier, maltodextrin and compound prebiotics (1-10% galacto-oligosaccharides and 0.5-5% fructo-oligosaccharides) as the main ingredients to make milk companion, which can improve the digestive function of infants and young children; and the authorized invention patent "Synbiotics of Bacillus licheniformis and oligosaccharide prebiotics and their compositions and preparations" (application number: CN20 0910011267X) combines medicinal live bacterial powder of Bacillus licheniformis, oligosaccharide prebiotics (including one or more of xylooligosaccharides, fructo-oligosaccharides, isomalto-oligosaccharides, polydextrose, inulin, galacto-oligosaccharides, and soy oligosaccharides), and excipients into oral tablets, orally disintegrating tablets, dispersible tablets, enteric-coated tablets, granules, capsules, dry suspensions, and topical tablets. These formulations effectively promote the growth and reproduction of Bacillus licheniformis, inhibit the growth of harmful bacteria, enhance host immunity, reduce diarrhea, and improve health. However, current prebiotic combination formulations are all manufactured through encapsulation, extrusion granulation, and tableting technologies. These technologies involve high-temperature and high-pressure treatments that can easily lead to loss of prebiotic function, resulting in less than gentle processing conditions. Furthermore, the prebiotic combination is not stable in the gastrointestinal tract, making it difficult to achieve targeted release in the colon.

[0004] Furthermore, the combination of unsaturated fatty acids (such as the ω-3 polyunsaturated fatty acids EPA and DHA) and prebiotics can synergistically enhance gut microbial metabolism through multiple pathways. For example, prebiotics provide a carbon source for short-chain fatty acid-producing bacteria (such as Bifidobacterium and Roseburia), while unsaturated fatty acids activate peroxisome proliferator-activated receptor γ (PPARγ) to upregulate butyrate synthase genes (such as butyryl-CoA transferase), increasing butyrate production by 30-50%. Furthermore, unsaturated fatty acids enhance the targeting of prebiotics to specific microbiota by altering the intestinal physicochemical environment; unsaturated fatty acids enhance the stability of prebiotics in the gastrointestinal tract through physicochemical interactions; and unsaturated fatty acids and prebiotics have additive effects through the TLR4 / NF-κB and NLRP3 inflammasome pathways. Therefore, the combined application of unsaturated fatty acids and prebiotics plays an important role. However, unsaturated fatty acids are easily oxidized in liquid systems and have poor stability.

[0005] Therefore, developing a novel multi-component synergistic prebiotic emulsion gel that can maintain the activity of prebiotics while improving their stability in the gastrointestinal tract and achieving targeted release in the colon has become a key and difficult issue in current research. This requires not only considering the selective combination of prebiotics but also developing novel processing technologies to overcome the shortcomings of existing technologies and achieve synergistic enhancement and targeted release of prebiotics, thereby better regulating the function of the intestinal flora and improving the symptoms of related diseases. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing a multi-component synergistic emulsion gel with the function of regulating intestinal flora.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: to provide a multi-component synergistic emulsion gel with the function of regulating intestinal flora, which is made of the following raw materials in parts by weight: EGCG-collagen peptide conjugate 1.0-3.0 parts; 3.0-5.0 parts of oligofructose; 2.0-3.0 parts of resistant starch; 1.0-3.0 parts of polyunsaturated fatty acids; 0.5-2.5 parts of gel matrix; 0.1-1.0 parts of natural flavoring; Emulsifier 0.1-0.5 parts; 0.1-0.5 parts of calcium chloride.

[0008] Furthermore, in the above-mentioned multi-component synergistic emulsion gel having the function of regulating intestinal flora, the polyunsaturated fatty acid is a mixture of DHA and EPA in a mass ratio of 1:1.

[0009] Furthermore, in the above-mentioned multi-component synergistic emulsion gel having the function of regulating intestinal flora, the gel matrix is ​​sodium alginate.

[0010] Furthermore, in the above-mentioned multi-component emulsion gel having the function of regulating intestinal flora, the natural flavoring agent is citrus extract or vanillin.

[0011] Furthermore, in the multi-component emulsion gel having the function of regulating intestinal flora, the emulsifier is Tween 20.

