EGCG (epigallocatechin gallate) and lycopene co-loaded low-fat plant-based fat as well as preparation method and application thereof

Abstract

The invention discloses a preparation method and application of low-fat plant-based fat co-loaded with EGCG (epigallocatechin gallate) and lycopene, and belongs to the technical field of fat product processing. The W / O / W and O / W / O type emulsion gel fats which have different fat properties and are co-loaded with EGCG and lycopene can be prepared, the preparation process is simple, the stabilizer is natural and green, efficient co-loading of hydrophilic EGCG and lipophilic lycopene is achieved, the fat digestibility of the plant-based fat can be controlled by regulating the structure and fat content of the plant-based fat, and the stability of the plant-based fat is improved. The product can be used for developing low-fat healthy non-dairy cream, ice cream, mayonnaise, custard sauce and other products; besides, degradation protection and fixed-point release of EGCG and lycopene can be achieved, bioavailability of EGCG and lycopene is improved, synergistic anti-inflammatory biological activity is shown, and development and manufacturing of novel nutrition-enriched fat-like and plant-based healthy fat products are greatly promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of oils and emulsified fat products, and specifically relates to a low-fat plant-based fat co-loaded with EGCG and lycopene, and a preparation method and application thereof. Background Art

[0002] After years of research, there are still many problems in the production and functional application of two-phase structured emulsions, especially gel-type two-phase emulsion fats. The production of two-phase emulsions usually requires the addition of hydrophilic and hydrophobic emulsifiers, so there may be abuse of synthetic emulsifiers (such as polyglycerol polyricinoleate, etc.). Therefore, in order to ensure food safety, improve food quality, promote healthy consumption, and achieve environmentally friendly food production, there is an urgent need to shift from traditional animal-derived fats to cleaner and more sustainable plant-based fats. Among them, functional fat foods have the advantages of anti-oxidation, stomach protection, and lipid-lowering, and are receiving more and more attention and favor in the food industry.

[0003] In recent years, researchers have also noted the health benefits of maintaining and enhancing dietary bioactive compounds through "synergy". "Synergy" refers to the joint action between two or more bioactive compounds. For example, epigallocatechin-3-gallate (EGCG) and lycopene have been shown to have synergistic effects in disease prevention and health promotion. EGCG is a hydrophilic antioxidant widely found in green tea and is the main component of tea polyphenols. Due to the presence of multiple phenolic hydroxyl groups in its structure, EGCG has strong antioxidant and anti-inflammatory activities and has a variety of beneficial effects on human health. Lycopene is a natural red chain hydrocarbon carotenoid with a chemical structure of 11 conjugated double bonds and 2 non-conjugated double bonds. Neither humans nor animals can produce lycopene. As an excellent antioxidant in nature, it can avoid cell damage and increase antioxidant enzymes in the body, such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). However, their co-delivery in food remains a challenge due to the solubility differences between the hydrophilicity of EGCG and the lipophilicity of lycopene.

[0004] In addition, the sensitivity of EGCG and lycopene to pH, light, oxygen, temperature and ionic strength leads to low bioavailability, poor digestive stability and poor intestinal absorption. In order to solve these problems, the delivery system needs to be specially designed to ensure that the nutrients are protected in the mouth, stomach and other parts and released at the target site. Therefore, as a carrier of nutritional functional ingredients, the design of the fat-like system needs to be customized according to the characteristics of the nutrients loaded. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] Different fat-like foods require different nutrient encapsulation and oil digestibility in different application scenarios. Currently, fat foods have problems such as single structure and limited nutritional and health functions. Studies have shown that the hydrophilic nutrient EGCG and the lipophilic nutrient lycopene have synergistic effects in disease prevention and health promotion. However, the poor bioavailability caused by differences in solubility and environmental instability limits their application in fat foods.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a low-fat plant-based fat that is co-loaded with EGCG and lycopene.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions: satisfying the following conditions:

[0010] (i) having a W1 / O / W2 or O1 / W / O2 dual-phase structure;

[0011] (ii) simultaneous co-loading and co-release of EGCG and lycopene;

[0012] (iii) The lycopene concentration in the oil phase was <5 mg / mL, and the EGCG concentration in the water phase was <8 mg / mL.

[0013] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing a low-fat plant-based fat co-loaded with EGCG and lycopene.

[0014] In order to solve the above technical problems, the present invention provides the following technical solutions: comprising:

[0015] EGCG powder is added to the aqueous solution of plant protein particles and stirred thoroughly to obtain an EGCG dispersed solution; lycopene powder is added to vegetable oil and subjected to ultrasonic treatment until completely dissolved to obtain a lycopene oil solution;

[0016] The lycopene oil solution, the EGCG dispersed solution and one or more of the solid fat and the vegetable protein particle aqueous solution are mixed and sheared and emulsified to obtain a low-fat plant-based fat with a dual-phase structure.

[0017] As a preferred embodiment of the method for preparing the low-fat plant-based fat co-loaded with EGCG and lycopene according to the present invention, the low-fat plant-based fat with a dual-phase structure comprises one of a W1 / O / W2 type emulsion gel or an O1 / W / O2 type emulsion gel.

