Compound emulsifier based on plukenetia volubilis linneo albumin as well as preparation method and application of compound emulsifier

Abstract

The invention provides a compound emulsifier based on plukenetia volubilis linneo albumin as well as a preparation method and application thereof, and relates to the field of foods. The method comprises the following steps: respectively preparing a plukenetia volubilis linneo albumin dispersion liquid, a polyphenol solution and a chitosan solution; mixing the plukenetia volubilis linneo albumin dispersion liquid with a polyphenol solution, adding laccase to carry out enzymatic cross-linking reaction, dialyzing a reaction product, and freeze-drying to obtain a binary compound; dissolving the binary compound, mixing the dissolved binary compound with a chitosan solution, adding laccase to carry out enzymatic cross-linking reaction, and dialyzing and freeze-drying a reaction product to obtain the compound emulsifier based on the sacha inchi albumin. Through the synergistic effect of the components, the stability, oxidation resistance, functionality and extrusion plasticity of a high internal phase emulsion constructed by taking the high internal phase emulsion as an emulsifier are improved, the high internal phase emulsion can be used as a core matrix of plant-based smear food, and the problems that a traditional matrix is poor in stability, single in function, poor in extrusion plasticity and the like are solved.

Description

Technical Field

[0001] This invention relates to the food industry, specifically to a composite emulsifier based on sacha inophylline protein, its preparation method, and its application. Background Technology

[0002] Plant-based foods are foods made from plant-based raw materials or their processed products as sources of protein, fat, etc., with or without the addition of other ingredients, and processed through certain techniques. The base material of these products contains no animal-derived ingredients and possesses one or more characteristics similar to a certain animal-derived food, such as form, flavor, and texture. Plant-based spreadable foods are an important category of plant-based foods, serving as a high-quality plant-based alternative to traditional animal-based spreadable foods (such as cream and cheese sauces). In recent years, the market demand for plant-based spreadable foods has grown rapidly due to their precise alignment with modern nutritional needs and environmentally conscious consumption trends. However, the core matrix of these products, namely high internal phase emulsions, faces several challenges: First, plant-based raw materials are rich in unsaturated fatty acids, which are prone to oxidation and rancidity, resulting in a short shelf life; second, existing emulsifiers (such as single plant proteins and chemical emulsifiers) are difficult to stabilize high oil-phase systems for long periods and have poor extrusion plasticity, affecting spreadability; third, traditional emulsifiers have limited functionality, restricting the functional upgrading of products.

[0003] Sacha inchi, a perennial woody vine belonging to the Euphorbiaceae family, is a specialty oilseed crop rich in unsaturated fatty acids and high-quality protein. Its oil extraction byproduct, fruit meal, is an ideal raw material for extracting sacha inchi protein. Sacha inchi protein, as an emerging plant protein, is rich in essential amino acids and has good biocompatibility. In addition to its excellent anti-inflammatory, antioxidant, and immunomodulatory functions, it also possesses certain emulsifying activity, showing great potential for application in the food emulsifier field. Therefore, promoting its development and application in the food industry aligns with the circular economy concept of high-value utilization of agricultural processing byproducts and conforms to the current development trend of plant proteins in the functional food sector.

[0004] However, studies have found that although sacha inophylline has a variety of excellent physiological functions, it has problems such as poor environmental responsiveness and easy oxidation and deterioration during long-term storage when used as an emulsifier. This limits its functionality to a certain extent, especially making it difficult to meet the high requirements of plant-based spreadable foods for the stability of high internal phase emulsions (such as lipid oxidation stability, pH stability, and thermal stability) and extrusion plasticity.

[0005] Therefore, how to use sacha inophylline protein to prepare a suitable emulsifier that can effectively improve the overall stability, antioxidant properties, functionality and extrusion plasticity of the high internal phase emulsion it constructs, and provide a new solution to overcome the difficulties of poor matrix stability, single function and poor extrusion plasticity of plant-based spreadable foods, has become an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the defects of high internal phase emulsion stability, poor extrusion plasticity, and single function when sacha inophylline is used as an emulsifier, which makes it difficult to meet the high requirements of plant-based spreadable foods. Thus, the present invention provides a composite emulsifier based on sacha inophylline, its preparation method and application.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing a composite emulsifier based on sacha inophylline protein, comprising the following steps: (1) Prepare sacha inophylline protein dispersion, polyphenol solution and chitosan solution respectively; (2) Mix the sacha inophylline protein dispersion with polyphenol solution, add laccase to carry out enzymatic cross-linking reaction, dialyze the reaction product and freeze dry to obtain binary complex; (3) After dissolving the binary complex, it was mixed with chitosan solution, and laccase was added to carry out enzymatic cross-linking reaction. The reaction product was dialyzed and freeze-dried to obtain a composite emulsifier based on sacha inophylline protein.

[0008] Further, in step (1), the preparation method of the sacha inophylline protein dispersion includes: mixing sacha inophylline protein with phosphate buffer at a mass-volume ratio of 5~15 mg / mL, stirring, hydrating, and obtaining sacha inophylline protein dispersion.

[0009] Further, saponin was mixed with phosphate buffer at a mass-to-volume ratio of 10 mg / mL.