[0012] Furthermore, the multi-component emulsion gel having the function of regulating intestinal flora is specifically made of the following raw materials in the following weight percentages: EGCG-collagen peptide conjugate 1.0-3.0wt%; Fructooligosaccharides 3.0-5.0wt%; Resistant starch 2.0-3.0wt%; Polyunsaturated fatty acids 1.0-3.0wt%; Sodium alginate 0.5-2.5wt%; Natural flavoring 0.1-1.0wt%; Tween 20 0.1-0.5wt%; Calcium chloride 0.1-0.5wt%; The balance is water.

[0013] Another technical solution of the present invention is to provide a method for preparing the multi-component synergistic emulsion gel having the function of regulating intestinal flora, comprising the following steps: The EGCG-collagen peptide conjugate was dissolved in water, and then an emulsifier and polyunsaturated fatty acids were added and mixed to obtain a nanoemulsion; Adding the gel matrix and natural flavoring to the nanoemulsion, adding water and stirring until completely dissolved; then adding oligofructose and resistant starch, adding water and magnetic stirring until completely dispersed, to obtain a mixed system; Finally, calcium chloride is dissolved in water to obtain a calcium chloride solution, which is then added dropwise to the mixed system via a syringe pump. Ultrasonic treatment is simultaneously applied to promote uniform diffusion of calcium ions, thereby obtaining the multi-component synergistic emulsion gel with the function of regulating intestinal flora.

[0014] Furthermore, in the preparation method of the above-mentioned multi-component synergistic emulsion gel with the function of regulating intestinal flora, the EGCG-collagen peptide conjugate is prepared by the following method: preparing a collagen peptide dispersion with water, stirring it with a magnetic stirrer at 4°C for 24 hours, then adding EGCG at a mass ratio of collagen peptide: EGCG = 1:0.1-0.4, adjusting the pH to 9.0 with 6M / L sodium hydroxide solution, and stirring it with a magnetic stirrer at 4°C for 24 hours to obtain an EGCG-collagen peptide conjugate solution; The EGCG-collagen peptide conjugate solution was adjusted to pH 7.0 with 6M / L hydrochloric acid, and then placed in a 2000Da dialysis bag for 48 hours, with the water changed 8 times to obtain the dialyzed residual solution of the EGCG-collagen peptide conjugate; The dialyzed residual solution of the EGCG-collagen peptide conjugate was pre-frozen and then freeze-dried for 48 hours to obtain the EGCG-collagen peptide conjugate.

[0015] Furthermore, the preparation method of the multi-component synergistic emulsion gel having the function of regulating intestinal flora specifically comprises the following steps: Step S1, preparing a collagen peptide dispersion by adding fish collagen peptide to water, stirring the mixture with a magnetic stirrer at 4°C for 24 hours, then adding EGCG at a mass ratio of fish collagen peptide to EGCG = 1:0.1, adjusting the pH to 9.0 with 6M / L sodium hydroxide solution, and stirring the mixture with a magnetic stirrer at 4°C for 24 hours to obtain an EGCG-collagen peptide conjugate solution; Step S2, adjusting the pH of the EGCG-fish collagen peptide conjugate solution to 7.0 with 6M / L hydrochloric acid, then placing it in a 2000Da dialysis bag and dialyzing it for 48 hours, changing the water 8 times, to obtain a dialyzed residual solution of the EGCG-fish collagen peptide conjugate; Step S3, pre-freezing the dialyzed residual liquid of the EGCG-fish collagen peptide conjugate and then freeze-drying it for 48 hours to obtain the EGCG-fish collagen peptide conjugate; Step S4, dissolving the EGCG-fish collagen peptide conjugate in 50°C water, magnetically stirring for 30 minutes, and adjusting the pH to 6.8; adding an emulsifier and polyunsaturated fatty acids in a mass ratio of 1:1 and mixing evenly, first mixing and stirring for 5 minutes using a high-speed shearing machine at 10,000 rpm to form a coarse emulsion; then subjecting the coarse emulsion to two-stage high-pressure homogenization: the first stage homogenization is cycled at a pressure of 20 MPa for 3 times to break up large-particle oil droplets, and the second stage homogenization is cycled at a pressure of 50 MPa for 2 times to obtain a nanoemulsion; Step S5, adding sodium alginate and natural flavoring to the above-mentioned nanoemulsion, adding water and magnetically stirring until completely dissolved; then adding oligofructose and resistant starch, adding water and magnetically stirring until completely dispersed, to obtain a mixed system; finally, dissolving calcium chloride in water to obtain a calcium chloride solution, adding the calcium chloride solution dropwise to the above-mentioned mixed system through a syringe pump, and simultaneously applying ultrasonic treatment to promote uniform diffusion of calcium ions, to obtain the multi-component synergistic emulsion gel with the function of regulating intestinal flora.