[0018] As a preferred embodiment of the method for preparing the low-fat plant-based fat co-loaded with EGCG and lycopene of the present invention, when the low-fat plant-based fat is a W1 / O / W2 type emulsion gel, the lycopene oil solution and the solid fat are heated and mixed evenly, and then mixed with the EGCG dispersion solution, and the W1 / O type primary emulsion is added to the aqueous solution of the plant protein particles after shear emulsification in an ice bath, and the W1 / O type primary emulsion is added to the aqueous solution of the plant protein particles, and the W1 / O / W2 type emulsion gel is obtained after secondary shear emulsification;

[0019] The added mass ratio of the lycopene oil solution, solid fat, EGCG dispersion solution and plant protein particle aqueous solution is (5-50): (5-70): (5-30): (20-70).

[0020] As a preferred embodiment of the method for preparing the low-fat plant-based fat co-loaded with EGCG and lycopene of the present invention, when the low-fat plant-based fat is an O1 / W / O2 type emulsion gel,

[0021] The lycopene oil solution and the EGCG dispersion solution are mixed, and an O1 / W type primary emulsion is obtained after a primary shear emulsification, and the primary emulsion is added to the heated and melted solid fat solution, and a secondary shear emulsification is performed in an ice bath to obtain an O1 / W / O2 type emulsion gel;

[0022] The added mass ratio of the lycopene oil solution, solid fat and EGCG dispersion solution is (5-30):(10-70):(10-70).

[0023] As a preferred embodiment of the method for preparing the low-fat plant-based fat co-loaded with EGCG and lycopene according to the present invention, the plant protein includes one of soy protein isolate, peanut protein isolate, pea protein isolate, mung bean protein isolate, oat protein isolate, quinoa protein isolate, broad bean protein isolate, and potato protein isolate; the vegetable oil includes one of soybean oil, rapeseed oil, peanut oil, sunflower seed oil, rice bran oil, corn oil, linseed oil, olive oil, wheat germ oil, cottonseed oil, almond oil, tea seed oil, and sesame oil; the solid fat includes one of palm oil, palm kernel oil, palm stearin, coconut oil, cocoa butter, shea butter, safflower resin, mango kernel oil, and ice grass butter.

[0024] As a preferred embodiment of the method for preparing the low-fat plant-based fat co-loaded with EGCG and lycopene according to the present invention, the amount of EGCG added is 1-5 mg / mL of the aqueous solution of plant protein particles; the amount of lycopene added is 1-3 mg / mL of the oil solution.

[0025] As a preferred embodiment of the method for preparing the low-fat plant-based fat co-loaded with EGCG and lycopene according to the present invention, the ultrasonic treatment comprises the following steps: the ultrasonic power is 200 to 500 W, and the ultrasonic time is 20 to 90 min; and the heating temperature is 50 to 80°C.

[0026] As a preferred embodiment of the method for preparing the low-fat plant-based fat co-loaded with EGCG and lycopene according to the present invention, the first shear emulsification condition is 8000-12000 rpm, 2-5 min; the second shear emulsification condition is 2000-5000 rpm, 2-5 min.

[0027] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a low-fat plant-based fat co-loaded with EGCG and lycopene for use in food and site-specific delivery of functional ingredients and cellular anti-inflammatory activity.

[0028] Beneficial effects of the present invention:

[0029] The plant-based fats co-loaded with lycopene and EGCG prepared by the present invention can be structurally regulated to obtain W / O / W and O / W / O type emulsion gel fats with different characteristics. The preparation method is simple, and the stabilizer is green and natural. It realizes high-efficiency co-loading of hydrophilic EGCG and lipophilic lycopene, and by regulating the structure and oil content of the fats, its oil digestibility and the co-release of EGCG and lycopene can be controlled, thereby improving the bioavailability of both, and then showing cellular anti-inflammatory activity. Specifically: the fats co-loaded with EGCG and lycopene with low oil digestibility can be used in products such as mayonnaise, ice cream and spreads, and a large amount of undigested fat can increase satiety and reduce calorie intake; hydrophilic EGCG and lipophilic lycopene are unstable to pH, heat, etc., and these functional active substances can be effectively avoided from being released in large quantities in the stomach and small intestine in the fats with a dual-phase structure, thereby smoothly reaching the large intestine, improving the bioavailability of both, and better exerting their synergistic anti-inflammatory biological effects. Low-fat plant-based fats co-loaded with EGCG and lycopene are good functional ingredient delivery systems, promoting the development and manufacture of new nutritionally fortified fats. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. Among them:

[0031] Figure 1 Schematic diagram of the structure of low-fat plant-based fats co-loaded with EGCG and lycopene;

[0032] Figure 2 The appearance of the low-fat plant-based fats co-loaded with EGCG and lycopene obtained in Examples 1 to 5 (0 day and after 28 days of storage at room temperature);

[0033] Figure 3 It is an optical micrograph of the low-fat plant-based fat co-loaded with EGCG and lycopene obtained in Examples 1 to 5;

[0034] Figure 4 The microstructure changes of the low-fat plant-based fats co-loaded with EGCG and lycopene obtained in Examples 1 to 3 during the in vitro simulated digestion process;

[0035] Figure 5 This is a decoration picture of the low-fat plant-based fat co-loaded with EGCG and lycopene obtained in Example 7. DETAILED DESCRIPTION

[0036] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0037] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0038] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0039] The raw materials used in the present invention are all commercially available unless otherwise specified.

