Pickering stabilizer containing all-natural polysaccharide and protein compound nanoparticles and Pickering emulsion

Abstract

The invention discloses a Pickering stabilizer containing all natural polysaccharide and protein compound nano particles and a Pickering emulsion containing all natural polysaccharide and protein compound nano particles. The preparation method of the Pickering stabilizer comprises the following steps: S1, extracting freeze-dried water extraction residues of pterocarya palustris by adopting an alkaline solution to obtain a filtrate; s2, adjusting the pH value of the filtrate to 6.8-7.3, and centrifuging to obtain a precipitate; and S3, dispersing the precipitate in a phosphate buffer solution, hydrating and shearing, and then performing ultrasonic treatment to obtain the product. The suspension prepared by the method disclosed by the invention can be used as a stabilizer to prepare an oil-in-water Pickering emulsion, and the preparation method specifically comprises the following steps: dispersing the suspension in water, and adjusting the pH value to 6-12; and then mixing with an oil phase, and carrying out ultrasonic treatment to obtain the Pickering emulsion. The suspension provided by the invention has the characteristics of simple preparation, eco-friendliness, sustainability and the like, and is expected to stabilize the Pickering emulsion in a food system.

Description

Technical Field

[0001] The present invention relates to a Pickering stabilizer containing nanoparticles of a natural polysaccharide and a protein complex and a Pickering emulsion, belonging to the field of deep processing of edible fungi. Background Art

[0002] Traditional emulsions are formed based on surfactants. Under specific ionic strength, mechanical treatment, and temperature, surfactants cannot effectively prevent emulsion destabilization, which results in many limitations in their use. In recent years, the excellent stability and wide application of food-grade Pickering have received great attention. The mechanism of Pickering emulsion stabilization is the irreversible adsorption of dispersed solid particles at the oil-water interface to form a physical barrier to prevent droplet flocculation and aggregation. Currently, many studies have shown that many types of inorganic particles can meet the partial wetting of common oils and be regarded as stabilizers for Pickering emulsions, such as silica, calcium carbonate, magnetic particles, and nanoparticles of gold and silver. However, the use of inorganic or synthetic nanomaterials in the food field poses significant safety hazards, and the search for and development of natural and non-toxic bio-based nanomaterials has become a research hotspot for food-grade Pickering emulsions. So far, it has been found that various fruit and vegetable processing by-products can be obtained nanofibers after continuous treatment with alkali solutions of different concentrations and then different mechanical treatments or strong acid hydrolysis. There are related studies on banana peels, pineapple peels, citrus fibers, apple pomace, ginkgo shells, etc.

[0003] At present, the food-grade particles found by research mainly fall into three categories: polysaccharide particles, protein particles, and composite particles. Most polysaccharide particles are extremely hydrophilic and difficult to be used as stabilizers for Pickering emulsions. Usually, physical or chemical modification is required to change their hydrophobicity. Protein particles have good amphiphilicity, but are easily restricted by temperature and pH during use. The combination of the two types of particles, polysaccharides and proteins, promotes complementary advantages. At the same time, a dense interfacial layer can be formed at the oil-water interface of the Pickering emulsion, serving as a good stabilizer for the Pickering emulsion. So far, most polysaccharide-protein composite particles have been prepared by methods such as chemical cross-linking, Maillard reaction, and physical mixing. For example, chestnut starch nanocrystals and macadamia nut isolate protein complex (SNC / MPI) were prepared by pH cycling; ovotransferrin-glucose conjugates and ovotransferrin-lactose conjugates were prepared by the Maillard reaction of ovotransferrin with glucose or lactose; zeatin / arabic gum composite colloidal nanoparticles (ZGAPs) with a core-shell structure were prepared by hydrogen bonding and electrostatic interaction. The chitosan-casein phosphopeptide nanocomposite stabilizes the emulsion at the oil-water interface and is formed by electrostatic interaction. Compared with the above composites, the preparation process of natural polysaccharide-protein composite particles is simple and environmentally friendly. However, there are few reports on the use of natural polysaccharide-protein composites as stabilizers for Pickering emulsions. Summary of the Invention

[0004] The object of the present invention is to provide a Pickering stabilizer (suspension) containing all-natural polysaccharide and protein composite nanoparticles, which is prepared by subjecting the water extraction residue of Pholiota squarrosa to hot alkali pretreatment and then using ultrasonic treatment; the present invention analyzes the morphology, particle size, Zeta potential, and contact angle of the suspension to explore the influence of ultrasonic amplitude on their physicochemical properties, so as to evaluate their potential for stabilizing O / W Pickering emulsions.

