A high-stability dha emulsion and a method for preparing the same

Abstract

The application provides a high-stability DHA emulsion and a preparation method thereof. The high-stability DHA emulsion comprises the following raw materials in parts by mass: 20-40 parts of DHA algal oil; 1-3 parts of phospholipid; 2-5 parts of emulsifier; 0.05-0.3 parts of fat-soluble antioxidant; 0.05-0.3 parts of water-soluble antioxidant; 2-3 parts of lactoferrin-reducing oligosaccharide conjugate; 0.5-2 parts of polysaccharide; and 55-75 parts of water. The high-stability DHA emulsion can be used as a nutritional fortifier for milk and dairy products, beverages and formula food.

Description

Technical Field

[0001] This application relates to the field of food processing technology, specifically to a highly stable DHA emulsion and its preparation method. Background Technology

[0002] DHA belongs to the Omega-3 series of polyunsaturated fatty acids and is an essential lipid nutrient for the human body. It plays a positive role in maintaining the normal function of the nervous system, promoting brain development in infants and young children, improving vision, and reducing the risk of cardiovascular disease.

[0003] With the increasing health awareness of consumers, the market demand for DHA-rich nutritional supplements and functional foods continues to grow; DHA has been widely used in milk and dairy products, beverages, and formula foods, becoming a key added ingredient.

[0004] In the existing technology, the addition of DHA products in various food systems is limited due to insufficient dispersibility and solubility, especially due to poor stability under beverage and dairy processing conditions;

[0005] Therefore, there is a need for a DHA formulation that has good solubility, uniform dispersion, and good stability in various food systems, in order to address the shortcomings of existing DHA products in terms of solubility, dispersibility, and application range, and improve their applicability in the food industry. Summary of the Invention

[0006] This invention provides a highly stable DHA emulsion and its preparation method.

[0007] In one aspect, this application provides a highly stable DHA emulsion, comprising, by weight, the following raw materials: 32 parts DHA algal oil; 1-3 parts phospholipids; 2-5 parts emulsifier; 0.05-0.3 parts fat-soluble antioxidant; 0.05-0.3 parts water-soluble antioxidant; 2-3 parts lactoferrin-reducing oligosaccharide conjugate; 0.5-2 parts polysaccharide; and 55-75 parts water.

[0008] Through the above technical solution, DHA algal oil and lipid-soluble antioxidants constitute the oil phase, while water and water-soluble antioxidants constitute the aqueous phase. Emulsifiers, phospholipids, lactoferrin-reducing oligosaccharide conjugates, and polysaccharides serve as interfacial components to stably disperse the droplets formed in the oil phase within the aqueous phase. As the main active ingredient, DHA algal oil requires layer-by-layer encapsulation by phospholipids, lactoferrin-reducing oligosaccharide conjugates, and polysaccharides to reduce the oxidation rate of DHA. Phospholipids, with their typical amphiphilic molecular structure, spontaneously align at the interface between the oil and aqueous phases, coating the surface of the droplets formed in the oil phase and improving the stability of the highly stable DHA emulsion. The combined use of lipid-soluble and water-soluble antioxidants can simultaneously scavenge free radicals in both the oil and aqueous phases, inhibiting DHA oxidation. The lactoferrin groups in the lactoferrin-reducing oligosaccharide conjugate can spontaneously migrate and become oriented. The components are concentrated at the oil-water interface. This interfacial layer achieves supplementary coating and densification of the primary monolayer formed by phospholipids through hydrophobic interactions between the hydrophobic residues of the lactoferrin groups and the phospholipid tail chains or the surface of the oil phase. The reducing oligosaccharide groups not only synergistically enhance the stability of the interfacial membrane with lactoferrin, but also form a steric barrier in the aqueous phase, inhibiting the contact and aggregation between oil droplets and improving the stability of the high-stability DHA emulsion. In the outer interfacial layer, polysaccharides form a loose but continuous hydration layer, providing a steric hindrance effect, further preventing close contact and aggregation between oil phase droplets, and synergistically enhancing the anti-agglomeration ability with lactoferrin-oligosaccharide conjugates. Through the synergistic effect of the above components, the high-stability DHA emulsion of this application can improve the stability of DHA and ensure its nutritional value, and is suitable as a nutritional fortifier in milk and dairy products, beverages, and formulated foods.

[0009] In some embodiments, the DHA algal oil in the highly stable DHA emulsion includes at least one of ordinary DHA algal oil, organic DHA algal oil, and winterized DHA algal oil.

[0010] Through the above-mentioned implementation methods, ordinary DHA algal oil has a stable source and is suitable for large-scale application; organic DHA algal oil meets organic food standards and satisfies the demand of the high-end market; winterized algal oil is treated at low temperature to remove high melting point impurities, resulting in better transparency and taste and higher oxidative stability. Different types of DHA algal oil can be flexibly selected or compounded according to market demand, which can expand the application scenarios and adaptability of the products.