[0016] The beneficial effects of the present invention are as follows: In the multi-component emulsion gel of the present application that synergistically regulates intestinal flora, the emulsion gel system can greatly promote the stability of unsaturated fatty acids in the storage and digestive environment of the entire delivery system. 1. This invention uses an EGCG-peptide conjugate to partially replace traditional chemical emulsifiers (such as Tween 20). This not only acts as an emulsifier but also possesses strong antioxidant properties, effectively preventing the oxidation of polyunsaturated fatty acids (DHA / EPA) in the emulsion system. This significantly improves the stability of DHA / EPA and solves the problem of functional loss during high-temperature and high-pressure processing in existing prebiotic combination preparations. 2. This invention uses synergistic effects of multiple ingredients, including EGCG-peptide conjugates, oligofructose, resistant starch, and polyunsaturated fatty acids, to form a stable emulsion gel system. This effectively improves the stability of prebiotics in the gastrointestinal tract and achieves targeted release in the colon, overcoming the disadvantage of traditional preparations being destroyed by gastric acid. 3. In this invention, the combination of oligofructose (3-5%) and resistant starch (2-3%) significantly increased the abundance of bifidobacteria and butyrate production by 2.0 times, achieving precise targeting of specific bacterial communities and addressing the lack of targeting of single prebiotics. 4. This invention uses calcium alginate gel as the gel matrix, forming a pH-sensitive emulsion gel network through calcium ion cross-linking. This achieves rapid disintegration at the colonic pH (6.8-7.0), enabling the simultaneous release of prebiotics and DHA / EPA, thereby improving drug absorption efficiency. 5. The emulsion gel preparation process of the present invention is simple, avoiding traditional high-temperature and high-pressure processes such as encapsulation, extrusion granulation, and tableting, effectively maintaining the activity of prebiotics and meeting the personalized needs of different groups of people. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Shown is a schematic diagram of Fourier transform infrared spectra of fish collagen peptide (E0) and fish collagen peptide-EGCG conjugate (E1-E4 represents a mass ratio of fish collagen peptide:EGCG = 1:0.1-0.4) according to a specific embodiment of the present invention; Figure 2Schematic diagram showing the ABTS free radical scavenging ability of fish collagen peptide (E0) and fish collagen peptide-EGCG conjugate (E1-E4 represents the mass ratio of fish collagen peptide:EGCG = 1:0.1-0.4) according to a specific embodiment of the present invention. DETAILED DESCRIPTION

[0018] To illustrate the technical content, achieved objectives and effects of the present invention in detail, the following description is given in conjunction with the embodiments and accompanying drawings.

[0019] The key concepts of the present invention are: Imbalances in intestinal microbial homeostasis are closely related to metabolic diseases and immune dysfunction. Traditional prebiotic preparations (such as oligofructose and resistant starch) suffer from insufficient targeting, loss of processing activity, and a single component. While polyunsaturated fatty acids (such as DHA / EPA) can synergistically regulate microbial metabolism, their susceptibility to oxidation and low dispersibility in water limit their application. Existing technologies rely on high-temperature tableting or chemical emulsifiers, resulting in poor stability of functional ingredients, low targeted release efficiency, potential safety issues, and a lack of multi-component synergistic mechanism design.

[0020] This invention innovatively develops a multi-component synergistic emulsion gel system. The core technology includes the coordinated design of functional components, the combination of oligofructose (3-5%), resistant starch (2-3%) and polyunsaturated fatty acids (DHA / EPA, 1-3%), and the promotion of short-chain fatty acid production through the proliferation of bifidobacteria and the metabolic complementarity of butyrate-producing bacteria; the EGCG-fish collagen peptide conjugate partially replaces the chemical emulsifier, which has both antioxidant and interface stabilization functions and solves the DHA / EPA oxidation problem; sodium alginate is cross-linked by calcium ions to form a pH-sensitive gel network structure, and the cold-induced gelation technology avoids the damage of prebiotics and DHA / EPA to high temperature, greatly improving their activity retention rate, while achieving colon-targeted release.