[0010] Furthermore, the phosphate buffer solution has a pH of 7.0 and a concentration of 0.01 mol / L.

[0011] Further, the stirring conditions are: 400~600 rpm for 1~3 h. Preferably, the stirring conditions are: 600 rpm for 2 h.

[0012] Furthermore, the hydration conditions are: temperature 4 ℃, time 6~10 h, preferably 8 h.

[0013] Furthermore, the preparation method of the sacha inophylline protein includes: pulverizing, sieving, and defatting sacha inophylline pulp; salting the defatted sacha inophylline pulp powder; centrifuging the salt solution and collecting the supernatant; dialyzing the supernatant; centrifuging the dialysate and freeze-drying it to obtain sacha inophylline protein powder.

[0014] Furthermore, the pulverized sacha inchi pulp is passed through a 40-80 mesh sieve, preferably through a 60 mesh sieve.

[0015] Further, the defatting conditions are as follows: the sieved sacha inophylline pulp powder is mixed with petroleum ether at a mass-to-volume ratio of 1:6 (w / v, g / mL) and refluxed at 50-70 °C for 4-8 h, preferably at 60 °C for 6 h.

[0016] Further, the salt-dissolving treatment conditions are as follows: defatted saponin fruit pulp powder is mixed with 2 mol / L NaCl solution at a mass-to-volume ratio of 1:20-30 (w / v, g / mL), and stirred at 400-600 rpm and 30 °C for 1-3 h. Preferably, the salt-dissolving treatment conditions are as follows: defatted saponin fruit pulp powder is mixed with 2 mol / L NaCl solution at a mass-to-volume ratio of 1:25 (w / v, g / mL), and stirred at 600 rpm and 30 °C for 2 h.

[0017] Furthermore, the centrifugation conditions for the salt solution are: centrifugation at 5000~10000 rpm for 15~25 min. Preferably, the centrifugation conditions for the salt solution are: centrifugation at 8000 rpm for 20 min.

[0018] Furthermore, the dialysis conditions were as follows: 6-8 kDa dialysis bag, dialysis at 4 °C for 48 h, with water changed every 6 h.

[0019] Furthermore, the centrifugation conditions for the dialysate are: centrifugation at 5000~10000 rpm for 20 min. Preferably, the centrifugation conditions for the dialysate are: centrifugation at 8000 rpm for 20 min.

[0020] Furthermore, the freeze-drying conditions were as follows: pre-freezing at -20 °C for 24 h, followed by vacuum freeze-drying at -50 °C and 10 Pa for 48 h.

[0021] Furthermore, in step (1), the polyphenol solution is obtained by dissolving the polyphenol compound in water.

[0022] Furthermore, the mass-to-volume ratio of the polyphenol compound to water is 1-3 mg / mL, preferably 2 mg / mL.

[0023] Furthermore, the polyphenolic compound is selected from epigallocatechin gallate or gallic acid.

[0024] Further, in step (1), the chitosan solution is obtained by dissolving chitosan in water.

[0025] Furthermore, the mass-to-volume ratio of chitosan to water is 1~5 mg / mL, preferably 3 mg / mL.

[0026] Further, in step (2), the sacha inophylline protein dispersion and polyphenol solution are mixed at a volume ratio of 1:1.

[0027] Further, in step (2), the amount of laccase added is 1~4 U / mL based on the volume of the mixture, preferably 2 U / mL.

[0028] Furthermore, in step (2), the conditions for the enzymatic cross-linking reaction are: 25 °C, stirring at 100~300 rpm for 24 h.

[0029] Furthermore, in step (2), the dialysis conditions are: 6~8 kDa dialysis bag, dialysis at 4 ℃ for 48 h, with water changed once every 6 h.

[0030] Furthermore, in step (2), the freeze-drying conditions are: pre-freezing at -20 ℃ for 24 h, and then vacuum freeze-drying at -50 ℃ and 10 Pa for 48 h.

[0031] Further, in step (3), the binary complex solution and the chitosan solution are mixed at a volume ratio of 1:0.5~1.5, preferably at a volume ratio of 1:1.

[0032] Further, in step (3), the binary complex is dissolved in acetate buffer; the mass-to-volume ratio of the binary complex to the acetate buffer is 10 mg / mL; and the concentration of the acetate buffer is 0.1 mol / L.

[0033] Furthermore, in step (3), the amount of laccase added is 1~4 U / mL based on the volume of the mixture, preferably 2 U / mL.

[0034] Furthermore, in step (3), the conditions for the enzymatic cross-linking reaction are: 25 °C, stirring at 100~300 rpm for 24 h.

[0035] Furthermore, in step (3), the dialysis conditions are: 6~8 kDa dialysis bag, dialysis at 4 ℃ for 48 h, with water changed once every 6 h.

[0036] Furthermore, in step (3), the freeze-drying conditions are: pre-freezing at -20 ℃ for 24 h, and then vacuum freeze-drying at -50 ℃ and 10 Pa for 48 h.

[0037] Secondly, the present invention provides a composite emulsifier based on saponin obtained by the preparation method described above.

[0038] Thirdly, the present invention provides the application of the composite emulsifier based on saponin obtained by the preparation method described above in the preparation of high internal phase Pickering emulsions.