[0011] In some embodiments, the lactoferrin-reducing oligosaccharide conjugate includes the following preparation steps:

[0012] S1: Disperse lactoferrin, reducing oligosaccharides, and potassium carbonate in water to obtain a dispersion;

[0013] S2: Freeze-dry the dispersion to obtain a powdered mixture;

[0014] S3: Mix the powdered mixture, electrolyte and water, and allow lactoferrin and reducing oligosaccharides to undergo Maillard reaction under controlled humidity conditions to obtain lactoferrin-reducing oligosaccharide conjugate.

[0015] Through the above-described implementation methods, the formation of the dispersion in step S1 allows lactoferrin and reducing oligosaccharides to fully contact and uniformly distribute in the aqueous phase. Potassium carbonate, as an alkaline regulator, can moderately increase the pH, providing a favorable initial reaction environment for the subsequent Maillard reaction, while avoiding excessive denaturation or aggregation of lactoferrin. In step S2, the freeze-drying step effectively removes moisture and maintains the spatial structural integrity of the protein and oligosaccharides, which is beneficial for the uniform reconstruction of components and the maintenance of interfacial activity in subsequent reactions. In step S3, the water activity of the reaction system is controlled by electrolytes and humidity control conditions, promoting the Maillard reaction between the ε-amino group in lactoferrin and the reducing glycosyl group of the reducing oligosaccharides to obtain a lactoferrin-reducing oligosaccharide conjugate with a coupling structure. It combines the hydrophobicity of lactoferrin with the hydrophilicity of reducing oligosaccharides, possesses excellent interfacial activity and layered adsorption capacity, and can construct a dense and flexible interfacial film in the emulsion system.

[0016] Furthermore, the lactoferrin-reducing oligosaccharide conjugate comprises the following preparation steps:

[0017] S1: Disperse 3 parts lactoferrin, 1.8-2.2 parts reducing oligosaccharides, and 0.02-0.06 parts potassium carbonate in 8-15 parts water to obtain a dispersion;

[0018] S2: Freeze the dispersion at -50~-35℃ for 3-6 hours, and then dry it at 20-30℃ to constant weight to obtain a powdered mixture;

[0019] S3: Mix 4 parts of powdered mixture, 0.8-1.5 parts of electrolyte and 0.5-0.8 parts of water, place in a sealed, humidity-controlled container with a saturated potassium carbonate solution at the bottom, and react at 50-60℃ for 16-36 hours to allow the lactoferrin and reducing oligosaccharide to undergo the Maillard reaction. Then dry at 20-30℃ to constant weight to obtain the lactoferrin-reducing oligosaccharide conjugate.

[0020] Through the above-described implementation methods, controlling the mass ratio of lactoferrin to reducing oligosaccharides at 3:1.8-2.2 is beneficial for forming a stable glycosylated coupling structure, balancing interfacial activity and steric hindrance, and improving the dispersibility and anti-agglomeration ability of the highly stable DHA emulsion. Potassium carbonate helps maintain the stability of lactoferrin and promotes the selective occurrence of the Maillard reaction between lactoferrin and reducing oligosaccharides in the initial stage, avoiding excessive cross-linking or browning. The mass ratio of water in the dispersion ensures the fluidity and reaction uniformity of the system, while also facilitating freeze-drying to form a loose structure, improving the reaction efficiency of the Maillard reaction between lactoferrin and reducing oligosaccharides and the dispersibility of the resulting lactoferrin-reducing oligosaccharide conjugate. The Maillard reaction between lactoferrin and reducing oligosaccharides requires suitable water activity, precise control of electrolytes and water volume, and regulation of the sealed humidity-controlled container holding the saturated potassium carbonate solution to promote the Maillard reaction between the ε-amino group in lactoferrin and the reducing glycosyl group of the reducing oligosaccharide, avoiding interference from side reactions.

[0021] In some embodiments, the reducing oligosaccharide includes 2'-fucosylated lactose and lactose-N-neotetrasaccharide; wherein the mass ratio of 2'-fucosylated lactose to lactose-N-neotetrasaccharide is (2-4):1.

