Application of Konjac Glucomannan in the Preparation of High Internal Phase Pickering Emulsion

Abstract

The present invention discloses the application of konjac glucomannan in the preparation of high internal phase Pickering emulsions, which relates to the technical field of food industry; the high internal phase Pickering emulsion uses myofibrillar protein as the protein raw material. The present invention also provides a method for preparing a high internal phase Pickering emulsion, which comprises the following steps: (1) A myofibrillar protein solution and a konjac glucomannan solution are mixed and homogenized to obtain a myofibrillar protein / konjac glucomannan mixed system; (2) Using the myofibrillar protein / konjac glucomannan mixed system as the aqueous phase, it is mixed with the oil phase and homogenized to prepare the high internal phase Pickering emulsion. The present invention finds through research that konjac glucomannan can improve the physical properties of high internal phase Pickering emulsions stabilized by myofibrillar protein by changing the interfacial adsorption and network structure.

Description

Technical Field

[0001] The present invention relates to the technical field of the food industry, and particularly to the application of konjac glucomannan in the preparation of high internal phase Pickering emulsions. Background Art

[0002] High internal phase emulsions (HIPEs) have been widely used in many fields due to their characteristics of high oil phase volume fraction (φ≥74%) and solid-like gel structure. Such as the potential substitution of solid fats and hydrogenated oils, the delivery of bioactive components, and the improvement of texture and oxidative stability (Yang, Li, & Tang, 2020). Although surfactants and inorganic particles have been used as emulsifiers for preparing HIPEs, their poor biocompatibility and environmental pollution have inhibited their utilization in the food industry (Li et al., 2020). Therefore, high internal phase Pickering emulsions (Pickering HIPEs) stabilized by food-grade particles (such as proteins, polysaccharides, protein / polysaccharide complexes) have received extensive attention (C. Zhou et al., 2022). However, most of these food-grade particles have strong hydrophilicity and need to be pretreated by esterification and acid hydrolysis to improve their emulsifying properties, which also brings complex processes and safety hazards, so their application in actual production is limited (S. Zhang, Geng, Shi, Ma, & Liu, 2022). At the same time, due to HIPEs having a high volume fraction of the dispersed phase, which violates the kinetic factors of emulsion formation, phase inversion is likely to occur (Kralchevsky, Ivanov, Ananthapadmanabhan, & Lips, 2005). In order to avoid the demulsification phenomenon caused by phase inversion, the commonly used method at present is to continuously stir while adding the dispersed phase to the continuous phase to prepare HIPEs (Tan, Pajoumshariati, Arshadi, & Abbaspourrad, 2019). However, the method of continuous stirring not only requires expensive equipment but also has a very complex preparation process (Su et al., 2018). Therefore, exploring a simple and economical method for preparing HIPEs has become a current research hotspot.

[0003] Li et al. (2020) found that meat proteins can form Pickering HIPEs over a wide pH range using a one-step homogenization process. Myofibrillar protein (MP) is the main protein in meat proteins, accounting for about 55%-60% of the total muscle proteins, and is considered the main endogenous emulsifier in meat products (Xiong et al., 2019). At the same time, MP has good amphiphilicity and low allergenicity, can adsorb on the surface of oil droplets to reduce the interfacial tension, and form an interfacial coating to form and stabilize emulsions (Sun, Ma, Fu, Dai, & Zhang, 2021). Therefore, MP has great potential in the wide application and large-scale production of Pickering HIPEs. However, emulsions stabilized by MP are prone to aggregation and flocculation. In recent years, there have been reports on improving the emulsifying properties of MP by ultrasound, but the high cost limits its industrial application (Xiong et al., 2019). Therefore, it is necessary to explore a simple, efficient, and low-cost method to improve the emulsifying properties of MP.

[0004] Konjac glucomannan (KGM) is derived from konjac tubers and is a water-soluble high-molecular-weight neutral polysaccharide with a molecular weight between 200 and 2000 kDa, which is composed of β-mannose and β-glucose linked by β-1,4-glycosidic bonds. KGM is inexpensive, has excellent water absorption, thickening, and gelation properties, and can reduce cholesterol levels in the blood and the incidence of colorectal cancer. Therefore, KGM is often used in restructured meat products to improve the gel strength of MP mixtures. There is currently no report on the preparation of high internal phase Pickering emulsions jointly prepared by KGM and MP. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of konjac glucomannan in the preparation of high internal phase Pickering emulsions to solve the problems existing in the above-mentioned prior art. The present invention studies and finds that konjac glucomannan can improve the physical properties of high internal phase Pickering emulsions stabilized by myofibrillar protein by changing the interfacial adsorption and network structure.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] The present invention provides the application of konjac glucomannan in the preparation of high internal phase Pickering emulsions, and the high internal phase Pickering emulsions use myofibrillar protein as the protein raw material.

[0008] Further, the application is to improve the physical properties of the high internal phase Pickering emulsions, and the physical properties include wettability, interfacial adsorption, and stability.

[0009] The present invention also provides a method for preparing high internal phase Pickering emulsions, including the following steps:

[0010] (1) The myofibrillar protein solution and konjac glucomannan solution are mixed and homogenized to obtain a myofibrillar protein / konjac glucomannan mixed system;

[0011] (2) Using the myofibrillar protein / konjac glucomannan mixed system as the aqueous phase, it is mixed with the oil phase and homogenized to prepare the high internal phase Pickering emulsion.

[0012] Furthermore, the mass-volume concentration of the myofibrillar protein solution is 2%.

[0013] Furthermore, the mass-volume concentration of the konjac glucomannan solution is 0.5 - 3%.

[0014] Furthermore, in step (1), the myofibrillar protein solution and the konjac glucomannan solution are mixed in a mass ratio of 1:1.

[0015] Furthermore, in step (2), the aqueous phase and the oil phase are mixed in a volume ratio of 25:75.

[0016] Furthermore, in step (2), the oil phase is perilla oil.

[0017] Furthermore, in steps (1) and (2), the rotation speed of the homogenization is 2800 rpm and the time is 1 min for both.

[0018] The present invention also provides a high internal phase Pickering emulsion prepared by the above preparation method.