[0040] Epigallocatechin-3-gallate (EGCG) and lycopene were purchased from Xi’an Kanghe Biotechnology Co., Ltd. and Xi’an Zelong Biotechnology Co., Ltd., respectively.

[0041] The preparation method of the vegetable protein particle aqueous solution comprises the following steps: preparing a vegetable protein isolate aqueous solution with a mass concentration of 3% to 10%, adjusting the pH to 7.0 after sufficient hydration, performing a heat denaturation treatment at 80 to 100° C. for 30 to 60 minutes, and obtaining the vegetable protein particle aqueous solution by homogenization treatment at a pressure of 30 MPa after cooling.

[0042] Example 1

[0043] This embodiment provides a method for preparing a low-fat plant-based fat co-loaded with EGCG and lycopene, specifically:

[0044] (1) Weighing 0.04 g of EGCG and adding it to 20 mL of quinoa protein particle (1 wt%) aqueous solution, stirring thoroughly to obtain an EGCG dispersion solution; weighing 0.04 g of lycopene powder and adding it to 20 mL of peanut oil, ultrasonically treating at 500 W for 30 min to obtain a lycopene oil solution;

[0045] (2) The lycopene oil solution in (1) was mixed with 10 mL of palm oil, and the clarified oil solution obtained after heating at 50° C. for 15 min was mixed with the EGCG dispersion in (1), and sheared and homogenized at 10,000 rpm for 2 min to obtain a W1 / O type primary emulsion, which was added to 50 mL of the vegetable protein particle aqueous solution, and sheared and homogenized at 8,000 rpm for 4 min to obtain a W1 / O / W2 type emulsion gel fat co-loaded with EGCG and lycopene, wherein the proportion of palm oil was 10%; the added mass ratio of lycopene oil solution, solid fat, EGCG dispersion solution and vegetable protein particle aqueous solution was 20:10:20:50.

[0046] (3) The lycopene oil solution in (1) and the EGCG dispersion in (1) were mixed, and sheared and homogenized at 10000 rpm for 2 min to obtain an O1 / W type primary emulsion, which was added to 10 mL of palm oil that had been heated and melted, and sheared and homogenized at 3000 rpm in an ice bath for 5 min to obtain an O1 / W / O2 type emulsion gel fat co-loaded with EGCG and lycopene, wherein the proportion of palm oil was 20%; the mass ratio of the lycopene oil solution, solid fat, and EGCG dispersion solution was 20:10:20.

[0047] Example 2

[0048] The difference between this embodiment and embodiment 1 is that the amount of palm oil added is adjusted to 20 mL, the amount of the vegetable protein particle aqueous solution added is adjusted to 40 mL, and the rest of the preparation process is the same as that of embodiment 1, to obtain a W1 / O / W2 type emulsion gel fat co-loaded with EGCG and lycopene, in which the proportion of palm oil is 20%.

[0049] The amount of palm oil added was adjusted to 20 mL to obtain an O1 / W / O2 type emulsion gel fat co-loaded with EGCG and lycopene, in which the proportion of palm oil was 33.3%.

[0050] Example 3

[0051] The difference between this embodiment and embodiment 1 is that the amount of palm oil added is adjusted to 30 mL, the amount of the vegetable protein particle aqueous solution added is adjusted to 30 mL, and the rest of the preparation process is the same as that of embodiment 1, and a W1 / O / W2 type emulsion gel fat co-loaded with EGCG and lycopene is obtained, in which the proportion of palm oil is 30%.

[0052] The amount of palm oil added was adjusted to 30 mL to obtain an O1 / W / O2 type emulsion gel fat co-loaded with EGCG and lycopene, in which the proportion of palm oil was 42.9%.

[0053] Example 4

[0054] The difference between this embodiment and embodiment 1 is that the amount of palm oil added is adjusted to 40 mL, the amount of the vegetable protein particle aqueous solution added is adjusted to 20 mL, and the rest of the preparation process is the same as that of embodiment 1, to obtain a W1 / O / W2 type emulsion gel fat co-loaded with EGCG and lycopene, in which the proportion of palm oil is 40%.

[0055] The amount of palm oil added was adjusted to 40 mL to obtain an O1 / W / O2 type emulsion gel fat co-loaded with EGCG and lycopene, in which the proportion of palm oil was 50%.

[0056] Example 5

[0057] The difference between this embodiment and embodiment 1 is that palm oil is adjusted to palm stearin (melting point 42° C.), and the rest of the preparation process is the same as that of embodiment 1, to obtain a W1 / O / W2 type emulsion gel fat co-loaded with EGCG and lycopene, in which the proportion of palm stearin is 20%.

[0058] Palm oil was adjusted to palm stearin (melting point 42° C.) to obtain an O1 / W / O2 type emulsion gel fat co-loaded with EGCG and lycopene, in which the proportion of palm oil was 20%.