[0022] Through the above-described embodiments, the lactoferrin-reduced oligosaccharide conjugate formed by the reducing oligosaccharide and lactoferrin can inhibit non-specific aggregation between proteins through the steric hindrance effect between the grafted reducing oligosaccharides, improve the dispersibility of the lactoferrin-reduced oligosaccharide conjugate in the oil and aqueous phases, effectively resist shear forces and temperature fluctuations, and improve the storage stability of high-stability DHA emulsions. The inventors discovered that unexpected performance optimization can be obtained when the reducing oligosaccharides are selected as 2'-fucosylated lactose and lactose-N-neotetrasaccharide in a mass ratio of (2-4):1. This may be because 2'-fucosylated lactose has a small molecular weight, low steric hindrance, and high nucleophilic reactivity, and can preferentially bind to the lactoferrin surface. The ε-amino group forms a Schiff base intermediate, improving coupling efficiency; the long chain and large steric hindrance of lactose-N-neotetrasaccharide can form a stronger hydration layer and spatial barrier on the outer layer of the coupling compound, effectively preventing enzyme molecules such as pepsin from contacting the lactoferrin backbone and delaying the enzymatic depolymerization process; 2'-fucosylated lactose forms hydrophobic association with the hydrophobic residues of lactoferrin through its fucose residues, enhancing the compactness and mechanical strength of the interfacial membrane; the combined use of both can effectively improve the stable generation of the coupling compound and optimize its interfacial stabilization effect; by controlling the mass ratio of 2'-fucosylated lactose to lactose-N-neotetrasaccharide at (2-4):1, uniform grafting and orderly arrangement of lactoferrin and oligosaccharides can be achieved in the coupling reaction, improving the construction selectivity and structural uniformity of the interfacial membrane.

[0023] In some embodiments, the electrolyte includes at least one of citric acid, sodium citrate, sodium chloride, and potassium citrate.

[0024] Through the above-described implementation methods, the introduction of electrolytes such as citric acid, sodium citrate, sodium chloride, and potassium citrate in the preparation process of lactoferrin-reducing oligosaccharide conjugates enables precise control of the water activity of the reaction system, playing a synergistic role in the coupling reaction and interfacial structure construction. By adjusting the ionic strength and osmotic pressure of the solution, these electrolytes can effectively reduce the free water content in the system, thereby controlling the Maillard reaction rate and coupling degree, preventing excessive Maillard reaction that could lead to non-specific cross-linking or conformational unfolding of lactoferrin, and ensuring that the functional structure and interfacial activity of lactoferrin are not damaged. Citric acid and its sodium salt can form a buffer system, stabilizing the pH value of the reaction system, maintaining the conformational stability of lactoferrin, and improving the selectivity of the coupling reaction. The electrolytes enhance the homogeneity of the lactoferrin-reducing oligosaccharide conjugate. Sodium citrate and potassium citrate, as weakly basic salts, possess both water activity regulation and metal ion complexation capabilities, thereby improving the antioxidant properties and storage stability of the conjugate. Sodium chloride, by increasing ionic strength, promotes the directional adsorption and orderly arrangement of proteins and oligosaccharides at the interface, contributing to the formation of a dense and flexible interfacial membrane structure. These electrolyte components work synergistically during conjugate preparation through mechanisms such as water activity regulation, pH buffering, conformational protection, and interface optimization, thereby improving the structural stability and functional performance of the lactoferrin-reducing oligosaccharide conjugate. This provides molecular-level support and functional assurance for the subsequent construction of the interface structure, regulation of physicochemical stability, and stress adaptability during processing in the high-stability DHA emulsion system.

[0025] In some embodiments, the highly stable DHA emulsion satisfies at least one of the following conditions:

[0026] 1) The emulsifier includes at least one of mono- and diglyceride fatty acid esters, sodium octenyl succinate starch, disodium dihydrogen pyrophosphate, sucrose fatty acid esters, polyglycerol fatty acid esters, Tween 80, Tween 60, Tween 40, and Tween 20.

[0027] 2) The fat-soluble antioxidants include at least one of ascorbyl palmitate, vitamin E, β-carotene, lutein, rosemary extract, theaflavins, propyl gallate, and coenzyme Q10;

[0028] 3) The water-soluble antioxidant includes at least one of L-ascorbic acid, sodium L-ascorbate, calcium L-ascorbate, potassium L-ascorbate, phytic acid, and sodium phytate;

[0029] 4) The polysaccharide includes at least one of carrageenan, locust bean gum, gum arabic, xanthan gum, and sodium alginate.

[0030] Through the above-described implementation methods, emulsifiers such as mono- and diglycerides of fatty acids, sucrose fatty acid esters, and polyglycerol fatty acid esters can synergistically work with proteins and lipids to enhance the density and flexibility of the interfacial film; sodium octenyl succinate starch and Tween-type emulsifiers provide additional steric hindrance and charge repulsion, potentially improving the stability of highly stable DHA emulsions under high-temperature conditions; lipid-soluble antioxidants are distributed in the oil phase, constructing an antioxidant barrier and delaying the oxidative degradation of DHA; water-soluble antioxidants can scavenge reactive oxygen species or chelate metal ions in the aqueous phase, inhibiting free radical chain reactions; polysaccharides can form a polymeric coating layer and a three-dimensional network structure on the surface of droplets in highly stable DHA emulsions, enhancing electrostatic repulsion and viscoelasticity, inhibiting aggregation, sedimentation, and oxidative diffusion, and improving the storage stability of highly stable DHA emulsions.