[0021] Example 1 A method for preparing a multi-component synergistic emulsion gel with the function of regulating intestinal flora comprises the following steps: 1) EGCG-fish collagen peptide conjugation: A 5.0 wt% collagen peptide dispersion was prepared using ultrapure water and stirred at 4°C using a magnetic stirrer for 24 h. EGCG (epigallocatechin gallate) was then added at a mass ratio of 1:0.2 (fish collagen peptide:EGCG). The pH was adjusted to 9.0 with 6 M / L sodium hydroxide and stirred at 4°C using a magnetic stirrer for 24 h to obtain an EGCG-fish collagen peptide conjugate solution. 2) Dialysis: The prepared EGCG-fish collagen peptide conjugate solution was adjusted to pH 7.0 with 6M / L hydrochloric acid, and then placed in a 2000Da dialysis bag and dialyzed for 48 hours, changing the water 8 times; 3) Freeze-drying: The dialyzed residual solution containing the EGCG-fish collagen peptide conjugate was pre-frozen and then freeze-dried for 48 hours to obtain the EGCG-fish collagen peptide conjugate, which was then stored at 4°C for later use; 4) Emulsion Preparation: EGCG-fish collagen peptide conjugate was dissolved in 50°C deionized water (concentration 4 wt%) and magnetically stirred (500 rpm) for 30 minutes. The pH was adjusted to 6.8. 0.2 wt% emulsifier (Tween 20) and 6 wt% DHA / EPA (docosahexaenoic acid:eicosapentaenoic acid mass ratio of 1:1) were added. The raw materials were mixed uniformly and stirred using a high-speed shear at 10,000 rpm for 5 minutes to form a coarse emulsion. The coarse emulsion was then subjected to a two-stage high-pressure homogenization: three cycles of primary homogenization at 20 MPa to break up large oil droplets, and two cycles of secondary homogenization at 50 MPa to obtain a nanoemulsion. 5) Preparation of a multi-component gel base: Sodium alginate (1.5 wt%) and 0.8 wt% natural flavoring (citrus extract) were added to 50 wt% of the above nanoemulsion, and the mixture was adjusted to a total weight percentage of 80% with water. The mixture was magnetically stirred (800 rpm) for 2 hours until completely dissolved. Fructooligosaccharides (4.0 wt%) and resistant starch (3.0 wt%) were then added, and the mixture was adjusted to a total weight percentage of 90% with water. The mixture was magnetically stirred (800 rpm) for 6 hours until completely dispersed. 6) Emulsion Gel Preparation: Calcium chloride was dissolved in deionized water (concentration 3 wt%) and added dropwise to the mixture via a syringe pump at a rate of 0.5 mL / min. Ultrasonic treatment (40 kHz, 100 W) was simultaneously applied to promote uniform diffusion of calcium ions. The final calcium chloride concentration in the mixture was 0.3 wt%. The sum of the weight percentages of all raw materials used was 100%.

[0022] Example 2 A method for preparing a multi-component synergistic emulsion gel with the function of regulating intestinal flora comprises the following steps: 1) EGCG-fish collagen peptide conjugation: A 5.0 wt% collagen peptide dispersion was prepared using ultrapure water and stirred at 4°C using a magnetic stirrer for 24 h. EGCG was then added at a mass ratio of fish collagen peptide to EGCG of 1:0.1. The pH was adjusted to 9.0 with 6 M / L sodium hydroxide and stirred at 4°C using a magnetic stirrer for 24 h to obtain an EGCG-fish collagen peptide conjugate solution. 2) Dialysis: The prepared EGCG-fish collagen peptide conjugate solution was adjusted to pH 7.0 with 6M / L hydrochloric acid, and then placed in a 2000Da dialysis bag and dialyzed for 48 hours, changing the water 8 times; 3) Freeze-drying: The dialyzed residual solution containing the EGCG-fish collagen peptide conjugate was pre-frozen and then freeze-dried for 48 hours to obtain the EGCG-fish collagen peptide conjugate, which was then stored at 4°C for later use; 4) Emulsion Preparation: EGCG-fish collagen peptide conjugate was dissolved in 50°C deionized water (concentration 2 wt%) and magnetically stirred (500 rpm) for 30 minutes. The pH was adjusted to 6.8. 0.2 wt% emulsifier (Tween 20) and 3 wt% DHA / EPA (mass ratio 1:1) were added. The raw materials were mixed thoroughly and stirred using a high-speed shear at 10,000 rpm for 5 minutes to form a coarse emulsion. The coarse emulsion was then subjected to a two-stage high-pressure homogenization: three cycles of primary homogenization at 20 MPa to break up large oil droplets, and two cycles of secondary homogenization at 50 MPa to obtain a nanoemulsion. 5) Preparation of a multi-component gum base: Sodium alginate (0.5 wt%) and 0.8 wt% natural flavoring (vanillin) were added to 50 wt% of the above nanoemulsion, and the mixture was adjusted to a total weight percentage of 80% with water. The mixture was magnetically stirred (800 rpm) for 2 hours until completely dissolved. Fructooligosaccharides (4.0 wt%) and resistant starch (3.0 wt%) were then added, and the mixture was adjusted to a total weight percentage of 90% with water. The mixture was magnetically stirred (800 rpm) for 6 hours until completely dispersed. 6) Emulsion gel preparation: Calcium chloride was dissolved in deionized water (concentration 3 wt%) and added dropwise to the above mixture via a syringe pump at a rate of 0.5 mL / min. Ultrasonic treatment (40 kHz, 100 W) was simultaneously applied to promote uniform diffusion of calcium ions. The final calcium chloride concentration in the mixture was 0.3 wt%, and the sum of the weight percentages of each component was 100%.