[0039] Fourthly, the present invention provides a high internal phase Pickering emulsion, the raw materials for which the preparation includes an aqueous phase raw material and an oil phase raw material, wherein the volume of the oil phase raw material accounts for 75% to 80% of the total volume of the emulsion raw material. The aqueous phase raw material includes: water and a composite emulsifier based on sacha inophylline obtained by the preparation method, wherein the ratio of water to composite emulsifier is 100 mL: 0.5~2.0 g; The oil phase raw material includes: oil.

[0040] Furthermore, the ratio of water to compound emulsifier is 100 mL: 1.0 g.

[0041] Furthermore, the oil is selected from at least one of soybean oil, olive oil, rapeseed oil, and corn oil.

[0042] Furthermore, the oil phase raw material also includes: fat-soluble active ingredients.

[0043] Furthermore, the fat-soluble active ingredient is selected from at least one of curcumin, β-carotene, and lycopene.

[0044] Furthermore, the mass ratio of the fat-soluble active ingredient to the oil is 0.1~2.0:100, preferably 0.5:100.

[0045] Fifthly, the present invention provides a method for preparing the high internal phase Pickering emulsion, comprising the following steps: mixing aqueous phase raw materials and oil phase raw materials in a certain proportion and homogenizing at 10,000 to 12,000 rpm for 1.5 to 3 min to obtain the high internal phase Pickering emulsion.

[0046] Furthermore, the homogenization conditions were as follows: homogenize at 12000 rpm for 2 min.

[0047] Sixthly, the present invention provides the application of the high internal phase Pickering emulsion or the high internal phase Pickering emulsion obtained by the preparation method described above in the preparation of plant-based spreadable foods or 3D printed foods.

[0048] The technical solution of this invention has the following advantages: This invention first utilizes laccase to catalyze the cross-linking of sacha inophylline with polyphenols to form a binary complex; then, through secondary catalysis by laccase, the binary complex is further cross-linked with chitosan, ultimately yielding a ternary composite emulsifier based on sacha inophylline.

[0049] First, the composite emulsifier obtained by this preparation method has excellent foaming properties and foam stability, and can construct a high internal phase Pickering emulsion with an oil phase ratio of ≥74%.

[0050] Secondly, the high internal phase Pickering emulsion constructed using this ternary composite emulsifier exhibits significantly enhanced pH stability, thermal stability, and antioxidant properties compared to emulsions stable by binary composites. Specifically, this emulsion maintains structural and performance stability over a wide pH range of 3–9 and under high temperature conditions (100 °C for 30 min), without exhibiting stratification, thus overcoming the environmental and processing sensitivity limitations of traditional plant protein emulsifiers.

[0051] Third, the high internal phase Pickering emulsion constructed using this ternary composite emulsifier can efficiently load and protect fat-soluble functional components such as curcumin, significantly improving the stability and bioavailability of fat-soluble active ingredients, thereby significantly increasing the nutritional added value of the product and providing key technical support for the functional upgrading of plant-based spreadable foods.

[0052] Fourth, the high internal phase Pickering emulsion constructed using this ternary composite emulsifier has good extrusion plasticity, providing new ideas for the development of functional formulations such as plant-based spreadable foods and 3D printed foods, and has good potential for industrial application.

[0053] Fifth, polyphenolic compounds, as secondary metabolites of plants, can alleviate oxidative stress by scavenging free radicals that cause cell damage, thereby intervening in the pathological processes of chronic diseases such as diabetes, atherosclerosis, and rheumatoid arthritis. Chitosan, a natural cationic polysaccharide obtained by deacetylation of chitin, is widely used in medicine, food, and other fields due to its unique biocompatibility and multiple functional activities. This invention utilizes the cross-linking of polyphenols and chitosan with sacha inophylline protein to obtain a ternary composite emulsifier that combines the excellent nutritional properties of plant protein, the strong antioxidant properties of polyphenols, and the bioactivity of chitosan, overcoming the limitation of single-function plant-based spreadable foods.

[0054] Sixth, the composite emulsifier based on sacha inophylline protein prepared by this invention is natural and safe, meets the "clean label" requirements of plant-based foods, and the main component, sacha inophylline protein, is extracted from the by-product cake after sacha inophylline oil extraction, realizing the high-value utilization of agricultural product processing by-products, which is in line with the concept of circular economy.

[0055] In summary, this invention utilizes the synergistic effect among sacha inophylline, polyphenols, and chitosan components to enhance the overall stability, antioxidant properties, functionality, and extrusion plasticity of high internal phase emulsions constructed using them as emulsifiers. It can be directly used as the core matrix for plant-based spreadable foods, solving the problems of poor stability in traditional matrices, such as easy stratification, poor processing resistance, and easy lipid oxidation and rancidity. It provides a solution that combines naturalness and functionality to overcome the difficulties of poor matrix stability, limited functionality, and poor extrusion plasticity in plant-based spreadable foods. Simultaneously, it achieves high-value utilization of agricultural product processing by-products and possesses multiple outstanding advantages, making it suitable for industrial-scale promotion and implementation. Attached Figure Description