[0031] Secondly, this application provides a method for preparing a highly stable DHA emulsion, comprising the following preparation steps:

[0032] Provide raw materials for the highly stable DHA emulsion according to any embodiment of the first aspect;

[0033] The raw materials are mixed to obtain a highly stable DHA emulsion.

[0034] Through the above-described implementation methods, the scientific selection and proportioning of raw materials ensure that each functional raw material can play a full role in subsequent steps, laying the foundation for the preparation of a highly stable DHA emulsion; the desired highly stable DHA emulsion is obtained by mixing the raw materials.

[0035] Furthermore, the method for preparing a highly stable DHA emulsion includes the following preparation steps:

[0036] P1: Mix phospholipids, 28-32 parts of water, emulsifiers and water-soluble antioxidants, and shear at 8000-12000 rpm for 8-12 minutes at 40-60℃ to obtain an aqueous dispersion;

[0037] P2: Mix DHA algal oil, fat-soluble antioxidant, and the aqueous dispersion, and shear at 3000-5000 rpm for 5-10 minutes at 40-60℃ to obtain a primary emulsion;

[0038] P3: Mix the primary emulsion, lactoferrin-reduced oligosaccharide conjugate, and 6-10 parts of water, and shear at 250-400 rpm for 10-20 minutes at 20-30°C to obtain the secondary emulsion.

[0039] P4: Mix the polysaccharide, remaining water, and the secondary emulsion, and shear at 250-400 rpm for 10-20 minutes at 20-30°C to obtain a highly stable DHA emulsion.

[0040] Through the above-described implementation methods, in step P1, phospholipids, water-soluble antioxidants, and emulsifiers are pre-dispersed in 28-32 parts of water, and sheared at 8000-12000 rpm for 8-12 minutes at 40-60°C to ensure the aqueous dispersion is fully homogenized, forming a stable aqueous precursor system and improving the adsorption efficiency and distribution uniformity of the emulsifier in the subsequent interfacial adsorption process. In step P2, DHA algal oil and lipid-soluble antioxidants are introduced into the aqueous dispersion to form a primary emulsion, which is beneficial for achieving pre-emulsification and antioxidant protection of the oil phase in the initial stage, reducing the oxidation risk of oxidation-sensitive components. In step P3, the primary emulsion, lactoferrin- The reducing oligosaccharide conjugate is mixed with 6-10 parts of water and sheared at 250-400 rpm for 10-20 minutes at 20-30℃. This avoids damage to the lactoferrin-reducing oligosaccharide conjugate structure, further enhances the density and flexibility of the interfacial membrane structure, and improves the physical stability and interfacial adsorption capacity of the emulsion droplets. In step P4, polysaccharides and remaining water are added to regulate the viscosity, rheology, and steric hindrance structure of the system. This stepwise mixing strategy achieves synergistic effects of emulsion particle size control, interfacial protection, and active ingredient stability by constructing interfacial structures and functional barriers in stages, thereby improving the storage stability and gastric environment tolerance of the highly stable DHA emulsion.

[0041] Thirdly, the application of highly stable DHA emulsion prepared according to any embodiment of the first aspect or the method described in any embodiment of the second aspect in milk and dairy products, beverages, and formulated foods.

[0042] Compared with the prior art, the beneficial effects of this application are at least as follows:

[0043] Through the synergistic effect of phospholipids, emulsifiers, lactoferrin-reducing oligosaccharide conjugates, and polysaccharides, an oil phase composed of DHA algal oil and fat-soluble antioxidants is formed in an aqueous phase system composed of water-soluble antioxidants and water, resulting in a highly stable DHA emulsion with uniform particle size. This improves dispersibility and solubility, reduces the risk of crystallization and phase separation, and enhances stability under heating conditions. The combination of fat-soluble and water-soluble antioxidants constructs a biphasic antioxidant system to delay DHA oxidation, thus enabling better application in milk and dairy products, beverages, and formulated foods. Detailed Implementation

[0044] The various embodiments or implementation schemes in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments.

[0045] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0046] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0047] In this manual, unless otherwise specified, "parts" refers to "parts by mass" and "rpm" refers to "revolutions per minute".

[0048] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that comply with the National Food Safety Standards and related regulations and are commercially available.