[0005] The present invention uses suspensions with different concentrations and pH values and soybean oil in different proportions to prepare Pickering emulsions, and characterizes the morphology of the emulsions by emulsion particle size and microscopic morphology, and understands the properties of the emulsions through rheological characteristics, storage, and heating stability. The present invention provides a theoretical guidance for the development of a new Pickering stabilizer PPCNs.

[0006] The present invention provides a method for preparing a suspension containing polysaccharide-protein composite nanoparticles, which comprises the following steps:

[0007] S1. Extract the freeze-dried water extraction residue of Pholiota squarrosa with an alkali solution to obtain a filtrate;

[0008] S2. After adjusting the pH value of the filtrate to 6.8-7.3, centrifuge to obtain a precipitate;

[0009] S3. Dispersing the precipitate in a phosphate buffer, hydrating and shearing the precipitate, and then performing ultrasonic treatment to obtain the suspension.

[0010] In the above preparation method, the preparation method of the freeze-dried water extraction residue of the Clitocybe squamae may include the following steps: drying the Clitocybe squamae fruiting body, crushing it with a grinder, and then passing it through an 80-mesh sieve, then extracting it in a water bath at 60-90°C with a solid-liquid ratio of 1:10-30, filtering it, and freeze-drying the filter residue in a freeze dryer.

[0011] In the above preparation method, in step S1, the alkaline solution is a sodium hydroxide aqueous solution with a mass concentration of 3.0-8.0%, preferably 4.5-5.5%.

[0012] The ratio of the water extraction residue of Clitocybe squamata to the alkaline solution is 1:10-30 g / mL, preferably 1:15-25 g / mL;

[0013] The extraction temperature is 35-45°C and the extraction time is 1.5-2.5h;

[0014] Repeat the extraction 2 to 3 times.

[0015] In the above preparation method, in step S2, glacial acetic acid is used to adjust the pH value;

[0016] The precipitate is freeze-dried and then dispersed;

[0017] In step S3, the concentration of the phosphate buffer is 3.0-7.0 mM, preferably 5 mM, and the pH value is 6.5-7.2, preferably 7;

[0018] The mass concentration of the precipitate in the phosphate buffer is 2.5-3.5%;

[0019] After overnight hydration, shear treatment was performed for 4 to 6 minutes.

[0020] In the above preparation method, in step S3, the ultrasonic treatment is performed in an ultrasonic processor under the condition of an ice water bath;

[0021] The conditions of the ultrasonic treatment are as follows:

[0022] The amplitude is 20% to 40%, the pulse is on for 1 to 3 seconds, off for 1 to 3 seconds, and lasts for 5 to 15 minutes;

[0023] Specifically, an ultrasonic processor (JY92-IIN, Ningbo, China) with a 6 mm diameter probe was inserted into a beaker (1.0 cm deep) for ultrasonic treatment.

[0024] The suspension prepared by the method of the present invention can be used as a stabilizer to prepare an oil-in-water Pickering emulsion, which can be specifically carried out according to the following steps:

[0025] Disperse the suspension in water and adjust the pH value to 6 - 12; then mix it with the oil phase and obtain the Pickering emulsion through ultrasonic treatment;

[0026] The mass concentration of the suspension in water after dispersion is 0.5 - 4%, preferably 2.5 - 3.5%;

[0027] The volume fraction of the oil phase in the Pickering emulsion is 10 - 60%, preferably 25 - 35%;

[0028] Adjust the pH value with hydrochloric acid and sodium hydroxide;

[0029] The oil phase is soybean oil or palm oil.

[0030] Clitocybe squamulosa, a wild edible and medicinal mushroom, is widely distributed in Wutai Mountain, Shanxi. At present, the research on Clitocybe squamulosa mainly focuses on the extraction and biological activity research of fruiting body crude polysaccharide (CSFP). For the subsequent utilization value of Clitocybe squamulosa and to reduce resource waste, the present invention uses the water extraction residue of Clitocybe squamulosa as a raw material to explore its application as a Pickering emulsion stabilizer.

