Restrictive enzymolysis pea protein isolate-chitosan Pickering emulsion as well as preparation method and application thereof

Abstract

The invention discloses a restriction enzymolysis pea protein isolate-chitosan Pickering emulsion which is composed of an oil phase and a water phase, and the water phase is composed of HPP, CS and water; the HPPI is obtained by carrying out enzymolysis on PPI through trypsin. According to the application of the restricted enzymolysis pea protein isolate-chitosan Pickering emulsion in preparation of a medicine embedding preparation, the medicine can be selected from astaxanthin. The invention further discloses the AST embedded Pickering emulsion and the Pickering emulsion yoghourt. According to the method disclosed by the invention, the PPI is subjected to enzymolysis by utilizing an enzymolysis technology to obtain the HPPI with more excellent emulsifying performance, the enzymolysis time is extremely short (2 minutes), the modification cost is favorably reduced in actual production, and the method is easy to realize in industrialization. The research of the invention provides theoretical support for developing a novel delivery system and widening the application range, and lays a foundation for the application of Pickering emulsion in future food.

Description

Technical Field

[0001] The present invention relates to a restricted enzymatic hydrolysis pea protein isolate-chitosan Pickering emulsion, a preparation method thereof and an application thereof, belonging to the technical field of emulsion preparation. Background Art

[0002] A Pickering emulsion is an emulsion stabilized by solid particles, which is gradually formed by high-speed shearing and high-pressure homogenization of an oil phase and an aqueous phase. The solid particles adsorb and accumulate at the interface to stabilize the oil droplets, forming a layer with high mechanical strength, thereby maintaining stability. The Pickering emulsion used in the food system generally refers to an emulsion stabilized by a particulate emulsifier with emulsifying effect extracted from natural ingredients.

[0003] Pea protein isolate (PPI) is an emerging protein isolated from pea seeds, which has a rich amino acid composition and good digestibility. PPI has good emulsifying and gelling properties and is often used as an emulsifier for Pickering emulsification. However, in the case of high ionic strength or when the pH is close to the isoelectric point of the protein, phenomena such as demulsification and creaming often occur in the emulsion. Therefore, it is necessary to perform specific treatment on PPI to improve its emulsifying ability. Chinese invention patent CN 114947107 A discloses the preparation and application of modified pea protein-chitosan nanoparticles, which uses a combined technology of pH shift-heating-ultrasonic wave to modify and obtain pea protein-chitosan composite particles. The particles enhance the hydrophobicity of pea protein, and through the homogenization emulsification technology, a stable pea protein-chitosan high internal phase Pickering emulsion is constructed, and the emulsion can be stored at 4 °C for more than 4 months. However, the method of pH shift is likely to change the acid-base environment of the food system itself, and it is necessary to repeatedly adjust the pH during the food processing, which not only increases the processing cost, but also brings unnecessary salts to the system, changes the original properties of the system, and ultimately affects the mineral element content, taste, etc.

[0004] Astaxanthin (AST) has important physiological functions such as antioxidant activity, anti-inflammatory activity, anti-diabetic, and anti-cancer activities, and plays an important role in maintaining human health. However, due to its poor water solubility, poor chemical stability, low bioavailability and other disadvantages, its application is limited. Using Pickering emulsion to encapsulate it to improve the bioavailability of astaxanthin is a feasible solution. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides a restricted enzymatic hydrolysis pea protein isolate-chitosan Pickering emulsion, a preparation method thereof and an application thereof.

[0006] The present invention is achieved by the following technical solutions: A restricted enzymatic hydrolysis pea protein isolate-chitosan Pickering emulsion consists of an oil phase and an aqueous phase. Among them, the aqueous phase is composed of restricted enzymatic hydrolysis pea protein isolate (HPPI), chitosan (Chitosan, CS) and water; the HPPI is obtained by enzymatic hydrolysis of PPI with trypsin.

[0007] Furthermore, the mass ratio of HPPI to CS is 1:1.2, the molecular weight of CS is 3 kDa, HPPI and CS combine to form nanoparticles, and the concentration of nanoparticles in the aqueous phase is 3% (mass percentage); the oil phase accounts for 70% (volume percentage).

[0008] Furthermore, the oil phase can be selected from corn oil.

[0009] The preparation method of the restricted enzymatic hydrolysis pea protein isolate-chitosan Pickering emulsion is as follows: (1) Preparation of HPPI: Dissolve PPI in water to obtain a PPI solution, add trypsin, carry out enzymatic hydrolysis, and centrifuge. The supernatant is the HPPI solution; (2) Preparation of HPPI-CS nanoparticles: Add CS to the HPPI solution and mix well to obtain an HPPI-CS nanoparticle solution; (3) Preparation of HPPI-CS Pickering emulsion: Add the oil phase to the HPPI-CS nanoparticle solution and homogenize to obtain the HPPI-CS Pickering emulsion.

[0010] Furthermore, in the step (1), the concentration of the PPI solution is 20 mg / ml, and the enzymatic hydrolysis conditions are: enzymatic hydrolysis for 2 min at a temperature of 30 °C, pH 9, and 600 r / min.

[0011] Furthermore, in the step (2), the molecular weight of CS is 3 kDa, and the mass ratio of HPPI to CS is 1:1.2.

[0012] Furthermore, in the step (3), the oil phase is selected from corn oil, the oil phase accounts for 70%, and the concentration of HPPI-CS nanoparticles in the HPPI-CS nanoparticle solution is 3%.

[0013] The application of the restricted enzymatic hydrolysis pea protein isolate-chitosan Pickering emulsion in the preparation of a formulation for encapsulating drugs.

[0014] Furthermore, the drug can be selected from astaxanthin.

[0015] A Pickering emulsion encapsulating AST consists of AST, an oil phase and an aqueous phase. Among them, the aqueous phase is composed of HPPI, CS and water; the HPPI is obtained by enzymatic hydrolysis of PPI with trypsin.

[0016] The preparation method of the Pickering emulsion encapsulating AST is as follows: Add AST to the oil phase to make the concentration of AST in the emulsion reach 0.4 mg / mL; mix the oil phase and the HPPI-CS nanoparticle solution, and disperse them at high speed to prepare the Pickering emulsion encapsulating AST.

[0017] A Pickering emulsion yogurt is composed of 100 mL of yogurt and 10 mL of the Pickering emulsion encapsulating AST.

[0018] In this invention, PPI and CS are used as raw materials for preparation. The emulsifying property of pea protein isolate is improved by restricted enzymolysis, and HPPI-CS nanoparticles are formed by the electrostatic adsorption of proteoglycan. The synthesized nanoparticles are characterized, and the nanoparticles are used to prepare Pickering emulsions. The internal structure and physical stability of the HPPI-CS Pickering emulsions are measured. Further, the Pickering emulsions are used to encapsulate AST, the stability and antioxidant property of AST in the emulsions are explored, and the in vitro and in vivo digestion of the emulsions and their application in yogurt are studied. The main conclusions are as follows: First, HPPI is prepared by restricted enzymolysis. The protein concentration, enzyme type, enzymolysis time, temperature, and pH during enzymolysis are screened by comparing the particle size, PDI, and Zeta potential of HPPI. The obtained enzymolysis conditions are: the concentration of PPI is 20 mg / ml, trypsin, enzymolysis time 2 min, 30 °C, and pH 9. The change in the protein secondary structure during enzymolysis is analyzed, and it is found that the α-helix increases, the β-sheet decreases, and the β-turn and random coil structures increase. The β-sheet is relatively stable, while the α-helix, β-turn, and random coil are relatively flexible and open, which makes HPPI more likely to adsorb to the oil-water interface and become a better emulsifier. Emulsions are prepared with HPPI, and it is found that HPPI is not sufficient to stabilize the emulsions.

[0019] Secondly, the particle size, PDI, Zeta potential, and appearance of the HPPI-CS nanoparticles are compared, and the molecular weight of CS and the mixing ratio of the HPPI solution and the CS solution are screened. The optimal formula is obtained as follows: the molecular weight of CS is 3 kDa, and the solution ratio of HPPI to CS is 1:1.2. The particle size of the HPPI-CS nanoparticles with the optimal formula is 217.23 nm, the Zeta potential is 28.9 mV, and the PDI is 0.40, indicating that the HPPI-CS nanoparticles have a small particle size, a high potential, and good dispersion. Infrared spectrum scanning of the nanoparticles shows that HPPI and CS are bound by hydrogen bonds, the contact angle is 57.5 ± 0.4°, and the pH with the best emulsifying property is 2 - 4. This provides high-quality nanoparticles for the following research.

[0020] Next, Pickering emulsions were prepared by high-speed homogenization using the above-prepared HPPI-CS nanoparticles. The Pickering emulsions were photographed by an electric fluorescence microscope, showing that the emulsions were O / W emulsions. The properties of the Pickering emulsions changed with the oil phase content. As the oil phase content increased, the droplet size of the Pickering emulsions gradually increased, and the G’, G’’, and viscosity of the emulsions also increased, making the emulsions tend to be stable. The G’ of all emulsions was greater than G’’, indicating that the structure of the emulsions was a gel-like network. The HPPI-CS Pickering emulsions still had strong stability after being stored for 45 days at different ionic concentrations (0 - 500 mM), pH values (2 - 7), and temperatures (-20 - 68 °C).

[0021] Subsequently, AST was encapsulated using the above-prepared HPPI-CS Pickering emulsions. Due to the property that AST is soluble in oil itself, it could be directly added to the oil phase to prepare the emulsion. After being stored for 45 days, the retention rate of AST in the encapsulated Pickering emulsions only decreased to 75.8%, showing high storage stability. At the same time, the encapsulated Pickering emulsions with AST also had good ionic, pH, and temperature stability. The Pickering emulsions exhibited strong antioxidant ability, and the antioxidant effect was better when the emulsions were stored at 4 °C, which was suitable for cold storage.

[0022] Finally, further research was conducted on the encapsulated Pickering emulsions with AST. After simulated gastrointestinal digestion, a high level of AST retention rate was shown in the oral and gastric phases. In the small intestine, a slow release phenomenon occurred in the emulsions. In addition, when the encapsulated Pickering emulsions with AST were added to yogurt, the retention rate of AST reached 76% during the shelf life of the yogurt, and the overall acceptance was relatively high.

[0023] In this invention, enzymatic hydrolysis technology was used to enzymatically hydrolyze PPI to obtain HPPI with more excellent emulsifying properties. After the enzymatic hydrolysis was completed, the enzyme was inactivated, and there was no need for separation, which would not affect the quality of the food in subsequent processing. Moreover, the enzymatic hydrolysis time was extremely short (2 min), which was beneficial to reducing the modification cost in actual production and was easy to be realized in industrialization, achieving good usage effects. The research of this invention provided theoretical support for the development of new delivery systems and the expansion of application ranges, and laid a foundation for the application of Pickering emulsions in future foods.

