Anti-flocculating submicron emulsion, its preparation method and application

Abstract

The application discloses an anti-flocculation submicron emulsion and a preparation method and application thereof. The preparation method comprises the following steps: taking a mixed solution containing a plant-based protein hydrolysate-EGCG compound and water as an aqueous phase, and taking walnut oil as an oil phase; and mixing the oil phase and the aqueous phase and performing shearing and high-pressure homogenization treatment to obtain the anti-flocculation submicron emulsion. In the application, the plant-based protein hydrolysate-EGCG compound is mixed with the walnut oil to perform shearing and homogenization, so that the anti-flocculation walnut oil submicron emulsion can be prepared. The anti-flocculation submicron emulsion has a good application prospect in the fields of liquid emulsified food, nutritional fortifier, embedding of bioactive substances, delivery of nutrients or controlled release of drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food engineering, and particularly relates to an anti-flocculation sub-micron emulsion and a preparation method and application thereof. BACKGROUND

[0002] Plant proteins, such as soy protein, pea protein, and chickpea protein, are being studied as substitutes for animal proteins in various foods due to their high yield, low cost, balanced amino acid structure, high lysine content, and rich nutritional value. In addition, plant proteins have been reported to exhibit some biological activities, such as antioxidant activity, blood pressure reduction, and regulation of intestinal flora. However, the widespread application of plant proteins in foods is still limited due to their poor solubility. Specifically, plant proteins have a relatively high globulin content, such as soybean glycinin and conglycinin, which leads to the extraction methods used to isolate them often promoting protein denaturation and aggregation. In addition, the poor solubility of plant proteins also affects their emulsifying properties. Hydrolyzing proteins to obtain protein hydrolysates can stretch the structure of the proteins, exposing reactive groups, thereby enhancing their solubility and emulsifying activity and other functional properties.

[0003] Emulsions refer to a heterogeneous liquid dispersion system in which one phase of liquid is dispersed in another phase of liquid in the form of small droplets, and have been widely used in the food industry. However, the flocculation problem of emulsions is still a major challenge in application. Flocculation refers to the aggregation of small droplets in emulsions to form larger aggregates, leading to a decrease in emulsion stability and affecting the texture and appearance of the product. Flocculation is usually caused by uneven particle size distribution, insufficient charge on the droplet surface, changes in environmental conditions, and improper selection and amount of emulsifiers. SUMMARY

[0004] The main purpose of the present application is to provide an anti-flocculation sub-micron emulsion and a preparation method and application thereof to overcome the deficiencies of the prior art.

[0005] To achieve the aforementioned purposes, the technical solutions adopted by the present application include:

[0006] The present application provides a preparation method of an anti-flocculation sub-micron emulsion, which comprises:

[0007] The mixture solution containing a plant-based protein hydrolysate-EGCG complex and water is used as the water phase, and walnut oil is used as the oil phase.

[0008] The oil phase and the water phase are mixed and subjected to shearing and high-pressure homogenization treatment to obtain the anti-flocculation sub-micron emulsion.

[0009] The present application also provides an anti-flocculation sub-micron emulsion prepared by the aforementioned preparation method.

[0010] The application also provides the use of the aforementioned anti-flocculation submicron emulsion in a liquid emulsified food.

[0011] Compared with the prior art, the application has the following beneficial effects:

[0012] (1) The application uses plant-based protein as raw material, which is cheap and easy to obtain, and expands the application range and channel of plant-based protein;

[0013] (2) The application avoids the use of expensive centrifuges or ultrasonic machines without introducing any organic reagents, and only common heating equipment and pH meters or pH test paper are needed in the preparation process. The protein hydrolysate is obtained only by heat alkali treatment. This method does not require chemical cross-linking agents, and is simple and easy to operate, and the treatment conditions are mild. After heat alkali treatment, the protein peptide chain is broken, and plant-based protein hydrolysate is generated, which exposes the reaction group and is beneficial to the covalent grafting reaction with EGCG, thereby improving the grafting degree of plant-based protein and EGCG, and enhancing the functional properties thereof;

[0014] (3) The application provides a preparation method of a plant-based protein hydrolysate-EGCG complex. By introducing natural polyphenol EGCG, the obtained protein covalent complex has better solubility and emulsifying properties, and at the same time, the protein hydrolysate is endowed with good antioxidant capacity. The complex has wide application in the fields of food and cosmetics;

[0015] (4) The plant-based protein hydrolysate-EGCG complex prepared by the application is a submicron emulsion with low flocculation degree, and the submicron emulsion has good storage stability. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments described in the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0017] Figure 1 is the preparation flowchart of the anti-flocculation walnut oil submicron emulsion prepared by the plant-based protein hydrolysate-EGCG complex in embodiment 1, embodiment 2 and embodiment 3 of the application;

[0018] Figure 2 is a schematic diagram of the grafting rate of the plant-based protein-EGCG complex and the plant-based protein hydrolysate-EGCG complex in embodiment 1, embodiment 2, embodiment 3, comparative example 1, comparative example 4 and comparative example 7 of the application;

[0019] Figure 3 FIG. 1 is a scanning electron microscope result diagram of the plant-based protein, plant-based protein hydrolysate, plant-based protein-EGCG complex, plant-based protein hydrolysate-EGCG complex, plant-based protein-EGCG physical mixture, and plant-based protein hydrolysate-EGCG physical mixture in Examples 1-3 and Comparative Examples 1-9 of the present application;

[0020] Figures 4A-4C FIG. 2 is a surface hydrophobicity result diagram of the plant-based protein, plant-based protein hydrolysate, plant-based protein-EGCG complex, plant-based protein hydrolysate-EGCG complex, plant-based protein-EGCG physical mixture, and plant-based protein hydrolysate-EGCG physical mixture in Examples 1-3 and Comparative Examples 1-9 of the present application, Figure 4A is soy protein, Figure 4B is pea protein, Figure 4C is chickpea protein;