[0049] Winterized DHA algal oil, selected from Vegalgal, Hubei Xinhe Biotechnology Co., Ltd. ® Products in the DHA algal oil (wintering type) series with DHA content of 50%;

[0050] Organic DHA algal oil, selected from Vegalgal, Hubei Xinhe Biotechnology Co., Ltd. ® Products in the organic DHA algal oil series with DHA content of 50%;

[0051] Ordinary DHA algal oil, selected from Vegalgal of Hubei Xinhe Biotechnology Co., Ltd. ® Products in the DHA algal oil series with DHA content of 50%;

[0052] Mono- and diglyceride fatty acid esters, selected from HP-C product of Danisco (China) Co., Ltd.;

[0053] Phospholipids were selected from PC15 product of Beijing Huaxia Houde Biotechnology Co., Ltd.

[0054] Preparation Example 1

[0055] Preparation of lactoferrin-reducing oligosaccharide conjugates:

[0056] S1: Disperse 3 parts lactoferrin, 1.5 parts 2'-fucosylated lactose, 0.5 parts lactose-N-neotetrasaccharide, and 0.03 parts potassium carbonate in 10 parts water to obtain a dispersion;

[0057] S2: Spread the dispersion to a thickness of 10 mm, freeze at -40°C for 5 h, and then vacuum dry at 25°C to constant weight to obtain a powdered mixture;

[0058] S3: Mix 4 parts of powdered mixture, 0.30 parts of sodium citrate, 0.15 parts of sodium chloride, and 0.60 parts of water in a sealed, humidity-controlled container. Place a saturated potassium carbonate solution at the bottom of the container and react at 55°C for 24 hours. Then, vacuum dry at 25°C to constant weight to obtain lactoferrin-reduced oligosaccharide conjugate.

[0059] Preparation Example 2

[0060] Preparation of lactoferrin-reducing oligosaccharide conjugates:

[0061] The preparation method is largely the same as in Example 1, except that: 1.2 parts of 2'-fucosylated lactose and 0.8 parts of lactose-N-neotetrasaccharide are used.

[0062] Preparation Example 3

[0063] Preparation of lactoferrin-reducing oligosaccharide conjugates:

[0064] The preparation was largely the same as in Example 1, except that 1.33 parts of 2'-fucosylated lactose and 0.67 parts of lactose-N-neotetrasaccharide were used.

[0065] Preparation Example 4

[0066] Preparation of lactoferrin-reducing oligosaccharide conjugates:

[0067] The preparation method is largely the same as in Example 1, except that: 1.6 parts of 2'-fucosylated lactose and 0.4 parts of lactose-N-neotetrasaccharide are used.

[0068] Preparation Example 5

[0069] Preparation of lactoferrin-reducing oligosaccharide conjugates:

[0070] The preparation method is largely the same as in Example 1, except that: 1.7 parts of 2'-fucosylated lactose and 0.3 parts of lactose-N-neotetrasaccharide are used.

[0071] Example 1

[0072] Raw material for a highly stable DHA emulsion:

[0073] 32 parts winterized DHA algal oil; 2.2 parts phospholipids; emulsifiers: 2.6 parts mono- and diglycerides of fatty acids, 0.2 parts sodium octenyl succinate starch, 0.2 parts disodium dihydrogen pyrophosphate; fat-soluble antioxidants: 0.15 parts ascorbate palmitate; water-soluble antioxidants: 0.06 parts L-ascorbic acid, 0.04 parts sodium L-ascorbate, 0.02 parts calcium L-ascorbate; 2.4 parts lactoferrin-reducing oligosaccharide conjugate from Preparation Example 1; polysaccharides: 0.6 parts carrageenan, 0.6 parts locust bean gum; 60 parts water;

[0074] A method for preparing a highly stable DHA emulsion:

[0075] P1: 2.2 parts phospholipid, 32 parts water, 2.6 parts mono- and diglyceride fatty acid esters, 0.2 parts sodium octenyl succinate starch, 0.2 parts disodium dihydrogen pyrophosphate, 0.06 parts L-ascorbic acid, 0.04 parts sodium ascorbate, and 0.02 parts calcium ascorbate were mixed and vacuum degassed at 10,000 rpm for 10 min at 45°C to obtain an aqueous dispersion.

[0076] P2: Mix 32 parts of winterized DHA algal oil, 0.15 parts of ascorbate palmitate, and the aqueous dispersion, and shear at 10,000 rpm for 10 minutes at 45°C to obtain a primary emulsion.

[0077] P3: Mix 2.4 parts of the lactoferrin-reduced oligosaccharide conjugate from Preparation Example 1 with 8 parts of water, and stir at 300 rpm for 15 min at 25°C to obtain a secondary emulsion.

[0078] P4: Mix 0.6 parts carrageenan, 0.6 parts locust bean gum, 20 parts water, and the secondary emulsion, stir at 350 rpm for 15 min at 25°C, homogenize three times at 120 MPa, and then sterilize by irradiation with 5 kGy gamma rays at 0°C to obtain a highly stable DHA emulsion.