[0031] The present invention uses the water extraction residue of C. squamulosa as a raw material and prepares a novel Pickering stabilizer through ultrasonic treatment. It is found through experiments that ultrasonic treatment can reduce the particle size from 430.73 nm to 304.43 nm, make the originally agglomerated particles more dispersed, and increase the absolute charge. In addition, ultrasonic treatment results in a decrease in the contact angle, indicating that wettability does not play a dominant role in improving the emulsifying performance of the suspension. Moreover, with the increase in the suspension concentration and the proportion of soybean oil phase, the stability of the emulsion also increases. pH is also an important factor affecting the emulsion stability, and the suspension has good stability under neutral and alkaline conditions. The obtained emulsions all exhibit shear thinning and gelation behaviors. The good storage stability and thermal stability of the emulsions promote the wider application value of the emulsions. The suspension of the present invention has the characteristics of simple preparation, ecological friendliness and sustainability, and is expected to stabilize Pickering emulsions in the food system. Description of the Drawings

[0032] Figure 1 SEM images of PPCs (A), PPCNs - 20 (B) and PPCNs - 40 (C) and infrared spectra of PPC S , PPCNs - 20 and PPCNs - 40 (D).

[0033] Figure 2 The effects of different concentrations of PPCNs-40 (1), oil phase volume fraction (2), and pH (3) on the average particle size (A), viscosity (B), and rheological properties (C) of Pickering emulsions; among them, the first group (A-1, B-1, C-1) is a PPCNs-40 suspension (pH 7) with an oil phase volume fraction of 30% v / v; the second group (A-2, B-2, C-2) is a PPCNs-40 suspension with a concentration of 3 wt% and pH = 7; the third group (A-3, B-3, C-3) is a PPCNs-40 suspension with a concentration of 3 wt% and an oil phase volume fraction of 30% v / v.

[0034] Figure 3 Pictures of Pickering emulsions prepared with different concentrations of PPCNs-40 suspensions, oil phase volume fractions, and pH; among them, A1-A5 are different concentrations of PPCNs-40 (1.0%, 1.5%, 2.0%, 2.5%, 3.0%), PPCNs-40 suspension (pH 7), and an oil phase volume fraction of 30%; B1-B5 are different oil phase volume fractions (10%, 20%, 30%, 40%, 50%), PPCNs-40 suspension (pH 7), and an oil phase volume fraction of 30%; C1-C5 are different pH values (pH 6, 7, 8, 10, 12), a PPCNs-40 suspension concentration of 3 wt%, and an oil phase volume fraction of 30% v / v. Specific implementation manners

[0035] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0036] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0037] In the following examples, the fruiting bodies of Lepiota cristata were purchased from the Shanxi Engineering and Technology Research Center for Edible Fungi. The fruiting bodies of Lepiota cristata were dried and then ground with a pulverizer and passed through an 80-mesh sieve. Subsequently, the filter residue obtained after filtration by water bath extraction at 80 °C with a solid-liquid ratio of 1:20 was freeze-dried in a freeze dryer and reserved. Soybean oil was purchased from a local supermarket. The rest of the chemicals were all of analytical grade and provided by Tianjin Chemical Reagent Co., Ltd.

[0038] Example 1. Preparation and performance testing of all-natural polysaccharide and protein complex nanoparticles (PPCNs) I. Preparation of PPCNs

[0039] The freeze-dried Lepista saeva aqueous extraction residue and 5% sodium hydroxide solution were extracted at a solid-liquid ratio of 1:20 (g / mL) at 40 °C for 2 h, then filtered and the extraction was repeated twice. The three filtrates were combined and the pH was adjusted to 7 with 36% glacial acetic acid. After centrifugation at 12000 rpm / min for 10 min using a high-speed centrifuge (Heraeus Multifuge X1R USA), the precipitate was washed three times with distilled water and then freeze-dried for storage.

[0040] Accurately weigh the dried PPCs and disperse them in 5 mM, pH = 7.0 phosphate buffer solution to a concentration of 3.0 wt%, and stir magnetically for 4 h and hydrate fully overnight at 4 °C. After overnight hydration, shear for 5 minutes, then insert an ultrasonic processor (JY92-IIN, Ningbo, China) with a 6 mm diameter probe into the beaker (1.0 cm deep) and cool with an ice-water bath. The ultrasonic treatment parameters are set to an amplitude of 20% and 40%, pulse on for 3 s, off for 3 s, and continue for 10 minutes. The treated suspensions are named PPCNs-20 and PPCNs-40 respectively. Before analysis, the obtained samples are stored at 4 °C.