[0059] Comparison

[0060] The O / W emulsion stabilized by synthetic emulsifier Tween 80 and the W / O solution stabilized by PGPR were used as the control for the EGCG and lycopene embedding efficiency, lipid fatty acid release rate and nutrient biorelease rate, specifically:

[0061] 0.6 g (3 wt%) Tween 80 was dispersed in 20 mL of water as the aqueous phase, mixed with 20 mL of peanut oil dissolved with lycopene (2 mg / mL), and sheared and homogenized at 10000 rpm for 2 min using a high-speed shearing device to obtain an O / W emulsion; 0.6 g (3 wt%) PGPR was dispersed in 20 mL of peanut oil as the aqueous phase, mixed with 20 mL of aqueous phase dissolved with EGCG (2 mg / mL), and sheared and homogenized at 10000 rpm for 2 min using a high-speed shearing device to obtain a W / O emulsion.

[0062] EGCG and lycopene encapsulation efficiency:

[0063] Method for determining the embedding rate of EGCG and lycopene in biphasic structured fats:

[0064]

[0065] In the formula, A total is the EGCG and lycopene initially added to the inner water phase and inner oil phase of the system, A outer It refers to the EGCG and lycopene that are not embedded in the external water phase or external oil phase in the system.

[0066] The results are shown in the following table.

[0067] Table 1 EGCG and lycopene embedding rate in different types of fat

[0068]

[0069] After testing, the embedding rate of lycopene in the control group O / W emulsion was 65.4%, and the embedding rate of EGCG in the control group W / O emulsion was 74.8%. As can be seen from Table 1, the W / O / W type and O / W / O type emulsion gel fats co-loaded with EGCG and lycopene obtained in the embodiment can achieve high-efficiency co-embedding of hydrophilic EGCG and lipophilic lycopene, and the embedding rate is significantly higher than that of the control group, both reaching more than 85%, indicating that the two stable interfaces of protein particles and fat crystals can provide excellent structural stability of the system, making it a good nutrient loading system. In addition, it is found that with the increase of oil, especially solid fat content, the embedding effect of nutrients is enhanced, which can be attributed to the increase in the amount of fat crystals, which is conducive to enhancing the thickness of its stable interface layer, thereby inhibiting the diffusion of nutrients. The type of solid fat used also affects the embedding effect of EGCG and lycopene. The increase in the melting point of solid fat promotes the formation of more solid fat crystals and enhances the network structure of the system, thereby better protecting the two nutrients.

[0070] Retention rate of EGCG and lycopene during storage:

[0071] Lycopene extraction and quantitative test method: 3 mL of dichloromethane / n-hexane (1:2, v / v) was added to 2 g of sample as solvent, and the mixture was shaken vigorously for 2 min, and then centrifuged at 3000 rpm for 20 min to form a two-phase system, and the upper organic layer was collected. Then, 3 ml of n-hexane was added for two extractions, and all polarities were combined. The absorbance of lycopene was measured at 472 nm using an ultraviolet spectrophotometer, and the prepared standard curve (R 2 =0.999) to calculate the mass of lycopene.

[0072] EGCG extraction and quantitative test method: The sample was placed in an ultrafiltration concentration centrifuge tube, diluted with an equal amount of 25% ethanol and 0.5% Tween 80 mixture, and centrifuged at 4000 rpm for 20 min at 4°C; the collected supernatant was filtered through a 0.22 μm syringe, and then measured using an ultraviolet spectrophotometer at 274 nm (maximum absorbance of EGCG) according to the established standard curve (R 2 =0.997) to quantify the amount of unencapsulated EGCG.

[0073] The fat is stored at room temperature, and the changes in the EGCG and lycopene contents therein are regularly measured using the above method to obtain the nutrient retention rate.

[0074] EGCG aqueous solution (2 mg / mL) and lycopene peanut oil solution (2 mg / mL) were used as controls for nutrient retention. The results are shown in the following table.

[0075] Table 2 Retention rate of EGCG and lycopene in different types of fat during storage (28 days)

[0076]

[0077] Figure 2 The following are the appearance pictures of the co-loaded nutrient fat in the embodiment at 0 day and after 28 days of storage at room temperature. It can be seen that the obtained product has good plasticity and can be inverted smoothly without any instability. Moreover, after long-term storage, the appearance state and color do not change significantly, and it has excellent stability.

[0078] After testing, the retention rate of lycopene in the control group O / W emulsion after 28 days was 35.4%, and the retention rate of EGCG in the control group W / O emulsion was 44.9%. As can be seen from Table 2, the retention rates of lycopene and EGCG obtained in the embodiment reached more than 80%, effectively inhibiting the degradation of hydrophilic and hydrophobic substances under storage conditions. Since the solidification phase effect of solid fat can slow down the diffusion of easily degradable compounds, the stability of embedded bioactive substances is enhanced. Therefore, we believe that enhancing the formation of gel network and the generation of thick interfacial film helps to improve stability. In addition, the antioxidant activity of lycopene and EGCG in the system may prompt them to protect each other.