[0056] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0057] Figure 1 This is a comparison chart of the foaming ability and foam stability of each emulsifier sample in Experiment Example 1 of this invention; Figure 2 This is a comparison chart of the POV results of each emulsion sample in Experiment Example 2 of this invention; Figure 3 This is a comparison chart of the MDA results of each emulsion sample in Experimental Example 2 of this invention; Figure 4 These are the particle size distribution and appearance of the emulsion sample from Example 1 of Experiment 3 of this invention at different pH values; Figure 5 These are the particle size distribution and appearance of the emulsion sample from Example 2 of Experiment 3 of this invention at different pH values; Figure 6 These are the particle size distribution and appearance of the emulsion sample from Comparative Example 1 in Experimental Example 3 of this invention at different pH values. Figure 7 These are the particle size distribution and appearance of the emulsion sample from Comparative Example 2 at different pH values ​​in Experimental Example 3 of this invention. Figure 8 These are the particle size distribution and appearance of the emulsion sample of Comparative Example 3 at different pH values ​​in Experimental Example 3 of this invention. Figure 9 This is a comparison diagram of the average particle size of each emulsion sample before and after heating in Experiment Example 4 of this invention; Figure 10 This is a comparison chart of the extrusion plasticity of each emulsion sample in Experimental Example 5 of this invention; Figure 11This is a comparison chart of the bioavailability of curcumin in various emulsion samples in Experimental Example 6 of this invention.

[0058] Figure 1 , 9 In the lowercase letters marked 11, different letters indicate significant differences between groups (P < 0.05), while identical or overlapping letters indicate no significant differences between groups. Figure 2 The lowercase letters in the text indicate the significance test results between POV values ​​of different samples at the same storage time, while the uppercase letters indicate the significance test results between POV values ​​of the same sample at different storage times. Different letters indicate that there is a significant difference between groups (P < 0.05), and the same or overlapping letters indicate that there is no significant difference between groups. Figure 3 The lowercase letters indicate the significance test results between MDA values ​​of different samples at the same storage time, while the uppercase letters indicate the significance test results between MDA values ​​of the same sample at different storage times. Different letters indicate that there is a significant difference between groups (P < 0.05), and the same or overlapping letters indicate that there is no significant difference between groups. Detailed Implementation

[0059] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0060] Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. All raw materials or instruments used are commercially available conventional products, including but not limited to those used in the embodiments of this application.

[0061] Example 1 This embodiment provides a method for preparing a composite emulsifier based on sacha inophylline protein, the specific steps of which are as follows: (1) Raw material preparation: a. Preparation of Sacha Inchi Albumin: Sacha Inchi pulp was mechanically pulverized and passed through a 60-mesh sieve to obtain uniform pulp powder, which was then sealed and stored in a dry environment for later use. The pulverized pulp powder was mixed with petroleum ether at a ratio of 1:6 (w / v, g / mL), placed in a Soxhlet extractor, and refluxed at 60 °C for 6 h to fully defatt the pulp. After thorough defatting, the pulp powder was dried under ventilation to obtain defatted pulp powder. The defatted pulp powder was mixed with 2 mol / L NaCl solution at a ratio of 1:25 (w / v, g / mL), and magnetically stirred at 600 rpm and 30 °C for 2 h. The mixture was then centrifuged at 8000 rpm for 20 min, and the supernatant was collected and filtered to remove the precipitate. The supernatant was placed in a 6-8 kDa dialysis bag and dialyzed at 4 °C for 48 h (replacing deionized water every 6 h). Impurities were then removed by centrifugation at 8000 rpm for 20 min. The supernatant was poured into a petri dish, pre-frozen at -20 °C for 24 h, and then transferred to a vacuum freeze dryer and dried at -50 °C for 10 h. Freeze-dry under Pa conditions for 48 h, grind to obtain sacha inophylline protein powder, and store at -20 ℃ for later use; b. Preparation of Sacha Inchi Protein Dispersion: Weigh Sacha Inchi protein powder, dissolve it in phosphate buffer (0.01 mol / L) at pH 7.0 at 10 mg / mL, stir magnetically at 600 rpm for 2 h, and then hydrate at 4 ℃ for 8 h to obtain Sacha Inchi Protein Dispersion. Store at 4 ℃. c. Preparation of polyphenol solution: Epigallocatechin gallate was dissolved in deionized water at a concentration of 2 mg / mL and stirred magnetically at 600 rpm for 30 min until completely dissolved to obtain a polyphenol solution. The solution was stored at 4 °C in the dark for later use. d. Preparation of chitosan solution: Dissolve chitosan in deionized water at 3 mg / mL, stir magnetically at 600 rpm for 30 min until completely dissolved, and store at 4℃. (2) Preparation of binary complexes: Sacha inophylline protein solution and polyphenol solution were mixed at a ratio of 1:1 (v / v), and laccase was added to a final concentration of 2 U / mL. The mixture was magnetically stirred at 300 rpm for 24 h at 25 °C, and the reaction system was kept in the dark throughout the process. The reaction solution was placed in a 6-8 kDa dialysis bag and dialyzed at 4 °C for 48 h (with deionized water replaced every 6 h) to remove unreacted polyphenols. The dialysate was pre-frozen at -20 °C for 24 h and then transferred to a vacuum freeze dryer and freeze-dried at -50 °C and 10 Pa for 48 h to obtain a binary complex powder. (3) The binary complex powder was dissolved at 10 mg / mL in pH 6.0 acetate buffer (0.1 mol / L), and mixed with chitosan solution at a ratio of 1:1 (v / v). The mixture was magnetically stirred at 600 rpm for 30 min until homogeneous. Laccase was added to the mixture to a final concentration of 2 U / mL. The mixture was magnetically stirred at 300 rpm for 24 h at 25 ℃. After the reaction was completed, the reaction solution was placed in a 6-8 kDa dialysis bag and dialyzed at 4 ℃ for 48 h (with deionized water replaced every 6 h). The dialysate was pre-frozen at -20 ℃ for 24 h and then transferred to a vacuum freeze dryer. The solution was freeze-dried at -50 ℃ and 10 Pa for 48 h to obtain the ternary complex powder based on saponin.