[0041] II. Performance testing of PPCNs

[0042] 1. Particle size, polydispersity index (PDI) and zeta potential of PPCNs

[0043] The particle size, PDI and zeta potential of freshly prepared PPCs, PPCNs-20, and PPCNs-40 suspensions were measured using a Nano-S90 dynamic light scattering particle size analyzer. The concentration of the suspension was diluted to 1 mg / ml, the refractive indices of the dispersed phase and the continuous phase (aqueous phase) were set to 1.47 and 1.33 respectively, and the absorption index was set to 0.001. All measurements were carried out at 25 °C, and each value represents the average and standard deviation (SD) of three repeated tests.

[0044] 2. Chemical composition

[0045] The polysaccharide content of PPCs, PPCNs-20, and PPCNs-40 was determined by the phenol-sulfuric acid method, the protein content was determined by the Kjeldahl method with a conversion factor of 6.25, the moisture content was determined by the national standard method GB 5009.3-2010, and the ash content was determined by the national standard method GB 5009.4-2016. All determinations for each sample were carried out three times.

[0046] 3. Infrared

[0047] PPCs, PPCNs-20, and PPCNs-40 in the range of 4000-400 cm were obtained using a TENSOR 27 Fourier transform infrared spectrometer (Beijing Bruker Technology Co., Ltd.) -1Infrared (IR) absorption spectra within the range. A sample was prepared by mixing it with KBr-disk at a ratio of 1:140.

[0048] 4. Three-phase contact angle

[0049] The PPCs, PPCNs-20, and PPCNs-40 suspensions were freeze-dried and then pressed into circular wafers several millimeters thick on an infrared press and immersed in peanut oil. Then, a drop of deionized water was dropped on each sample, and the geometry of the droplet was recorded and analyzed using an optical contact angle tensiometer (LSA100, Germany).

[0050] 5. Microscopic morphology

[0051] The morphologies of freeze-dried PPCs, PPCNs-20, and PPCNs-40 were characterized by SEM (JSM-IT800, Japan), and SEM images were taken at different magnifications.

[0052] III. Performance test results of PPCNs

[0053] 1. SEM images of PPCs, PPCNs-20, and PPCNs-40

[0054] Figure 1 Figures A - C in it are SEM images of PPCs, PPCNs-20, and PPCNs-40 respectively. It can be seen that ultrasonic treatment has a significant impact on the morphological characteristics of PPCs. From Figure 1 Figure A in it, it can be seen that PPCs are in an aggregated state, which is consistent with the research result of Chang et al. who found that non-ultrasonic-treated starch aqueous slurry is in an aggregated state. Both PPCNs-20 and PPCNs-40 are in a leaf shape, and PPCNs-40 is partially in a rod shape with a higher degree of rupture. Similarly, after ultrasonic treatment of the starch aqueous slurry, the original aggregated morphology becomes more dispersed and the particle size is smaller. Since the Pickering emulsion stabilizer is spherical with an aspect ratio of 1, while the aspect ratios of various irregular particles are always greater than 1. Particles with a high aspect ratio have the ability to wet both phases, thus enabling the preparation of Pickering emulsions with better stability characteristics. The SEM images of PPCNs partially show a leaf shape and a rod shape, so it is speculated that the aspect ratio of PPCNs is greater than 1. Particles with a high aspect ratio have the ability to wet both phases, which makes it possible to prepare Pickering emulsions with better stability performance. In addition, the emulsification efficiency of rod-shaped cellulose nanocrystals (CNCs) is higher than that of oval CNCs. It is speculated that PPCNs-40 with a partially rod shape has a higher emulsification ability than PPCNs-20.