[0021] Figures 5A-5C FIG. 3 is a solubility result diagram of the plant-based protein, plant-based protein hydrolysate, plant-based protein-EGCG complex, plant-based protein hydrolysate-EGCG complex, plant-based protein-EGCG physical mixture, and plant-based protein hydrolysate-EGCG physical mixture in Examples 1-3 and Comparative Examples 1-9 of the present application, Figure 5A is soy protein, Figure 5B is pea protein, Figure 5C is chickpea protein;

[0022] Figures 6A-6C FIG. 4 is a thermal stability result diagram of the plant-based protein, plant-based protein hydrolysate, plant-based protein-EGCG complex, plant-based protein hydrolysate-EGCG complex, plant-based protein-EGCG physical mixture, and plant-based protein hydrolysate-EGCG physical mixture in Examples 1-3 and Comparative Examples 1-9 of the present application, Figure 6A is soy protein, Figure 6B is pea protein, Figure 6C is chickpea protein;

[0023] Figures 7A-7C FIG. 5 is a result diagram of the emulsifying activity index (EAI) and emulsion stability index (ESI) of the plant-based protein, plant-based protein hydrolysate, plant-based protein-EGCG complex, plant-based protein hydrolysate-EGCG complex, plant-based protein-EGCG physical mixture, and plant-based protein hydrolysate-EGCG physical mixture in Examples 1-3 and Comparative Examples 1-9 of the present application, Figure 7A is soy protein, Figure 7B is pea protein, Figure 7C is chickpea protein;

[0024] Figures 8A-8C The results of the antioxidant test (ABTS radical scavenging rate) of the plant-based protein, plant-based protein hydrolysate, plant-based protein-EGCG complex, plant-based protein hydrolysate-EGCG complex, plant-based protein-EGCG physical mixture, and plant-based protein hydrolysate-EGCG physical mixture in Examples 1-3 and Comparative Examples 1-9 of the present application are shown in the following graph, Figure 8A The plant-based protein is soy protein, Figure 8B The plant-based protein is pea protein, Figure 8C The plant-based protein is chickpea protein.

[0025] Figures 9A-9I The results of the average particle size, flocculation index, and particle size distribution of the walnut oil emulsion dispersed in distilled water and 1% SDS solution of the plant-based protein, plant-based protein hydrolysate, plant-based protein-EGCG complex, and plant-based protein hydrolysate-EGCG complex stabilized walnut oil emulsion in Examples 1-3, Comparative Example 1, Comparative Example 4, and Comparative Example 7 of the present application are shown in the following graph;

[0026] Figure 10 The emulsion morphology of the anti-flocculated walnut oil submicron emulsion stabilized by the plant-based protein, plant-based protein hydrolysate, plant-based protein-EGCG complex, and plant-based protein hydrolysate-EGCG complex in Examples 1-3, Comparative Example 1, Comparative Example 4, and Comparative Example 7 of the present application after storage in a 4°C refrigerator. DETAILED DESCRIPTION

[0027] In view of the defects of the prior art, the present inventors have obtained the technical solution of the present application through long-term research and a large number of practices. The technical solution of the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0028] Specifically, as one aspect of the technical solution of the present application, the preparation method of an anti-flocculated submicron emulsion includes:

[0029] The mixed solution containing the plant-based protein hydrolysate-EGCG complex and water is used as the water phase, and the walnut oil is used as the oil phase.

[0030] The oil phase and the water phase are mixed and subjected to shearing and high-pressure homogenization treatment to obtain the anti-flocculated submicron emulsion.

[0031] In some preferred embodiments, the plant-based protein hydrolysate is mixed with EGCG to obtain the plant-based protein hydrolysate-EGCG complex through alkali induction.

[0032] In some preferred embodiments, the preparation method specifically comprises:

[0033] dispersing the plant-based protein hydrolysate-EGCG complex in water to form a plant-based protein hydrolysate-EGCG complex dispersion as an aqueous phase;

[0034] and slowly adding walnut oil as an oil phase to the aqueous phase under stirring, and shearing at 8000-10000 rpm (e.g. 9000 rpm) for 2-4 min (e.g. 3 min), and then homogenizing by a high-pressure homogenizer to obtain an anti-flocculating submicron emulsion.

[0035] Further, the homogenizing condition comprises: three times of fixed cycles at 40-60 MPa.

[0036] Further, the concentration of the plant-based protein hydrolysate-EGCG complex in the plant-based protein hydrolysate-EGCG complex dispersion is 0.5-1.5 wt%.

[0037] Further, the volume ratio of the aqueous phase to the oil phase is 95:5-85:15.

[0038] Further, the volume ratio of the aqueous phase to the oil phase is 70:30-90:10.

[0039] In some preferred embodiments, the preparation method of the plant-based protein hydrolysate-EGCG complex comprises:

[0040] subjecting the plant-based protein to heat-alkali treatment to obtain a plant-based protein hydrolysate;

[0041] mixing the plant-based protein hydrolysate with EGCG and subjecting to alkali induction treatment to obtain a plant-based protein hydrolysate-EGCG complex.

[0042] In some preferred embodiments, the preparation method of the plant-based protein hydrolysate-EGCG complex comprises:

[0043] dispersing the plant-based protein in water and stirring overnight to fully hydrate the plant-based protein to obtain a plant-based protein dispersion;

[0044] and adjusting the pH value of the plant-based protein dispersion to 11.5-12.5, and heating by water bath to 75-85°C for 25-35 min, and then cooling, adjusting the pH value to 6.5-7.5 to obtain a plant-based protein hydrolysate.

[0045] Further, the concentration of the plant protein in the plant-based protein dispersion is 0.5-1.5 w / v%.

[0046] Further, the plant-based protein includes any one or a combination of soybean protein isolate, pea protein isolate, chickpea protein isolate, and is not limited thereto.

[0047] In some preferred embodiments, the method for preparing the plant-based protein hydrolysate-EGCG complex includes mixing a plant-based protein hydrolysate solution with an EGCG solution and adjusting the pH value to 8.5-9.5, stirring the reaction under dark conditions at 200-400 rpm for 24 h, and then performing purification and drying treatment to obtain the plant-based protein hydrolysate-EGCG complex.

[0048] Further, the plant-based protein hydrolysate-EGCG complex has a particle size of 0.588-1.266 μm.