[0019] The present invention discloses the following technical effects:

[0020] The present invention studied the effects of KGM at different concentrations (0, 0.25, 0.5, 1, 1.5%, w / v) on the structure and interfacial adsorption of MP and on the microstructure and stability of MP-emulsified high internal phase Pickering emulsions (Pickering HIPEs). The results showed that the addition of KGM promoted the unfolding of the MP structure and improved the wettability of MP. Especially when 0.5% KGM was added, the three-phase contact angle (θ o / w)Reaching 89.41°, this is beneficial to reducing the energy barrier for the penetration and rearrangement of MPs at the interface. Adsorption kinetics and the protein adsorption rate (AP%) indicate that KGM (≤1%) promotes the adsorption of MP molecules onto the oil-water interface. KGM promotes the "condensation" of MPs and forms a dense MP network structure by hydrogen bonding with MPs, which increases the steric hindrance and prevents oil droplet aggregation. KGM (≤1%, especially 0.5%) reduces the droplet size, relaxation time (T2), and degree of lipid oxidation of Pickering HIPEs and improves the storage stability and rheological properties. However, KGM (>1%) leads to a decrease in interfacial MPs due to competitive adsorption and forms a continuous hydrogel, resulting in an unaggregated MP network structure, which further causes a decline in emulsion performance. Therefore, KGM can regulate the physical properties of MP-stabilized Pickering HIPEs by changing the interfacial adsorption and network structure. Description of the Drawings

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 Fourier infrared spectra curves of MPs and MP-KGM complexes (a) and the corresponding secondary structure ratios of MPs at different KGM concentrations (b);

[0023] Figure 2 For the three-phase contact angles (θ o / w ) of MPs, MP-KGM complexes, and KGM;

[0024] Figure 3 For the adsorption dynamics of MPs, MP-KGM, and KGM at the oil-water interface; where a is the time dependence of the interfacial pressure (π); b is the dependence of π on the square root of time (t 1 / 2 ), and the theoretical model represents a typical graph, where k diff refers to the diffusion rate; c-h are the time dependences of ln[(π 1800 -π t ) / (π 1800 -π0)], and the theoretical model represents a typical graph, where K P and K R refer to the penetration and rearrangement rates, respectively;

[0025] Figure 4Effect of the concentration of KGM on the performance of Pickering HIPEs; where, a is the droplet size distribution; b is the visual appearance (row 1: fresh emulsion, row 2: stored at 25 °C for 30 days, from left to right, the concentrations of KGM are 0%, 0.25%, 0.5%, 1.0% and 1.5% respectively); c is the optical microscope image, diluted three times with deionized water, scale bar: 100 μm;

[0026] Figure 5 SEM images of Pickering HIPEs prepared using n - hexane as the oil phase (75 wt%);

[0027] Figure 6 Percentage of interfacial adsorption of MP on the constructed Pickering HIPEs interface at different concentrations of KGM; error bars represent standard deviation (n = 3); different lowercase letters above the error bars indicate significant differences between different samples (p < 0.05);

[0028] Figure 7 Relaxation curves (a) and T 21 、T 22 and T 23 relaxation percentages (b) of MP - stabilized Pickering HIPEs at different concentrations of KGM;

[0029] Figure 8 Rheological properties and visual appearance of MP - stabilized Pickering HIPEs at different KGM concentrations; where, a1 is the apparent viscosity of Pickering HIPEs at a shear rate of 0.1 to 100 rad / s; a2 is the frequency - sweep curve at a fixed strain (1%), frequency range from 0.1 to 100 rad / s; b is the appearance of Pickering HIPEs (from left to right, the concentrations of KGM are 0%, 0.25%, 0.5%, 1.0% and 1.5% in turn);

[0030] Figure 9Appearance and MDA content of bulk perilla seed oil, Tween-80-stabilized emulsions, and Pickering HIPEs emulsified with MP containing different KGM concentrations under accelerated oxidation conditions; where, a is the appearance images of Pickering HIPEs during storage at 50 °C for 30 days (from left to right are bulk perilla seed oil, emulsion stabilized by Tween-80, Pickering HIPEs stabilized by MP, MP-KGM 0.25%, MP-KGM 0.5%, MP-KGM 1%, and MP-KGM 1.5%); b is the effect of KGM concentration on the lipid oxidation stability of Pickering HIPEs during storage; T80 represents the emulsion stabilized by Tween-80; all groups contain the same oil content (75%);

[0031] Figure 10 The mechanism of the effect of adding KGM on the formation of MP-stabilized Pickering HIPEs. Detailed implementation manners

[0032] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0035] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0036] As used herein, terms such as "comprising", "including", "having", "containing", etc. are all open-ended terms, meaning including but not limited to.

[0037] The polysaccharide synergy provides a simple and feasible strategy for improving the stability of protein emulsions. When proteins adsorb at the interface, structural dissociation often occurs, making the emulsion prone to flocculation and stratification. In contrast, emulsions prepared from protein / polysaccharide composite particles often have more excellent physical properties and storage stability. The adsorption state of proteins at the oil-water interface, such as wettability, adsorption rate, and thickness, is a crucial factor affecting emulsion stability. The interaction between proteins and polysaccharides (mainly non-covalent interactions such as electrostatic interactions, hydrophobic interactions, van der Waals forces, etc.) can regulate the adsorption state of proteins at the oil-water interface by modifying and improving the structural and functional properties of proteins, thereby improving emulsion stability. Polysaccharides can also form complexes with proteins and adsorb at the interface to form a protein-polysaccharide bilayer interfacial layer, thus increasing emulsion stability. The thermodynamic incompatibility between proteins and polysaccharides also promotes the concentration of proteins at the interface, enhancing emulsion stability. In addition, the state of the continuous phase is also an important factor affecting emulsion performance. The thickening effect of polysaccharides can limit the approach of droplets to prevent flocculation and coalescence. Therefore, adding polysaccharides may be a simple and effective strategy to improve the stability of MP-stabilized emulsions.

[0038] In this invention, Pickering HIPEs were prepared using KGM / MP complexes, and the effects of KGM on the properties of MP / KGM mixtures in aqueous solutions, the adsorption properties at the oil-water interface, and the properties of Pickering HIPEs stabilized by MP / KGM mixtures were investigated. In addition, the relationship between MP / KGM interactions in aqueous solutions or at the oil-water interface and the properties of Pickering HIPEs was revealed, and the mechanism by which KGM affects MP-stabilized Pickering HIPEs was discussed. This invention provides a low-cost method for improving the overall stability of MP high internal phase emulsions, increases their application potential in nutrient delivery systems, and is beneficial to the high-value application of MP.

[0039] Example 1

[0040] 1 Materials and Methods

[0041] 1.1 Materials

[0042] Frozen chicken breast was purchased from Shandong Chaohe Food Co., Ltd. (Shandong, China). The chicken breast was sourced from male white - feather chickens at 42 days of age, with each piece weighing approximately 0.25 kg. It was stored at - 18°C and used within 1 month. Konjac glucomannan powder (food - grade) was purchased from Hubei Qiangsen Konjac Technology Co., Ltd. (Hubei, China), with a purity of 98% and a viscosity of 37300 mPa·s. Perilla seed oil was purchased from Hebei Jiafeng Vegetable Oil Co., Ltd. All other chemical reagents were of analytical grade.

[0043] 1.2 Preparation of myofibrillar protein (MP)

[0044] Take out the chicken breast stored at - 18°C, thaw it at 4°C for 20 minutes, remove connective tissue, fat and other impurities, and cut it into minced meat. Then add 4 - fold volume of buffer A (containing 0.1 mol / L KCl, 1 mmol / L MgCl2, 7 mmol / L KH2PO4, 18 mmol / L K2HPO4, 1 mmol / L EGTA, pH 7.0), homogenize twice in an ice - bath (12000 r / min, 30 s), freeze - centrifuge (2000×g, 15 min, 4°C), discard the supernatant and collect the precipitate, and repeat the above operations twice. Collect the precipitate to obtain the crude protein, mix it with 4 - fold volume of buffer B (containing 0.1 mol / L NaCl, 1 mol / L HCl, pH 6.0), and perform homogenization and centrifugation as above, repeat twice. After the third homogenization, filter the liquid through 4 - layer gauze, and then centrifuge. The obtained precipitate is pure myofibrillar protein. Use a BCA protein assay kit (Nanjing Jiancheng Bioengineering Institute) and refer to its instruction manual to determine the concentration of MP. Store the extracted MP in a refrigerator at 4°C and use it within 48 hours.