[0024] All the terms and phrases used in this invention have the general meanings well-known to those skilled in the art. Description of the Drawings

[0025] Figure 1 : Effects of different protein concentrations on the (A) particle size, (B) Zeta potential, and (C) PDI of HPPI.

[0026] Figure 2: Effects of different enzyme types and hydrolysis time on the (A) particle size, (B) Zeta potential, and (C) PDI of HPPI.

[0027] Figure 3 : DH of HPPI at different hydrolysis times.

[0028] Figure 4 : Effects on the secondary structure of HPPI at different hydrolysis times.

[0029] Figure 5 : Effects of different hydrolysis temperatures on the (A) particle size, (B) Zeta potential, and (C) PDI of HPPI.

[0030] Figure 6 : Effects of different hydrolysis pH on the (A) particle size, PDI, (B) Zeta potential, and (C) PDI of HPPI.

[0031] Figure 7 : Effects of different chitosan molecular weights on the (A) particle size, PDI, and (B) Zeta potential of HPPI-CS nanoparticles.

[0032] Figure 8 : Changes in (A) particle size and PDI, (B) Zeta potential, and (C) appearance at different mixing ratios of HPPI solution and CS solution.

[0033] Figure 9 : Changes in the contact angle of nanoparticles before and after hydrolysis.

[0034] Figure 10 : Fluorescence spectra of HPPI-CS nanoparticles with different hydrolysis times.

[0035] Figure 11 : Infrared spectra of HPPI, CS, and HPPI-CS.

[0036] Figure 12 : Effects of nanoparticle solutions with different pH values on the emulsifying activity index (EAI) and emulsion stability index (ESI).

[0037] Figure 13 : Electrokinetic fluorescence microscopy images of HPPI-CS Pickering emulsions.

[0038] Figure 14 : Average droplet sizes of HPPI-CS Pickering emulsions stabilized with different oil phase contents (φ = 0.4 - 0.7) after storage for 0 d and 45 d.

[0039] Figure 15 : Storage modulus (G’), loss modulus (G’’), and (B) viscosity of HPPI-CS Pickering emulsions with different oil phase contents.

[0040] Figure 16 : (A) CI and (B) morphologies of HPPI-CS Pickering emulsions with different oil phase contents (φ = 0.4 - 0.7) at 0 d, 30 d, and 45 d.

[0041] Figure 17 : (A) Storage modulus (G’), loss modulus (G’’), (B) average droplet size, and (C) morphologies of HPPI-CS Pickering emulsions stabilized by different sodium chloride concentrations (0 - 500 mM) at 0 d, 30 d, and 45 d.

[0042] Figure 18 : (A) Storage modulus (G’), loss modulus (G’’), (B) average droplet size, and (C) morphologies of HPPI-CS Pickering emulsions stabilized at different pH values (2 - 7) at 0 d, 30 d, and 45 d.

[0043] Figure 19 : (A) Average droplet size and (B) morphologies of HPPI-CS Pickering emulsions at different temperatures (-20 °C, 4 °C, 20 °C, 37 °C, 68 °C) at 0 d, 30 d, and 45 d.

[0044] Figure 20 : Standard curve of AST.

[0045] Figure 21 : Changes in the retention rate of AST and DPPH radical scavenging rate after 45 days of storage, where A: retention rate; B: DPPH radical scavenging rate.

[0046] Figure 22 : Appearance, retention rate, and radical scavenging rate of AST at different ionic concentrations (0 - 500 mM) after 45 days of storage, where A: appearance; B: retention rate and radical scavenging rate.

[0047] Figure 23 : Appearance, retention rate, and DPPH radical scavenging rate of AST at different pH values (2 - 7) after 45 days of storage, where A: appearance; B: retention rate and radical scavenging rate.

[0048] Figure 24 : Appearance, retention rate, and radical scavenging rate of AST at different temperatures (-20 °C, 4 °C, 20 °C, 37 °C, 68 °C) after 45 days of storage, where A: appearance; B: retention rate and radical scavenging rate.

[0049] Figure 25 : Content changes of AST in Pickering emulsions and fluorescence microscopy images during simulated digestion, where A: content changes of AST; B: fluorescence microscopy images.

[0050] Figure 26 : Retention rate of AST in Pickering emulsion yogurt during shelf life.

[0051] Figure 27 : pH change of Pickering emulsion yogurt during shelf life.

[0052] Figure 28 : Change in water holding capacity of Pickering emulsion yogurt during shelf life.

[0053] Figure 29 : Sensory evaluation personnel analyze the sensory characteristics of different types of yogurt. Detailed implementation mode

[0054] The present invention will be further described below in conjunction with embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0055] The instruments, reagents, and materials involved in the following embodiments are all conventional instruments, reagents, and materials existing in the prior art and can be obtained through regular commercial channels without special instructions. The experimental methods, detection methods, etc. involved in the following embodiments are all conventional experimental methods and detection methods existing in the prior art without special instructions.

[0056] Experiment 1 Preparation and characterization of restricted enzymolysis pea protein isolate-chitosan nanoparticles In this experiment, nanoparticles were formed by the electrostatic adsorption of restricted enzymolysis of proteins and polysaccharides, and the effects of enzymolysis temperature, pH, time, pea protein isolate concentration, chitosan molecular weight, and solution ratio on the particle size, Zeta potential, and PDI of the nanoparticles were explored to determine the optimal preparation formula. Infrared spectroscopy scanning and fluorescence spectroscopy scanning were performed on the nanoparticles to determine the protein-polysaccharide synthesis situation, and the contact angle of the nanoparticles was measured for subsequent further research on emulsions.

[0057] 1.1 Experimental materials Pea protein isolate (pea protein concentration ≥ 90%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; chitosan (MW 2 - 50 KDa) was purchased from Hefei Bomei Biotechnology Co., Ltd.

[0058] 1.2 Experimental methods 1.2.1 Preparation and characterization of hydrolysates of pea protein isolate (HPPI) 1.2.1.1 Preparation of HPPI Dissolve 2 wt% of PPI in water, stir magnetically at 600 r / min for 30 min, then enzymatically hydrolyze with 0.1 wt% trypsin at 600 r / min at 30 °C for 2 min. Subsequently, inactivate at 95 °C for 10 min. After cooling in a water bath, adjust the pH to 9.5 with 1 M sodium hydroxide solution, stir magnetically at 600 r / min for 2 h, then adjust the pH to 7 with 1 M hydrochloric acid solution, seal with plastic wrap, and stir magnetically at 600 r / min overnight. Then centrifuge the HPPI solution at 7500 r / min for 30 min and collect the supernatant.

[0059] 1.2.1.2 Determination of HPPI particle size, polydispersity index (PDI), and Zeta potential Use a Nanosizer ZS90 (ZEN-3690) instrument to measure the average particle size distribution, PDI, and Zeta potential of HPPI nanoparticles. Set the instrument parameters as follows: measurement temperature 25 °C; dispersant water; equilibration time 20 s. Dilute all samples 20-fold and take 1 mL of each sample for determination at 25 °C.

[0060] 1.2.1.3 HPPI degree of hydrolysis determination method Determine the degree of hydrolysis (DH) of HPPI at each time point according to the method described by Zhang et al. Dilute the HPPI (400 μL) solution 4-fold and mix it with 3.00 mL of OPA reagent, incubate at 25 °C for 2 min, and then read the absorbance (OD sample ) at 340 nm using a spectrophotometer. Standard samples (OD standard ) and blank samples (OD blank ) are measured using 0.9516 mmol / L serine and distilled water instead of HPPI, respectively. Calculate DH using the following equation: where V is the sample volume (L), X is the sample weight (g), P is the protein content (%), α is 0.970, β is 0.342, h is the number of hydrolyzed peptide bonds, and for PPI, htot is 7.55.

[0061] (1-1); (1-2); (1-3).

[0062] The OPA reagent is prepared as follows: (1) Dissolve 7.620 g of sodium tetraborate decahydrate and 200 mg of sodium dodecyl sulfate (SDS) in 150 mL of deionized water. Wait for complete dissolution.

[0063] (2) Dissolve 160 mg of o-phthalaldehyde 97% (OPA) in 4 mL of ethanol.

[0064] (3) Then quantitatively transfer the OPA ethanol solution into the above solution by rinsing with deionized water.

[0065] (4) Add 176 mg of 99% dithiothreitol (DTT) to the solution by rinsing with deionized water.

[0066] (5) Prepare the solution to 200 mL with deionized water.

[0067] 1.2.1.4 Circular dichroism (CD) analysis The secondary structure changes of its hydrolysis can be analyzed by the circular dichroism of the protein. The curve was measured using a J-815 CD spectrometer (Jasco, Tokyo). Dilute the enzymolysis samples at different enzymolysis times to a mass concentration of 0.1 mg / mL. Instrument parameters: detection wavelength is 190 - 240 nm, scanning speed is 100 nm / min, data interval is 0.5 nm, and bandwidth is 1.00 nm.

[0068] 1.2.2 Preparation and characterization of HPPI-CS nanoparticles 1.2.2.1 Preparation of HPPI-CS nanoparticles Dissolve 0.5 wt% of chitosan (CS) solid in 1% acetic acid solution and stir overnight at a speed of 600 r / min. Slowly drip different ratios of CS solution (HPPI:CS = 4:1, 3:1, 2:1, 1:1, 1:1.2, 1:2, 1:3, 1:4) into the HPPI solution and magnetically stir at a rate of 600 r / min for 30 min. Finally, filter the HPPI-CS solution through a 0.45 μm filter membrane to remove insoluble substances and unbound CS. Freeze-dry the HPPI-CS solution for 2 d, then grind the freeze-dried sample into powder and place it in a desiccator for subsequent experiments.

[0069] 1.2.2.2 Contact angle In this study, a contact angle measuring instrument DCAT21 was used to measure the contact angles of HPPI-CS and PPI-CS nanoparticles. The experimental procedure is as follows: First, dilute the sample to a final concentration of 10 mg / mL. Then, take an appropriate amount of the sample, spread it evenly on a clean glass plate, and dry it in an oven at 60 °C. Next, deposit the uniform sample film on a smooth glass substrate. Finally, drop water droplets onto the sample film to determine the contact angle.