[0062] This embodiment provides a method for preparing a high internal phase Pickering emulsion based on the aforementioned composite emulsifier, the specific steps of which are as follows: Curcumin was dissolved in soybean oil at 0.5% (w / w) and stirred at 55 °C in the dark until completely dispersed to obtain the oil phase raw material; the ternary complex powder was dissolved in water at 1 g / 100 mL to obtain the aqueous phase raw material; the oil phase raw material and the aqueous phase raw material were mixed at a volume ratio of 75:25 and homogenized at 12000 rpm for 2 min to obtain a yellow high internal phase Pickering emulsion, which was stored at 4 °C in the dark.

[0063] Example 2 This embodiment provides a method for preparing a composite emulsifier based on sacha inophylline protein. The specific steps are the same as in Example 1, except that epigallocatechin gallate is replaced with gallic acid.

[0064] This embodiment provides a method for preparing a high internal phase Pickering emulsion based on the aforementioned composite emulsifier, and the specific steps are as described in Example 1.

[0065] Comparative Example 1 This comparative example provides a method for preparing a composite emulsifier based on sacha inophylline protein. The specific steps are the same as in Example 1, except that step (3) is omitted.

[0066] This comparative example provides a method for preparing a high internal phase Pickering emulsion based on the aforementioned composite emulsifier. The specific steps are the same as in Example 1, except that binary composite emulsifier powder is used instead of ternary composite powder.

[0067] Comparative Example 2 This comparative example provides a method for preparing a composite emulsifier based on sacha inophylline protein. The specific steps are the same as in Example 1, except that epigallocatechin gallate is replaced with gallic acid, and step (3) is omitted.

[0068] This comparative example provides a method for preparing a high internal phase Pickering emulsion based on the aforementioned composite emulsifier. The specific steps are the same as in Example 1, except that binary composite emulsifier powder is used instead of ternary composite powder.

[0069] Comparative Example 3 This comparative example provides a method for preparing sacha inophylline protein emulsifier, and the specific steps are the same as those in step (1) of Example 1 for preparing sacha inophylline protein.

[0070] This comparative example provides a method for preparing a high internal phase Pickering emulsion based on the aforementioned sacha inophylline protein emulsifier. The specific steps are the same as in Example 1, except that sacha inophylline protein is used instead of the ternary complex powder.

[0071] Comparative Example 4 This comparative example provides a method for preparing a composite emulsifier based on sacha inophylline protein. The specific steps are the same as in Example 1, except that chitosan is replaced with Dendrobium officinale polysaccharide (commercially available BR grade reagent with a purity of 90%).

[0072] This comparative example provides a method for preparing a high internal phase Pickering emulsion based on the aforementioned composite emulsifier, with specific steps as described in Example 1.

[0073] Experimental Example 1: Foaming Ability and Foam Stability of Emulsifiers 1. Experimental Samples Emulsifier samples prepared in Examples 1-2 and Comparative Examples 1-3.

[0074] 2. Experimental Methods Dissolve the emulsifier sample in water at a concentration of 1 g / 100 mL to obtain an emulsifier solution (i.e., the initial dispersion system). Pour 30 mL of the emulsifier solution into a graduated beaker, and shear the mixture using a high-speed disperser at 12000 rpm for 2 min. Measure the volume of the dispersion system. Then, allow the dispersion system to stand at room temperature for 30 min, and record its volume again. Calculate the foaming capacity (FC) and foam stability (FS) of the sample using the following formulas:

[0075] Where V1 is the initial volume of the dispersion system, V2 is the volume of the dispersion system measured immediately after shearing, and V3 is the volume of the dispersion system measured after standing at room temperature for 30 min.

[0076] 3. Experimental Results like Figure 1As shown, the foaming ability and foam stability of Examples 1-2 are significantly better than those of Comparative Examples 1-3. This may be because the interaction between chitosan and sacha inophylline protein-polyphenols further unfolds the protein structure, leading to increased protein structure flexibility and surface hydrophobicity, which in turn facilitates the formation of an air-water interface film. Good foaming ability and foam stability are also more conducive to the preparation of plant-based spreadable foods.

[0077] Experimental Example 2: Lipid Oxidation Stability of Emulsions 1. Experimental Samples Pickering emulsion samples with high internal phase prepared in Examples 1-2 and Comparative Examples 1-3.