[0055] 2. Average particle size and PDI of PPCs, PPCNs-20, and PPCNs-40

[0056] The average particle size and PDI of PPCs and PPCNs suspensions were measured using Nano - S90. As shown in Table 1, the average particle size of PPCNs was significantly smaller than that of PPCs. With the increase of ultrasonic amplitude, the average particle size of PPCNs gradually decreased. The smaller size of PPCNs may be due to the fact that ultrasound promoted the fragmentation and dissociation of PPCs. The PDI of high - quality colloidal suspensions ranges from 0.1 to 0.5, while that of low - quality samples with large particles or aggregates is greater than 0.7, indicating a very wide particle size distribution. The PDI value of PPCs without ultrasonic treatment was about 0.8, indicating a wide particle size distribution of PPCs and larger particles or aggregates (Table 1). However, after ultrasonic treatment, the PDI value decreased, indicating that PPCNs - 40 was more uniform and the large particles decreased.

[0057] 3. Surface properties of PPC, PPCNs - 20 and PPCNs - 40

[0058] Table 1 shows the zeta potential of PPCs, PPCNs - 20 and PPCNs - 40 suspensions. They were all negative, but the zeta potential value of the PPCs suspension was the lowest. With the increase of ultrasonic amplitude, the zeta potential value increased. The reason may be that ultrasound promoted the stirring of the suspension and increased the contact between PPCs and oxygen, resulting in partial oxidation of the particles and promoting the generation of surface negative charges. In the present invention, with the increase of ultrasonic amplitude, PPCNs - 40 with a high zeta potential value formed a stable layer at the oil - water interface, which was beneficial to reducing the aggregation of emulsion droplets and improving the emulsion stability.

[0059] In addition, the present invention also investigated the effect of mechanical treatment on the hydrophobicity of PPCNs through the contact angle (θ O / W ) between soybean oil and PPCNs suspension. The θ O / W values of PPCNs treated with different ultrasonic amplitudes were all less than 90°, which was related to the polysaccharide components they contained. The θ O / W values of PPCNs in Table 1 decreased with the increase of ultrasonic amplitude, which may be due to the fact that high - frequency ultrasonic cavitation increased the hydrophilicity of PPCNs. PPCNs - 40 prepared in the present invention can be used as a good stabilizer for stabilizing O / W Pickering emulsions.

[0060] 4. Chemical compositions and structures of PPCs, PPCNs - 20 and PPCNs - 40

[0061] The chemical compositions of PPCs, PPCNs - 20 and PPCNs - 40 are shown in Table 2. It can be seen that PPCs and PPCNs are mainly composed of proteins and polysaccharides. There was no significant difference in protein content with the increase of ultrasonic amplitude. The Fourier transform infrared spectra of PPCs, PPCNs - 20 and PPCNs - 40 are shown in Figure 1Figure D. 3000 - 3750 cm -1 The broad absorption peak at corresponds to the stretching vibration of hydrogen bonds (-OH), 2920 - 2850 cm -1 The two peaks at correspond to the symmetric and asymmetric stretching vibrations of saturated terminal methyl groups (-CH3), respectively. 1000 - 1400 cm -1 The wavelength reflects the stretching vibrations of -OH side chains and glycosidic bonds (C-O-C), 1700 - 1600 cm -1 and 1550 cm -1 The absorption peaks at the wavelengths correspond to the characteristic absorption peaks of protein amide I and amide II, respectively, indicating the presence of proteins in the PPCs and PPCNs samples. There are no obvious differences in the infrared curves of PPCs and PPCNs, indicating that the basic structures of proteins and polysaccharides in PPCs and PPCNs have not changed significantly due to ultrasonic treatment.

[0062] Table 1 Average particle size (Z-average), polydispersity index (PDI), and surface properties (ζ-potential and contact angle) of PPC, PPCNs-20, and PPCNs-40 suspensions (pH = 7.0)

[0063]

[0064] Table 2 Chemical compositions of PPC, PPCNs-20, and PPCNs-40

[0065]

[0066]

[0067] Example 2. Preparation and performance testing of Pickering emulsions

[0068] I. Preparation of Pickering emulsions stabilized by PPCNs-40

[0069] Pickering emulsions were prepared by a one-step emulsification method. The specific steps are as follows:

[0070] Disperse PPCNs-40 in deionized water to reach the specified concentration of 0.5 - 4% wt%, adjust the pH of the PPCNs-40 suspension to 6 - 12 using 1 mol / l hydrochloric acid and sodium hydroxide, and then mix with an appropriate volume of soybean oil to obtain a two-phase system with an oil phase volume fraction (Φ) between 10% and 60%. Each sample was treated in a JY92-IIN (ultrasonic cell disruptor) at an ultrasonic amplitude of 40% for 3 minutes to form a homogeneous emulsion. A pulse of on for three seconds / off for three seconds was used to suppress sample heating. At the same time, the sample was immersed in an ice-water bath to absorb the heat generated during the ultrasonic process, controlling the sample temperature not to exceed 25°C.