[0070] 1.2.2.3 Fluorescence spectroscopyscanning The fluorescence spectra of HPPI-CS nanoparticles at different enzymatic hydrolysis times were measured using a fluorescence spectrophotometer F-4600. Instrument parameters were set as follows: excitation wavelength was 280 nm, emission wavelength range was 290 - 460 nm, scanning speed was 240 nm / min, and excitation and emission slit widths were 5 nm.

[0071] 1.2.2.4 Infrared spectrum scanning The chemical structures of CS, HPPI, HPPI-CS, and HPPI-CS nanoparticles at different enzymatic hydrolysis times were determined using a Fourier transform infrared spectrometer Nicolet iS10. First, potassium bromide dried at 65 °C for 12 h was pressed into a thin slice to collect the background. Subsequently, potassium bromide was mixed with the freeze-dried and ground sample in a ratio of 20:1. Scanning was performed in the wavelength range of 400 - 4000 cm -1 -1.

[0072] 1.2.2.5 Emulsifying property determination 4.5 mL of the mixture at pH 2, 3, 4, 5, 6, and 7 was placed in a 50 mL centrifuge tube and mixed with 10.5 mL of corn oil to analyze the emulsifying properties. The mixture was homogenized at 12000 rpm for 2 min. 50 μL of the emulsion was taken from the bottom of the centrifuge tube at 0 min and 10 min at room temperature, respectively. The emulsion was mixed with 5 mL of 0.1 wt% sodium dodecyl sulfate (SDS) solution, and the absorbance was measured at 500 nm. The emulsifying activity index (EAI) and emulsion stability index (ESI) were calculated according to the following equations: (1 - 4); (1 - 5).

[0073] Where C is the initial protein concentration (g / mL), φ is the oil volume fraction of the emulsion, which is 0.20 (v / v), n is the dilution factor of 100, EAI0 is the EAI at 0 min after homogenization, ΔEAI is the change in EAI between 0 min and 10 min of static storage at room temperature, and Δt is 10 min.

[0074] 1.2.3 Statistical analysis All measured values were measured at least three times, and the data were expressed as mean ± standard deviation. One-way analysis of variance and other statistical analyses were performed using Origin 2019 software and SPSS 25. The difference between the data was p < 0.05.

[0075] 1.3 Results and discussion 1.3.1 Characterization of HPPI 1.3.1.1 Effect of enzymatic hydrolysis on PPI and screening of enzymatic hydrolysis protein concentration Although previous studies have shown that limited enzymatic hydrolysis can enhance the emulsifying properties of rice bran protein and others, there are few reports on the enhancement of the emulsifying properties of PPI by limited enzymatic hydrolysis. Therefore, in this paper, the effects of enzymatic hydrolysis on PPI were investigated by measuring the changes in particle size, Zeta potential, and PDI before and after enzymatic hydrolysis. As Figure 1 can be seen, in terms of the enzymatic hydrolysis effect, at different protein concentrations, the particle size of the unhydrolyzed protein increased from 181.73 ± 2.41 nm to 283.97 ± 11.46 nm, while the particle size of the protein after trypsin hydrolysis increased from 139.43 ± 4.69 nm to 224.73 ± 3.70 nm. At the same protein concentration, the particle size after hydrolysis was significantly reduced. This is because hydrolysis causes some protein chains to break, resulting in a decrease in particle size. Therefore, the protein hydrolyzed by trypsin is more suitable for preparing stable Pickering emulsions in terms of particle size. At low protein concentrations, PDI also showed a decreasing trend. When the PDI value is low, it indicates that the sample has good uniformity and stability. Therefore, the protein after enzymatic hydrolysis is more uniform and stable. The negative charge carried by the protein after hydrolysis decreased slightly. This may be due to the fact that enzymatic hydrolysis causes the internal peptide chains of the protein to unfold, and the hydroxyl groups in the peptide chains are protonated, resulting in a decrease in negative charge.

[0076] Moreover, as the substrate protein concentration increases, the enzymatic reaction rate usually also increases, resulting in more products being formed. When the protein concentration is too high, the enzyme system may tend to reach a saturated state. Therefore, the selection of protein concentration is very important for the results of enzymatic hydrolysis. In this study, PPI at different concentrations (5 - 30 mg / mL) was used for enzymatic hydrolysis, and compared with PPI before enzymatic hydrolysis, the effects of enzymatic hydrolysis on protein particle size, Zeta potential, and PDI were observed to screen the optimal protein concentration for enzymatic hydrolysis. As Figure 1 can be observed, as the protein concentration increases, the particle size of HPPI increased from 173.00 ± 2.10 nm to 224.73 ± 3.70 nm, and PDI also showed the same trend of change, increasing from 0.20 to 0.47, and the Zeta potential gradually decreased from -24.93 ± 0.9 to -17.47 ± 1.2 mV. This may be because as the concentration of HPPI increases, due to the reduction of pores and the influence of electrostatic attraction, the enzymatic hydrolysis products aggregate with each other to form large clusters, resulting in an increase in the particle size and dispersion coefficient of HPPI. This aggregation may cause the charged groups on the surface of the enzymatic hydrolysis products to be buried inside the aggregates, thus reducing the absolute value of the Zeta potential. This phenomenon may be affected by the concentration effect and the interaction between enzymatic hydrolysis products, further affecting the properties and performance of HPPI.

[0077] Therefore, based on the data results of the particle size, Zeta potential, and PDI of HPPI, HPPI with a concentration of 20 mg / mL was selected for subsequent experiments.

[0078] 1.3.1.2 Screening of Enzymatic Hydrolysis Types and Time The influence of enzymes on protein hydrolysis mainly depends on the enzyme type, applicable substrate, and hydrolysis conditions. Common protein hydrolases include pepsin, trypsin, etc., and different enzymes will have different effects on protein substrates during hydrolysis. For example, some studies have shown that at the same degree of hydrolysis, trypsin can more effectively reduce the size of soy protein aggregates than alkaline protease. Therefore, in the protein hydrolysis process, it is necessary to comprehensively consider the characteristics and applicability of enzymes to achieve the best hydrolysis effect and product quality. The influence of hydrolysis time on protein hydrolysis effect is equally crucial. During protein hydrolysis, the length of hydrolysis time directly affects the degree of hydrolysis and the characteristics of the product. Therefore, in this study, trypsin and pepsin were used as research objects to study their effects on the particle size, Zeta potential, and PDI of HPPI at different hydrolysis times (0.5 min, 2 min, 10 min, 120 min).

[0079] By comparing the effects of trypsin and pepsin on the particle size, PDI, and Zeta potential of HPPI at different hydrolysis times, it was found that at the same hydrolysis time, the particle size and PDI of HPPI hydrolyzed by pepsin were larger than those of HPPI hydrolyzed by trypsin, and the Zeta potential was smaller. This may be because pepsin has more hydrolysis sites for HPPI and a faster hydrolysis rate. After PPI is hydrolyzed, due to the hydrophobic attraction of polypeptides at high degrees of hydrolysis, large aggregates are formed, resulting in an increase in particle size and PDI. The negatively charged free -COOH is wrapped inside the aggregates, resulting in a decrease in the absolute value of the Zeta potential due to the reduction of negative charges. Therefore, trypsin hydrolysis is superior to pepsin hydrolysis in terms of particle size, PDI, and Zeta potential. So, trypsin was selected as the target hydrolysis enzyme. According to the subsequent DH results, it can be seen that the hydrolysis time needs to be controlled within 10 min. When the hydrolysis time is 2 min, the particle size of HPPI decreases significantly, and both PDI and Zeta potential meet the requirements. Therefore, 2 min was selected as the hydrolysis time.

[0080] 1.3.1.3 Determination of Degree of Hydrolysis at Different Hydrolysis Times The degree of hydrolysis (DH) represents the proportion of cleaved peptide bonds in a protein hydrolysate and is a commonly used parameter to determine the properties of the hydrolysate. In the related functional improvement of protein hydrolysis, DH... When DH is too large, a high concentration of hydrolyzed protein can enter the continuous phase instead of adsorbing at the oil - water interface, and it may cause the protein to lose its optimal special globular structure, which has an adverse effect on emulsification. This indicates that using protein hydrolysis to increase functionality is limited to low DH values. According to Figure 3It can be seen that DH increases with the increase of enzymatic hydrolysis time. When the enzymatic hydrolysis time exceeds 10 min, the change of DH tends to be gentle. This may be because in the initial stage of the enzymatic hydrolysis reaction, the substrate is abundant, and there are more peptide bonds and enzymatic hydrolysis sites that trypsin can act on. Therefore, the enzymatic hydrolysis efficiency is the highest, and the hydrolysis degree curve shows an obvious upward trend. As the enzymatic hydrolysis reaction proceeds, PPI is gradually cleaved into polypeptides, and the cleavage sites available for proteases gradually decrease, resulting in a slowdown in the growth of the hydrolysis degree. In addition, the accumulation of enzymatic hydrolysis products may cause competitive inhibition of proteases, thereby affecting the activity of proteases, which is also one of the reasons for the slow growth of the hydrolysis degree curve. Generally, when the hydrolysis degree of protein is within 20%, it can have good emulsifying properties. Therefore, the enzymatic hydrolysis time within 10 min meets the preparation requirements.

[0081] 1.3.1.4 CD spectrum analysis CD is a method widely used to analyze the secondary structure of proteins and peptides. Through the CD spectrum, the secondary structure of proteins at different hydrolysis times can be determined. Figure 4 The research results show that after 120 min of hydrolysis, compared with PPI, the α-helix of HPPI increases from 33.2% to 34.6%, the β-sheet decreases from 13.8% to 9.4%, while the β-turn and random coil structures increase from 22.8% to 23.9% and from 30.2% to 32.1% respectively. The β-sheet is relatively stable, while the α-helix, β-turn and random coil are relatively flexible and open. During the hydrolysis process, the secondary structure of the protein is unfolded, and the β-sheet is transformed into α-helix, β-turn and random coil, resulting in the formation of an ordered and flexible structure of the protein, which makes the nanoparticles more easily adsorbed to the oil-water interface and become better emulsifiers. These findings not only help to deeply understand the dynamic changes of protein structure, but also provide important references for further optimizing the application of nanoparticles in the emulsification process.