[0045] 1.3 Preparation of myofibrillar protein solution, konjac glucomannan solution, myofibrillar protein / konjac glucomannan mixed system (MP / KGM)

[0046] The MP prepared in 1.2 was dissolved in phosphate buffer solution (PBS) (0.6 M NaCl, 50 mM Na2HPO4 / NaH2PO4, pH 7.0) to adjust the MP concentration to 2% (w / v) to obtain an MP suspension, which was stored at 4°C overnight to ensure full hydration. 0.5, 1, 2 and 3 g of KGM powder samples were dispersed in 100 g of the same buffer solution, and then homogenized at 2800 rpm for 1 min using an XHF-DY high-speed disperser (Ningbo Xinzhi Biotechnology Co., Ltd., China) to obtain 0.5%, 1%, 2% and 3% (w / v) KGM solutions, respectively, and cooled at 4°C for later use; the KGM solution was mixed with the MP suspension (2%) at a mass ratio of 1:1 and homogenized at 2800 rpm for 1 min in an ice bath to obtain an MP / KGM mixed system. The MP concentration in the MP / KGM mixed system was 1%, while the KGM concentrations were 0.25%, 0.5%, 1% and 1.5%, respectively.

[0047] 1.4 Characterization of MP / KGM mixture

[0048] 1.4.1 Determination by Fourier transform infrared spectroscopy (FTIR)

[0049] The MP / KGM mixed system obtained in 1.3 and the MP suspension before and after homogenization were dried in a Lab-1-50 freeze dryer (Beijing Boycon Laboratory Instrument Co., Ltd., China) for 48 hours. The freeze-dried MP and MP-KGM complex were fully mixed with dry KBr (1:50, w / w), ground, and pressed into 1 mm thick slices using a tablet press (Tianjin Jinbeier Technology Co., Ltd., China). Then, a Fourier transform infrared spectrometer (Frontier, PerkinElmer, USA) was used to measure the concentration of the sample at 4000-400 cm -1 All spectra in the area were scanned 64 times with a resolution of 4 cm -1 The absorption spectrum of amide I analysis is 1700-1600cm -1 The secondary structure information of amide I was obtained by fitting the second-order derivative peak using PeakFit software (version 4.12, SPSS, Chicago, USA). The secondary structure of MP was calculated as the percentage of α-helix, β-sheet, β-turn and random coil structures. Each sample was analyzed three times.

[0050] 1.4.2 Three-phase contact angle (θ o / w )

[0051] The three-phase contact angles (θ) of MP suspension and MP / KGM mixed system o / w)Measurements were carried out using an SPCAX3 contact angle measuring instrument (Beijing Hake Test Instrument Factory, China). The particulate powder obtained by freeze-drying was compressed into a tablet with a thickness of 2 mm and a diameter of 13 mm. Then the tablet was immersed in perilla seed oil, which was placed in an optical glass cuvette. Deionized water (2 μL) was gently placed on the surface of the tablet using a high-precision syringe. After equilibration for 1 minute, the falling image was captured with a high-speed camera, and the contact angle was calculated using the baseline circle. The measured values were averaged for at least three droplets.

[0052] 1.4.3 Determination of dynamic interfacial pressure

[0053] The interfacial pressure (π) was determined using an OCA-20 contact angle measuring instrument (Dataphysics Instruments GmbH, Germany). The MP, MP-KGM, KGM (1.5%) suspensions were placed in a syringe and allowed to stand for at least 30 minutes to stabilize the temperature at 25 °C. Then a drop of the sample solution (15 μL) was delivered into an optical glass cuvette containing perilla seed oil and allowed to stand at the tip for 1800 s to achieve sample adsorption. The falling images were continuously captured from a charge-coupled device (CCD) camera and digitized. The interfacial pressure was defined as π = γ0 - γ, where γ0 is the interfacial tension of 50 mM PBS (the same as used in Section 1.3), and γ is the time-dependent interfacial tension of the sample.

[0054] 1.5 Preparation of high internal phase Pickering emulsions (Pickering HIPEs)

[0055] When preparing Pickering HIPEs, the MP / KGM mixed system (prepared in 1.3) was used as the aqueous phase, while perilla seed oil was used as the oil phase. The volume fraction of the oil phase was 75% (v / v), and the volume fraction of the aqueous phase was 25% (v / v). The oil-water mixture was homogenized at 2800 rpm for 1 minute using an XHF-DY high-speed disperser (Ningbo Xinzhi Biotechnology Co., Ltd., China) to form Pickering HIPEs. The entire homogenization process was carried out in an ice bath and stopped every 30 s to prevent temperature rise.

[0056] 1.6 Characterization of Pickering HIPEs

[0057] 1.6.1 Determination of droplet size distribution (DSD)

[0058] The droplet size distribution region of Pickering HIPEs was measured using a laser diffraction particle size analyzer (LS 13320, Beckman, California, USA) after diluting 20-fold with deionized water. The refractive indices (RI) of perilla seed oil and water were 1.467 and 1.330, respectively, and the absorption parameter was 0.001.

[0059] 1.6.2 Observation of Microstructure

[0060] Optical microscope measurement: Pickering HIPEs were in a gel state and were diluted 3-fold with deionized water before the experiment and gently shaken by vortex mixing. 10 mL of the sample was dropped onto the center of a glass slide and covered with a coverslip, and then the micrographs of the emulsion were observed using an optical microscope (DMEX20, Ningbo Shunyu Instrument Co., Ltd.) to estimate the droplet size and aggregation state.

[0061] Scanning electron microscopy (SEM) measurement: To better observe the cross-linked network of MP / KGM in the aqueous phase, volatile hexane was used instead of perilla oil as the oil phase to prepare Pickering HIPEs (containing 75% cyclohexane). After the emulsion was formed, the fresh emulsion was freeze-dried for 24 hours using a freeze dryer (LGJ-18, Shanghai Yuming Instrument Co., Ltd.). Next, gold was sprayed, and then observation and photography were carried out using an SEM (SU8010, Hitachi, Japan) with an accelerating voltage of 20 kV.