[0078] 2. Experimental Methods Freshly prepared emulsion samples were stored at 45 °C for 30 days to accelerate oxidation. Key oxidation parameters, including peroxide value (POV) and malondialdehyde (MDA), were measured every 5 days.

[0079] POV determination: 0.3 g of the emulsion sample was mixed with 1.7 mL of isooctane / isopropanol (3:1, v / v) and centrifuged at 12000 rpm for 2 min. 0.2 mL of the supernatant was collected and mixed with 2.8 mL of methanol / n-butanol (2:1, v / v), 0.1 mL of ammonium thiocyanate (3.94 mol / L), and 0.1 mL of FeCl2 solution (0.072 mol / L). The mixture was reacted in the dark for 20 min, and the absorbance at 510 nm was recorded. The POV value of the sample was calculated based on the plotted standard curve. The standard curve was plotted as follows: using calibrated hydrogen peroxide stock solution, it was diluted with isooctane / isopropanol solvent (3:1, v / v) to prepare a series of standards with concentrations (0–1 mmol / kg). The absorbance at 510 nm was measured following the steps described above. A standard curve was plotted with concentration on the x-axis and absorbance on the y-axis.

[0080] MDA value determination: Add 0.3 g of emulsion sample to 1.7 mL of deionized water and 5 mL of 0.02 mol / L TBA solution and mix thoroughly; heat at 90 °C for 20 min, then centrifuge the mixture at 5000 rpm for 20 min; collect the supernatant, measure the absorbance at 532 nm, and calculate the POV value of the sample according to the plotted standard curve. The standard curve is plotted as follows: Use accurately weighed 1,1,3,3-tetraethoxypropane and dilute it with distilled water to prepare a series of standard solutions with concentrations (1, 2, 4, 8, 16 μmol / kg), and measure the absorbance of the standard solutions of different concentrations at 532 nm according to the above steps. Plot the standard curve with concentration as the abscissa and absorbance as the ordinate.

[0081] 3. Experimental Results POV is a key indicator for measuring the initial oxidative state of lipids in an emulsion. For example... Figure 2 As shown, in the high-temperature accelerated storage test, the POV values ​​of each group of emulsions gradually increased over time. After 15 days of storage at 45 °C, the POV value of Comparative Example 3 was significantly higher than that of the other groups. By day 30, the POV value of Comparative Example 3 reached as high as 136.6 μmol / g, while the POV value of the high internal phase emulsion stabilized by the ternary composite emulsifier in Example 1 was only 98.5 μmol / g.

[0082] MDA measures the degree of lipid peroxidation in an emulsion, specifically reflecting the content of end products of lipid peroxidation. For example... Figure 3 As shown, after 30 days of storage, Comparative Example 3 exhibited the highest MDA content, reaching 244.3 nmol / g, while the MDA content of Example 1 was only 163.3 nmol / g. This indicates that Examples 1-2 (emulsions constructed from ternary composite emulsifiers) possess superior antioxidant capabilities and can effectively slow down the lipid oxidation process.

[0083] Experimental Example 3: pH Stability of Emulsion 1. Experimental Samples Emulsifier samples prepared in Examples 1-2 and Comparative Examples 1-3.

[0084] 2. Experimental Methods The emulsifier sample powder was dissolved in water at a concentration of 1 g / 100 mL to obtain an aqueous solution. 0.02% sodium phosphate was added to inhibit microorganisms. Under continuous stirring, 1 mol / L HCl solution or 1 mol / L NaOH solution was added dropwise to the aqueous solution, and the pH value was precisely adjusted to 3.0, 5.0, 7.0, and 9.0 using a pH meter. Curcumin standard was dissolved in soybean oil at a concentration of 0.5% (w / w) and stirred at 55 °C in the dark until completely dispersed to ensure complete dissolution and form an oil phase solution. The oil phase solution and the aqueous phase solution were mixed at a volume ratio of 75:25 and homogenized at 12000 rpm for 2 min to obtain a high internal phase Pickering emulsion. The appearance and particle size changes of the emulsion at different pH values ​​were observed and measured.

[0085] 3. Experimental Results like Figure 4 and Figure 5 As shown, Examples 1 and 2, using the ternary complex as an emulsifier, did not exhibit phase separation in the pH range of 3–9, and showed excellent pH stability due to their small particle size and narrower particle size distribution. Figure 6 and Figure 7As shown, Comparative Examples 1 and 2, using binary complexes as emulsifiers, still exhibit droplet aggregation and particle size distribution broadening issues at pH 5 (near isoelectric point) and pH 9 (phenolic hydroxyl oxidation / ionization). Figure 8 As shown, in Comparative Example 3, which used unmodified sacha inophylline as an emulsifier, significant emulsion phase separation occurred due to isoelectric point aggregation under acidic conditions of pH 3-5. Curcumin was precipitated and the droplet size increased significantly, indicating weak interfacial stability.

[0086] Experimental Example 4: Thermal Stability of Emulsion 1. Experimental Samples Pickering emulsion samples with high internal phase prepared in Examples 1-2 and Comparative Examples 1-4.

[0087] 2. Experimental Methods Measure 15 mL of freshly prepared emulsion sample into a centrifuge tube, heat at 100 °C for 30 min, cool at room temperature, take pictures of the emulsion sample before and after heat treatment, and measure the particle size change before and after heat treatment.