[0082] 1.3.1.5 Screening of enzymatic hydrolysis temperature The effect of enzymatic hydrolysis temperature on proteins is very important because temperature can affect the activity of enzymes and the structure of proteins. Therefore, when selecting the enzymatic hydrolysis temperature, it is necessary to consider the optimal temperature of the enzyme, the stability of the protein and the requirements of the enzymatic hydrolysis reaction to ensure the best enzymatic hydrolysis effect and product characteristics. Considering the influence of temperature on enzymes and proteins comprehensively, reasonable temperature control is a key factor in the protease hydrolysis process. Therefore, in this experiment, four different enzymatic hydrolysis temperatures (20 - 50 °C) were selected to explore their effects on HPPI. As Figure 5As shown in the figure, with the increase of enzymatic hydrolysis temperature, the particle size of HPPI increased from 178.0 ± 1.5 nm to 215.0 ± 4.7 nm, and then decreased to 211.0 ± 4.6 nm. The PDI showed the same change trend, increasing from 0.28 to 0.31 and then decreasing to 0.27. There was no significant difference in Zeta potential. This is because with the increase of enzymatic hydrolysis temperature, the hydrophobic interaction force of the protein was destroyed, the protein peptide chain was stretched, and denaturation may occur, resulting in the formation of larger particles or clusters, thus increasing the particle size and PDI. When the temperature was too high, trypsin was inactivated, thus destroying the enzymatic hydrolysis reaction. And the nanoparticles for preparing Pickering emulsion require a smaller particle size and a mild enzymatic hydrolysis reaction. Therefore, considering the cost and application effect, 30 °C was selected as the enzymatic hydrolysis temperature for subsequent experiments.

[0083] 1.3.1.6 Screening of Enzymatic Hydrolysis pH The effect of enzymatic hydrolysis pH on protein properties is also very important. pH can affect the activity of hydrolase and the structure of protein, and then affect the hydrolysis process and product characteristics of protein. In this experiment, different enzymatic hydrolysis pH values (6, 7, 8, 9) were adjusted to study the effects of changes in enzymatic hydrolysis pH on the particle size, PDI and Zeta potential of HPPI. The results are as Figure 6 shown. The research shows that with the decrease of enzymatic hydrolysis pH, the particle size of HPPI increased from 189.2 ± 7.89 nm to 1406.7 ± 142.50 nm, the PDI increased from 0.25 to 0.52, and the Zeta potential decreased from -18.8 ± 0.40 mV to -22.1 ± 0.65 mV. The changes in particle size, PDI and Zeta potential were mainly because when approaching the isoelectric point (about 4.5) of pea protein isolate, the solubility of HPPI decreased, protein aggregation led to an increase in particle size and an expansion of the molecular size distribution range, resulting in an increase in PDI. At this time, the positive and negative charges of HPPI gradually cancelled each other out, and the net charge decreased, thus leading to a decrease in Zeta potential. And when the pH was too high, trypsin was inactivated. Therefore, the enzymatic hydrolysis pH of 9 was selected.

[0084] 1.3.2 Preparation of HPPI Pickering Emulsion Since previous research reports have shown that stable emulsions can be formed by hydrolyzing protein particles, in this experiment, HPPI was tried to prepare an emulsion. An emulsion was prepared under the conditions of 3 wt% HPPI and an oil phase content of 70%. The results showed that the emulsion prepared from HPPI underwent creaming at the initial stage and demulsification within 24 h. Therefore, HPPI was not sufficient to stabilize the Pickering emulsion, and chitosan needed to be added as a stabilizer to ensure the stability of the emulsion.

[0085] 1.3.3 Preparation and Characterization of HPPI-CS Nanoparticles 1.3.3.1 Screening of Chitosan Molecular Weight Many studies have demonstrated that the molecular weight of chitosan (CS) has a great influence on its functionality, such as solubility, viscosity, and surface activity. Different CS molecular weights exhibit diverse polysaccharide properties. Therefore, in this experiment, four different molecular weight CSs (2 - 50 kDa) were selected to explore their effects on HPPI-CS nanoparticles. As Figure 7 shown, as the CS molecular weight increased, the particle size of HPPI-CS nanoparticles decreased from 376 ± 32.0 nm to 212 ± 7.2 nm, then increased to 560 ± 18.2 nm and decreased to 249 ± 5.5 nm. The PDI showed the same trend of change, decreasing from 0.54 to 0.50, then increasing to 0.55 and decreasing to 0.44. The Zeta potential changed as Figure 7 shown, increasing from 28 ± 0.6 mV to 33 ± 2.9 mV, then decreasing to 29 ± 1.4 mV. This indicates that CS with different molecular weights has a great influence on the properties of HPPI-CS nanoparticles. This is mainly because CS with a larger molecular weight may produce larger particles, as longer chain segments can provide more cross-linking points and interactions, thus causing aggregation during particle formation. For example, chitosan with a molecular weight of 30 kDa had the highest values for particle size, PDI, and Zeta potential, indicating that the nanoparticles formed by this chitosan were larger and more dispersed, and the stronger electrostatic repulsion could maintain the stability of the nanoparticle solution. However, too large particle size and PDI may not be able to stabilize the emulsion well.

[0086] Therefore, considering the data results of the particle size, Zeta potential, and PDI of the nanoparticles, chitosan with a molecular weight of 3 kDa was selected for subsequent experiments.

[0087] 1.3.3.2 Effect of the mixing solution ratio of HPPI solution and CS solution on HPPI-CS nanoparticles HPPI and CS are stable through electrostatic interaction, so it is necessary to determine the binding ratio of HPPI and CS. The experimental results are as Figure 8As shown, as the ratio of HPPI:CS decreased, the particle size of HPPI-CS nanoparticles decreased from 7351±127.1 nm to 153±4.0 nm, the PDI increased from 0.374 to 1 and then decreased to 0.363±0.051, and the Zeta potential increased from 10.88±2.44 mV to 30.97±0.55 mV. The particle size of the hydrolyzed protein was 177.3 nm, and as the proportion of chitosan solution increased, the particle size of the solution increased rapidly. This may be due to the acetic acid in the CS solution causing the pH to decrease, and the unbound HPPI reaching the isoelectric point, thus rapidly aggregating. As the proportion of chitosan solution increased, more and more HPPI bound to CS. CS was positively charged, and the pH continued to decrease beyond the isoelectric point, resulting in a decrease in particle size, an increase and then a decrease in PDI, and an increase in Zeta potential. The appearance of HPPI-CS nanoparticles (1:1.2) did not change after 30 days of storage, while the solution with larger particle size precipitated after 30 days. This may be because the electrostatic repulsion was not sufficient to stabilize the HPPI-CS nanoparticles in the system for 30 days. When the binding ratio of HPPI to CS was 1:1.2, the HPPI-CS nanoparticles exhibited good particle size, Zeta potential, PDI, and stability, meeting the requirements for preparing Pickering emulsions. Therefore, 1:1.2 was selected for the next step of research.

[0088] 1.3.3.3 Contact angle analysis The measurement of the contact angle can be used to evaluate the hydrophobicity and hydrophilicity of HPPI-CS nanoparticles. The nanoparticles used to stabilize Pickering emulsions should be partially wetted by both the oil phase and the water phase. By measuring the contact angle, if the contact angle is between 53° and 82°, it can be judged that the HPPI-CS nanoparticles have medium wettability, meeting the conditions for preparing Pickering emulsions. And the closer the contact angle between the particles and the water phase is to 90°, the higher the energy required for particle desorption, and the more stable the emulsion. Through contact angle analysis, as Figure 9 shown, the contact angle of PPI-CS was 34.1±0.5°, and the contact angle of HPPI-CS nanoparticles was 57.5±0.4°. The results showed that after restricted hydrolysis, HPPI-CS met the condition of medium wettability for preparing Pickering emulsions. In addition, for Pickering emulsions stabilized by nanoparticles, when the contact angle is between 15° and 90°, an oil-in-water (O / W) emulsion is usually formed. This indicates that the Pickering emulsion stabilized by HPPI-CS is an O / W emulsion.

[0089] 1.3.3.4 Fluorescence spectroscopy analysis The tertiary structure of proteins is mainly maintained by hydrophobic interactions. Changes in the endogenous fluorescence intensity can reflect changes in hydrophobic groups, thus affecting the tertiary structure of proteins. As Figure 10As shown, with the increase of enzymatic hydrolysis time, the fluorescence intensity first increases and then gradually decreases, accompanied by a red shift, indicating that the exposure degree of aromatic groups in water first increases and then decreases. This is because enzymatic hydrolysis leads to the unfolding of protein structure, exposing more aromatic groups in the initial stage. Subsequently, under high enzymatic hydrolysis conditions, peptide segments interact through hydrophobic attraction, and hydrophobic groups are buried in larger aggregates, resulting in a decrease in fluorescence intensity. The red shift phenomenon after enzymatic hydrolysis indicates local changes in the hydrophobic region of the protein, and the environment of chromophore groups (such as tryptophan residues) changes from non-polar to polar. Similar changes in fluorescence intensity were also observed during the enzymatic hydrolysis of wheat gluten.

[0090] 1.3.3.5 Infrared chromatographic analysis Infrared spectroscopy can provide information on the vibrations and rotations between atoms inside the molecules of a substance. The types of chemical bonds, the presence and environment of functional groups, etc. can be determined through the positions and intensities of absorption peaks, thus helping to determine the structure of unknown substances. Therefore, in this study, a Fourier transform infrared spectrometer was used to study whether HPPI and CS are connected and by what chemical bond through measuring the chemical structures of HPPI, CS, and HPPI-CS. As Figure 11 can be seen, restricted enzymatic hydrolysis of pea protein isolate has a characteristic peak at 3293.61 cm -1 Chitosan has a characteristic peak at 3419.04 cm -1 and 3293.61 cm -1 , 3419.04 cm -1 These two characteristic peaks reflect the changes in the O-H group. As Figure 11 can be seen, these two characteristic peaks show stretching vibrations, indicating that CS and HPPI are combined in the form of hydrogen bonds.

[0091] 1.3.3.6 Emulsifying properties The effects of different pH values of the nanoparticle solution on the EAI and ESI of Pickering emulsions are as Figure 12 shown. The pH of the nanoparticle solution has a significant effect on the emulsifying characteristics of Pickering emulsions. At pH 3, the EAI is the highest, reaching 5.60 m 2 / g. As the pH increases, the EAI gradually decreases. When the pH of the nanoparticle solution approaches the isoelectric point, the ESI of the Pickering emulsion is lower. This is because when the pH of the nanoparticle solution approaches the isoelectric point of 4.5, the number of positively charged and negatively charged groups is basically equal. Therefore, under this pH condition, the net charge carried by the nanoparticles is close to 0, resulting in a very low electrostatic repulsion between the nanoparticles. And the electrostatic repulsion between droplets is the reason for maintaining the stability of droplets. Therefore, under this condition, the droplets begin to aggregate, and the EAI and ESI decrease. According to the above results, it shows that the emulsifying properties of the nanoparticles are better when the pH is 2 - 4.