[0062] 1.6.3 Determination of Protein Adsorption Percentage (AP%)

[0063] An equal volume of deionized water was added to Pickering HIPEs, and after stirring, it was left to stand for 5 hours until all the samples were stratified. The aqueous phase was extracted from the bottom using a syringe, and the protein concentration (Cf) of the aqueous phase was determined using a BCA (bicinchoninic acid) protein assay kit. This process was repeated 3 times. The protein content in the initial Pickering HIPEs and the total volume of the aqueous phase in the sample were recorded as MI and Vf respectively. The adsorption percentage (AP%) was calculated as follows in Equation (1):

[0064] AP% = (MI - C f ×V f ) / MI × 100% (1)

[0065] 1.6.4 Determination of Low-Field Nuclear Magnetic Resonance (LF-NMR)

[0066] The transverse relaxation of Pickeing HIPEs was measured using an LP-NMR analyzer (NMI20-040 V-I, Newmai Analytical Instruments Co., Ltd., Suzhou, China), which has a proton frequency of 21 MHz at 32 °C. The Pickering HIPEs were placed in a sample bottle and then inserted into a nuclear magnetic resonance tube with a diameter of 25 mm, and signals were collected using a Carr-Purcell-Meiboom-Gill (CPMG) sequence. The parameter settings were as follows: P1 (90° pulse), P2 (180° pulse), and TW (π value) were 7.52 μs, 15.52 μs, and 5000 ms, respectively. The number of scans (NS) and the number of echoes were 8 and 18000, respectively. The obtained data were fitted using Multiexp Inv analysis software (Newmai Analytical Instruments Co., Ltd., Suzhou, China).

[0067] 1.6.5 Rheological measurement

[0068] The rheological properties of Pickering HIPEs were analyzed using a rotational rheometer (Mars60, Haake, Germany) with a plate-plate geometry (diameter of 20 mm) according to the method described previously. The Pickering HIPEs were evenly applied to the measurement cell and waited for 5 minutes to reach the set temperature (25 °C). The linear viscoelastic region (LVR) was determined by dynamic strain scanning. At a constant frequency of 1 Hz, the strain was increased logarithmically from 0.1% to 100%. The frequency sweep was performed from 0.1 to 100 rad / s at a fixed strain of 1%. When the shear rate increased from 0.1 to 100 s -1 , the apparent viscosity was recorded. The elastic modulus (G′) and loss modulus (G″) were measured using oscillatory frequency sweep measurements from 0.1 to 100 rad / s.

[0069] 1.6.6 Determination of lipid oxidation

[0070] For comparison, the present invention also studied the lipid oxidation of bulk perilla seed oil and Tween 80 (1% wt)-stabilized emulsion (containing 75% oil). Fresh Pickering HIPEs samples (8 g) were placed in open glass tubes exposed to air and stored in an oven (GZX-9140MBE, Shanghai Boxun Industry Co., Ltd., China) at 50 °C for 30 days to accelerate the oxidation of perilla seed oil. An aliquot of 0.1 g of Pickering HIPEs was taken out regularly and the content of MDA (malondialdehyde) was determined using an enzyme-linked immunosorbent assay (ELISA) reader (Spectra MAX190, Molecular Devices, USA). The determination method of MDA followed the protocol described by the manufacturer of the MDA kit, which was purchased from Nanjing Jiancheng Bioengineering Co., Ltd., China.

[0071] 1.7 Data processing

[0072] Use Origin 2021 and Office 2019 to draw charts. Use IBM SPSS Statistics 26 for significance analysis.

[0073] 2 Results

[0074] 2.1 Effect of KGM concentration on the properties of MP / KGM complex

[0075] 2.1.1 Fourier transform infrared spectroscopy (FTIR)

[0076] FTIR spectra reflect the intermolecular and intramolecular interactions of functional groups and are often used to study the structural changes of MP. The FTIR of MP suspensions at different KGM concentrations is shown in Figure 1 a below. No new peaks were found in the MP-KGM complex, indicating that no new groups were generated between MP and KGM. The amide A band of MP has a maximum peak at 3432 cm -1 , which is caused by the N-H stretching vibration. Adding 1% KGM blue-shifted the amide A band of MP to 3441 cm -1 , which means that fewer N-H groups participate in hydrogen bond formation, resulting in a decrease in the number of hydrogen bonds and the structure of MP tending to unfold. The amide I band of MP has a maximum peak at 1632 cm -1 , which is mainly related to C-N stretching and C=O stretching vibrations. Compared with pure MP, the amide I band of the MP / KGM complex shows a blue shift, and the blue shift is the largest when the KGM addition amount is 0.5% KGM. This indicates that hydrogen bonds are formed between MP and KGM molecules, generating an MP-KGM bridging structure, and the most MP-KGM bridging structures are obtained at a 0.5% KGM addition amount.

[0077] The secondary structure changes of MP with different concentrations of KGM added are shown in Figure 1As shown in Figure b. The results showed that the secondary structure of MP was mainly β-sheet at different KGM concentrations. As the addition amount of KGM increased from 0% to 0.5%, α-helix (14.6% - 13.4%) and random coil (22.0% - 16.2%) gradually decreased, while the content of β-sheet (37.3% - 51.4%) increased, indicating the unfolding of the MP structure. The thermodynamic incompatibility between proteins and polysaccharides may be the reason for the unfolding of the MP structure. The unfolding of MP exposes more hydrophobic groups buried in the helical structure, which will promote the hydrophobic aggregation between MPs to form a network structure. The exposure of hydrophobic groups also promotes the interaction between proteins and oil and promotes protein adsorption at the interface. Compared with the pure MP group, the α-helix (14.5% - 15.1% - 17.1%) in the MP-KGM 1% and MP-KGM 1.5% groups slightly increased, probably because a small part of the unfolded MPs re-aggregated. However, the random coil (22.0% - 19.3% - 13.6%) decreased significantly, and the β-sheet (37.3% - 45.8% - 45.0%) increased significantly. It shows that generally KGM (>0.5%) promotes the unfolding of MP, but the promotion degree is weakened to a certain extent.

[0078] Generally speaking, adding KGM, especially 0.5% KGM, promotes the unfolding of the MP structure and the exposure of hydrophobic groups, forming an MP-KGM bridging structure, which is beneficial to increasing the surface hydrophobicity of MP and promoting the formation of a network structure.

[0079] 2.1.2 Wettability of the MP / KGM mixed system at the oil-water interface

[0080] The wettability of particles in oil and water can be characterized by the three-phase contact angle (θ o / w ). This parameter has a significant impact on the quality of emulsifiers and can be used to judge the type and feasibility of Pickering emulsions. The θ o / w of MP, MP / KGM complexes and KGM in perilla seed oil is shown in Figure 2 . The θ o / w of MP is 73.69°, less than 90°, indicating that the hydrophilicity of MP is dominant. The θ o / w of KGM is 43.20°, showing strong hydrophilicity, which is attributed to the large number of hydroxyl groups on the surface of KGM molecules. The θ o / w of all MP-KGM groups is greater than that of MP. When the addition amount of KGM increases from 0% to 0.5%, θ o / w increases from 73.69° to 89.41° correspondingly, and θ o / w is close to 90°, that is, the wettability of the particles is close to neutral, which means that the particles are more firmly adsorbed at the oil / water interface and can also form steric hindrance to prevent oil droplet aggregation. As the KGM concentration continues to increase (>0.5%), θ o / wshowed a slightly decreasing trend. θ o / w and the secondary structure ratio of MP ( Figure 1 in b) had a similar trend with the change of KGM concentration. 0.25 - 0.5% KGM caused the MP structure to unfold, exposing more hydrophobic amino acids, thus leading to the increase of θ o / w When KGM > 0.5, part of the MP re - aggregated, which led to the burial of some hydrophobic groups, thus resulting in the slight decrease of θ o / w Generally speaking, the addition of KGM improved the hydrophobicity of MP, and the MP - KGM 0.5% group had nearly perfect amphiphilicity.