[0071] II. Testing of Pickering Emulsion

[0072] 1. Property Characterization

[0073] 1-1. Determination of Average Emulsion Particle Size

[0074] The particle size and PDI of the fresh emulsion were measured at 25 °C using a dynamic light scattering particle size analyzer (Nano-ZS90, UK). After diluting the emulsion to 1 mg / mL, the refractive index of the dispersed phase was 1.47 and that of the continuous phase was 1.33. The absorption index was set to 0.001. The measurement was carried out three times for statistical analysis.

[0075] 1-2. Optical Microscope

[0076] The microstructure of the emulsion was observed using an optical microscope (Nikon Eclipse Ni-U, Japan). The emulsion was dropped on a microscope slide, covered with a coverslip, and the excess water was blotted with absorbent paper, and then observed with a 40-fold magnifying glass.

[0077] 1-3. Rheological Properties

[0078] The rheological properties of the O / W Pickering emulsion stabilized by PPCNs-40 were analyzed at 25 °C using a rotational rheometer MCR-102 (Anton Paar GmbH, Austria). The steady-state shear test was carried out using a cone plate with a diameter of 50 mm, and the shear rate (γ) ranged from 0.1 s-1 to 100 s-1 . In addition, under the condition of an angular frequency of 10 rad / s, a strain sweep test was carried out in the range of an oscillatory strain from 0.01% to 100%. A constant linear viscoelastic strain of 5% was used. The storage modulus (G′) and loss modulus (G″) were measured in the range of 0.1 - 100 rad / s.

[0079] 2. Stability

[0080] 2-1. Storage Stability

[0081] The fresh emulsion was stored at 4 °C for 15 days, and the stability of the emulsion was monitored by visual observation. The stability of the emulsion can be estimated by the creaming index (CI) of the emulsion, and its calculation formula is (1): CI(%) = Hs / Ht × 100% (1)

[0082] where H s is the height of the supernatant layer, and H t is the total height of the emulsion.

[0083] 2-2. Thermal Stability

[0084] The fresh emulsion was stored at 4 °C for 2 h, and then the temperature of the incubator was set at 20, 40, 60, 80, and 100 °C for 30 min. After cooling to room temperature, visual observation and optical microscopy were used for comparative analysis.

[0085] 3. Statistical analysis

[0086] In this invention, ORIGIN 2021 software was used for image drawing and processing. SPSS Statistics 26 was used for statistical analysis, and analysis of variance (ANOVA) and least significant difference test (p < 0.05) were adopted. To ensure the accuracy of the experiment, each experimental group was carried out in parallel with the other three groups, and all data were expressed as mean ± standard deviation.

[0087] III. Test results of Pickering emulsion

[0088] 1. Emulsion particle size

[0089] As Figure 1 shown in Figure S in

[0090] Figure 2 Figure A in Figure 2 Figure A-1 in Figure 2 Figure 2 Figure 2In Figure A-3, when determining the concentration of PPCNs-40 and the oil phase fraction, the average particle size of the emulsion prepared with PPCNs-40 under acidic conditions is significantly larger than that under neutral and alkaline conditions. This may be because neutral and alkaline conditions increase the solubility of the proteins contained therein, thereby improving the emulsifying ability of PPCNs-40. However, the Pickering emulsion stabilized by medium-ground starch granules shows good stability under neutral conditions, and when modified to acidic and alkaline conditions, the emulsion droplet size increases. While the droplet size of the Pickering emulsion prepared with citrus fiber under acidic and alkaline conditions decreases significantly, indicating that different types of materials have different sensitivities to pH values, and the average particle size of the Pickering prepared within different pH value ranges is also different.