[0092] 1.4 Experimental summary In this experiment, HPPI was prepared by restricted enzymatic hydrolysis of PPI to improve its emulsifying properties. Nanoparticles were prepared through electrostatic adsorption with CS. The optimal concentration of enzymatically hydrolyzed protein, type of enzyme, time, temperature, pH, as well as the optimal CS molecular weight and the mixing ratio of HPPI and CS solution for forming HPPI-CS nanoparticles were screened out. Through experimental research, the preparation formula of the nanoparticles was determined as follows: the concentration of PPI was 20 mg / mL, trypsin, enzymatic hydrolysis time was 2 min, temperature was 30 °C, pH was 9, the molecular weight of CS was 3 kDa, and the mixing ratio of HPPI and CS solution was 1:1.2. The particle size of the HPPI-CS nanoparticles with the optimal formula was 217.23 ± 15.91 nm, the Zeta potential was 28.9 ± 0.70 mV, and the PDI was 0.40. Emulsion was prepared with HPPI, and it was found that HPPI was not sufficient to stabilize the emulsion. Subsequently, the contact angle of the nanoparticles was measured, which was 57.5 ± 0.4°, suitable for preparing O / W Pickering emulsion for subsequent emulsion research. By measuring the fluorescence intensity of HPPI-CS with different enzymatic hydrolysis times, it was known that enzymatic hydrolysis led to the unfolding of the protein structure, exposing more aromatic groups in the initial stage. Subsequently, under high enzymatic hydrolysis conditions, peptide segments interacted through hydrophobic attraction, and hydrophobic groups were buried in larger aggregates. At the same time, infrared spectroscopy analysis was carried out on CS, HPPI, and HPPI-CS nanoparticles. Through the analysis of the chemical structure and interaction of protein-polysaccharide, it was determined that CS was combined with HPPI through hydrogen bonds. Finally, it was determined that the emulsifying properties of the nanoparticles were better when the pH was 2 - 4.

[0093] Experiment 2 Preparation and Characterization of HPPI-CS Pickering Emulsion In this experiment, HPPI-CS nanoparticles studied in Experiment 1 were used to prepare Pickering emulsion. An electric fluorescence microscope was used to characterize the emulsion type and internal structure, and the storage, ionic, pH, and temperature stabilities of the emulsion were studied by measuring the internal and external morphology, average droplet size, and rheological properties of the emulsion.

[0094] 2.1 Experimental Materials HPPI-CS nanoparticles were prepared according to Experiment 1; corn oil was purchased from Jinlongyu Group Co., Ltd.

[0095] 2.2 Experimental Methods 2.2.1 Preparation of HPPI-CS Nanoparticles Prepared according to the method of Experiment 1.

[0096] 2.2.2 Preparation of HPPI-CS Pickering Emulsion The HPPI-CS suspension was prepared by dispersing 3 wt% freeze-dried nanoparticles into ultrapure water. The HPPI-CS Pickering emulsion was prepared by adding different volumes of corn oil (φ = 0.5, 0.6, 0.7) to the nanoparticle solution and homogenizing it at 12,000 rpm for 2 min. The freshly prepared emulsion was stored at 4 °C.

[0097] 2.2.3 Characterization of HPPI-CS Pickering emulsion 2.2.3.1 Average particle size of Pickering emulsion The average particle size of the Pickering emulsion was measured using a laser diffraction particle size analyzer. 300 mL of the emulsion to be measured was added to ultrapure water until an appropriate light shielding rate was achieved. During the measurement, it was stirred at a speed of 12,000 r / min. All samples were measured three times at room temperature, and the volume-average diameter (D4,3) was recorded.

[0098] 2.2.3.2 Creaming index of HPPI-CS Pickering emulsion The creaming index (CI) is one of the key indicators for evaluating the stability of the emulsion. 10 g of the freshly prepared emulsion was stored in a 25 mL glass bottle and kept at 4 °C. The height of the water layer H s , and the total height H of the emulsion t were measured at room temperature after the emulsion was stored for 0 d and 30 d, and the creaming index was calculated according to the following formula.

[0099] (2-1).

[0100] 2.2.3.3 Microstructure of HPPI-CS Pickering emulsion The microstructure of the HPPI-CS Pickering emulsion was observed using an electric fluorescence microscope (NIKON / NI-E). 50 μL of the prepared Pickering emulsion (c = 3 wt%, φ = 0.7) was diluted 20 times with ultrapure water, then 1.2 mL of 1% Nile blue acetone solution and 1.0 mL of 0.1% Nile red acetone solution were added. After vortex stirring for 1 min, it was observed using a microscope.

[0101] 2.2.3.4 Rheological properties of HPPI-CS Pickering emulsion The rheological properties of HPPI-CS Pickering emulsions were determined at 25 °C by a rheometer (Anton Paar Co., Austria) with different oil volume fractions (50% - 70%), pH values (2 - 6), and sodium chloride concentrations (0 mM, 50 mM, 100 mM, 300 mM, 500 mM). First, the strain was determined to be 0.1% by dynamic strain scanning method. The frequency curves of the loss modulus (G’’) and storage modulus (G’) were recorded in the frequency range of 0.1 - 100 rad / s. The viscosity changes of emulsions with different oil volume fractions (40% - 70%) were measured at a shear rate of 0.1 to 100 s -1 under.

[0102] 2.2.4 Stability analysis 2.2.4.1 Temperature stability Pickering emulsions were prepared according to the above method. The emulsions were stored at -20 °C, 4 °C, 20 °C, 37 °C, 68 °C, and after 2 h, they were stored at 4 °C and their appearance changes were observed.

[0103] 2.2.4.2 Ionic strength stability Different concentrations (0, 50, 100, 250, and 500 mM) of sodium chloride were added to the HPPI-CS solution and Pickering emulsions were prepared according to the above method. All emulsions were stored at 4 °C and their appearance changes were observed.

[0104] 2.2.4.3 pH stability The pH of the HPPI-CS nanoparticle solution was adjusted to 2, 3, 4, 5, 6, 7 with HCl solution and NaOH solution, and corn oil (φ = 0.7) was added to the suspension according to the above method 2.2.2 to prepare Pickering emulsions. All emulsions were stored at 4 °C and their appearance changes were observed.

[0105] 2.2.5 Statistical analysis All measured values were measured at least three times, and the data were expressed as mean ± standard deviation. One-way ANOVA and other statistical analyses were performed using Origin 2019 software and SPSS 25. The difference between the data was p < 0.05.

[0106] 2.3 Results and discussion 2.3.1 Type and internal structure of Pickering emulsions The interfacial structure was observed by an electric fluorescence microscope to determine the type of HPPI-CS Pickering emulsions. As Figure 13As shown, the electrofluorescence microscope image shows that the green part (oil phase) is encapsulated within the spherical region (emulsion droplet). The red part (HPPI-CS nanoparticles) is uniformly dispersed inside and at the edge of the spherical region, encapsulating the oil phase to prevent the aggregation of Pickering emulsions and maintain their stability, indicating that the HPPI-CS Pickering emulsion is of the O / W type. This is consistent with the contact angle measurement results.

[0107] 2.3.2 Influence of Oil Phase Content on Emulsion During the preparation of Pickering emulsions, the oil phase content can affect the properties and stability of the emulsion. Specifically, changes in the oil phase content can affect the average droplet size, rheological properties, etc. of Pickering emulsions. In this experiment, the concentration of HPPI-CS nanoparticles was fixed at 3 wt%, and the influence of different oil volume fractions on the droplet size and rheological properties of the emulsion was investigated.

[0108] The average droplet size of Pickering emulsions prepared with different oil phase contents was measured using a laser particle size analyzer. From Figure 14 it can be seen that at a fixed HPPI-CS nanoparticle content, as the oil phase content increased from 0.4 to 0.7, the average droplet size increased from 32.08 ± 0.18 μm to 42.06 ± 0.22 μm. The difference in droplet size caused by emulsions prepared with different oil phase contents may be due to the insufficient number of nanoparticles to stabilize the additional oil droplets. At higher oil contents, emulsion droplets with larger sizes and smaller interfacial areas were prepared to completely cover the oil-water interface. This verifies the importance of the oil phase content for Pickering emulsions, and the average droplet diameter of the emulsion can be changed by adjusting the oil phase content, thereby changing the interfacial properties of the emulsion. After the Pickering emulsion was stored at 4 °C for 45 days, its average droplet size changed slightly at low oil phase contents, while there was almost no change at high oil phase contents (60% - 70%), which shows the good stability of the HPPI-CS Pickering emulsion at high oil phase contents.

[0109] For food-grade Pickering emulsions, the rheological properties of the emulsion are crucial for studying its properties and performance. In this study, the storage modulus (G’), loss modulus (G’’), and viscosity of emulsions with different oil phase contents were determined. G’ reflects elastic properties, and G’’ reflects viscous properties. In all samples, G’ > G’’, indicating that the elastic characteristics of the emulsion are stronger than the viscous characteristics, and the gel-like behavior dominates the HPPI-CS Pickering emulsion. In addition, as the oil phase content increased, G’ and G’’ also increased. This is because emulsions with high oil phase contents have high droplet strength, resulting in stronger resistance to deformation and higher moduli.

[0110] Figure 15It shows the correlation between the apparent viscosity of the emulsion, the applied shear rate, and the oil phase content. As the shear rate increases, the viscosity of the emulsion decreases and finally stabilizes. This is because the internal structure of the emulsion is destroyed, resulting in a decrease in viscosity, thus showing the shear thinning phenomenon. This phenomenon is attributed to the deformation of the emulsion droplets and the influence of their ordered arrangement and the continuous phase after shearing. At the same time, the viscosity of the emulsion increases with the increase in the oil phase content. This is because in Pickering emulsions with a higher oil phase content, the droplet spacing gradually decreases, thereby enhancing their anti-deformation ability and resulting in a higher viscosity.

[0111] 2.3.3 Storage stability Analyzing the stability of the emulsion is crucial, and the separation phenomenon of the emulsion can be evaluated by CI. Unstable emulsions will gradually undergo phase separation, forming immiscible oil phase layer and water phase layer. According to Figure 16 A, when the oil phase content is 40%, 50%, and 60%, the emulsion shows different degrees of separation. This separation is due to the phase separation of the oil phase layer and the water phase layer caused by density differences. However, as the oil phase content increases, the degree of separation decreases, and the CI decreases from 35.22% to 8.84%. When the oil phase content reaches 70%, the emulsion no longer undergoes separation, and the creaming index is 0. Through Figure 16 B, it can be found that the HPPI-CS Pickering emulsion with 70% oil phase content remains stable after being stored at 4 °C for 45 days. This excellent stability may be due to the fact that as the oil phase content increases, the nanoparticles pack more closely, forming a dense gel network structure, which restricts the migration of oil droplets, resulting in a weaker fluidity of the Pickering emulsion and preventing phase separation. Thus, the CI decreases.