[0081] 2.2 Effect of KGM concentration on interfacial adsorption performance

[0082] 2.2.1 Dynamic interfacial pressure

[0083] The adsorption characteristics of proteins at the oil - water interface have a crucial impact on their emulsifying ability. The changes of the dynamic interfacial pressure (π) of MP, MP - KGM complex, and KGM with time (t) are shown as Figure 3 in a. The π of all samples including pure KGM increased rapidly at the initial stage of adsorption, indicating that both MP molecules and KGM molecules can spontaneously adsorb onto the oil - water interface. Although KGM is hydrophilic, there are a small number of acetyl groups in the molecular chain of KGM, endowing them with amphiphilicity, and KGM can also be adsorbed as an emulsifier on the oil - water interface during the preparation of emulsions. After that, the growth rate of π decreased, which was due to the gradual saturation of proteins and polysaccharides adsorbed on the interface and the increase of adsorption electrostatic energy barriers. However, the curve still did not reach equilibrium until the end of the experiment, and π was still rising slowly and slightly, which may be because the molecular weights of proteins and polysaccharides are large and the adsorption equilibrium is slow, and proteins have disulfide bonds, and a molar energy barrier is also required during their adsorption. However, previous studies have shown that protein adsorption cannot reach equilibrium even after several days.

[0084] The initial interfacial pressure (π0) of pure KGM was larger than that of pure MP, because the large - molecular - weight KGM exerted a greater pressure on the oil - water interface. The growth rate of π of KGM was less than that of MP, which may be related to the strong hydrophilicity, large molecular weight, and high viscosity of KGM, which limit its diffusion rate to the interface.

[0085] The curve of π for 0.25 - 0.5% MP-KGM is similar to that of pure MP, and both π and its growth rate of the mixed system increase with the increase of KGM concentration. This indicates that at this concentration, MP mainly covers the interface, and the addition of KGM promotes the adsorption of MP on the oil-water interface, resulting in an increase in π. Polysaccharides can affect protein interfacial adsorption in the following aspects: (Ⅰ) affecting protein properties such as structure, hydrophobicity, and electrostatics; (Ⅱ) forming complexes through hydrophobic or electrostatic interactions; (Ⅲ) being related to the properties of polysaccharides themselves such as charge polarity and viscosity; (Ⅳ) thermodynamic instability caused by solvent exclusion. According to FTIR( Figure 1 ) and contact angle( Figure 2 ), KGM promotes the unfolding of the MP structure, making the wettability of MP on the interface close to neutral, which promotes the adsorption of MP on the interface. In addition, the repulsive effect of the thermodynamic incompatibility between MP and KGM drives MP to diffuse from the continuous phase to the oil-water interface, resulting in a larger π, which is also called the interfacial concentration effect, and this effect is enhanced with the increase of KGM concentration.

[0086] However, the π curve of 1% - 1.5% MP-KGM is similar to that of pure KGM and π0 is much larger than that of 0 - 0.5% MP-KGM. KGM is a non-ionic polysaccharide and does not form electrostatic complexes with MP. Both are dispersed in the aqueous phase in a free state. Once the interface is formed, competitive adsorption may occur. Therefore, combined with the results of AP% (section 1.5), it can be speculated that in 1 - 1.5% MP-KGM, high-molecular-weight KGM adsorbs and winds around the surface of oil droplets, covering and competing for some of the adsorption sites of MP on the oil-water interface, resulting in a sharp increase in π and a decrease in the interfacial MP content.

[0087] 2.2.2 Diffusion to the perilla seed oil-water interface

[0088] Protein adsorption to the oil-water interface undergoes three processes: (Ⅰ) diffusion (from the aqueous phase to the interface); (Ⅱ) penetration (the protein unfolds on the interface); (Ⅲ) rearrangement (hydrophobic amino groups are exposed and rearranged in the oil phase, while hydrophilic amino acids are in the aqueous phase). According to the method described in previous studies, the diffusion rate (K diff ) is obtained from the slope of the linear plot of π against t 1 / 2 in the initial stage of adsorption (b in Figure 3 ).

[0089] As shown in Table 1, the K diffIncreased with the increase of KGM concentration (0 - 0.5%), and reached the maximum value at 0.5% KGM concentration, indicating that adding medium concentration of KGM can increase the diffusion rate of MP to the oil - water interface. The possible reason is that the improved hydrophobicity of MP and the repulsive force of KGM jointly act as the driving force to promote the diffusion of MP to the interface. As the KGM concentration continues to increase (>0.5%) diff it decreased sharply, which was caused by the diffusion resistance and / or steric hindrance due to the high viscosity of the mixed system caused by high - concentration KGM.

[0090] 2.2.3 Penetration and rearrangement at the perilla seed oil - water interface

[0091] After diffusing into the interface region, the penetration and rearrangement of MP molecules at the interface became the dominant process. The penetration and rearrangement of proteins at the interface can be described using the first - order phenomenological equation (Equation (2)):

[0092] [(π f - π t ) / (π f - π0)]= - k i t (2)

[0093] where k i is the first - order rate constant; π f , π0, π t are the interfacial pressures at the final time (1800 s), any time (t), and initial time (0 s), respectively.

[0094] The plot of the above equation has two linear ranges. The slope of the first linear region is the penetration rate constant (K P ), and the slope of the second linear region is the rearrangement rate constant (K R ), which are listed in Table 1. Figure 3 Figures c - h respectively depict the typical plots of the above equation for all the studied samples. Adding KGM increased K P and K R , and the maximum value was obtained at 0.5% KGM concentration, indicating that KGM accelerated the penetration and rearrangement rates of MP at the oil - water interface. The increase in K P might be due to the fact that KGM made the wettability of MP close to neutral, which reduced the energy barrier for MP to penetrate at the oil - water interface. Faster penetration might reduce the particle size by preventing oil droplet collision. The K P and K R of the MP - KGM 1.5% group were greater than those of MP, indicating that the penetration and rearrangement of MP at the interface were not limited by high viscosity. The K R of all samples was significantly higher than K P , indicating that the structural rearrangement of interfacial MP contributed more to the formation of the interfacial film than the penetration behavior. K RIt depends to a large extent on the conformational flexibility of the protein. According to the analysis of the MP structure (section 1.1), KGM promotes the unfolding of MP, enhances its conformational flexibility, and helps with the rearrangement and expansion of MP at the interface. The K of the MP-KGM 0.5% group R is much larger than that of other groups, which may be related to its contact angle approaching 90°. The near-perfect amphiphilicity minimizes the free energy required for the protein to rearrange at the interface. In addition, K R is related to the multi-layer absorption of the protein at the oil-water interface. Therefore, the addition of KGM, especially 0.5% KGM, may promote the formation of a multi-layer MP adsorption layer at the oil-water interface.

[0095] Based on the above analysis, it can be concluded that the KGM concentration significantly affects the adsorption kinetics of MP. 0.25 - 0.5% KGM promotes the adsorption process of MP, improving the equilibrium interfacial pressure, diffusion rate, permeation rate, and rearrangement rate. However, 1 - 1.5% KGM competes for some MP adsorption sites and reduces the diffusion rate.