[0049] Further, the volume ratio of the plant-based protein hydrolysate solution to the EGCG solution is 10:2-10:4.

[0050] Further, the purification treatment is performed by dialysis, wherein the dialysis temperature is 4-8℃; and the molecular weight of the dialysis bag used for dialysis is 8000-16000 Da.

[0051] Further, the drying treatment is performed by vacuum freeze-drying.

[0052] In some preferred embodiments, the volume ratio of the plant-based protein to EGCG is 10:3.

[0053] In some more specific embodiments, the method for preparing the plant-based protein hydrolysate-EGCG complex includes the following steps: obtaining a plant-based protein hydrolysate solution by heat alkali treatment, mixing the plant-based protein hydrolysate solution with an EGCG solution in equal volume, adjusting the pH, stirring the reaction under dark conditions at 300 rpm for 24 h, dialyzing the sample after the reaction for 72 h and performing vacuum freeze-drying to obtain the plant-based protein hydrolysate-EGCG complex.

[0054] Further, the plant-based protein is dispersed into water, stirred at 300 rpm overnight to make it fully hydrated, to obtain a plant-based protein dispersion, the pH value of the plant-based protein dispersion is adjusted to 11.5-12.5 to make it in an alkaline environment, and then heated in a water bath to 75-85℃ for 25-35 min and cooled, the pH is adjusted to 7.0 to obtain the plant-based protein hydrolysate, which is stored at 0-4℃.

[0055] Further, EGCG is dispersed in water to form an EGCG solution, the plant-based protein hydrolysate solution is mixed with the EGCG solution, and the pH value of the reaction system is maintained at 9.0, and the plant-based protein hydrolysate-EGCG complex is obtained by reacting in the dark for 24 h, and then the plant-based protein hydrolysate-EGCG complex is dialyzed, dried, and stored at 0-4°C.

[0056] Another aspect of the embodiments of the present application also provides the anti-flocculation submicron emulsion prepared by the preparation method.

[0057] Another aspect of the embodiments of the present application also provides the use of the anti-flocculation submicron emulsion in liquid emulsified food.

[0058] The anti-flocculation submicron emulsion in the present application has a good application prospect in the fields of liquid emulsified food, nutritional fortifier, embedding of bioactive substances, delivery of nutrients, or controlled release of drugs.

[0059] The technical solutions of the present application will be further described in detail below in combination with several preferred embodiments and the accompanying drawings. The embodiments are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0060] The pea protein isolate (purity 76.63%) and the soybean protein isolate (purity 75.03%) used in the following embodiments are purchased from Shanghai Yuan Ye Co., Ltd., the chickpea protein isolate (purity 72.45%) is purchased from Shaanxi Jinkangtai Biological Technology Co., Ltd.; epigallocatechin gallate is purchased from Beijing Beishi Zongheng Technology Development Co., Ltd., and the purity is ≥98%; the ferric chloride reagent is purchased from Tianjin Kemio Chemical Reagent Co., Ltd.; the medium-chain triglyceride (MCT) is purchased from Xi'an Haishifu Biological Technology Co., Ltd.; and other reagents are all analytical pure (AR).

[0061] Example 1 DETAILED DESCRIPTION

[0063] 1. Preparation of alkali heat-treated soybean protein and modified soybean protein-EGCG complex

[0064] (1) Alkali heat-treated soybean protein

[0065] At room temperature, 1.0 g of soybean protein (SPI) is dissolved in 100 mL of distilled water and stirred overnight for hydration. The dissolved protein dispersion is adjusted to pH 12.0 using 1M NaOH solution, so that the protein dispersion is in an alkaline environment, and then heated in a water bath at 80°C for 30 min. After cooling, the pH is adjusted to 7.0 to obtain an alkali heat-treated soybean protein hydrolysate.

[0066] (2) Preparation of protein hydrolysate-EGCG complex

[0067] The alkali heat-treated soy protein hydrolysate obtained in step (1) was mixed with EGCG solution (10 mg / mL) at a ratio of protein hydrolysate solution: EGCG solution = 10:3 (V / V), and the pH of the mixed solution was adjusted to 9.0, and reacted at 26°C for 24 h in the dark. After treatment, all samples were dialyzed in dialysis bags (8000-14000 Da) at 4°C for 48 h to remove free EGCG. During this period, clean deionized water was changed every 6 h. All samples after dialysis were freeze-dried and stored in a -20°C refrigerator, and the treated sample was designated as TSPI-EGCG.

[0068] (3) Preparation of walnut oil emulsion

[0069] A stable O / W type walnut oil emulsion was prepared using the plant-based protein hydrolysate-EGCG complex prepared above, and the specific steps included:

[0070] The plant-based protein hydrolysate-EGCG complex dispersion was the water phase, and the walnut oil was the oil phase. The concentration of the plant-based protein hydrolysate-EGCG complex in the water phase was 1.0 wt%; the volume fraction of the oil phase was 10%. The oil phase was slowly added to the water phase under stirring with a high-speed emulsifying homogenizer, sheared at 9,000 rpm for 3 minutes, and then treated with a high-pressure homogenizer at 50 MPa for 3 times to prepare a walnut oil submicron emulsion. The emulsion was then stored at 0-4°C.

[0071] Example 2

[0072] Example 2 differed from Example 1 in that 10 mg / mL of pea protein dispersion (PPI) was used for alkali heat treatment and then reacted with EGCG in step (1), and the rest of the conditions remained the same as in Example 1. The complex obtained was named TPPI-EGCG.

[0073] Example 3

[0074] Example 3 differed from Example 1 in that 10 mg / mL of chickpea protein dispersion (CPI) was used for alkali heat treatment and then reacted with EGCG in step (1), and the rest of the conditions remained the same as in Example 1. The complex obtained was named TCPI-EGCG.

[0075] Comparative Example 1

[0076] As in Example 1, the only difference was that the alkali heat-treated soy protein dispersion was not used to react with the EGCG solution, and the complex obtained was named SPI-EGCG.