[0079] Figure 3 It is a microscopic picture of the fat-like substance in the embodiment. It can be seen that both W1 / O / W2 type and O1 / W / O2 type emulsion gel fat-like substances are successfully formed, and a fairly clear multiphase structure is observed, in which dense inner chamber droplets are evenly distributed in the outer phase. For W1 / O / W2 type fat-like substances, the primary W1 / O emulsion is first stabilized by the fat crystals generated in situ during cooling in the solid fat, and then the external oil-water phase interface (O / W2) is stabilized by the plant protein particles. Increasing the number of fat crystals in the oil is conducive to the increase and uniform distribution of the number of spherical water droplets in the oil. At the same time, the high-viscosity water droplets stabilized by the fat crystals will hinder their aggregation and deformation, which is conducive to the stability of the internal water phase droplets in the system and the improvement of the encapsulation efficiency of the two nutrients. For O1 / W / O2 type fat-like substances, the O1 / W interface is first stabilized by the plant protein particles, and then the fat crystals generated in situ during cooling in the solid fat adsorb and stabilize the external water-oil interface (W / O2) to form a stable coating layer. Similarly, the increase of solid fat in the outer oil phase can enhance the gel network structure. Therefore, W / O / W and O / W / O fats with excellent structures for co-encapsulating hydrophilic EGCG and lipophilic lycopene can be successfully prepared by relying only on fat crystals and plant protein particles. Interestingly, due to the different structures of W / O / W and O / W / O fats, such as the location of different phases, they have different fat properties (including fat properties and encapsulation properties). The presence of various phases provides more dimensions, allowing for finer texture and nutrient loading effects to be controlled by modifying various structural units.

[0080] In vitro simulated digestion process:

[0081] An in vitro digestion model was established according to the method in the literature (Carrageenan-induced conjugated oatprotein isolate microgel particles as structure modulators in fat analogues and their digestion behaviors. Journal of Colloid and Interface Science. 2024.), the fat was digested in vitro in a simulated manner, and the structure of the fat at different stages was measured. The free fatty acid release rate was measured according to the literature (Encapsulation of vitamin D3 in pickering emulsions stabilized by nanofibrillated mangosteen cellulose: Impact on in vitro digestion and bioaccessibility. Food Hydrocolloids, 2018, 83, 153-164.);

[0082]

[0083] Where V NaOH is the volume of sodium hydroxide solution consumed during the titration, m NaOH is the molar mass of the sodium hydroxide solution, M Lipid is the molecular weight of the oil, W Lipid It’s the quality of the oil.

[0084] The bioavailability of EGCG and lycopene in lipids was determined according to the literature (Tailoring the properties of double-crosslinked emulsion gels using structural design principles: Physical characteristics, stability, and delivery of lycopene. 2021, Biomaterials.);

[0085] Bio-accessibility (%)=Cmicelle / Cinitial×100

[0086] Where, Cmicell e is the nutrient content in the micellar phase obtained after digestion, Cinitia l is the nutrient content in the initial sample.

[0087] The results are shown in the following table.

[0088] Table 3 Release rate of free fatty acids of different fats after in vitro digestion

[0089]

[0090] The fatty acid release rate of the control group O / W emulsion was 84.9%. The main difference between Examples 1 to 4 is that the solid fat content gradually increases. For Examples 1 to 4, it can be seen that as the solid fat content increases, the fat digestibility of the fat analog becomes lower, which means that the fat digestibility can be adjusted by changing the oil phase content. And the O1 / W / O2 type structural fat analog has a significantly lower fat digestibility than the W1 / O / W2 type fat analog. The presence of more fat crystals in the outer oil phase can cause an interfacial barrier and hinder lipid digestion, because digestion mainly involves the interfacial area where lipase binds to the droplet surface. Fat analogs with higher viscosity can inhibit the diffusion of lipase and the diffusion of lipolysis products, thereby delaying lipid digestion. In addition, through the microstructural observation of different digestion stages of Examples 1 to 3 ( Figure 4 ), we found that after oral processing, the co-loaded nutrient fats all retained the typical "droplet-in-droplet" structure well. And the structure is maintained during the gastric digestion stage, the degree of emulsion aggregation is low, and the high salt and low pH environment in the stomach has little effect on structural damage, which also shows that the EGCG and lycopene embedded in this stage have a good protective effect. When these fats enter the small intestine stage, it can be clearly seen that there are still a large number of undigested emulsion droplets, which is attributed to the gelled oil phase agglomerating into blocks, reducing the surface contact area between lipase and lipids. In addition, the two-phase structure will also greatly reduce the speed and degree of digestion. This means that compared with Examples 1 and 2, Example 3 has a stronger fat-like structure. By changing the fat-like structure and fat content, the fat digestibility can be effectively adjusted.

[0091] The main difference between Example 1 and Example 5 is the difference in the solid fat used. Compared with the product prepared from palm oil (melting point 34°C) used in Example 1, the W / O / W and O / W / O type fats stabilized by palm stearin (melting point 42°C) with co-loaded nutrients in Example 5 have significantly lower fat digestibility, indicating that the stronger network structure and interface layer strength given to the system by high melting point fat crystals can inhibit the lipid digestion rate and the degree of structural damage. Because the change in the type of solid fat used makes the system show different fat digestibility.