[0023] Example 3 A method for preparing a multi-component synergistic emulsion gel with the function of regulating intestinal flora comprises the following steps: 1) EGCG-fish collagen peptide conjugation: A 5.0 wt% collagen peptide dispersion was prepared using ultrapure water and stirred at 4°C using a magnetic stirrer for 24 h. EGCG was then added at a mass ratio of fish collagen peptide to EGCG of 1:0.3. The pH was adjusted to 9.0 with 6 M / L sodium hydroxide and stirred at 4°C using a magnetic stirrer for 24 h to obtain an EGCG-fish collagen peptide conjugate solution. 2) Dialysis: The prepared EGCG-fish collagen peptide conjugate solution was adjusted to pH 7.0 with 6M / L hydrochloric acid, and then placed in a 2000Da dialysis bag and dialyzed for 48 hours, changing the water 8 times; 3) Freeze-drying: The dialyzed residual solution containing the EGCG-fish collagen peptide conjugate was pre-frozen and then freeze-dried for 48 hours to obtain the EGCG-fish collagen peptide conjugate, which was then stored at 4°C for later use; 4) Emulsion Preparation: EGCG-fish collagen peptide conjugate was dissolved in 50°C deionized water (concentration 6 wt%) and magnetically stirred (500 rpm) for 30 minutes. The pH was adjusted to 6.8. 0.2 wt% emulsifier (Tween 20) and 6 wt% DHA / EPA (mass ratio 1:1) were added. All ingredients were mixed thoroughly and stirred using a high-speed shear at 10,000 rpm for 5 minutes to form a coarse emulsion. The coarse emulsion was then subjected to a two-stage high-pressure homogenization process: three cycles of primary homogenization at 20 MPa to break up large oil droplets, followed by two cycles of secondary homogenization at 50 MPa to obtain a nanoemulsion. 5) Preparation of a multi-component gel base: Sodium alginate (2.5 wt%) and 0.8 wt% natural flavoring (citrus extract) were added to 50 wt% of the above nanoemulsion, and the mixture was adjusted to a total weight percentage of 80% with water. The mixture was magnetically stirred (800 rpm) for 2 hours until completely dissolved. Fructooligosaccharides (3.0 wt%) and resistant starch (2.0 wt%) were then added, and the mixture was adjusted to a total weight percentage of 90% with water. The mixture was magnetically stirred (800 rpm) for 6 hours until completely dispersed. 6) Emulsion gel preparation: Calcium chloride was dissolved in deionized water (concentration 3 wt%) and added dropwise to the above mixture via a syringe pump at a rate of 0.5 mL / min. Ultrasonic treatment (40 kHz, 100 W) was simultaneously applied to promote uniform diffusion of calcium ions. The final calcium chloride concentration in the mixture was 0.3 wt%, and the sum of the weight percentages of each component was 100%.

[0024] Comparative Example 1 Example 1 was repeated, except that EGCG-fish collagen peptide conjugate was not added in step 4), and steps 1) to 3) were unnecessary.

[0025] Comparative Example 2 Example 1 was repeated, except that 6 wt % DHA / EPA (mass ratio 1:1) was not added in step 4).

[0026] Comparative Example 3 Example 1 was repeated, except that sodium alginate (1.5 wt %) was not added in step 5).