[0112] These results provide an important reference for further optimizing the emulsion formulation. Therefore, in order to ensure the long-term stability of subsequent Pickering emulsions, an oil phase content of 70% is selected for subsequent experiments.

[0113] 2.3.4 Ion stability From the above experimental studies, it can be known that the Pickering emulsion can maintain its own stability when the oil phase content is 70%. However, since the application of the emulsion often requires external stimuli, and sodium chloride is a substance commonly present in food systems, it is necessary to measure the stability of the Pickering emulsion under different ionic strengths. From Figure 17 A, it can be seen that G’ and G’’ increase with the increase in ionic strength. This may be due to the shielding of electrostatic forces by salt ions, resulting in the aggregation of nanoparticles, leading to the formation of a highly cross-linked gel network in the emulsion, thereby increasing both G’ and G’’ of the Pickering emulsion. In order to more accurately determine the effect of salt ion strength on the droplet size of the emulsion, the average droplet size of the emulsion was measured. As Figure 17As shown in Figure B, as the ionic strength increased from 0 mM to 500 mM, the size of the droplets was observed to increase from 33.77 ± 0.13 μm to 40.03 ± 0.10 μm. This change was due to the ionization of sodium chloride added to the nanoparticle suspension, which produced Cl - , and these Cl - interacted with CS, further weakening the interaction between the COO - of HPPI and CS, resulting in the instability of HPPI-CS nanoparticles and a decrease in nanoparticle concentration. When the nanoparticle concentration decreased, the emulsion was prone to coalescence, leading to an increase in droplet size. The appearance of the Pickering emulsion is shown in Figure 17 Figure C. When the concentration of NaCl in the HPPI-CS Pickering emulsion was in the range of 0 - 500 mM, the emulsion did not phase separate and remained stable during a 45-day storage period. Therefore, the results indicate that the HPPI-CS Pickering emulsion can tolerate high salt ions.

[0114] 2.3.5 pH Stability Since the pH of the environment where the nanoparticles are located can vary with the storage environment and the actual application conditions of the emulsion, it is of great significance to study the stability of the emulsion at different nanoparticle pH values. In this study, the pH of different nanoparticle solutions was adjusted (2 - 7). As Figure 18 can be seen, G’ and G’’ increased with increasing pH because the increase in pH reduced the positive charge of CS, thus reducing the electrostatic interaction between HPPI and CS and leading to the aggregation of nanoparticles.

[0115] At pH values of 2.0 and 3.0, the HPPI-CS Pickering emulsion did not show phase separation. While remaining stable, the average droplet size was relatively small, 9.97 ± 0.08 μm and 10.52 ± 0.04 μm respectively. Gradually increasing the pH value to 4.0 to 7.0, the emulsion showed slight phase separation, and the average droplet size increased from 18.58 ± 0.33 μm to 53.36 ± 0.17 μm. This was because the positive charge of HPPI increased, resulting in a weakening of the electrostatic force between it and CS, so that the droplets aggregated at higher pH values. However, regardless of the pH conditions, the particle size of the emulsion did not change significantly after 45 days of storage, indicating that from the perspective of the droplet size of the emulsion, the emulsion had good storage stability in different pH environments.

[0116] The appearance of the Pickering emulsion is shown in Figure 18As shown, when the pH is in the range of 2 - 4, the emulsion remains stable within a storage period of 45 days. However, when the pH is in the range of 5 - 7, a small amount of phase separation and precipitation occur in the emulsion. This is because when the pH deviates from the isoelectric point (about pH 5), the net charge of HPPI-CS nanoparticles increases, which promotes their solubility, inhibits the interfacial tension, and enhances the emulsion stability. When the pH approaches the isoelectric point of pea protein isolate, the net charge of the nanoparticles decreases, resulting in a weakening of the electrostatic repulsion between the nanoparticles and causing molecular aggregation, leading to a decrease in emulsion stability and thus droplet aggregation. Therefore, the HPPI-CS Pickering emulsion has good stability at pH 2 - 4.

[0117] 2.3.6 Temperature Stability Temperature, as a stimulus of the external environment, is equally important for the storage conditions of Pickering emulsions. The Pickering emulsion was stored at different temperatures (-20°C, 4°C, 20°C, 37°C, 68°C) for 2 h and then stored at 4°C for 45 days. The change in the size of the emulsion droplets is as Figure 19 shown. When the temperature is -20°C, the emulsion breaks due to freeze-thaw, resulting in a larger particle size of the emulsion. As the temperature increases, the diameter of the emulsion droplets increases. This may be because the Brownian motion of the nanoparticles is enhanced, promoting the redistribution of the nanoparticles at the oil-water interface and increasing the possibility of the oil phase being exposed, thus increasing the tendency of droplet aggregation. Compared with storage at room temperature, the change in the size of the emulsion droplets at other temperatures is not significant, indicating that the HPPI-CS Pickering emulsion has good temperature stability.

[0118] The appearance of the emulsion is as Figure 19 shown in Figure B. Slight demulsification occurs in the emulsion at -20°C. This may be because the internal structure of the emulsion is damaged after freezing, resulting in demulsification and stratification. The Pickering emulsion remains stable at -4°C, 20°C, and 37°C without creaming or precipitation. This indicates that the Pickering emulsion can maintain good stability under refrigeration or at room temperature. However, after the emulsion is treated at 68°C for 2 h, the color of the emulsion changes from milky white to light yellow. This can be attributed to Ostwald ripening in the emulsion, but no phase separation occurs and it remains stable. This indicates that the HPPI-CS Pickering emulsion can be used for pasteurization. In summary, the HPPI-CS Pickering emulsion has good temperature stability.

[0119] 2.4 Experimental Summary In this experiment, Pickering emulsions were prepared by using the HPPI-CS nanoparticles studied in Experiment 1, and the type of HPPI-CS Pickering emulsions, the effects of oil phase content on Pickering emulsions, as well as their rheological properties, storage, ionic, pH, and temperature stabilities were investigated. The experimental results showed that the HPPI-CS Pickering emulsion was an O / W type emulsion, and the HPPI-CS nanoparticles could effectively stabilize the oil phase to prepare Pickering emulsions. By adjusting the oil phase content, the droplet size and rheological properties of the emulsion could be flexibly adjusted. As the oil phase content increased, the CI% decreased, the droplet size and stability gradually increased, and at the same time, G’, G’’, and viscosity also increased. G’ of all samples was greater than G’’, indicating that the emulsion was composed of a gel-like network, showing shear thinning and non-Newtonian fluid properties. Through appearance observation, droplet size, and rheological property characterization, after storage for 45 days under different ionic concentrations, pH, and temperature conditions, the HPPI-CS Pickering emulsion still maintained good stability, which provided the possibility for further studying the potential of Pickering emulsions in delivering functional substances.

[0120] Experiment 3 Embedding and Application Characteristics of AST Pickering Emulsion In this experiment, the retention rate of AST in the emulsion was determined by ultraviolet spectrophotometry using the above-mentioned HPPI-CS Pickering emulsion. The emulsion was studied for its storage, ionic, pH, and thermal stabilities, and the antioxidant properties of the encapsulated AST Pickering emulsion, as well as the retention rate and absorption rate of AST during in vitro digestion, were explored. The encapsulated AST Pickering emulsion was applied to yogurt to evaluate its basic physicochemical properties and sensory characteristics.

[0121] 3.1 Materials and Instruments 3.1.1 Experimental Materials The HPPI-CS Pickering emulsion was prepared according to Experiment 2; AST (98%) was purchased from Solarbio Co., Ltd. The yogurt products were purchased from Inner Mongolia Yili Industrial Group Co., Ltd.

[0122] 3.2 Experimental Methods 3.2.1 Preparation of HPPI-CS Nanoparticles: Prepared according to the method of Experiment 2.

[0123] 3.2.2 Preparation of HPPI-CS Pickering Emulsion: Prepared according to the method of Experiment 2.

[0124] 3.2.3 Preparation of Encapsulated AST Pickering Emulsion AST was added to the oil phase to make the concentration of AST in the emulsion reach 0.4 mg / mL. 70% of the oil phase was mixed with 3 wt% of the nanoparticle solution and prepared into the encapsulated AST Pickering emulsion by high-speed dispersion. The fresh emulsion was stored at 4°C for analysis.

[0125] 3.2.4 Determination of AST Content 3.2.4.1 Plotting of the AST Standard Curve The AST standard stock solution (0.5 mg / mL) was diluted to AST standard solutions with different concentrations (0.01, 0.02, 0.05, 0.1, 0.125, 0.25 mg / mL) by dichloromethane and diluted 100 times. After shaking well, the absorbance was measured at 480 nm with a UV spectrophotometer to plot the standard curve, as Figure 20 shown.

[0126] 3.2.5 Storage Stability of AST The Pickering emulsion embedded with AST was stored at 4 °C for 45 d, with the groups of Tween-80 (TW-80) + astaxanthin and corn oil + astaxanthin as controls. The retention rate was calculated according to the following formula: (3-1); where C is the content of AST in the Pickering emulsion embedded with AST, and C0 is the content of AST in the Pickering emulsion.

[0127] 3.2.6 Ion Stability of AST Sodium chloride solutions with different concentrations (0, 50, 100, 300, and 500 mM) were added to the 3 wt% nanoparticle solution, and the Pickering emulsion embedded with AST was prepared according to the method described in 3.2.3 above. All emulsions were stored at 4 °C for 45 d. The AST retention rate was determined according to the above formula (3-1).

[0128] 3.2.7 pH Stability of AST The pH of the 3 wt% nanoion suspension was adjusted to 2, 3, 4, 5, 6, and 7 with 1 M hydrochloric acid and 0.1 M sodium hydroxide solutions, and the Pickering emulsion embedded with AST was prepared according to the method described in 3.2.3 above. All emulsions were stored at 4 °C for 20 d. The AST retention rate was determined according to the above formula (3-1).

[0129] 3.2.8 Thermal Stability of AST The Pickering emulsion embedded with AST was prepared according to the method described in 3.2.3 above. The emulsion was stored at -20 °C, 4 °C, 20 °C, 37 °C, and 68 °C. After 2 h, it was stored at 4 °C and its appearance change was observed. The AST retention rate was determined according to the above formula (4-1).