[0077] Comparative Example 2

[0078] The same as Example 1, except that the soy protein dispersion solution not subjected to the alkali heat treatment was mixed with the EGCG solution, and the resulting complex was named SPI-EGCG-MIX after the pH was adjusted to 7.0 and directly dialyzed and freeze-dried.

[0079] Comparative Example 3

[0080] The same as Example 2, except that the pea protein dispersion solution not subjected to the alkali heat treatment was mixed with the EGCG solution, and the resulting complex was named PPI-EGCG-MIX after the pH was adjusted to 7.0 and directly dialyzed and freeze-dried.

[0081] Comparative Example 4

[0082] The same as Example 2, except that the pea protein dispersion solution not subjected to the alkali heat treatment was mixed with the EGCG solution, and the resulting complex was named PPI-EGCG-MIX after the pH was adjusted to 7.0 and directly dialyzed and freeze-dried.

[0083] Comparative Example 5

[0084] The same as Example 2, except that the pea protein dispersion solution not subjected to the alkali heat treatment was mixed with the EGCG solution, and the resulting complex was named PPI-EGCG-MIX after the pH was adjusted to 7.0 and directly dialyzed and freeze-dried.

[0085] Comparative Example 6

[0086] The same as Example 2, except that the pea protein dispersion solution not subjected to the alkali heat treatment was mixed with the EGCG solution, and the resulting complex was named PPI-EGCG-MIX after the pH was adjusted to 7.0 and directly dialyzed and freeze-dried.

[0087] Comparative Example 7

[0088] The same as Example 3, except that the chickpea protein dispersion solution not subjected to the alkali heat treatment was mixed with the EGCG solution, and the resulting complex was named CPI-EGCG-MIX after the pH was adjusted to 7.0 and directly dialyzed and freeze-dried.

[0089] Comparative Example 8

[0090] The same as Example 3, except that the chickpea protein dispersion solution not subjected to the alkali heat treatment was mixed with the EGCG solution, and the resulting complex was named CPI-EGCG-MIX after the pH was adjusted to 7.0 and directly dialyzed and freeze-dried.

[0091] Comparative Example 9

[0092] The same as Example 3, the only difference is that the solution of heat-alkali treated chickpea protein hydrolysate is mixed with the solution of EGCG, and then the pH is adjusted to 7.0, followed by direct dialysis treatment and freeze-drying. The prepared complex is named TCPI-EGCG-MIX.

[0093] 2. Protein-EGCG grafting degree:

[0094] A 0.2 mol / L Folin phenol reagent was prepared, and 0.5 mL of 1 mg / mL protein-EGCG sample was mixed with 2.5 mL of Folin phenol reagent. After 5 min in the dark, 2 mL of sodium carbonate solution (7.5%, w / v) was added, vortexed for 2 min, and then placed in the dark for 2 h. The absorbance of the mixture at 760 nm was measured by ultraviolet-visible spectrophotometer, with plant-based protein not reacted with EGCG as a blank control. The EGCG content in each complex sample was determined according to the EGCG standard curve. The grafting degree was calculated using the following formula:

[0095] Grafting degree (%) = (C / C0) x 100

[0096] In the formula, C is the EGCG content in the complex, and C0 is the EGCG content in the reactant before dialysis.

[0097] Figure 2 is the EGCG grafting rate of the plant-based protein hydrolysate-EGCG complex, plant-based protein-EGCG complex in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 4, Comparative Example 7. Under the same environment, SPI, PPI, CPI and TSPI, TPPI, TCPI with the same protein content form covalent complexes with an equal amount of EGCG. The results show that the binding rate of TSPI, TPPI, TCPI with EGCG is 77.35%, 75.51%, and 74.19%, respectively, which is 35%, 46%, and 19% higher than that of SPI, PPI, and CPI, respectively. Obviously, TSPI, TPPI, and TCPI have higher binding rates, which may be related to the change in protein structure and the exposure of reactive sites. Heat-alkali treatment causes protein hydrolysis to generate protein hydrolysate, resulting in the unfolding of protein structure, increasing the number of free sulfhydryl groups and the content of free amino groups, so that TSPI, TPPI, and TCPI have more reactive groups and can more easily react with EGCG, thereby increasing the grafting degree.

[0098] 3. Microstructure of the complex

[0099] The microstructure of different samples was observed by scanning electron microscope. The freeze-dried sample was fixed on double-sided tape, and then coated with a 30 nm gold sputter coating layer for 45 s to make it conductive, and then scanned and observed at 5 kV.

[0100] The microstructure of the plant-based proteins, plant-based protein hydrolysates, plant-based protein-EGCG complexes, plant-based protein hydrolysate-EGCG complexes, plant-based protein-EGCG physical mixtures, plant-based protein hydrolysate-EGCG physical mixtures in Examples 1-3, Comparative Examples 1-9 were observed using a scanning electron microscope, and the results are shown in FIGS. 1-9. Figure 3 As shown in FIGS. 1-9, SPI, PPI, and CPI all contained globular proteins of varying sizes with some slight depressions on the surface. Compared to SPI, PPI, and CPI, TSPI, TPPI, and TCPI exhibited fragmented protein pieces, indicating that the proteins were hydrolyzed under alkaline heat treatment to generate protein hydrolysates. SPI-EGCG-MIX, PPI-EGCG-MIX, and CPI-EGCG-MIX were very similar in morphology to SPI, PPI, and CPI, and TSPI-EGCG-MIX, TPPI-EGCG-MIX, and TCPI-EGCG-MIX were very similar in morphology to TSPI, TPPI, and TCPI, which indicated that non-covalent binding of EGCG to the proteins had little effect on the overall structure of the proteins. However, due to electrostatic interactions, when the concentration of EGCG was high, EGCG could act as a ligand to link proteins and / or protein-EGCG complexes, forming dimers and aggregates of proteins. As can be seen from the SEM images, EGCG acted as a ligand to link proteins, forming insoluble particles, which led to protein precipitation. In contrast, the particles of the covalent complexes were larger and more irregular in shape than the particles in SPI, PPI, and CPI, which indicated that covalent reactions could change the particle size and morphology of the proteins. These results indicated that covalent reactions with EGCG changed the size and morphology of the protein-based particles. The increase in particle size was partially attributed to the formation of protein polymers linked together by EGCG.