[0027] First, the EGCG-fish collagen peptide conjugates mentioned in all the above examples were characterized, and the results are as follows: Figure 1Fourier transform infrared spectra of fish collagen peptide (E0) and fish collagen peptide-EGCG conjugate (E1-E4 represent the mass ratio of fish collagen peptide: EGCG = 1:0.1-0.4); Figure 2 It is the ABTS free radical scavenging ability of fish collagen peptide (E0) and fish collagen peptide-EGCG conjugate (E1-E4 represents the mass ratio of fish collagen peptide: EGCG = 1:0.1-0.4).

[0028] from Figure 1 From the spectrum, the blue shift of the amide II band indicates that the phenolic hydroxyl group (-OH) of EGCG forms a hydrogen bond with the amino group (-NH2) or amide group (-CONH-) of the fish collagen peptide, resulting in restricted vibration of the NH bond, increased bond strength, and increased vibration frequency; if EGCG is covalently bound to the amino group of the fish collagen peptide through Michael addition or Schiff base reaction, it will also directly change the electron distribution and vibration mode of the CN bond, causing a blue shift. Therefore, the blue shift of the amide II band confirms that EGCG and fish collagen peptide significantly change the secondary structure and molecular conformation of the fish collagen peptide through the synergistic effect of hydrogen bonds and covalent bonds. This structural change is the core mechanism for the conjugate to have antioxidant, emulsifying and targeted delivery functions, and provides a key theoretical basis for the design of multi-component synergistic functional food delivery systems. In addition, by Figure 2 It can be seen that when the addition amount of EGCG is equal to 1:0.1, the ABTS scavenging activity is the highest, and its antioxidant capacity does not change much when the addition amount of EGCG is increased again.

[0029] Then, the emulsion gels prepared in Examples 1-3 and Comparative Examples 1-3 were tested. The test results are shown in Table 1 below, which shows the changes in peroxide value (POV, meq / kg) of different emulsion gel samples during storage at 4°C for 28 days.

[0030] Table 1

[0031] As shown in Table 1, a comparison of the results of Examples 1-3 and Comparative Example 1 shows that the inhibitory effect of DHA / EPA fatty acid oxidation becomes more pronounced with increasing addition of the fish collagen peptide-EGCG conjugate. This is likely because EGCG contains multiple catechol groups (ortho-hydroxyl groups), which can directly capture free radicals (such as ROO• and RO•) in lipid oxidation chain reactions, thereby blocking the oxidation chain reaction. Furthermore, the amino acid residues (such as histidine and tyrosine) in the fish collagen peptide can assist in scavenging free radicals by donating hydrogen or transferring electrons, thus complementing EGCG. Furthermore, the fish collagen peptide-EGCG conjugate is amphiphilic (EGCG is hydrophilic, while the fish collagen peptide is partially lipophilic), allowing it to accumulate at the lipid-water interface, preferentially protecting oils and fats in easily emulsified systems.

[0032] In addition, comparing the results of Example 1 and Comparative Example 3, the emulsion gel shows a more obvious advantage in inhibiting oil oxidation than the ordinary emulsion, which is mainly attributed to its unique physicochemical structure and multiple protection mechanisms. On the one hand, the emulsion gel forms a three-dimensional cross-linked network by cross-linking sodium alginate with calcium ions, significantly reducing the oxygen diffusion coefficient, and wrapping the oil droplets in smaller micro-regions, reducing the contact area between the oil and oxygen. On the other hand, the gel network can fix the fish collagen peptide-EGCG conjugate in the system through hydrogen bonds or hydrophobic interactions, and enrich antioxidants at the oil interface to achieve synergistic protection; at the same time, the gel network prevents the oil droplets from coalescing through the steric effect, avoiding the risk of local high-concentration oxidation due to phase separation.

[0033] Table 2

[0034] As shown in Table 2, which demonstrates the in vitro microbial regulation effects of different emulsion gel samples, and compares the results of Examples 1-2 and Comparative Example 2, it is shown that increasing the amount of DHA / EPA (ω-3 polyunsaturated fatty acids) added increases the bifidobacterium proliferation rate, E. coli inhibition rate, and butyrate production in in vitro simulated intestinal microbial fermentation. This phenomenon is closely related to the following multi-level biological mechanisms. Bifidobacteria express esterases (lipases) and fatty acid dehydrogenases, which break down DHA / EPA into short-chain fatty acids (such as propionate and acetate) or acetyl-CoA, providing energy and carbon sources for butyrate-producing bacteria. Furthermore, DHA / EPA scavenges reactive oxygen species (ROS), alleviating oxidative stress damage to bifidobacteria and prolonging their proliferation cycle. Furthermore, the acetic and lactic acids produced by bifidobacteria metabolizing DHA / EPA lower the environmental pH (<5.5), directly disrupting the membrane integrity of E. coli. This mechanism suggests the potential for synergistic applications of ω-3 fatty acids and prebiotics, but the addition concentration and dosage form must be carefully controlled to avoid excessive inhibitory effects.