[0096] Table 1 Effects of KGM concentration on the kinetic parameters (Kdiff, KP, KR) of MP adsorption at the oil-water interface (pH 7.0)

[0097]

[0098] Note: a Linear regression coefficient.

[0099] 2.3 Effects of KGM concentration on the performance of Pickering HIPEs

[0100] 2.3.1 Droplet size distribution, microstructure, and visual stability of Pickering HIPEs

[0101] The droplet size and dispersion state of the emulsion are usually used to evaluate the emulsifying ability of the emulsifier. The effect of KGM concentration on the droplet size distribution of freshly prepared Pickering HIPEs is as Figure 4 shown in a. In the absence of added KGM, the droplet distribution of Pickering HIPEs emulsified by pure MP shows two main peaks (27 μm and 53 μm respectively), indicating that the droplets are larger and unevenly distributed, which is similar to the results of the optical microscope images ( Figure 4 in c). With the addition of KGM (0.25 - 0.5%), the droplet distribution peak shifts to the left and shows a single peak (36 μm), which means smaller and more uniform droplet sizes, reflecting stronger emulsifying ability. KGM may reduce the droplet size through the following several ways: (Ⅰ) KGM promotes the adsorption of MP at the interface ( Figure 3 in a and Figure 6), thus reducing the interfacial tension and promoting the droplet decomposition during emulsification. MP adsorption also leads to the formation of a thicker interfacial protein adsorption layer, creating a steric hindrance to prevent the coalescence of adjacent oil droplets; (II) KGM promotes the formation of a dense MP network structure in the continuous phase ( Figure 5 ), and the oil droplets are embedded in the network to form a steric hindrance, thereby inhibiting the flocculation and aggregation of oil droplets; (III) KGM in the aqueous phase forms intermolecular hydrogen bonds with MP adsorbed on the surface of oil droplets ( Figure 1 in a)), connecting the oil droplets, resulting in the relative displacement between droplets not being easy and preventing aggregation; (IV) the high viscosity of the continuous phase caused by KGM prevents the oil droplets from approaching and coalescing with each other.

[0102] However, as the KGM concentration further increases (1 - 1.5%), the droplet distribution returns to two peaks again. And the average droplet size of the emulsion at 1.5% KGM is larger than that of pure MP, and large-sized droplets are observed in the optical microscope images, indicating that the droplets coalesce, resulting in an increase in the droplet size. This can be attributed to the less interfacial MP ( Figure 6 ) and the loose MP network structure ( Figure 5 ) when adding 1.5% KGM, which leads to a reduction in steric hindrance and makes the oil droplets easier to collide and coalesce with each other. In addition, a high KGM concentration may induce the depletion of polysaccharides between the narrow gaps of adjacent droplets, thus leading to bridging flocculation or coalescence.

[0103] The appearance images of fresh Pickering HIPEs and Pickering HIPEs stored at 25 °C for 30 days are as shown in Figure 4 b. Macroscopic phase separation occurred in the Pickering HIPEs added with 1.5% KGM, indicating that the amount of MP is insufficient to stabilize the newly formed interface, which is consistent with the results of the interfacial protein adsorption rate ( Figure 6 ). After storing for 30 days, phase separation occurred in the Pickering HIPEs stabilized by pure MP, the degree of phase separation in the Pickering HIPEs added with 1.5% KGM increased, trace amounts of oil were separated out in the Pickering HIPEs added with 0.25% and 1% KGM, while almost no oil was separated out in the emulsion with 0.5% KGM. The results show that an appropriate amount of KGM (0.25 - 1%, especially 0.5%) promotes the formation of a compact interfacial adsorption layer and network structure, thus preventing coalescence and flocculation during storage, proving that Pickering HIPEs have a good shelf life. This observation is in good agreement with the droplet size distribution and optical microscope images.

[0104] 2.3.2 Scanning electron microscopy (SEM) of Pickering HIPEs.

[0105] To observe the effect of KGM on the MP network structure in the aqueous phase, volatile cyclohexane was used instead of perilla oil to prepare Pickering HIPEs stabilized by pure MP, MP-KGM 0.5%, and MP-KGM 1.5% for SEM imaging. As Figure 5 shown, the gray and white areas are the MP network structures in the aqueous phase of Pickering HIPEs, and the black areas are the pores left after freeze-drying Pickering HIPEs to remove hexane and water. Large and irregular pore sizes or even continuous pore sizes (indicated by red arrows) were observed in Pickering HIPEs stabilized only by MP. The network structure was loose and soft, indicating uneven oil droplet distribution and even aggregation, which was consistent with the droplet size distribution results ( Figure 4 in a)). After adding 0.5% KGM, the pore size decreased and the distribution became more regular, and the network structure became more compact and dense. This phenomenon may be because KGM promotes the unfolding of the MP structure and the exposure of hydrophobic groups ( Figure 1 in b)), thereby promoting the formation of a network structure by hydrophobic cross-linking of MP. KGM rich in hydroxyl groups competes with MP to form hydrogen bonds with water molecules, reducing the hydrogen bonds between MP and water molecules, and thus promoting the formation of more hydrogen bonds between MP molecules. The formation of hydrogen bonds between MP is beneficial to promoting the aggregation of MP and the formation of a network structure. In addition, the KGM-MP bridging structure formed by hydrogen bonds ( Figure 1 in a)). This substructure replaces the water pores and fills the protein network, making the network structure denser. In addition, it was observed that the pores left by the sublimation of droplets in Pickering HIPEs stabilized by 0.5% MP-KGM were polyhedral in shape because the droplets were tightly packed together above the packaging limit.

[0106] However, when the KGM addition amount reached 1.5%, more continuous pore sizes (indicated by red arrows) appeared, and the network structure was divided into several clusters by the continuous pore sizes. This indicates more serious oil droplet aggregation, which can be attributed to the fact that KGM competes for adsorption sites, resulting in insufficient interfacial MP to wrap the oil droplets ( Figure 6 ). It may also be because a large amount of KGM absorbs water in the aqueous phase to form a continuous KGM hydrogel that penetrates the MP network structure, which physically hinders the cross-linking of MP hydrophobic groups and separates the MP network into clusters.

[0107] 2.3.3 Protein adsorption rate of Pickering HIPEs

[0108] To verify the adsorption behavior of MP at the interface at different KGM concentrations, the percentage of MP adsorbed at the interface was measured ( Figure 6)。As the concentration of KGM increased from 0% to 0.5%, the adsorption percentage of MP at the interface increased from 54.28% to 69.84%, indicating enhanced interfacial adsorption capacity and emulsifying capacity, which may be related to the increased content of exposed hydrophobic groups (Section 1.1.1), near-neutral wettability (Section 1.1.2), and interfacial protein concentration caused by thermodynamic incompatibility. When the KGM concentration was greater than 0.5%, the AP% value showed a downward trend, confirming the hypothesis proposed in Section 1.2.1 that high-concentration KGM (>0.5%) competes for the adsorption sites of MP at the interface. Competitive adsorption has also been reported between KGM and myosin. It should be noted that the AP% value of the MP-KGM 1% group, although starting to decline, was still greater than that of the pure MP group, while the AP% value of the MP-KGM 1.5% group was less than that of the pure MP group, indicating that the weakening effect of competitive adsorption of KGM on MP adsorption at the interface at a KGM concentration of 1% was less than the promoting effect of hydrophobic group exposure and thermodynamic incompatibility, and the result was opposite at 1.5% KGM. This result was completely consistent with the results of interfacial adsorption in Section 2.2. In other words, MP or MP / KGM mixtures exhibited similar interfacial adsorption properties in aqueous solutions or emulsion systems, and adding an appropriate amount of KGM could improve the interfacial adsorption activity of MP in different systems.