[0101] 4. Particle size and zeta potential of complexes

[0102] The average particle size, polydispersity index (PDI), and Zeta potential of the samples were measured using a Malvern particle size analyzer. All measurements were performed at 25 °C, and each sample was measured three times under the same conditions. Table 1 is a comparison of the particle size, PDI values, and zeta potential changes of the plant-based proteins, plant-based protein hydrolysates, plant-based protein-EGCG complexes, plant-based protein hydrolysate-EGCG complexes, plant-based protein-EGCG physical mixtures, plant-based protein hydrolysate-EGCG physical mixtures in Examples 1-3, Comparative Examples 1-9.

[0103] Table 1 Average particle size, PDI, and potential of different samples

[0104]

[0105] Note: Different letters in the same column indicate a significant difference (P < 0.5)

[0106] As can be seen from Table 1, the particle size of protein was significantly reduced after alkaline heat treatment, because protein hydrolysis occurred during the alkaline heat treatment. The PDI of TSPI, TPPI, TCPI and TSPI-EGCG, TPPI-EGCG, TCPI-EGCG were all smaller than that of SPI, PPI, CPI and SPI-EGCG, PPI-EGCG, CPI-EGCG, respectively, indicating that the particle size distribution of TSPI, TPPI, TCPI and TSPI-EGCG, TPPI-EGCG, TCPI-EGCG was narrower, and the particle size of the samples was more uniform. After grafting with EGCG, the particle size of the samples increased, because EGCG was grafted to the surface of the protein. It is worth noting that the particle size of SPI-EGCG-MIX, PPI-EGCG-MIX, CPI-EGCG-MIX and TSPI-EGCG-MIX, TPPI-EGCG-MIX, TCPI-EGCG-MIX was very large, because EGCG at a higher concentration could connect different protein molecules as a ligand, leading to the formation of protein aggregates. In terms of zeta potential, the absolute value of the zeta potential of SPI-EGCG, PPI-EGCG, CPI-EGCG and TSPI-EGCG, TPPI-EGCG, TCPI-EGCG showed an increasing trend compared with SPI, PPI, CPI and TSPI, TPPI, TCPI. This is mainly because in alkaline environment, quinone substances are negatively charged, which can combine with the positively charged groups of protein, thereby enhancing the negative potential of the complex system, leading to an increase in the absolute potential. And the absolute zeta potential of SPI-EGCG, PPI-EGCG, CPI-EGCG and TSPI-EGCG, TPPI-EGCG, TCPI-EGCG all exceeded 30 mV, which can prevent particle aggregation caused by electrostatic repulsion, suggesting its potential as an effective emulsifier.

[0107] 5. Surface hydrophobicity of the complexes:

[0108] Surface hydrophobicity (H0) of proteins was measured using 8-anilino-l-naphthalene sulfonic acid (ANS). Different protein concentrations (0.04, 0.08, 0.12, 0.16 and 0.20 mg / mL) and ANS reagent (8 mM) were prepared in 0.01 M phosphate buffer (pH 7.0). 4 mL of protein (0.04-0.2 mg / mL) was mixed with 50 μL of ANS reagent (8 mM), and the fluorescence intensity was measured using a fluorescence spectrometer (RF-6000, Shimadzu, Japan). The excitation and emission wavelengths were 365 nm and 484 nm, respectively. The relative fluorescence intensity of the sample was defined as the fluorescence intensity of the sample minus the fluorescence intensity of the blank reagent. The slope of the line obtained by plotting the relative fluorescence intensity of the sample against the protein concentration was the surface hydrophobicity index (H0).

[0109] Figures 4A-4C Surface hydrophobicity of the plant-based proteins, plant-based protein hydrolysates, plant-based protein-EGCG complexes, plant-based protein hydrolysate-EGCG complexes, plant-based protein-EGCG physical mixtures, and plant-based protein hydrolysate-EGCG physical mixtures in Examples 1-3 and Comparative Examples 1-9, Figure 4A SPI is soy protein, Figure 4B PPI is pea protein, Figure 4C CPI is chickpea protein. Compared with SPI, PPI and CPI, the surface hydrophobicity (H0) of TSPI, TPPI and TCPI after alkaline heat treatment increased significantly, because alkaline heat treatment led to partial hydrolysis of the proteins, generating protein hydrolysates, and the secondary structure of the protein hydrolysates was stretched, resulting in exposure of the hydrophobic groups and regions to a more polar surrounding environment. However, the surface hydrophobicity index (H0) of the covalent complexes and the physical mixtures decreased significantly (p < 0.05). This can be because (1) the hydrophilic hydroxyl groups of EGCG were introduced; (2) the hydrophobic groups on the surface of the plant-based proteins were covered; and (3) the previously hidden hydrophilic groups of the plant-based proteins were exposed.

[0110] 6. Solubility of complexes

[0111] The solubility of the protein samples was determined by the Coomassie brilliant blue method. The samples were dissolved in deionized water to obtain a sample solution with a protein concentration of 3.0 mg / mL, and the pH value of the solution was adjusted to 2-12 using 1 M HC1 and NaOH. The sample was stirred at a constant speed (300 rpm) for 30 min to ensure complete dissolution, and then centrifuged at 8000 rpm for 20 min. The protein content in the supernatant was determined by the Coomassie brilliant blue method. The solubility of the protein was the ratio of the protein concentration in the supernatant to the total protein concentration.