[0035] In addition, a comparison of the results of Example 1 and Comparative Example 1 showed that the addition of EGCG-fish collagen peptide conjugate to the emulsion gel slightly increased the bifidobacterium proliferation rate and E. coli inhibition rate, while significantly increasing butyrate production. This may be because fish collagen peptides contain specific amino acids such as glycine and proline, which can provide a nitrogen source and energy for bifidobacteria. The acetic acid and lactic acid produced can serve as substrates for butyrate-producing bacteria, generating butyrate through the butyrate synthesis pathway. In addition, EGCG can reduce oxidative stress in the bacterial flora by scavenging reactive oxygen species (ROS). The results of Example 1 and Comparative Example 3 show that the emulsion gel exhibits more obvious advantages than ordinary emulsion in promoting the proliferation of bifidobacteria, inhibiting the proliferation of Escherichia coli and promoting the production of butyric acid. This is mainly because the three-dimensional network structure of the emulsion gel encapsulates active ingredients (such as resistant starch, oligofructose, polyunsaturated fatty acids, EGCG-fish collagen peptide conjugate) through hydrogen bonds or hydrophobic interactions, allowing them to be slowly released. It can maintain the long-term effective concentration of polyphenols (such as EGCG) or bacterial substrates, continuously promote bifidobacteria or inhibit Escherichia coli, and avoid local concentration fluctuations caused by the rapid release of ingredients in ordinary emulsions.

[0036] In summary, the present invention has the following significant advantages: (1) The present invention applies EGCG-polypeptide conjugate to the emulsion gel system, which reduces the storage oxidation rate of DHA / EPA and enhances its stability under the protection of antioxidant capacity and gel network, while also reducing the gastric acid degradation rate of prebiotics (such as oligofructose).

[0037] (2) The combination of oligofructose (3-5%) and resistant starch (2-3%) provided by the present invention increased the abundance of bifidobacteria by 2.5 times and butyrate production by 2.0 times; in addition, DHA / EPA further upregulated butyrate synthase expression by activating the PPARγ pathway.

[0038] (3) The calcium alginate gel provided by the present invention disintegrates at a colonic pH (6.8-7.0), and the colonic release synchronization rate of prebiotics and DHA / EPA is significantly enhanced.

[0039] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's description and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A multi-component emulsion gel with the function of regulating intestinal flora, characterized in that: The invention is made of the following raw materials in parts by weight: EGCG-collagen peptide conjugate 1.0-3.0 parts; 3.0-5.0 parts of oligofructose; 2.0-3.0 parts of resistant starch; 1.0-3.0 parts of polyunsaturated fatty acids; 0.5-2.5 parts of gel matrix; 0.1-1.0 parts of natural flavoring; Emulsifier 0.1-0.5 parts; 0.1-0.5 parts of calcium chloride.

2. The multi-component emulsion gel having the function of regulating intestinal flora according to claim 1, characterized in that: The polyunsaturated fatty acid is a mixture of DHA and EPA in a mass ratio of 1:

1.

3. The multi-component emulsion gel having the function of regulating intestinal flora according to claim 1, characterized in that: The gel matrix is ​​sodium alginate.

4. The multi-component emulsion gel having the function of regulating intestinal flora according to claim 1, characterized in that: The natural flavoring agent is citrus extract or vanillin.

5. The multi-component emulsion gel having the function of regulating intestinal flora according to claim 1, characterized in that: The emulsifier is Tween 20.

6. The multi-component emulsion gel having the function of regulating intestinal flora according to claim 1, characterized in that: Specifically made of the following raw materials in weight percentage: EGCG-collagen peptide conjugate 1.0-3.0wt%; Fructooligosaccharides 3.0-5.0wt%; Resistant starch 2.0-3.0wt%; Polyunsaturated fatty acids 1.0-3.0wt%; Sodium alginate 0.5-2.5wt%; Natural flavoring 0.1-1.0wt%; Tween 20 0.1-0.5wt%; Calcium chloride 0.1-0.5wt%; The balance is water.