[0079] Example 2

[0080] For the preparation of a highly stable DHA emulsion:

[0081] Similar to Example 1, except that winterized DHA algal oil is replaced with organic DHA algal oil.

[0082] Example 3

[0083] For the preparation of a highly stable DHA emulsion:

[0084] Similar to Example 1, except that the winterized DHA algal oil is replaced with ordinary DHA algal oil.

[0085] Example 4

[0086] For the preparation of a highly stable DHA emulsion:

[0087] The formula is largely the same as Example 1, except that mono- and diglyceride fatty acid esters are replaced with Tween 20.

[0088] Example 5

[0089] For the preparation of a highly stable DHA emulsion:

[0090] The formulation is largely the same as Example 1, except that the lactoferrin-reducing oligosaccharide conjugate used in the formulation was prepared in Preparation Example 2.

[0091] Example 6

[0092] For the preparation of a highly stable DHA emulsion:

[0093] The formulation is largely the same as Example 1, except that the lactoferrin-reducing oligosaccharide conjugate used in the formulation was prepared in Preparation Example 3.

[0094] Example 7

[0095] For the preparation of a highly stable DHA emulsion:

[0096] The formulation is largely the same as Example 1, except that the lactoferrin-reducing oligosaccharide conjugate used in the formulation was prepared in Preparation Example 4.

[0097] Example 8

[0098] For the preparation of a highly stable DHA emulsion:

[0099] The formulation is largely the same as Example 1, except that the lactoferrin-reducing oligosaccharide conjugate used in the formulation was prepared in Preparation Example 5.

[0100] Comparative Example 1

[0101] Similar to Example 1, except that in step P3, 1.29 parts of lactoferrin, 0.65 parts of 2'-fucosylated lactose, 0.21 parts of lactose-N-neotetrasaccharide, 0.013 parts of potassium carbonate, 0.158 parts of sodium citrate, and 0.079 parts of sodium chloride are used to replace 2.4 parts of lactoferrin-reducing oligosaccharide conjugate. That is, the raw materials in the preparation process of lactoferrin-reducing oligosaccharide conjugate are replaced with lactoferrin-reducing oligosaccharide conjugate in equal mass ratios. The remaining steps are the same as in Example 1.

[0102] Comparative Example 2

[0103] The procedure is largely the same as in Example 1, except that in step P4, 1.2 parts of lactoferrin-reducing oligosaccharide conjugate are used instead of 0.6 parts of carrageenan and 0.6 parts of locust bean gum. The remaining steps are the same as in Example 1.

[0104] Comparative Example 3

[0105] The procedure is largely the same as in Example 1, except that in step P3, 1.2 parts of carrageenan and 1.2 parts of locust bean gum are used instead of 2.4 parts of lactoferrin-reducing oligosaccharide conjugate. The remaining steps are the same as in Example 1.

[0106] Test section

[0107] Stability test:

[0108] Centrifugation stability: 3000 rpm, centrifugation for 30 min;

[0109] 4℃ stability: Let stand at 4℃ until oil droplets appear on the surface or an odor is produced;

[0110] 25℃ stability: Let stand at 25℃ until oil droplets appear on the surface or an odor is produced;

[0111] Stability at 85℃: Allow to stand at 85℃ until oil droplets appear on the surface or an odor is produced.

[0112] The test results are shown in Table 1:

[0113] Table 1

[0114]

[0115] As shown in Table 1, the high-stability DHA emulsions obtained in each embodiment exhibited superior stability at all temperatures compared to the comparative example. This indicates that the high-stability DHA emulsion formulation and preparation method provided in this application can improve the physical stability and antioxidant properties of the emulsion through the synergistic effect of phospholipids, emulsifiers, lactoferrin-reducing oligosaccharide conjugates, and polysaccharides. This may be due to the effect of the formed phospholipid-lactoferrin-reducing oligosaccharide conjugate-polysaccharide network. Phospholipid molecules have a typical amphiphilic structure, spontaneously oriented to form a phospholipid bilayer coating DHA algal oil. The lactoferrin groups of the lactoferrin-reducing oligosaccharide conjugate simultaneously contain hydrophobic residues and charged groups, which can... Spontaneous adsorption occurs on the outer side of the phospholipid layer. The reducing oligosaccharide groups provide steric hindrance, reducing the risk of flocculation and aggregation in highly stable DHA emulsions. The polar groups such as carboxyl or hydroxyl groups carried on the reducing oligosaccharide groups can regulate the charge density on the surface of each droplet in the highly stable DHA emulsion, enhance electrostatic repulsion, and further improve the dispersion stability of the emulsion. The polysaccharides have a large number of hydroxyl and anionic groups, which bind to the lactoferrin-reducing oligosaccharide coupling interface through hydrogen bonds, electrostatic interactions, and van der Waals forces to form a polysaccharide polymer chain coating layer. At the same time, lipid-soluble and water-soluble antioxidants scavenge free radicals inside the oil droplets and in the continuous phase, respectively, forming a synergistic antioxidant system and reducing oxidation-induced interfacial damage.