[0112] The solubility of the plant-based proteins, plant-based protein hydrolysates, plant-based protein-EGCG complexes, plant-based protein hydrolysate-EGCG complexes, plant-based protein-EGCG physical mixtures, and plant-based protein hydrolysate-EGCG physical mixtures in Examples 1-3 and Comparative Examples 1-9 was detected, and the detection results are shown in Table 1. Figures 5A-5C Figure 5A SPI was soy protein, Figure 5B PPI was pea protein, Figure 5C CPI was chickpea protein. Since SPI, PPI, and CPI all belong to legume proteins, their main components are 11S globulin and 7S globulin, and the isoelectric points of 11S globulin and 7S globulin are both between 4.5 and 5.2, so it was observed that the solubility of SPI, PPI, and CPI was the lowest at pH 4.0. The solubility of TSPI, TPPI, and TCPI was significantly higher than that of SPI, PPI, and CPI in the entire pH range, because the proteins were partially hydrolyzed in the alkaline heat treatment. In addition, the hydrolysis process exposed more ionizable groups in SPI, PPI, and CPI, further improving their solubility. After SPI, PPI, and CPI formed covalent complexes with EGCG, their solubility was significantly improved in the pH range studied, because the presence of the polar EGCG phenolic hydroxyl group enhanced the hydrogen bonding of the complex with water molecules. In addition, it can be observed that the solubility of all physical mixtures (SPI-EGCG-MIX, PPI-EGCG-MIX, CPI-EGCG-MIX, TSPI-EGCG-MIX, TPPI-EGCG-MIX, and TCPI-EGCG-MIX) was lower than that of the original protein samples (SPI, PPI, CPI, TSPI, TPPI, and TCPI), because in the non-covalent complex, EGCG and proteins mainly interact through hydrophobic interactions and hydrogen bonds, serving as a bridge between polymer protein molecules to form macromolecular protein polymers, making their solubility lower. It is worth noting that when the pH environment becomes alkaline, the solubility of the non-covalent complex increases, not only because when the pH value is alkaline, the surface charge density of the protein molecule increases, the hydrophilicity increases significantly, resulting in higher solubility, but also because when the pH is in an alkaline environment, EGCG in the sample is oxidized into a quinone, and part of the protein-EGCG non-covalent complex and the plant-based protein hydrolysate-EGCG non-covalent complex becomes a protein-EGCG non-covalent complex and a plant-based protein hydrolysate-EGCG complex, which has higher solubility.

[0113] 7. Thermal stability of complexes

[0114] ​The thermal denaturation temperature and enthalpy of different lyophilized samples were determined by differential scanning calorimetry analysis. 2.0 mg of lyophilized sample was added to an aluminum pan and sealed, and empty sealed aluminum pan was used as blank control. The heating rate was set at 10 °C / min, and the heating range was 40-180 °C. The initial denaturation temperature, thermal denaturation temperature and enthalpy of each sample were calculated according to the instrument analysis software.

[0115] The thermal stability of the plant-based proteins, plant-based protein hydrolysates, plant-based protein-EGCG complexes, plant-based protein hydrolysate-EGCG complexes, plant-based protein-EGCG physical mixtures, and plant-based protein hydrolysate-EGCG physical mixtures in Examples 1-3 and Comparative Examples 1-9 were detected, and the detection results are shown in Table 1. Figures 6A-6C Figure 6A SPI is soy protein, Figure 6B PPI is pea protein, Figure 6C CPI is chickpea protein. All thermal analysis graphs showed a broad endothermic peak, which was attributed to the thermal denaturation of protein subunits. The Td value is the peak thermal denaturation temperature, and a high value usually indicates that a globular protein has high thermal stability. After the protein is treated by alkali heat, its denaturation temperature decreases, which may be due to the exposure of internal thiol or hydrophobic groups. After the protein is treated by alkali heat, the protein compact structure supported by disulfide bonds is reduced, and as the protein hydrolysis proceeds, the energy required for complete denaturation is continuously reduced. The protein-EGCG complex has better thermal stability due to the addition of EGCG, and the grafting with EGCG increases the denaturation temperature of the protein by 5.47-48.04 °C. In addition, compared with the protein-EGCG complex (SPI-EGCG, PPI-EGCG, CPI-EGCG), the plant-based protein hydrolysate-EGCG complex (TSPI-EGCG, TPPI-EGCG, TCPI-EGCG) shows higher denaturation temperature and better thermal stability, which may be related to the higher grafting degree of the plant-based protein hydrolysate-EGCG complex.

[0116] 8. Emulsifying activity of the complex:

[0117] Protein and protein-EGCG complex solutions with a concentration of 5 mg / mL were prepared, and then 1.5 mL of medium-chain triglyceride (MCT) was added. The mixture was homogenized at a speed of 15000 rpm for 5 min at room temperature to form an emulsion. 40 μL of liquid was taken from the bottom of the homogenate at 0 and 10 min, respectively, and diluted 50 times with 0.1% SDS solution. The absorbance value of the sample was measured at 500 nm, and was recorded as A0, A 10 , respectively. The emulsifying activity (EAI) and emulsion stability (ESI) of the sample were calculated according to the following formula. 0.1% SDS solution was used as a blank control. ​

[0118] EAI(m 2 / g) = (2 x 2.303 x A0) / (C x 10 4 x Φ) x 100

[0119] ESI(%) = A 10 / A0 x 100%

[0120] Wherein, A0and A 10 are the absorbance of the solution taken out at 400 nm at 0 min and 10 min, respectively; C is the protein concentration (5 g / mL); Φ is the proportion of oil phase (20%).

[0121] The emulsifying activity index (EAI) and emulsion stability index (ESI) of the plant-based proteins, plant-based protein hydrolysates, plant-based protein-EGCG complexes, plant-based protein hydrolysate-EGCG complexes, plant-based protein-EGCG physical mixtures, and plant-based protein hydrolysate-EGCG physical mixtures in Examples 1-3 and Comparative Examples 1-9 were detected, and the detection results are shown in Table 1. Figures 7A-7C Figure 7A SPI is soybean protein, Figure 7B PPI is pea protein, Figure 7C CPI is chickpea protein. Compared with SPI, PPI and CPI, the EAI of TSPI, TPPI and TCPI increased significantly (p < 0.05), because the partial structure of the treated TSPI, TPPI and TCPI samples was unfolded, which led to an increase in the exposure of hydrophobic amino acid side chain groups, which allowed more oil droplets to be adsorbed, thereby obtaining greater surface activity.