[0109] 2.3.4 State and distribution of water in Pickering HIPEs

[0110] LF-NMR is a method for studying the physical state and distribution of water in emulsion gels, which is closely related to the internal structure of the emulsion and the bonds between water and proteins. The denser the network structure in the aqueous phase of the emulsion, the easier it is for water to be trapped. As shown in Figure 7 a below, three proton signals (i.e., T 21 , T 22 and T 23 ) were observed in the T2 relaxation curve of Pickering HIPEs, where the fastest proton signal T 21 (0.1 - 10 ms) corresponded to the bound water tightly bound to macromolecules; T 22 (15 - 350 ms) represented the immobilized water trapped in the network structure in the aqueous phase of Pickering HIPEs; T 23 (400 - 3000 milliseconds), the slowest proton signal, could be designated as the mobile water in the aqueous phase. In addition to the hydrogen protons of water molecules, the hydrogen protons of glycerol molecules in the emulsion would also show a T2 characteristic peak at approximately 10 - 500 ms, close to T 22 , so the relaxation signal T 22 might be partly due to the emulsified oil phase. The proportions of these 3 proton populations in Pickering HIPEs were respectively designated as PT 21 / PT 22and PT 23 , and presented in Figure 7 b of

[0111] As Figure 7 shown in a and b of 21 , the addition of KGM shifts the T 21 peak of Pickering HIPEs emulsified by MP to the left, while PT 23 and T 22 first decreases and then gradually increases with the increase of KGM addition amount. It indicates that KGM absorbs the water in MP, converting the water bound to MP into bound water bound to KGM and immobilized water trapped in the KGM structure. This result confirms the hypothesis in the SEM analysis that KGM competes with MP for forming hydrogen bonds with water, thus promoting the formation of intermolecular hydrogen bonds in MP and strengthening the network structure. At the same time, the absorption of part of the water in MP by KGM will produce "concentrated" MP, which promotes the aggregation of hydrophobic groups, forming a denser MP network structure and an interfacial MP adsorption layer in the aqueous phase and at the interface respectively. T 23 and T 22 and the peak values of 23 and PT 22 gradually shift to the left with the increase of KGM concentration (0%-1%), while PT 23 and PT 22 decrease and increase respectively, reflecting the decrease in the water mobility in the emulsion system. This is due to the strong water absorption of KGM, which forms bound water by forming hydrogen bonds with water molecules and traps some free water in the KGM structure to convert it into immobilized water. At the same time, according to the above analysis, KGM promotes the formation of a denser network structure ( Figure 5 ) and an interfacial protein layer ( Figure 6 ) that act as steric hindrance, which will further limit the mobility of water and oil molecules. The phenomenon of KGM restricting the mobility of water has also been reported in the KGM and myosin gel system.

[0112] Further increasing the KGM concentration to 1.5%, the peak values of T 23 and T 22 slightly shift to the right, and PT 23 and PT 22 increase and decrease respectively. This may be because the formation of a continuous hydrogel by KGM hinders the aggregation of the MP network, resulting in the conversion of some immobilized water into free water. It should be noted that although the water mobility rate of the MP-KGM 1.5% group is slower than that of the pure MP group, its emulsion stability ( Figure 4 ) and rheology ( Figure 8 a) are worse than those of the pure MP group. This shows that the water migration rate in the aqueous phase is not the main decisive factor affecting the properties of Pickering HIPEs, which may be because the proportion of water in the Pickering HIPEs system is very small.

[0113] 2.3.5 Rheological properties of Pickering HIPEs

[0114] The rheological properties are closely related to the physical properties of Pickering HIPEs (such as appearance and storage stability). Shear rheological measurements were used to provide information on the effect of KGM addition on the mechanical properties of Pickering HIPEs. As Figure 8 shown in a1, the apparent viscosity of all Pickering HIPEs decreased significantly with increasing shear rate, showing shear-thinning behavior, indicating pseudoplastic non-Newtonian fluids. This is attributed to the destruction of the network structure of Pickering HIPEs and the decomposition of oil droplet clusters under shear force. It can be seen that with the increase in KGM addition (0 - 1%), the apparent viscosity increased significantly, and the highest viscosity was obtained at 0.5% KGM addition. When adding a higher concentration of KGM (1.5%), the apparent viscosity decreased sharply to be smaller than that of MP. Consistent with the results of SEM( Figure 5 ) and protein interfacial adsorption rate( Figure 6 ), but inconsistent with the low water migration rate phenomenon shown by the LF-NMR results( Figure 7 ) at 1.5% KGM addition. This effect indicates that KGM mainly affects Pickering HIPEs by changing the MP network structure and interfacial protein adsorption, and has little relationship with the viscosity of the aqueous phase.

[0115] The viscoelasticity and network structure strength of Pickering HIPEs can be characterized by the storage modulus (G′) and loss modulus (G″). As Figure 8 shown in a2, within the entire frequency range, G′ of all Pickering HIPEs was greater than G″, and there was no crossover with the increase in the angular frequency range, indicating that these Pickering HIPEs can be considered as strongly gel-like solid viscoelastic materials with covalent bonding. Adding KGM (0.25 - 1%) increased the values of G′ and G″ and obtained the maximum value at 0.5% KGM addition, indicating better plasticity and a more rigid system of Pickering HIPEs. The main reasons include: (Ⅰ) Denser interfacial protein coating( Figure 6 ): Emulsions with elastic adsorption layers usually mean increased rheological properties and a rigid system; (Ⅱ) Smaller droplet size( Figure 4 in a): Small and dense droplets squeeze each other, strengthening the physical cross-linking between MPs at the interface, thus enhancing the network structure strength within Pikering HIPEs; (Ⅲ) Stronger droplet-droplet interaction: KGM promotes the unfolding of MP and the exposure of hydrophobic groups( Figure 1 in b), strengthening the hydrophobic interaction between MPs on the interfaces of adjacent droplets; (Ⅳ) Stronger network structure( Figure 5)。However, when the addition amount (1.5%) is more, the G′ and G″ values decrease significantly and are even lower than those of the pure MP group. This may be because the competitive adsorption of KGM leads to a reduction in interfacial MP, resulting in the aggregation of oil droplets. It may also be that excessive KGM destroys the network structure of MP in the aqueous phase by forming a continuous hydrogel. These will all lead to a decrease in the rigidity of Pickering HIPEs.