[0116] As can be seen from Examples 1-3, when winterized DHA algal oil is selected, its stability at various temperatures is better than that of ordinary DHA algal oil and organic DHA algal oil. This may be because winterized DHA algal oil is obtained by low-temperature crystallization and separation, which removes some impurities such as saturated fatty acids and waxes.

[0117] As can be seen from Examples 1 and 4, the stability of Example 4 at 4°C and 25°C is slightly lower than that of Example 1, while the stability at 85°C is basically the same as that of Example 1. This may be because Tween 20 can reduce interfacial tension and exhibits film toughness and tolerance that is basically the same as that of mono- and diglyceride fatty acid esters under thermal stress at 85°C. However, its polyoxyethylene chain is more prone to oxidation during long-term storage at 4°C and 25°C. It is also possible that the phospholipid-lactoferrin-reducing oligosaccharide conjugate-polysaccharide network undergoes partial rearrangement, resulting in a slight decrease in stability.

[0118] As shown in Implementation 1 and Examples 5-8, during the preparation of lactoferrin-reducing oligosaccharide conjugates, the stability at all temperatures significantly decreased when the mass ratio of 2'-fucosylated lactose to lactose-N-neotetrasaccharide was less than 2:1 or greater than 4:1. This may be because the 2'-fucosylated lactose molecule is rich in sterically hindered groups, possessing excellent hydration capabilities, and can form a dense and stable hydration layer on the surface of the droplets, thereby effectively inhibiting close contact and aggregation between droplets and improving the steric stability of the system. In contrast, lactose-N-neotetrasaccharide enhances the surface charge density of the interfacial film through its molecular charge distribution characteristics, thereby improving the stability of the system between droplets. Electrostatic repulsion inhibits flocculation and aggregation. When 2'-fucosylated lactose and lactose-N-neotetrasaccharide are grafted onto lactoferrin in an appropriate ratio, they exhibit a synergistic effect in terms of steric hindrance and electrostatic repulsion, optimizing the structural compactness and charge barrier performance of the interfacial membrane, thereby constructing a lactoferrin-reduced oligosaccharide conjugate interfacial membrane with excellent stability. When the ratio of the two is unbalanced, the spatial structure and charge distribution of the lactoferrin-reduced oligosaccharide conjugate interfacial membrane tend to be uneven, resulting in damage to the integrity of the lactoferrin-reduced oligosaccharide conjugate interfacial membrane, a decrease in the ability to regulate the interaction forces between milk droplets, and ultimately a reduction in the stability of the highly stable DHA emulsion.

[0119] As can be seen from Example 1 and Comparative Example 1, the stability of Comparative Example 1 at all temperatures is inferior to that of Example 1. This may be because the single stacking of the lactoferrin-reducing oligosaccharide conjugate component leads to a decrease in the rigidity and uniformity of the membrane layer. The lack of the synergistic flexibility and steric hindrance of the covalent bond between lactoferrin and reducing oligosaccharide makes it easier for droplets in the high-stability DHA emulsion to aggregate and cause the interfacial membrane structure to break down.

[0120] According to Example 1 and Comparative Example 2, the stability of Comparative Example 2 at all temperatures is inferior to that of Example 1. This may be because the lack of an outer polysaccharide network causes the disappearance of viscoelasticity and diffusion barriers between droplets in the high-stability DHA emulsion, resulting in enhanced interaction, obvious aggregation and stratification, and decreased stability under thermal stress.

[0121] According to Example 1 and Comparative Example 3, the stability of Comparative Example 3 at all temperatures is inferior to that of Example 1. This may be because directly replacing lactoferrin-reduced oligosaccharide conjugate with polysaccharide lacks the synergistic interfacial adsorption capacity of lactoferrin-reduced oligosaccharide conjugate, resulting in the lack of an effective protective layer on the surface of the droplets in the high-stability DHA emulsion, high interfacial tension, loose membrane structure, and easy aggregation of droplets.