[0122] Compared with SPI, PPI, CPI, TSPI, TPPI and TCPI, the EAI value of the covalent complexes (SPI-EGCG, PPI-EGCG, CPI-EGCG, TSPI-EGCG, TPPI-EGCG and TCPI-EGCG) increased, and the improvement of the emulsifying performance of the complexes may be attributed to the increase in their water solubility, which is essential for good emulsification. In addition, the covalent connection of hydrophilic EGCG with plant-based proteins may reduce the aggregation of oil droplets by increasing the spatial repulsive force between oil droplets. Compared with SPI-EGCG, PPI-EGCG and CPI-EGCG, TSPI-EGCG, TPPI-EGCG and TCPI-EGCG showed stronger emulsifying performance, which may be because it has the highest grafting degree.

[0123] 9. Antioxidant activity of the complexes:

[0124] ​The antioxidant capacity of plant-based proteins, plant-based protein hydrolysates, plant-based protein-EGCG complexes, plant-based protein hydrolysates-EGCG complexes, plant-based protein-EGCG physical mixtures, and plant-based protein hydrolysates-EGCG physical mixtures in Examples 1-3 and Comparative Examples 1-9 was evaluated using the ABTS free radical scavenging method. The test results are as follows: Figures 8A-8C As shown, Figure 8A It is soy protein. Figure 8B It is made from pea protein. Figure 8C The sample was chickpea protein. ABTS working solution was prepared in advance and stored at room temperature in the dark. Before analysis, the sample was diluted with deionized water to an absorbance of 0.70 ± 0.02. 1 mL of sample solution was pipetted into a centrifuge tube, 3 mL of diluted ABTS solution was added, and the mixture was stirred thoroughly. After reacting at room temperature in the dark for 18 min, the absorbance at 734 nm was measured. Deionized water was used as a blank control instead of the sample solution. The formula for calculating the ABTS free radical scavenging rate is as follows:

[0125] ABTS free radical scavenging activity (%) = (1-A m / A n )×100

[0126] Among them, A m An is the absorbance of the sample at 734 nm, and An is the absorbance of the blank sample at 734 nm.

[0127] The antioxidant properties of plant-based proteins, plant-based protein hydrolysates, plant-based protein-EGCG complexes, plant-based protein hydrolysates-EGCG complexes, plant-based protein-EGCG physical mixtures, and plant-based protein hydrolysates-EGCG physical mixtures from Examples 1-3 and Comparative Examples 1-9 were tested. The results are shown below. Figures 8A-8Cantioxidant activity. Overall, the antioxidant activity of SPI, PPI, and CPI was relatively weak, while the antioxidant activity of TSPI, TPPI, and TCPI was significantly lower than that of SPI, PPI, and CPI, which was mainly due to the significant increase in protein carbonyl formation during heat treatment, resulting in a decrease in antioxidant activity. Secondly, the antioxidant activity of covalent complexes SPI-EGCG, PPI-EGCG, CPI-EGCG, and TSPI-EGCG, TPPI-EGCG, TCPI-EGCG was significantly improved (p<0.05). In addition, the antioxidant activity of TSPI-EGCG, TPPI-EGCG, and TCPI-EGCG was slightly stronger than that of SPI-EGCG, PPI-EGCG, and CPI-EGCG, which may be related to the higher grafting degree of TSPI-EGCG, TPPI-EGCG, and TCPI-EGCG. Compared with TSPI, TPPI, and TCPI, and SPI, PPI, and CPI, the antioxidant activity of TSPI-EGCG, TPPI-EGCG, and TCPI-EGCG was increased by 294%-350%, while the antioxidant activity of SPI-EGCG, PPI-EGCG, and CPI-EGCG was only increased by 208%-294%. This shows that under the same conditions, TSPI-EGCG, TPPI-EGCG, and TCPI-EGCG have higher antioxidant activity than SPI-EGCG, PPI-EGCG, and CPI-EGCG due to the higher binding rate of EGCG.

[0128] 10. Flocculation index of emulsion

[0129] The flocculation index (FI, %) of fresh emulsion was calculated based on the D 43 values measured in distilled water and 1% (w / v) SDS, according to the following formula:

[0130] FI (%) = (D w / D s -1) x 100

[0131] In the formula, D w is the D 43 in distilled water; D s is the D 43 in 1% (w / v) SDS.

[0132] The flocculation degree of the emulsion was further evaluated by measuring the flocculation index of the fresh emulsion. The particle size of the walnut oil emulsion stabilized by the plant-based protein, plant-based protein hydrolysate, plant-based protein-EGCG complex, and plant-based protein hydrolysate-EGCG complex in Examples 1-3, Comparative Example 1, Comparative Example 4, and Comparative Example 7 was measured in distilled water and 1% (w / v) SDS to evaluate the flocculation degree of the emulsion, and the results are shown in Table 5. Figures 9A-9IFigure 9A Average particle size of emulsions stabilized by soy protein samples in distilled water and 1% SDS, Figure 9B Flocculation index of soy protein in distilled water and 1% SDS, Figure 9C Particle size distribution of soy protein in distilled water and 1% SDS; Figure 9D Average particle size of emulsions stabilized by pea protein samples in distilled water and 1% SDS, Figure 9E Flocculation index of pea protein in distilled water and 1% SDS, Figure 9F Particle size distribution of pea protein in distilled water and 1% SDS; Figure 9G Average particle size of emulsions stabilized by chickpea protein samples in distilled water and 1% SDS, Figure 9H Flocculation index of chickpea protein in distilled water and 1% SDS, Figure 9I Particle size distribution of chickpea protein in distilled water and 1% SDS). As shown in Figure 9A , Figure 9D , Figure 9G can be seen, for all plant protein species, the emulsions stabilized by plant-based protein hydrolysate-EGCG complexes have sub-micron emulsions, and the emulsions stabilized by plant-based protein hydrolysate-EGCG complexes have the lowest particle size and the lowest flocculation index compared to other emulsifiers, indicating that the covalent binding of EGCG and plant-based protein hydrolysate can effectively reduce the flocculation degree of the emulsion. After dilution in SDS solution, the droplet size of the emulsions stabilized by other emulsifiers is significantly reduced, indicating that the emulsion has undergone serious reversible flocculation.