[0116] Figure 8 Figure b shows the appearance of Pickering HIPEs emulsified by MP at different KGM concentrations. It can be seen that the Pickering HIPEs with 0.25 - 1%, especially 0.5% KGM addition, exhibit stronger plasticity, and the Pickering HIPEs with 1.5% KGM become softer, which is completely consistent with the rheological results.

[0117] 2.3.6 Lipid oxidation of Pickering HIPEs

[0118] Lipid oxidation is an important factor affecting product quality. Figure 9 Figures a and b show the appearance and MDA content of bulk perilla seed oil, Tween-80 stabilized emulsion, and Pickering HIPEs emulsified by MP with different KGM concentrations under accelerated oxidation conditions, respectively. It can be seen that the color of Pickering HIPEs gradually darkens with the increase of storage time, which may be caused by lipid oxidation or may be related to the oxidation of myofibrillar protein in Pickering HIPEs induced by peroxyl radicals, during which carbonylation and S-S are generated.

[0119] MDA is an important substance for measuring the oxidation degree of oils and emulsions. As shown in Figure b, the MDA content of all samples increased rapidly during the storage period of 0 - 7 days, and the addition of 0.25% and 0.5% KGM slowed down the increase of MDA content. The increase of MDA content was slow during 14 - 21 days and there was no significant difference among groups (p > 0.05). After 30 days of storage, the increase of MDA in Pickering HIPEs containing 0.5% KGM was significantly inhibited (p < 0.05), while the MDA content of the remaining groups increased rapidly and there was no significant difference (p > 0.05). The results show that the oil oxidizes rapidly in the initial and late stages of storage, and the addition of 0.5% KGM can effectively inhibit the oil oxidation in Pickering HIPEs. On the contrary, Tween-80 has no inhibitory effect. According to the previous text, this may be because the most MP is adsorbed on the surface of oil droplets at 0.5% KGM concentration ( Figure 6 ) and the wettability is close to 90° ( Figure 2 ), so MP constructs a strong and thick interfacial layer around the droplets, thus forming a physical barrier to inhibit the penetration and diffusion of lipid oxidation initiators into the interior of oil droplets.

[0120] In addition, as demonstrated by the rheological results ( Figure 8 b) in Figure 5 ) and the SEM images (

[0121] ), the Pickering HIPEs containing 0.5% KGM are in a gel-like state and the oil is embedded inside the dense network structure to partially prevent oxidation. The mechanism of Tween-80 stabilized emulsion is to reduce the interfacial tension and no adsorption layer barrier is formed on the surface of the droplets to block pro-oxidants. Moreover, the Tween-80 stabilized emulsion is in a flowing state, so the oil droplets are easily oxidized by pro-oxidants.

[0122] 2.4 Stability mechanism of MP / KGM composite stabilized Pickering HIPEs Figure 10 as follows.

[0123] The mechanism of pure MP stabilized HIPEs is that MP adsorbs on the surface of homogenized and chopped oil droplets, reducing the interfacial tension and forming steric hindrance. However, MP is not sufficient to completely wrap the oil droplets, resulting in the aggregation of oil droplets with larger and unevenly distributed emulsion droplets.

[0124] When 0.25 - 1% KGM is added, KGM promotes the unfolding of the MP structure, exposes more hydrophobic groups, and improves the wettability of MP, which reduces the energy barrier for the penetration and rearrangement of MP at the interface. KGM promotes the adsorption of more MP at the oil-water interface. The dense MP interfacial layer acts as a barrier to prevent the collision and aggregation of oil droplets, so the formed emulsion droplets are small and uniform. In addition, KGM absorbs water from MP, resulting in the "concentration" of MP, strengthening the hydrogen bonds and hydrophobic interactions between MPs, and promoting a denser MP network structure. At the same time, KGM forms substructures with MP through hydrogen bonds and fills the voids in the MP network, enhancing the strength of the network structure. The dense network structure further increases the steric hindrance to limit the aggregation of oil droplets. The dense interfacial adsorption layer and network structure improve the stability and rheological properties of Pickering HIPEs and act as a barrier to prevent the oxidation of oils by pro-oxidants. This enhancement effect is more obvious at the addition amount of 0.5% KGM.

[0125] When the addition amount of KGM reached 1.5%, the high viscosity of KGM reduced the diffusion rate of MP from the aqueous phase to the interface, which provided an opportunity for KGM to compete for some adsorption sites of MP, resulting in less MP adsorbed on the interface. Therefore, the oil droplets aggregated with each other to become larger and uneven, which further led to the demulsification of the emulsion. At the same time, KGM transferred a large amount of water from MP to form a continuous hydrogel network permeating in the MP network. This hindered the hydrophobic aggregation of the MP network and destroyed the integrity of the network, resulting in a decrease in steric hindrance. The reduced interfacial adsorbed MP and the unaggregated network structure led to the coalescence of the oil droplets in the emulsion and demulsification, which further led to the decline of the emulsion stability and rheological properties and did not alleviate lipid oxidation.

[0126] In summary, KGM significantly affects the physical properties of MP-stabilized Pickering HIPEs by influencing interfacial adsorption and network structure formation. KGM, especially 0.5% KGM, promotes the unfolding of the MP structure and improves the wettability of MP. The change in interfacial adsorption indicates that 0.25 - 0.5% KGM promotes the adsorption of MP at the oil-water interface, and 1 - 1.5% KGM may compete for some adsorption sites of MP on the interface. The interfacial protein adsorption rate further confirmed this and showed that 1% KGM has a greater promoting effect on the adsorption of MP at the interface than the weakening effect caused by its competitive adsorption. 0.5% KGM makes the MP network structure (SEM) denser, while excessive KGM (1.5%) absorbs a large amount of water to form a continuous hydrogel permeating in the MP network structure, resulting in the fragmentation of the MP network structure. Correspondingly, when 0.25 - 1% KGM is added, the droplet size of Pickering HIPEs decreases, and the storage stability and rheological properties increase, and the opposite changes are observed at the addition amount of 1.5% KGM. Therefore, by using the food-grade polysaccharide KGM, the emulsification function of MP can be improved without using expensive enzymes or chemical modifications.

[0127] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for improving the physical properties of a high internal phase Pickering emulsion using myofibrillar protein as a protein raw material, characterized in that, The method comprises the following steps: (1) Mix a myofibrillar protein solution and a konjac glucomannan solution and then homogenize them to obtain a myofibrillar protein / konjac glucomannan mixed system; (2) Use the myofibrillar protein / konjac glucomannan mixed system as the aqueous phase, mix it with an oil phase and then homogenize to prepare the high internal phase Pickering emulsion; The mass-volume concentration of the myofibrillar protein solution is 2%; The mass-volume concentration of the konjac glucomannan solution is 0.5%; In step (1), the myofibrillar protein solution and the konjac glucomannan solution are mixed in a mass ratio of 1:1; In step (2), the aqueous phase and the oil phase are mixed in a volume ratio of 25:75; The physical properties include wettability, interfacial adsorption and stability; In step (2), the oil phase is perilla oil.

2. The method according to claim 1, characterized in that, In steps (1) and (2), the rotation speed of the homogenization is 2800 rpm and the time is 1 min for both.

3. A high internal phase Pickering emulsion prepared by the method according to claim 1 or 2.

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