[0122] After centrifugation stability testing, no oil droplets were observed on the surface of the above-described embodiments and comparative examples, and no odor was generated.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application 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 therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A highly stable DHA emulsion, characterized in that, Includes the following quantities of raw materials: 32 parts of DHA algal oil; 1-3 parts phospholipids; 2-5 parts emulsifier; 0.05-0.3 parts of fat-soluble antioxidant; 0.05-0.3 parts of water-soluble antioxidant; 2-3 parts of lactoferrin-reducing oligosaccharide conjugate; Polysaccharide 0.5-2 parts; 55-75 parts water; The lactoferrin-reducing oligosaccharide conjugate comprises the following preparation steps: S1: Disperse lactoferrin, reducing oligosaccharides, and potassium carbonate in water to obtain a dispersion; S2: Freeze-dry the dispersion to obtain a powdered mixture; S3: Mix the powdered mixture, electrolyte, and water, and allow lactoferrin and reducing oligosaccharides to undergo a Maillard reaction under controlled humidity conditions to obtain a lactoferrin-reducing oligosaccharide conjugate; the reducing oligosaccharide includes 2'-fucosylated lactose and lactose-N-neotetrasaccharide; wherein the mass ratio of 2'-fucosylated lactose to lactose-N-neotetrasaccharide is (2-4):1; the electrolyte includes at least one of citric acid, sodium citrate, sodium chloride, and potassium citrate.

2. The highly stable DHA emulsion according to claim 1, characterized in that, The DHA algal oil in the highly stable DHA emulsion includes at least one of ordinary DHA algal oil, organic DHA algal oil, and winterized DHA algal oil.

3. The highly stable DHA emulsion according to claim 1, characterized in that, Place The preparation steps for the lactoferrin-reducing oligosaccharide conjugate are as follows: S1: Disperse 3 parts lactoferrin, 1.8-2.2 parts reducing oligosaccharides, and 0.02-0.06 parts potassium carbonate in 8-15 parts water to obtain a dispersion; S2: Freeze the dispersion at -50~-35℃ for 3-6 hours, and then dry it at 20-30℃ to constant weight to obtain a powdered mixture; S3: Mix 4 parts of powdered mixture, 0.8-1.5 parts of electrolyte and 0.5-0.8 parts of water, place in a sealed, humidity-controlled container with a saturated potassium carbonate solution at the bottom, and react at 50-60℃ for 16-36 hours to allow the lactoferrin and reducing oligosaccharide to undergo the Maillard reaction. Then dry at 20-30℃ to constant weight to obtain the lactoferrin-reducing oligosaccharide conjugate.

4. The highly stable DHA emulsion according to any one of claims 1 to 3, characterized in that, The highly stable DHA emulsion meets at least one of the following conditions: 1) The emulsifier includes at least one of mono- and diglyceride fatty acid esters, sodium octenyl succinate starch, disodium dihydrogen pyrophosphate, sucrose fatty acid esters, polyglycerol fatty acid esters, Tween 80, Tween 60, Tween 40, and Tween 20. 2) The fat-soluble antioxidants include at least one of ascorbyl palmitate, vitamin E, β-carotene, lutein, rosemary extract, theaflavins, propyl gallate, and coenzyme Q10; 3) The water-soluble antioxidant includes at least one of L-ascorbic acid, sodium L-ascorbate, calcium L-ascorbate, potassium L-ascorbate, phytic acid, and sodium phytate; 4) The polysaccharide includes at least one of carrageenan, locust bean gum, gum arabic, xanthan gum, and sodium alginate.

5. A method for preparing a highly stable DHA emulsion, characterized in that, The preparation steps include the following: Provide raw materials for the highly stable DHA emulsion according to any one of claims 1-4; The raw materials are mixed to obtain a highly stable DHA emulsion.

6. A method for preparing the highly stable DHA emulsion as described in claim 1, characterized in that, The preparation steps include the following: P1: Mix phospholipids, 28-32 parts of water, emulsifiers and water-soluble antioxidants, and shear at 8000-12000 rpm for 8-12 minutes at 40-60℃ to obtain an aqueous dispersion; P2: Mix DHA algal oil, fat-soluble antioxidant, and the aqueous dispersion, and shear at 3000-5000 rpm for 5-10 minutes at 40-60℃ to obtain a primary emulsion; P3: Mix the primary emulsion, lactoferrin-reduced oligosaccharide conjugate, and 6-10 parts of water, and shear at 250-400 rpm for 10-20 minutes at 20-30°C to obtain the secondary emulsion. P4: Mix the polysaccharide, remaining water, and the secondary emulsion, and shear at 250-400 rpm for 10-20 minutes at 20-30°C to obtain a highly stable DHA emulsion.

7. The application of the highly stable DHA emulsion according to any one of claims 1-4 or the highly stable DHA emulsion prepared according to the method of claim 5 or 6 in the preparation of formulated foods.

8. The application of the highly stable DHA emulsion according to any one of claims 1-4 or the highly stable DHA emulsion prepared according to the method of claim 5 or 6 in the preparation of milk and dairy products and beverages.

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