[0088] 3. Experimental Results like Figure 9 As shown, Comparative Examples 2, 3, and 4 showed a significant increase in particle size after heat treatment. In contrast, the particle size of Examples 1, 2, and 1 remained basically unchanged before and after heat treatment, demonstrating better thermal stability. This indicates that the constructed high internal phase emulsion can withstand the high temperature environment during processing.

[0089] Experimental Example 5: Plasticity of Emulsion Extrusion 1. Experimental Samples Pickering emulsion samples with high internal phase prepared in Examples 1-2 and Comparative Examples 1-4.

[0090] 2. Experimental Methods The emulsion sample was extruded into a "five-pointed star" shape using a piping tip. The shape appearance was photographed immediately after extrusion (0 min) and after standing for 20 min to characterize its extrusion plasticity and stability.

[0091] 3. Experimental Results like Figure 10As shown, the emulsion sample (Comparative Example 3) prepared using unmodified sacha inophylline as an emulsifier exhibited generally poor extrusion performance, and its shape changed significantly after 20 minutes of extrusion, indicating poor plasticity and stability. The emulsion samples (Comparative Examples 1 and 2) prepared using the binary complex obtained through enzymatic crosslinking as an emulsifier showed some improvement in the extrusion and stability of the matrix, but the appearance showed signs of softening and enlargement after 20 minutes. The emulsion samples (Examples 1 and 2) prepared using the ternary complex based on sacha inophylline obtained through enzymatic crosslinking as an emulsifier significantly improved the plasticity and stability of the matrix. The results of Comparative Example 4 clearly show that although both are ternary complex systems, the extrusion plasticity and stability of the emulsion using Dendrobium officinale polysaccharide were poor, even inferior to the effect of Comparative Example 1 (binary complex system), which further proves the outstanding effect of the ternary complex provided by the present invention as an emulsifier. The improved extrusion plasticity and stability are helpful for extrusion coating or 3D printing in food production, and are of great significance for the application of high internal phase emulsion functional transport systems in plant-based spreadable foods.

[0092] Experimental Example 6: Biological Accessibility of Curcumin 1. Experimental Samples Pickering emulsion samples with high internal phase prepared in Examples 1-2 and Comparative Examples 1-3.

[0093] 2. Experimental Methods Constructing an in vitro three-stage digestion model: (1) Oral digestion stage 30 mL of freshly prepared emulsion was mixed with artificial saliva at a ratio of 1:1 (v / v). Before mixing, both the emulsion and artificial saliva were preheated to 37 °C. The pH of the mixture was adjusted to 6.8 ± 0.1 using 1 mol / L NaOH solution. The mixture was then continuously oscillated at 100 rpm for 3 min at a constant temperature of 37 °C to simulate the oral chewing process.

[0094] (2) Stomach digestion stage Take 20 mL of the sample after oral digestion and mix it with an equal volume of gastric juice. Before mixing, ensure that the temperature of both the sample and the gastric juice is 37 ℃. Immediately adjust the pH of the mixture to 2.5±0.1 using 1 mol / L NaOH solution. Then, continuously shake at 100 rpm for 2 h at a constant temperature of 37 ℃. After digestion, take a portion of the sample and adjust the pH to 7.0 with 1 mol / L NaOH solution to terminate pepsin activity.

[0095] (3) Small intestine digestion stage Take 30 mL of the digested gastric sample and place it in a 100 mL beaker. Adjust the pH to 7.0 ± 0.1 using 1 mol / L NaOH or HCl solution. Then, add 1.5 mL of small intestinal fluid (5.5 g CaCl2 and 32.87 g NaCl dissolved in 150 mL deionized water) and 3.5 mL of bile salt solution (53.57 mg / mL). After precisely adjusting the pH to 7.0, immediately add 2.5 mL of lipase suspension (24 mg / mL). All reagents were kept at 37 °C before addition. The mixture was continuously shaken at 100 rpm for 2 h at a constant temperature of 37 °C, during which the pH was maintained within the range of 7.0 ± 0.1 by titration with 0.25 mol / L NaOH solution. Centrifuge the small intestinal digestion fluid at 8000 rpm for 30 min at 4 °C. The collected supernatant represents the micelles formed during digestion. The absorbance of the supernatant was measured at a wavelength of nm using a UV spectrophotometer, and the mass concentration of curcumin was calculated based on the standard curve.

[0096] The method for constructing the curcumin standard curve is as follows: Accurately weigh 6 mg of curcumin standard and dissolve it in 100 mL of ethanol. Accurately pipette 0, 500, 1000, 1500, 2000, and 2500 μL of each standard solution and dilute to 15 mL. Measure the absorbance of the standard solutions at different concentrations (0, 2, 4, 6, 8, and 10 μg / mL) at a wavelength of 425 nm. Plot the standard curve with concentration on the x-axis and absorbance on the y-axis. Calculate the bioavailability of curcumin using the following formula:

[0097] In the formula: c1 is the concentration of curcumin in the emulsion, μg / mL; c2 is the concentration of curcumin in the micelles, μg / mL.