[0133] The particle size distribution of different emulsions was also evaluated, and the results are shown in Figure 9C , Figure 9F , Figure 9I When dispersed in distilled water, the emulsion showed a bimodal distribution. After dilution with SDS, the absorption peak height of the emulsion at 1 μm increased significantly. Therefore, the distribution peak of the original emulsion near the larger particle size is caused by the flocculation of droplets. Compared with other complexes, the flocculation index of plant-based protein hydrolysate-EGCG complex is the smallest, which is mainly due to the strong absolute value of the zeta potential of the plant-based protein hydrolysate-EGCG complex after alkaline heat treatment, which can maintain the stability of the emulsion and inhibit its aggregation by providing steric repulsion and electrostatic interaction.

[0134] 11. Storage stability of emulsions

[0135] An appropriate amount of emulsion was taken into a measuring bottle and stored in a 4°C refrigerator, and the appearance of the emulsion was recorded every other week.

[0136] ​The plant-based protein, plant-based protein hydrolysate, plant-based protein-EGCG complex, and plant-based protein hydrolysate-EGCG complex in Example 1-3, Comparative Example 1, Comparative Example 4, and Comparative Example 7 were stored in a 4℃ refrigerator, and the emulsion morphology was observed every other week, and the results are shown in Table 1. Figure 10 From the visual observation, all the samples of the stable emulsion were milky white at the beginning, and there was no oil or water separation, which indicated that the emulsion was relatively stable at this time. However, the plant-based protein and plant-based protein hydrolysate stable emulsion had serious oil-water separation after 28 days of storage, and the water phase was completely separated from the emulsion. The plant-based protein hydrolysate-EGCG complex stable emulsion still maintained a good appearance after 28 days, and the emulsion morphology was still uniform. This is mainly because the plant-based protein hydrolysate-EGCG complex after alkaline heat treatment has a strong absolute value of zeta potential, which can maintain the stability of the emulsion and inhibit its aggregation by providing steric repulsion and electrostatic interaction.

[0137] In summary, the present application provides a plant protein hydrolysate-EGCG complex and the preparation and application of the stable anti-flocculation submicron emulsion thereof. The plant-based protein hydrolysate is obtained by alkaline heat treatment, natural EGCG is introduced, and the plant-based protein hydrolysate and EGCG are covalently reacted by alkaline induction to obtain the plant-based protein hydrolysate-EGCG complex. The plant-based protein hydrolysate-EGCG complex stable walnut oil emulsion is a submicron emulsion, has a low flocculation index, and greatly improves the 4℃ storage stability. The present application provides a new method for the preparation of a new food functional factor delivery carrier, and can improve the absorption efficiency of hydrophobic bioactive substances. In addition, the preparation method of the present application is novel, the preparation process is simple, and the industrial production is easy to realize. The present application has wide application in the fields of bioactive substance embedding, nutrient delivery, and drug controlled release.

[0138] In the present application, soybean protein, pea protein, and chickpea protein are selected as raw materials, and only after simple alkaline heat treatment, the solubility, emulsification activity, and other functional properties of these proteins are significantly improved. The plant-based protein hydrolysate-EGCG complex prepared in the present application has significantly improved emulsification activity, solubility, thermal stability, and antioxidant activity, which provides a new method for the preparation of a new food functional factor delivery carrier. The emulsion stabilized by the prepared plant-based protein hydrolysate-EGCG complex is a submicron emulsion, has a low flocculation index, and good storage stability.

[0139] In addition, the present inventors also refer to the foregoing examples, and other raw materials, process operations, and process conditions described in the present specification for testing, and all ideal results are obtained.

[0140] It should be understood that the technical solutions of the present application are not limited to the above specific implementation cases, and any technical modification made according to the technical solutions of the present application without departing from the purpose of the present application and the scope protected by the claims falls within the protection scope of the present application.

Claims

1. A process for the preparation of an anti-flocculating submicron emulsion, characterized in that, The method comprises the following steps: dispersing plant-based protein in water and stirring overnight to fully hydrate the plant-based protein to obtain a plant-based protein dispersion; adjusting the pH value of the plant-based protein dispersion to 11.5-12.5, and heating to 75-85 ℃ through a water bath for 25-35 min, and then cooling, adjusting the pH value to 6.5-7.5 to obtain a plant-based protein hydrolysate solution; wherein the concentration of plant protein in the plant-based protein dispersion is 0.5-1.5 w / v %; mixing the plant-based protein hydrolysate solution with an EGCG solution and adjusting the pH value to 8.5-9.5, stirring at 200-400 rpm in the dark for 24 h, and then purifying and drying to obtain a plant-based protein hydrolysate-EGCG complex; wherein the volume ratio of the plant-based protein hydrolysate solution to the EGCG solution is 10:2-10:4, and the concentration of the EGCG solution is 10 mg / mL; dispersing the plant-based protein hydrolysate-EGCG complex in water to form a plant-based protein hydrolysate-EGCG complex dispersion as an aqueous phase; wherein the concentration of the plant-based protein hydrolysate-EGCG complex in the plant-based protein hydrolysate-EGCG complex dispersion is 0.5-1.5 wt %; and slowly adding walnut oil as an oil phase to the aqueous phase under stirring, shearing at 8000-10000 rpm for 2-4 min, and then homogenizing by a high-pressure homogenizing device to prepare an anti-flocculation submicron emulsion; wherein the homogenizing conditions include: fixed cycle for three times at 40-60 MPa; and the volume ratio of the aqueous phase to the oil phase is 95:5-85:

15.

2. The method of claim 1, wherein: The plant-based protein includes any one of soybean protein isolate, pea protein isolate, and chickpea protein isolate.

3. The method of claim 1, wherein: The particle size of the plant-based protein hydrolysate-EGCG complex is 0.588-1.266 μm.

4. The method of claim 1, wherein: The purification treatment is performed by dialysis, wherein the dialysis temperature is 4-8 ℃; and the molecular weight of the dialysis bag used for dialysis is 8000-16000 Da.

5. The method of claim 1, wherein: The drying treatment is performed by vacuum freeze-drying.

6. The anti-flocculation submicron emulsion prepared by the preparation method of any one of claims 1-5.

7. Use of the anti-flocculation submicron emulsion of claim 6 in liquid emulsified food.

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