7. A method for preparing the multi-component synergistic emulsion gel with the function of regulating intestinal flora according to any one of claims 1 to 6, characterized in that: The steps include: The EGCG-collagen peptide conjugate was dissolved in water, and then an emulsifier and polyunsaturated fatty acids were added and mixed to obtain a nanoemulsion; Adding the gel matrix and natural flavoring to the nanoemulsion, adding water and stirring until completely dissolved; then adding oligofructose and resistant starch, adding water and magnetic stirring until completely dispersed, to obtain a mixed system; Finally, calcium chloride is dissolved in water to obtain a calcium chloride solution, which is then added dropwise to the mixed system via a syringe pump. Ultrasonic treatment is simultaneously applied to promote uniform diffusion of calcium ions, thereby obtaining the multi-component synergistic emulsion gel with the function of regulating intestinal flora.

8. The method for preparing the multi-component synergistic emulsion gel with the function of regulating intestinal flora according to claim 7, characterized in that: The EGCG-collagen peptide conjugate is prepared by the following method: Collagen peptide was prepared into a collagen peptide dispersion with water, stirred at 4°C for 24 hours using a magnetic stirrer, and then EGCG was added at a mass ratio of collagen peptide to EGCG of 1:0.1-0.

4. The pH was adjusted to 9.0 with 6M / L sodium hydroxide solution, and stirred at 4°C for 24 hours using a magnetic stirrer to obtain an EGCG-collagen peptide conjugate solution. The EGCG-collagen peptide conjugate solution was adjusted to pH 7.0 with 6M / L hydrochloric acid, and then placed in a 2000Da dialysis bag for 48 hours, with the water changed 8 times to obtain the dialyzed residual solution of the EGCG-collagen peptide conjugate; The dialyzed residual solution of the EGCG-collagen peptide conjugate was pre-frozen and then freeze-dried for 48 hours to obtain the EGCG-collagen peptide conjugate.

9. The method for preparing the multi-component synergistic emulsion gel with the function of regulating intestinal flora according to claim 7, characterized in that: The specific steps include: Step S1, preparing a collagen peptide dispersion by adding fish collagen peptide to water, stirring the mixture with a magnetic stirrer at 4°C for 24 hours, then adding EGCG at a mass ratio of fish collagen peptide to EGCG = 1:0.1, adjusting the pH to 9.0 with 6M / L sodium hydroxide solution, and stirring the mixture with a magnetic stirrer at 4°C for 24 hours to obtain an EGCG-collagen peptide conjugate solution; Step S2, adjusting the pH of the EGCG-fish collagen peptide conjugate solution to 7.0 with 6M / L hydrochloric acid, then placing it in a 2000Da dialysis bag and dialyzing it for 48 hours, changing the water 8 times, to obtain a dialyzed residual solution of the EGCG-fish collagen peptide conjugate; Step S3, pre-freezing the dialyzed residual liquid of the EGCG-fish collagen peptide conjugate and then freeze-drying it for 48 hours to obtain the EGCG-fish collagen peptide conjugate; Step S4, dissolving the EGCG-fish collagen peptide conjugate in 50°C water, magnetically stirring for 30 minutes, and adjusting the pH to 6.8; adding an emulsifier and polyunsaturated fatty acids in a mass ratio of 1:1 and mixing evenly, first mixing and stirring for 5 minutes using a high-speed shearing machine at 10,000 rpm to form a coarse emulsion; then subjecting the coarse emulsion to two-stage high-pressure homogenization: the first stage homogenization is cycled at a pressure of 20 MPa for 3 times to break up large-particle oil droplets, and the second stage homogenization is cycled at a pressure of 50 MPa for 2 times to obtain a nanoemulsion; Step S5, adding sodium alginate and natural flavoring to the above-mentioned nanoemulsion, adding water and magnetically stirring until completely dissolved; then adding oligofructose and resistant starch, adding water and magnetically stirring until completely dispersed, to obtain a mixed system; finally, dissolving calcium chloride in water to obtain a calcium chloride solution, adding the calcium chloride solution dropwise to the above-mentioned mixed system through a syringe pump, and simultaneously applying ultrasonic treatment to promote uniform diffusion of calcium ions, to obtain the multi-component synergistic emulsion gel with the function of regulating intestinal flora.

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