[0098] 3. Experimental Results like Figure 11 As shown, the bioavailability of curcumin in the emulsion samples of Examples 1 and 2 was 51.36% and 45.64%, respectively, which was significantly higher than that in the emulsion samples of Comparative Examples 1-3. This indicates that the ternary composite emulsifier based on sacha inophylline obtained by two-step enzymatic crosslinking has the potential to improve both system stability and the utilization of active ingredients, providing an important reference for overcoming the limitations of product functionalization upgrades.

[0099] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a composite emulsifier based on sacha inophylline protein, characterized in that, Includes the following steps: (1) Prepare sacha inophylline protein dispersion, polyphenol solution and chitosan solution respectively; (2) Mix the sacha inophylline protein dispersion with polyphenol solution, add laccase to carry out enzymatic cross-linking reaction, dialyze the reaction product and freeze dry to obtain binary complex; (3) After dissolving the binary complex, it was mixed with chitosan solution, and laccase was added to carry out enzymatic cross-linking reaction. The reaction product was dialyzed and freeze-dried to obtain a composite emulsifier based on sacha inophylline protein.

2. The method for preparing the composite emulsifier based on sacha inophylline protein according to claim 1, characterized in that, In step (1), The preparation method of the sacha inophylline protein dispersion includes: mixing sacha inophylline protein with phosphate buffer at a mass-to-volume ratio of 5-15 mg / mL, stirring, and hydrating to obtain the sacha inophylline protein dispersion; the pH value of the phosphate buffer is 7.0 and the concentration is 0.01 mol / L; the stirring conditions are: 400-600 rpm for 1-3 h; the hydration conditions are: 4℃ for 6-10 h. The polyphenol solution is obtained by dissolving a polyphenol compound in water; the mass-to-volume ratio of the polyphenol compound to water is 1-3 mg / mL; the polyphenol compound is selected from epigallocatechin gallate or gallic acid; The chitosan solution is obtained by dissolving chitosan in water; the mass-to-volume ratio of chitosan to water is 1~5 mg / mL.

3. The method for preparing the composite emulsifier based on sacha inophylline protein according to claim 1, characterized in that, In step (2), Sacha indica protein dispersion and polyphenol solution were mixed at a volume ratio of 1:

1. The amount of laccase added is 1~4 U / mL based on the volume of the mixture; Enzymatic cross-linking reaction conditions: stirring at 25 ℃ and 100~300 rpm for 24 h; Dialysis conditions: 6~8 kDa dialysis bag, dialysis at 4 ℃ for 48 h, water changed once every 6 h; Freeze-drying conditions: Pre-freeze at -20 ℃ for 24 h, then freeze-dry under vacuum at -50 ℃ and 10 Pa for 48 h.

4. The method for preparing the composite emulsifier based on sacha inophylline protein according to claim 1, characterized in that, In step (3), The binary complex was dissolved in acetate buffer; the mass-to-volume ratio of the binary complex to the acetate buffer was 10 mg / mL; the concentration of the acetate buffer was 0.1 mol / L. The binary complex solution and the chitosan solution were mixed at a volume ratio of 1:0.5~1.5; The amount of laccase added is 1~4 U / mL based on the volume of the mixture; Enzymatic cross-linking reaction conditions: stirring at 25 ℃ and 100~300 rpm for 24 h; Dialysis conditions: 6~8 kDa dialysis bag, dialysis at 4 ℃ for 48 h, water changed once every 6 h; Freeze-drying conditions: Pre-freeze at -20 ℃ for 24 h, then freeze-dry under vacuum at -50 ℃ and 10 Pa for 48 h.

5. The composite emulsifier based on sacha inophylline obtained by the preparation method according to any one of claims 1 to 4.

6. The application of the composite emulsifier based on saponin obtained by the preparation method according to any one of claims 1 to 4 in the preparation of high internal phase Pickering emulsions.

7. A high internal phase Pickering emulsion, characterized in that, The raw materials for preparation include aqueous phase raw materials and oil phase raw materials, wherein the volume of the oil phase raw materials accounts for 75% to 80% of the total volume of the emulsion raw materials. The aqueous phase raw material includes: water and a composite emulsifier based on sacha inophylline obtained by the preparation method according to any one of claims 1 to 4, wherein the ratio of water to composite emulsifier is 100 mL: 0.5~2.0 g; The oil phase raw material includes: oil.

8. The high internal phase Pickering emulsion according to claim 7, characterized in that, The oil is selected from at least one of soybean oil, olive oil, rapeseed oil, and corn oil; The oil phase raw material also includes: fat-soluble active ingredients; The fat-soluble active ingredient is selected from at least one of curcumin, β-carotene, and lycopene; The mass ratio of the fat-soluble active ingredient to the oil is 0.1~2.0:

100.

9. The method for preparing the high internal phase Pickering emulsion according to claim 7 or 8, characterized in that, The process includes the following steps: mixing aqueous and oil phase raw materials in a certain proportion and homogenizing at 10,000 to 12,000 rpm for 1.5 to 3 minutes to obtain a high internal phase Pickering emulsion.

10. The use of the high internal phase Pickering emulsion of claim 7 or 8 or the high internal phase Pickering emulsion obtained by the preparation method of claim 9 in the preparation of plant-based spreadable foods or 3D printed foods.

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