[0092] Table 4 Bioavailability of EGCG and lycopene after in vitro digestion of different types of fat

[0093]

[0094] The bioavailability of lycopene in the O / W emulsion of the control group was 23.4%, and the bioavailability of EGCG in the W / O emulsion was 21.5%. As can be seen from Table 4, compared with the control group, the fat obtained in the embodiment can realize the co-release of hydrophilic EGCG and lipophilic lycopene while realizing the co-loading of the two, thereby improving their bioavailability. The dual structures of O / W / O and W / O / W can effectively protect and slowly release lycopene and EGCG, and it is found that the co-loading of lycopene and EGCG has no significant effect on the release of each other. The dual-phase structure can make the lycopene in the O1 / W / O2 fat and the EGCG in the W1 / O / W2 have a more obvious sustained release, because they are dissolved in the inner and outer chambers at the same time, and the active compounds contained in the outer droplets are ready for early release and diffusion through the membrane, while the amount in the inner droplet chamber ensures long-term release. In addition, with the increase of the oil phase content, the bioavailability of EGCG and lycopene in the O / W / O type and W / O / W type fats decreases accordingly. Compared with O / W / O type lipids, W / O / W type lipids have higher bioavailability of lycopene and EGCG. The gelation of the outer oil phase not only delays the release of free fatty acids, but also reduces the transport efficiency of lycopene to micelles. The hydrophilicity of EGCG makes it easy for the molecule to diffuse into the hydrophilic release medium. Therefore, lipids co-loaded with EGCG and lycopene can be used as a universal and effective nutritional fortification delivery system.

[0095] Lipids affect the inflammatory response of HepG2 cells induced by lipopolysaccharide (LPS):

[0096] After the co-loaded EGCG and lycopene lipids were digested in the intestinal phase of in vitro digestion, the obtained micelle samples were diluted with serum-free medium. HepG2 cells were divided into blank group (O / W / O and W / O / W), LPS model group (O / W / O and W / O / W), EGCG loading group (O / W / O and W / O / W), lycopene loading group (O / W / O and W / O / W), and EGCG and lycopene co-loaded group (O / W / O and W / O / W). HepG2 cells in the logarithmic growth phase were seeded in 96-well plates and incubated at 37°C, 5% CO 2 The cells were cultured in an incubator for 48 h, then washed with PBS, 100 μL of the diluted digested sample was added to 3 wells and cultured for 4 h, washed twice with PBS, and then 100 μL (1 μg / mL) of LPS was added and cultured for 12 h. Finally, the cell viability was determined using the MTT method with a cell counting kit.

[0097] The micellar phases after in vitro digestion of the products of the control group (without LPS induction), the control group (LPS induction, no nutrient embedding in Example 1), the control group (only EGCG embedding in Example 1), the control group (only lycopene embedding in Example 1) and Examples 1 to 3 were collected and used to explore the anti-inflammatory activity of cells according to the above method. The results are shown in Table 5.

[0098] Table 5 Effects of in vitro digestion products of different types of fat on the cell survival rate of LPS-induced HepG2 cells

[0099]

[0100] Lipopolysaccharide is one of the main components of the cell wall of Gram-negative bacteria and is a strong stimulant that causes inflammatory reactions. As can be seen from Table 5, when EGCG and lycopene are not embedded in W / O / W type and O / W / O type fats, the effect of LPS on cell viability is significant, the cell survival rate is low, and the loaded nutrients have a good protective effect on LPS-induced inflammation. At the same time, compared to the fats (control group) loaded with EGCG or lycopene alone, the cell survival rate of the co-loaded EGCG and lycopene in Example 1 is high, indicating that EGCG and lycopene in fats have a synergistic anti-inflammatory effect. From the results analysis of Example 1, Example 2 and Example 3, with the increase of oil content, the cell survival rate of LPS-induced cells under the action of bioactive substances shows a decreasing trend, which may be due to the reduction of the content of nutrients in the micelle phase after multi-stage digestion in vitro.

[0101] Example 6

[0102] The difference between this embodiment and embodiment 1 is that the protein particles are adjusted to be peanut protein isolate (2wt%), the palm oil is adjusted to be coconut oil, and the rest of the preparation process is the same as that of embodiment 1, to obtain a W1 / O / W2 type emulsion gel fat co-loaded with EGCG and lycopene, wherein the proportion of coconut oil is 20%; and an O1 / W / O2 type emulsion gel fat co-loaded with EGCG and lycopene, wherein the proportion of coconut oil is 33.3%.

[0103] The results showed that both types of dual-phase structures were successfully formed and had excellent nutrient retention rates. The encapsulation rates of lycopene and EGCG in the W1 / O / W2 emulsion gel were 93.05% and 91.48%, respectively, and the encapsulation rates of lycopene and EGCG in the O1 / W / O2 emulsion gel fat were 94.89% and 91.12%, respectively.

[0104] Example 7

[0105] The difference between this embodiment and embodiment 3 is that the protein particles are adjusted to potato protein isolate (2wt%), the palm oil is adjusted to cocoa butter (melting point 38°C), and the rest of the preparation process is the same as that of embodiment 3, and the W1 / O / W2 type emulsion gel fat co-loaded with EGCG and lycopene is obtained, wherein the proportion of cocoa butter is 30%; the O1 / W / O2 type emulsion gel fat co-loaded with EGCG and lycopene is obtained, wherein the proportion of cocoa butter is 32.9%.