[0091] 2. Microscopic Morphology

[0092] Microscope images of PPCNs-40 suspensions with different concentrations and pH values and Pickering emulsions with different oil phase fractions are as Figure 3 shown. Most of the Pickering emulsion droplets are spherical. In Figure 3 Figure A, as the concentration of PPCNs-40 increases, the droplet size of the Pickering emulsion decreases and becomes more uniform. This may be because PPCNs-40 has a strong covering ability at the oil-water interface, which can protect the dispersed oil droplets from agglomeration. In Figure 3 Figure B, as the proportion of soybean oil increases, the droplet size of the emulsion decreases, but when the oil phase is greater than 30%, the droplet size increases. This may be because the concentration of PPCNs-40 in the emulsion is relatively low and is not sufficient to completely cover the surface of the oil droplets, resulting in flocculation and aggregation of the oil droplets. In Figure 3 Figure C, the Pickering emulsion droplets prepared from the PPCNs-40 suspension with a pH value of 6 have various shapes, and the aggregation of the oil droplets leads to an increase in the average particle size of the emulsion. When the pH value is 7-12, the emulsion prepared from the PPCNs-40 suspension is spherical and evenly distributed. These observations are consistent with the average particle size, indicating that the Pickering emulsion prepared after ultrasonic treatment with a pH value of 7, a PPCNs-40 suspension (3 wt%), and an oil phase volume fraction of 30% has a smaller particle size and a more uniform distribution.

[0093] 3. Rheological Properties

[0094] The study of the rheology of Pickering emulsions can not only provide in-depth understanding of their flow characteristics but is also crucial for processing applications. As can be seen from Figure 2 Figure B, as the shear rate increases, the viscosity of all emulsions shows a downward trend, presenting non-Newtonian fluid and shear thinning phenomena within the test range. In Figure 2In Figure B-1, the viscosity of the emulsion increases with the increase in the concentration of the PPCNs-40 suspension. As the concentration of PPCNs-40 increases, more nanoparticles surround the oil droplets, forming a three-dimensional network structure, thus increasing the viscosity of the emulsion. In Figure 2 In Figure C-1, throughout the frequency range, G' is always greater than G", and the emulsion exhibits typical gel properties. At the same frequency, G' and G" increase with the increase in the concentration of PPCNs-40, indicating that the increase in the concentration of PPCNs-40 enhances the network structure of the emulsion and further improves the stability of the emulsion. In Figure 2 In Figure B-2, increasing the proportion of oil makes the Pickering emulsion droplets pack more densely and increases the viscosity of the emulsion. In Figure 2 In Figure C-2, when the oil phase is between 10% and 50%, G' is always greater than G", indicating that the emulsion remains gel-like within this range. The rheological properties of the Pickering emulsion prepared from the PPCNs-40 suspension show irregular changes at different pH values ( Figure 2 Figure B-3 and Figure 2 Figure C-3). However, the emulsion exhibits typical gel-like non-Newtonian fluid properties within all pH ranges.

[0095] 4. Emulsion stability

[0096] To determine the stability of Pickering emulsions prepared from PPCNs-40 suspensions during storage, a certain degree of phase separation occurred when the emulsions were left standing, and a clear water layer appeared below the emulsions. The larger the clear water layer, the less stable the emulsion. The results showed that as the concentration of PPCNs-40 increased, the CI value of the Pickering emulsion decreased, but it still had good stability after long-term storage. As the oil phase fraction increased, the CI of the Pickering emulsion gradually decreased, indicating that the stability of the emulsion gradually increased. Similarly, when the oil-water mass ratio varied between 1:9 and 5:5, the CI of the Pickering emulsion stabilized by pH-responsive cellulose nanoparticles gradually decreased, indicating that the emulsion stability gradually increased. The results showed the emulsion CI of Pickering emulsions stabilized by PPCNs-40 suspensions with different pH values after being stored at room temperature for 15 days. In the first 3 days of storage, the Pickering emulsion prepared from the suspension under acidic conditions was extremely prone to stratification, and the CI increased with the prolongation of storage time. The emulsions prepared with PPCNs-40 suspensions under neutral and alkaline conditions had good storage stability, with slight stratification occurring after 10 days and remaining stable after 30 days. A brown layer appeared at the bottom of the emulsion prepared with the suspension under strongly alkaline conditions after 5 days of storage, which might be due to the deposition of PPCNs-40 particles. Similarly, the Pickering emulsion stabilized by alkali-extracted peanut polysaccharide-protein complex also showed a turbid water layer related to the excess particles in the emulsion after 20 days.