[0130] 3.2.9 Determination of Antioxidant Activity Further investigate the effect of the embedded AST Pickering emulsion on the free radical scavenging rate (DPPH). After mixing 1 mL of the Pickering emulsion with an equal volume of DPPH ethanol solution (0.1 mM), react for 30 min in the dark. After centrifuging at 1000 g for 5 min, measure the absorbance value of the sample at 517 nm. The calculation formula for the DPPH free radical scavenging rate is as follows: (3-2); Among them, A2 is the absorbance of the emulsion plus the DPPH ethanol solution, A1 is the absorbance of the emulsion plus ethanol, and A0 is the absorbance of the DPPH ethanol solution.

[0131] 3.2.10 In vitro simulated digestion In the oral stage, preheat 10 mL of the emulsion and 10 mL of simulated saliva to 37 °C respectively, then mix them together, and adjust the pH value of the mixture to 6.8 using 1 M sodium hydroxide solution. Next, magnetically stir the mixture at 100 rpm at 37 °C for 10 min, then observe the microstructure of the sample and measure the retention rate of AST.

[0132] In the gastric stage, the sample (20 mL) obtained in the oral stage is mixed with the same volume of simulated gastric juice preheated to 37 °C. Then, adjust the pH of the mixture to 2.5 using 1 M hydrochloric acid solution, and stir and incubate at 37 °C at a rate of 100 rpm for 2 h. Take samples every 30 min, calculate the AST retention rate using the above method, and observe the microstructure of the mixture.

[0133] In the intestinal stage, the sample (20 mL) obtained in the gastric stage is mixed with the same volume of simulated intestinal fluid preheated to 37 °C, and adjust the pH value to 6.8 using 1 M NaOH solution. Then, stir the mixture at a rate of 100 rpm at 37 °C for 3 h. Take samples every hour, calculate the AST retention rate using the above method and calculate the release rate of AST by dissolving the sample in dichloromethane. Also observe the microstructure of the mixture.

[0134] 3.2.11 Yogurt application 3.2.11.1 Preparation of Pickering emulsion yogurt Add 10 mL of the embedded AST Pickering emulsion to 100 mL of yogurt as the experimental group, and the original yogurt as the control group. All yogurts are stored at -4 °C for further analysis.

[0135] 3.2.11.2 Storage stability of AST in yogurt Determine the retention rate of AST in the HPPI-CS Pickering emulsion yogurt embedded with AST after storage for 0, 10, 20, 30, 40, 45 d according to the method in 3.2.5 above.

[0136] 3.2.11.3 Determination of the pH value of yogurt Use a pH meter to measure the pH values of the above two kinds of yogurt respectively.

[0137] 3.2.11.4 Determination of the water-holding capacity of yogurt Add 10 mL of embedded AST Pickering emulsion to 100 mL of yogurt as the experimental group, AST + yogurt as control group 1, and yogurt as control group 2. Place 20 mL of the three kinds of yogurt in a centrifuge tube, and record the mass as m1. After centrifuging the yogurt at 5000 r / min for 10 min, remove the supernatant, and record the mass as m2. The water-holding capacity of the yogurt is measured according to the formula: (3-3); 3.2.11.5 Sensory evaluation of yogurt Twenty trained normal sensory evaluators taste the three kinds of yogurt and evaluate the odor, texture, viscosity, color and overall acceptance respectively.

[0138] 3.2.12 Statistical analysis All measured values are measured at least three times, and the data are expressed as mean ± standard deviation. Use Origin 2019 software and SPSS 25 for various statistical analyses such as one-way ANOVA. The difference between the data is p < 50.05.

[0139] 3.3 Results and discussion 3.3.1 Storage stability of AST Due to the instability and easy oxidation of AST, the stability of AST during storage is crucial. Dissolve AST in corn oil as the oil phase, use HPPI-CS nanoparticles to stabilize the mixed oil phase to form HPPI-CS Pickering emulsion, and through the comparison with the TW-80 group and the Oil group, explore the change of the retention rate of AST in the HPPI-CS Pickering emulsion with time extension and the change of the DPPH free radical scavenging rate.

[0140] The retention rate of AST after 45 days of storage is as Figure 21As shown in the figure. In the first 5 days, only a slight loss of AST occurred in the HPPI-CS Pickering emulsion group, while the retention rates in the TW80 group and the Oil group decreased by 16.8% and 26.4% respectively. During the subsequent storage time, the retention rate of AST decreased in each group. However, after 45 days of storage, the astaxanthin retention rate in the HPPI-CS Pickering emulsion group was still as high as 75.8%, while those in the TW80 group and the Oil group were 45.8% and 37.5% respectively. The loss rate of AST in the remaining groups was higher than that in the emulsion prepared with HPPI-CS nanoparticles. The results of the free radical scavenging rate were consistent with the AST retention rate, as Figure 21 shown. These results indicate that the HPPI-CS Pickering emulsion can encapsulate AST, and the dense and thick particle layer around the oil droplets provides an adequate barrier to prevent the degradation and oxidation of AST. This may be due to the dense nanoparticle layer around the oil droplets. In addition, the HPPI-CS Pickering emulsion has strong anti-deformation ability and forms a gel network structure, resulting in a delayed diffusion of pro-oxidants or free radicals, and the emulsion can better maintain its own stability and the stability of the encapsulated substances.

[0141] 3.3.2 Ion stability of AST Since there are different concentrations of ions in food and the gastrointestinal environment of the human body, it is very important to study the effect of ionic strength on the retention rate of AST in the Pickering emulsion encapsulating AST. In this study, the effect of ionic strength on the retention rate of AST was evaluated by adding different concentrations (0 - 500 mM) of sodium chloride. As Figure 22 shown, after 45 days of storage, the retention rate of AST in the Pickering emulsions prepared with different oil phase contents was affected by the ionic strength. As the ionic strength increased (0 - 500 mM), the retention rate of AST in the emulsion was above 60%. Interestingly, when the ionic strength reached 500 mM, the retention rate also decreased to 60.9%, and the results of the DPPH free radical scavenging rate were consistent with the decreasing trend of the retention rate. This is because the addition of sodium chloride introduced Cl - , and the competition between Cl - and CS weakened the electrostatic attraction between HPPI and CS, thus reducing the concentration of HPPI-CS nanoparticles, and ultimately leading to the aggregation and coalescence of droplets, resulting in a decrease in the stability of the emulsion. Generally speaking, however, the HPPI-CS Pickering emulsion has strong ionic stability, which helps to protect AST from loss. As the concentration of sodium chloride increased, the surface charge decreased, and the repulsion between droplets decreased, thus affecting the stability of the lycopene microemulsion. As Figure 22 can be seen, there was no obvious change in the appearance of the emulsion after 45 days of storage in an environment with different ionic concentrations, further confirming that the Pickering emulsion encapsulating AST has good ionic stability.

[0142] 3.3.3 pH Stability of AST Due to the complexity of food application systems, the pH of the environment where Pickering emulsions are located can vary. Emulsions need to ensure stability under different environmental conditions and a high retention rate of AST. Therefore, it is of great significance to explore the stability of AST in Pickering emulsions stabilized by HPPI-CS nanoparticles at different pH values. By adjusting the pH value of the HPPI-CS nanoparticle solution, Pickering emulsions encapsulating AST with different pH values were prepared. After 45 days of storage, the retention rate of AST and the DPPH radical scavenging rate in Pickering emulsions prepared with different oil phase contents are as Figure 23 shown. As the pH value increased, the retention rate of AST in the emulsion increased from 62.5% to 74.0%, and then decreased to 59.8%. The results of the DPPH radical scavenging rate showed the same trend as the retention rate change. This is because under over-acidic and neutral conditions, the electrostatic repulsion between nanoparticles is weak, resulting in droplet aggregation, which weakens the protective effect of Pickering emulsions on AST. At a pH of 4, the retention rate of AST by Pickering emulsions is relatively high, which may be because within this pH range, HPPI and CS bind more tightly and the electrostatic repulsion is stronger, so the stability of Pickering emulsions is better. Generally speaking, HPPI-CS Pickering emulsions have a high AST retention rate at different pH values because high-oil-phase emulsions have stronger anti-deformation ability and a denser gel network. These results indicate that HPPI-CS Pickering emulsions exhibit good performance in terms of pH stability and help protect AST from damage. As Figure 23 can be seen, after 45 days of storage in different pH environments, the appearance of the emulsion did not show obvious changes, further confirming that the Pickering emulsion encapsulating AST has good pH stability.

[0143] 3.3.4 Thermal Stability of AST In food processing, heat treatment is a common method. This experiment aimed to study the thermal stability of Pickering emulsions loaded with AST under different temperature conditions. Five temperature conditions were set: frozen (-20 °C), refrigerated (4 °C), room temperature (20 °C), body temperature (37 °C), and pasteurization temperature (68 °C). The effects of heat treatment on the AST-loaded emulsion were studied, and the retention rate of AST and the DPPH radical scavenging rate were measured. The results are as Figure 24 shown. The experimental results showed that after different temperature treatments, the retention rate of AST was above 55%, indicating that the Pickering emulsion has good thermal stability and effectively protects AST in the emulsion from temperature damage. This is mainly because the particle layer around the oil phase can provide a strong barrier effect, inhibiting the droplet aggregation of Pickering emulsions, and thus maintaining the thermal stability of the emulsion. As Figure 24It can be seen that the appearance of the emulsion did not change significantly at different temperatures, further verifying that the encapsulated AST Pickering emulsion has good thermal stability.

[0144] 3.3.5 In vitro simulated digestion Through in vitro simulated digestion, the retention and release of AST during the in vivo digestion process can be understood, which is very important for the research of delivery systems.

[0145] The results are as Figure 25 shown. In the oral phase, the emulsion still had a high retention rate (98.3 ± 0.3%), which was due to the short residence time of the emulsion in the oral cavity and the relatively gentle oral environment. The slight aggregation that occurred in the emulsion droplets can be attributed to the electrostatic interaction between the HPPI-CS-coated oil droplets and the viscous proteins, as well as the bridging flocculation between the HPPI-CS-coated oil droplets and other particles, resulting in the formation of aggregates.

[0146] In the gastric phase, the retention rate of AST in the HPPI-CS-stabilized Pickering emulsion was as high as 88.5%, while under the same conditions, the retention rate of AST in the nanoemulsion stabilized by soy protein isolate-sodium alginate was only 60.76%. This difference may be attributed to two reasons: First, the dense protective layer formed by HPPI-CS can effectively prevent the action of the acidic environment and pepsin on AST, reducing the decomposition and oxidation of AST. Second, the Pickering emulsion has good stability, and the oil droplets in the emulsion can be stably dispersed in the aqueous phase, preventing the direct contact and reaction of AST with other components, thus maintaining a high retention rate. Therefore, in the gastric phase, the HPPI-CS-stabilized Pickering emulsion can effectively protect AST.