[0106] The results showed that the fat hardness of the nutrient-loaded ingredients stabilized by potato protein isolate and cocoa butter was higher, and it had excellent decorating support ( Figure 5 ), the embedding rates of lycopene and EGCG in W1 / O / W2 emulsion gel were 94.85% and 95.48%, respectively, and the embedding rates of lycopene and EGCG in O1 / W / O2 emulsion gel fat were 94.01% and 96.52%, respectively.

[0107] Comparative Example 1

[0108] The difference between this comparative example and Example 1 is that step (1) in Example 1 is changed to: weigh 0.15 g of lycopene powder and add it to 20 mL of peanut oil, and then ultrasonicate at 500 W for 60 min to obtain a lycopene oil solution; the remaining steps are consistent with Example 1.

[0109] The results showed that when the lycopene content (5 mg / mL in the oil phase) was too high, it reduced the ability of fat crystals to stabilize the W / O emulsion, and thus could not form a biphasic lipid, and thus could not successfully and efficiently co-load EGCG and lycopene. Therefore, only when a certain amount of lycopene was present could a co-encapsulated low-fat plant-based lipid with good structure and properties be formed.

[0110] Comparative Example 2

[0111] The difference between this comparative example and Example 1 is that step (1) in Example 1 is changed to: weigh 0.16 g EGCG and add it into 20 mL quinoa protein granule (1 wt %) aqueous solution; the remaining steps are consistent with Example 1.

[0112] The results showed that when the EGCG content (8 mg / mL in the aqueous phase) was too high, the interaction between EGCG and plant protein reduced the emulsification stabilization ability of the plant protein, resulting in the inability to stabilize the oil-water interface, and thus failing to form a lipid with a biphasic structure, and thus failing to successfully and efficiently co-load EGCG and lycopene.

[0113] Comparative Example 3

[0114] The difference between this comparative example and Example 1 is that step (2) in Example 1 is changed to: mixing the lycopene oil solution in (1) with 10 mL of peanut oil, and the other operations remain the same;

[0115] The step (3) in Example 1 was modified as follows: the obtained O1 / W primary emulsion was added into 10 mL of peanut oil, and the other operations remained the same.

[0116] The test found that due to the absence of solid fat added to the oil phase, a stable interface of the crystalline network could not be formed, and thus W / O / W and O / W / O fats that co-loaded EGCG and lycopene could not be formed.

[0117] Comparative Example 4

[0118] The step (2) in Example 1 is modified as follows: the lycopene oil solution in (1) is mixed with 10 mL of palm oil, and heated at 50° C. for 15 min to obtain a clear oil solution, the EGCG dispersion in (1) is mixed with 50 mL of the plant protein particle aqueous solution to obtain an aqueous phase, and the aqueous phase and the oil phase are mixed and sheared and homogenized at 10000 rpm for 5 min to obtain an O / W type emulsion co-loaded with nutrients;

[0119] The step (3) in Example 1 was changed to: the lycopene oil solution in (1) was mixed with 10 mL of palm oil after heating and melting as the oil phase, which was mixed with the EGCG dispersion in (1), and sheared and homogenized at 10000 rpm in an ice bath for 5 min to obtain a W / O type emulsion co-loaded with nutrients.

[0120] The results showed that if the aqueous solution of plant protein particles containing EGCG and the oil solution containing lycopene were used to directly construct O / W and O / W emulsions, the embedding rates of EGCG and lycopene in the formed O / W type fats were 13.4% and 90.2%, respectively, and the embedding rates of EGCG and lycopene in the W / O type were 88.9% and 9.6%, respectively. Therefore, unlike the plant-based fats with a dual-phase structure prepared by the two-step method of the present invention, it cannot simultaneously achieve high-efficiency co-loading of EGCG and lycopene.

[0121] Comparative Example 5

[0122] In step (1) of Example 1, lycopene was not added to the oil solution, and the other steps remained the same, thereby obtaining W / O / W type and O / W / O type plant-based fats loaded with only EGCG.

[0123] The test found that the retention rates of EGCG in W / O / W type and O / W / O type fats after 28 days were 73.2% and 77.8%, respectively, which were significantly lower than the EGCG and lycopene co-encapsulation system in Example 1. This indicates that the co-encapsulation of lycopene and EGCG can effectively enhance the retention effect of fat emulsion gel on EGCG.

[0124] Comparative Example 6

[0125] In step (1) of Example 1, EGCG was not dissolved in the plant protein particle solution, and the other steps remained the same, thereby obtaining W / O / W type and O / W / O type plant-based fats loaded with only lycopene.

[0126] The test found that the retention rates of lycopene in W / O / W and O / W / O fats after 28 days were 71.7% and 83.3%, respectively, indicating that the co-encapsulation of lycopene and EGCG can effectively enhance the retention effect of fat emulsion gel on lycopene.

[0127] In summary, the plant-based fats co-loaded with EGCG and lycopene prepared by the present invention can achieve high-efficiency co-loading of the two nutrients, and can obtain W / O / W type and O / W / O type fats with different dual-phase structures according to functional characteristics, and the preparation process is simple, green and natural, and conforms to the development trend of health. It has a good protective effect on nutrients when stored at room temperature and has good structural stability.