[0097] The results showed the effect of temperature on the stability of PPCNs-40 stabilized emulsions. As the temperature increased (20 °C - 100 °C), the droplet size of the PPCNs-40 stabilized emulsion increased. In addition, no foaming or oil separation occurred in the emulsions after heat treatment, which meant that the PPCNs-40 stabilized emulsions could be subjected to heat treatments such as steaming, pasteurization, and sterilization.

[0098] The present invention provides a novel nanoparticle, which can be used as an excellent Pickering stabilizer. The nanoparticle can be easily prepared from the residue after hot water extraction of *Pholiota squarrosa* by ultrasonic treatment. The nanoparticle mainly composed of polysaccharide and protein has different shapes according to different ultrasonic amplitudes. Among them, PPCNs-40 shows a dispersed leaf-like and rod-like shape, with a relatively small average particle size and a relatively large zeta potential, having great potential for preparing oil-in-water (O / W) Pickering emulsions. All Pickering emulsions exhibit shear-thinning and gel-like behaviors. The concentration of PPCNs-40 and the proportion of soybean oil have a great influence on the average particle size, microstructure and viscosity of the emulsion. In addition, the pH value is also an important factor affecting the stability of the emulsion. The emulsion has better stability under neutral and alkaline conditions. When the concentration of PPCNs-40 is 3 wt%, the pH value is 7, and the volume fraction of soybean oil is 30%, the Pickering emulsion has good storage stability and thermal stability.

[0099] The present invention not only has important significance for the development of environmentally friendly nanoparticles for stabilizing Pickering emulsions, but also provides a strategy for improving the added value of *Pholiota squarrosa*.

Claims

1. A method for preparing a suspension containing polysaccharide-protein complex nanoparticles, comprising the following steps: S1. Extract the freeze-dried aqueous extract residue of Lepiota cristata with an alkali solution to obtain a filtrate; S2. After adjusting the pH value of the filtrate to 6.8 - 7.3, centrifuge to obtain a precipitate; S3. Disperse the precipitate in a phosphate buffer solution, hydrate and shear, and then perform ultrasonic treatment to obtain the suspension.

2. The preparation method according to claim 1, characterized in that: In step S1, the alkali solution is an aqueous sodium hydroxide solution with a mass concentration of 4.5 - 5.5%; The ratio of the aqueous extract residue of Lepiota cristata to the alkali solution is 1:10 - 30 g / mL; The extraction temperature is 35 - 45 °C, and the time is 1.5 - 2.5 h; Repeat the extraction 2 - 3 times.

3. The preparation method according to claim 1 or 2, characterized in that: In step S2, adjust the pH value with glacial acetic acid; The precipitate is freeze-dried and then dispersed; In step S3, the concentration of the phosphate buffer solution is 3.0 - 7.0 mM, and the pH value is 6.5 - 7.2; The mass concentration of the precipitate in the phosphate buffer solution is 2.5 - 3.5%; After hydrating overnight, shear for 4 - 6 min.

4. The preparation method according to any one of claims 1-3, characterized in that: In step S3, the ultrasonic treatment is carried out in an ultrasonic processor under the condition of an ice-water bath; The conditions of the ultrasonic treatment are as follows: The amplitude is 20% - 40%, the pulse is turned on for 1 - 3 seconds, turned off for 1 - 3 seconds, and lasts for 5 - 15 minutes.

5. A suspension prepared by the method according to any one of claims 1 - 4.

6. Use of the suspension according to claim 5 in the preparation of Pickering emulsion as a stabilizer.

7. A Pickering emulsion containing the suspension according to claim 5.

8. The Pickering emulsion according to claim 7, wherein: The volume fraction of the oil phase in the Pickering emulsion is 10 - 60%.

9. A method for preparing the Pickering emulsion according to claim 7 or 8, comprising the following steps: Disperse the suspension in water, adjust the pH value to 6 - 12; then mix with the oil phase and obtain the Pickering emulsion by ultrasonic treatment.

10. The preparation method according to claim 9, characterized in that: The mass concentration of the suspension in water after dispersion is 0.5 - 4%; Adjust the pH value with hydrochloric acid and sodium hydroxide; The oil phase is soybean oil or palm oil.

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