[0147] In the intestinal phase, the release rate of AST encapsulated in the HPPI-CS Pickering emulsion gradually increased to 28.8% within the first 2 h and then rapidly increased to 56% within the next 1 h. This is mainly because trypsin is present in the intestine, which can effectively decompose the proteins in the emulsion and cause their degradation, resulting in the destruction of the nanoparticles and thus the release of AST. In addition, the pH of the simulated intestinal fluid is 6.8, which is within the optimal pH range for the action of trypsin. Therefore, this environment helps to promote the reaction between the proteins and enzymes in the HPPI-CS Pickering emulsion, ultimately leading to degradation. In addition, the phenomenon of emulsion rupture was observed in the fluorescence microscope images, further confirming that the HPPI-CS Pickering emulsion can effectively release AST in the intestine.

[0148] 3.3.6 Yogurt application 3.3.6.1 Storage stability of AST Yogurt is recognized by consumers due to its rich taste and various biological functions, including promoting digestion, solving oral problems, and changing the flora in the gastrointestinal tract. According to previous studies, the pH of yogurt is around 4.0, and the Pickering emulsion encapsulating AST prepared in this experiment has good stability at pH 4.0. Therefore, consumers can ingest sufficient amounts of AST while enjoying yogurt. Due to the instability of AST, it is crucial to ensure a high retention rate of AST during shelf life. In this experiment, the retention rate of AST in yogurt was monitored every 5 days. The results are as Figure 26 shown. The results show that after a 45-day shelf life, the retention rate of AST is 71.2%. After adding the Pickering emulsion encapsulating AST to yogurt, the retention rate of AST still remains at a relatively high level. This may be because the pH of yogurt is about 4.0, at which pH the electrostatic repulsion between nanoparticles increases and the gel network structure becomes tighter. Therefore, the Pickering emulsion can be effectively applied to yogurt to maintain the good stability of AST. These findings provide important references for further research on how to optimize the retention of AST in yogurt, which helps to improve the quality and nutritional value of yogurt products.

[0149] 3.3.6.2 Changes in the pH of Yogurt The pH of yogurt is a key indicator for evaluating the quality of yogurt after storage. This is because acidic substances are produced after the oxidation of AST, resulting in a decrease in pH. Therefore, observing the change in the pH of yogurt can also show the degree of AST oxidation. After 45 days of storage, the pH of the three groups of samples all showed an obvious downward trend, as Figure 27 shown. Among them, the pH of the experimental group was the highest, at 3.76, while the pH of control group 1 was the lowest, at 3.62. This may be because over time, the fermenting bacteria in yogurt convert lactose into lactic acid, resulting in a decrease in pH; in control group 1, the oxidation of AST also causes a decrease in pH. The pH of the experimental group decreased more slowly, indicating that preparing the Pickering emulsion and encapsulating AST can provide a certain degree of protection, making AST more stable during storage. These findings provide useful references for further research on how to improve the quality of yogurt and maintain the stability of AST. Through the exploration of this experiment, future research can focus on how to effectively control the change in pH in dairy products to optimize the quality of yogurt and maintain the activity of AST.

[0150] 3.3.6.3 Changes in the Water-Holding Capacity of Yogurt The water-holding capacity of yogurt is one of the important indicators reflecting the ability of its internal protein structure to retain water, directly reflecting the tightness of the gel structure in yogurt. The stronger the water-holding capacity, the tighter the gel structure in yogurt, the stronger the ability to retain water, thus reducing the possibility of whey separation during storage, which is beneficial to improving the quality and taste of yogurt. Therefore, the purpose of this experiment was to study the change of its gel structure during the shelf life by exploring the water-holding capacity performance of yogurt and yogurt added with emulsion during 45 days of storage at 4 °C. The results are as Figure 28 shown. The research shows that after 45 days of storage, the water-holding capacity of the emulsion group reached 80.5%, while that of pure yogurt was only about 69.5%. This may be because the addition of Pickering emulsion caused some water to be absorbed, delaying the whey separation process of yogurt. At the same time, due to the high viscosity and gel properties of Pickering emulsion itself, its addition to yogurt will also increase the viscosity of the whole yogurt, thus reducing the whey separation of yogurt. These results suggest that the addition of Pickering emulsion may help improve the quality of yogurt, providing a beneficial reference basis for future applications. Therefore, future research can further explore how to optimize the addition method and proportion of emulsion to further improve the quality and taste of yogurt to meet the needs of consumers.

[0151] 3.3.6.4 Sensory evaluation of yogurt This study analyzed in detail the effects of adding emulsion to yogurt on its odor, texture, viscosity, color and overall acceptance. The results are as Figure 29 shown. The analysis results show that the addition of emulsion had a significant impact on the sensory evaluation parameters of yogurt. In terms of taste and appearance, the addition of emulsion did not show obvious differences from the original yogurt; however, the yogurt added with Pickering emulsion embedded with astaxanthin was slightly inferior to the original yogurt in terms of odor, taste and overall acceptance. This indicates that the addition of Pickering emulsion brought a slight greasy taste to a certain extent. Generally speaking, Pickering emulsion yogurt was still accepted to a certain extent, but further research and optimization are still needed to improve its sensory characteristics and enhance consumer satisfaction. Therefore, future research should focus on how to improve the emulsion addition formula to better integrate it into yogurt and enhance the quality and market competitiveness of the product.

[0152] 3.4 Experimental summary In this experiment, HPPI-CS Pickering emulsions studied in Experiment 2 were used to encapsulate AST to prepare Pickering emulsions encapsulating AST. The storage, ionic, pH, thermal stability, and antioxidant properties of the Pickering emulsions encapsulating AST were investigated. The results showed that after 45 days of storage, the retention rate of AST in the emulsion only decreased to 75.8%. The retention rate of the Pickering emulsion was above 62% within the pH range of 2 - 6. At different temperatures, except for demulsification caused by high temperature at 68°C and freeze-thaw at -20°C, the AST content was basically unaffected at other temperatures and remained at a high level. With the extension of storage time, the DPPH radical scavenging rate of the antioxidant property of the Pickering emulsion gradually decreased, but the emulsion still had a high antioxidant capacity. In addition, the antioxidant effect of the emulsion stored at 4°C was stronger than that at 20°C, indicating that it is best to choose cold storage when selecting the storage temperature. Therefore, it was shown that the Pickering emulsion encapsulating AST had good storage, ionic, pH, thermal stability, and antioxidant capacity, indicating that the HPPI-CS Pickering emulsion was a good encapsulation system for AST.

[0153] Subsequently, the digestion and absorption characteristics and applications of the Pickering emulsion encapsulating AST were investigated. The emulsion showed high stability in simulated saliva and gastric juice, and the retention rate of AST in the Pickering emulsion was 88.5% after passing through the oral and gastric phases. After 3 h of simulated small intestine digestion, a slow release phenomenon of AST in the emulsion occurred, and the release rate was 56%, indicating that the emulsion had a good sustained release effect. At the same time, the application of the Pickering emulsion encapsulating AST in yogurt was explored. During the 45-day shelf life of the Pickering emulsion yogurt, the retention rate of AST was 76%, showing a significant increase compared with 34.9% of the control group. Compared with the two control groups, the pH value of the emulsion group was the highest and the pH value decreased the least, indicating that it had a good AST protection effect. The water holding capacity of the yogurt group was 69.5%, while that of the emulsion group reached 80.5%, indicating that the addition of the Pickering emulsion would absorb some water, delay the precipitation of yogurt whey, and enhance the quality of yogurt. The sensory evaluation personnel had a high overall acceptance of the yogurt added with the emulsion. This shows that the Pickering emulsion has a great possibility of being applied to yogurt, laying a foundation for the future application of the Pickering emulsion.

[0154] The above embodiments are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope disclosed herein. Modifications that are obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A restricted enzymatically hydrolyzed pea protein isolate-chitosan Pickering emulsion, characterized in that: It consists of an oil phase and a water phase, wherein the water phase is composed of HPP, CS and water; the HPPI is obtained by enzymatic hydrolysis of PPI with trypsin.

2. The restricted enzymatically hydrolyzed pea protein isolate-chitosan Pickering emulsion according to claim 1, characterized in that: The mass ratio of HPPI to CS is 1:1.2, the molecular weight of CS is 3 kDa, HPPI and CS are combined to form nanoparticles, the concentration of nanoparticles in the water phase is 3%; the oil phase accounts for 70%.

3. The restricted enzymatically hydrolyzed pea protein isolate-chitosan Pickering emulsion according to claim 1, characterized in that: The oil phase is selected from corn oil.

4. The method for preparing the restricted enzymatically hydrolyzed pea protein isolate-chitosan Pickering emulsion according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Preparation of HPPI: PPI is dissolved in water to obtain a PPI solution, trypsin is added, the mixture is hydrolyzed, and centrifuged. The supernatant is the HPPI solution. (2) Preparation of HPPI-CS nanoparticles: Add CS to the HPPI solution and mix well to obtain an HPPI-CS nanoparticle solution; (3) Preparation of HPPI-CS Pickering emulsion: Add the oil phase into the HPPI-CS nanoparticle solution and homogenize to obtain the HPPI-CS Pickering emulsion.

5. The method for preparing the restricted enzymatically hydrolyzed pea protein isolate-chitosan Pickering emulsion according to claim 4, characterized in that: In the step (1), the concentration of the PPI solution is 20 mg / mL, and the enzymatic hydrolysis conditions are: temperature 30°C, pH 9, and enzymatic hydrolysis at 600 r / min for 2 min.

6. The method for preparing the restricted enzymatically hydrolyzed pea protein isolate-chitosan Pickering emulsion according to claim 4, characterized in that: In the step (2), the molecular weight of CS is 3 kDa, and the mass ratio of HPPI to CS is 1:1.2; Or: In the step (3), the oil phase is selected from corn oil, the oil phase accounts for 70%, and the concentration of HPPI-CS nanoparticles in the HPPI-CS nanoparticle solution is 3%.

7. Use of the restricted enzymatically hydrolyzed pea protein isolate-chitosan Pickering emulsion according to any one of claims 1 to 3 in the preparation of a drug-embedded preparation.

8. The use according to claim 7, characterized in that: The drug is selected from astaxanthin.

9. A Pickering emulsion for embedding AST, characterized in that: It consists of AST, oil phase and water phase, wherein the water phase is composed of HPPI, CS and water; the HPPI is obtained by enzymolysis of PPI with trypsin.

10. A Pickering emulsion yogurt, characterized in that: The method comprises yogurt and the Pickering emulsion for embedding AST as claimed in claim 9.

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