[0128] In addition, lipids can resist harsh digestive environments, allowing nutrients to be released in the small intestine and better absorbed and utilized by the human body. The fat digestibility and nutrient bioavailability of co-loaded nutrient lipids can be adjusted by changing the lipid structure, oil phase content, and the type of solid fat used. O / W / O type lipids, increased oil phase content, and increased melting point of solid fats are beneficial to reducing fat digestion and calorie intake, and can be applied to healthy foods such as mayonnaise and ice cream. W / O / W type lipids release more EGCG and lycopene when digested in vitro, which is beneficial to the slow and efficient co-release of the two and improves their bioavailability. Furthermore, lipids co-loaded with EGCG and lycopene can effectively inhibit cell damage and show synergistic biological effects in cell anti-inflammatory activity. This greatly expands the customized design and application of new functional fat foods.

[0129] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A low-fat plant-based fat co-loaded with EGCG and lycopene, characterized in that: The following conditions are met, (i) Having a W1 / O / W2 or O1 / W / O2 dual-phase structure; (ii) simultaneous co-loading and co-release of EGCG and lycopene; (iii) The lycopene concentration in the oil phase was <5 mg / mL, and the EGCG concentration in the water phase was <8 mg / mL.

2. The method for preparing low-fat plant-based fat co-loaded with EGCG and lycopene according to claim 1, characterized in that: include, EGCG powder is added to the aqueous solution of plant protein particles and stirred thoroughly to obtain an EGCG dispersed solution; Lycopene powder is added to vegetable oil and subjected to ultrasonic treatment until completely dissolved to obtain a lycopene oil solution; The lycopene oil solution, the EGCG dispersed solution and one or more of the solid fat and the vegetable protein particle aqueous solution are mixed and sheared and emulsified to obtain a low-fat plant-based fat with a dual-phase structure.

3. The method for preparing low-fat plant-based fat co-loaded with EGCG and lycopene according to claim 2, characterized in that: The low-fat plant-based fat with a dual-phase structure includes one of a W1 / O / W2 type emulsion gel or an O1 / W / O2 type emulsion gel.

4. The method for preparing low-fat plant-based fat co-loaded with EGCG and lycopene according to claim 2, characterized in that: When the low-fat vegetable-based fat is a W1 / O / W2 type emulsion gel, The lycopene oil solution and the solid fat are heated and mixed uniformly, and then mixed with the EGCG dispersion solution, and after a primary shear emulsification in an ice bath, the obtained W1 / O type primary emulsion is added to the aqueous solution of the plant protein particles, and a secondary shear emulsification is performed to obtain a W1 / O / W2 type emulsion gel; The added mass ratio of the lycopene oil solution, solid fat, EGCG dispersion solution and plant protein particle aqueous solution is (5-50): (5-70): (5-30): (20-70).

5. The method for preparing low-fat plant-based fat co-loaded with EGCG and lycopene according to claim 2, characterized in that: When the low-fat plant-based fat is an O1 / W / O2 type emulsion gel, The lycopene oil solution and the EGCG dispersion solution are mixed, and an O1 / W type primary emulsion is obtained after a primary shear emulsification, and the primary emulsion is added to the heated and melted solid fat solution, and a secondary shear emulsification is performed in an ice bath to obtain an O1 / W / O2 type emulsion gel; The added mass ratio of the lycopene oil solution, solid fat and EGCG dispersion solution is (5-30):(10-70):(10-70).

6. The method for preparing low-fat plant-based fat co-loaded with EGCG and lycopene according to claim 2, characterized in that: The plant protein includes one of soybean protein isolate, peanut protein isolate, pea protein isolate, mung bean protein isolate, oat protein isolate, quinoa protein isolate, broad bean protein isolate, and potato protein isolate; the vegetable oil includes one of soybean oil, rapeseed oil, peanut oil, sunflower seed oil, rice bran oil, corn oil, linseed oil, olive oil, wheat germ oil, cottonseed oil, almond oil, tea seed oil, and sesame oil; the solid fat includes one of palm oil, palm kernel oil, palm stearin, coconut oil, cocoa butter, shea butter, balsam pear resin, mango kernel oil, and ice grass fat.

7. The method for preparing low-fat plant-based fat co-loaded with EGCG and lycopene according to claim 2, characterized in that: The added amount of EGCG is 1-5 mg / mL of the plant protein particle aqueous solution; the added amount of lycopene is 1-3 mg / mL of the oil solution.

8. The method for preparing low-fat plant-based fat co-loaded with EGCG and lycopene according to claim 2, characterized in that: The ultrasonic treatment comprises an ultrasonic power of 200 to 500 W and an ultrasonic time of 20 to 90 min.

9. The method for preparing low-fat plant-based fat co-loaded with EGCG and lycopene according to claim 4 or 5, characterized in that: The primary shear emulsification condition is 8000-12000 rpm, 2-5 min; the secondary shear emulsification condition is 2000-5000 rpm, 2-5 min; the heating temperature is 50-80°C.

10. Use of the low-fat plant-based fat co-loaded with EGCG and lycopene as claimed in claim 1 in food and site-specific delivery of functional ingredients and cellular anti-inflammatory activity.

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