Soy dietary fiber/soy protein isolates nanoparticles to stabilize Pickering emulsion for astaxanthin delivery

Soy dietary fiber/soy protein isolates nanoparticles to stabilize Pickering emulsion for astaxanthin delivery | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Soy dietary fiber/soy protein isolates nanoparticles to stabilize Pickering emulsion for astaxanthin delivery Yue Qiu, Feifan Fu, Jiajun Chen, Aodong Yue, Tingting Yang, Ligen Zou, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6808307/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 18 You are reading this latest preprint version Abstract Astaxanthin (AST), a powerful antioxidant, faces challenges due to its susceptibility to decomposition, poor solubility and low bioavailability. In this study, green edible plant-Pickering emulsion was prepared by soy dietary fiber (SDF) and soy protein isolate (SPI) complex nanoparticles (SSPs) and used for AST delivery. The SSPs characterized by their negatively charged fibrous rod-like structures, are primarily held together by hydrogen bonding. When the SDF/SPI mass ratio of SSPs is closed to 1:1, it exhibit good dispersibility and water-oil amphiphilicity. The storage stability test and rheological measurement showed that Pickering emulsions stabilized by SSPs have high viscosity and fluid stability, with a higher creaming index than the emulsions by separate SDF/SPI material. DPPH and ABTS free radical scavenging of AST in the 1:1 ratio of SDF and SPI stabilized Pickering emulsion, were higher( 65.5 ± 5.7% and 97.6 ± 0.5% respectively). In vitro digestion indicated that the bioaccessibility and chemical stability of AST in Pickering emulsion was 20.56% and 22.34% higher than free astaxanthin in oil (AST-O) group. Thus, Pickering emulsion stabilized by SDF/SPI complex particles is a potential delivery system for AST in the food industry. Pickering emulsion Astaxanthin Anti-oxidation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Astaxanthin (AST), a kind of β-carotene with strong antioxidant properties. It also possesses biological activities such as anti-inflammatory and anti-cancer and widely used in food and medicine (Chen et al., 2024) . Astaxanthin has been widely extracted from red algae, shrimp and crab shells. However, AST application is restricted due to low bioavailability, poor water solubility and chemical instability ( Zhou et al., 2018 ). Currently, many methods were being adopted to improve the utilization of astaxanthin, including micro-encapsulation (Zhang et al., 2021 ), liposomes (Pan et al., 2018) , nanoparticles ( Zhang et al., 2021 ) and emulsions. Emulsions are now widely used because of easy production, low cost and high stability. Pickering emulsion is a new type of emulsion that replace traditional emulsifiers with solid, insoluble colloidal particles. Additionally, Pickering emulsion is freeze-thaw stable, resistant to oxidation, and resistant to Ostwald Ripening ( Zhang et al., 2021 ). Since Pickering emulsion is made of combination of compact nanoparticle materials, the gap between them facilitates slow-release of encapsulated actives and allow for precise delivery. By dispersing and adsorbing nanoparticles at the interface of the immiscible two-phase interface and inhibiting droplet aggregation, a stable emulsion system is successfully formed for the encapsulation and delivery of biologically active substances (Zhang et al., 2022) . Pickering emulsion has been used successfully to encapsulate polyphenols and β-carotenoids. Du used cinnamic acid-modified acid-ethanol hydrolyzed starch-stabilized Pickering emulsion to encapsulate curcumin with 96.2% encapsulation rate, and better bioavailability and antioxidant activity ( Du et al., 2024 ). Ge et al. (2022) prepared Pickering emulsion stabilized by zein/Adzuki bean seed coat polyphenol nanoparticles to enhance the stability and bioaccessibility of astaxanthin. Nowadays, more green edible materials such as polysaccharides and proteins are being used to stabilize Pickering emulsions. Polysaccharides can act as a barrier against enzymatic degradation, while proteins can provide affinity for binding bioactive compounds with strong emulsifying activity (Hou et al., 2022) . Soy dietary fiber (SDF) is one kind of polysaccharide mainly extracted from plants that cannot be digested and absorbed by the body (Liu et al., 2024) . SDF, extracted from soybean processing by-products, contains hydroxyl and carboxyl groups, providing excellent gelling and emulsifying properties. These features enable SDF to enhance food texture and stability, making it highly promising for food industry applications (Yang et al., 2024) . Soy protein isolate (SPI) is one of the most frequent and widely used proteins in food because of its low cost and relatively balanced nutritional profile ( Pinho et al., 2024 ). At the same time, SPI is an organic polymer consisting of a series of peptide-bonded amino acids that rely on their amphiphilicity and thermally induced self-assembly properties to act as novel Pickering emulsion stabilizers (Ezo et al., 2022) . To the best of our knowledge, the application of soy dietary fiber (SDF) and soy isolate protein (SPI) complexes as solid particle emulsifiers for encapsulated delivery of AST has not been explored. The purpose of this study was to use the SDF-SPI nanocomplex as a stabilizer to prepare Pickering emulsions that encapsulated and delivered AST. The microstructure, chemical bonding, and hydrophilicity of SDF/SPI with varying complex particle ratios were characterized to explain the effect of composite particles on emulsion stability. Additionally, the storage stability and centrifugal stability of Pickering emulsions was determined. Furthermore, after simulating in vitro digestion, the bioavailability and chemical stability of AST was determined. This study could facilitate the design of a novel delivery system based on Pickering emulsions to encapsulate and protect bioactive for various applications. 2. Materials and methods 2.1 Materials Soy dietary fiber was obtained from Henan Wanbang Industrial Company, Zhengzhou, China. Soy protein isolate was provided by Rhawn, shanghai, China. Soy oil was purchased from Yihaikerry Co., ltd, Shanghai, China. Astaxanthin was obtained from Shanghai Macklin Biochemical Co., ltd, Shanghai, China. All other chemical reagents used were of analytical grade. 2.2 Preparation of SDF/SPI complex particles (SSPs) Mixed with different SDF:SPI (m/m) ratios of 3:1, 1:1, and 1:3, 4g mixed particles were selected and named as SSPs 3:1, SSPs 1:1, and SSPs 1:3, respectively.The 4 g separate SDF and SPI were prepared as controls. And then each of the five sets of samples was added to 100 mL of ultrapure water. The aqueous samples were then homogenized (IKA, Germany) at a high speed of 12,000 rpm for 5 min and ultrasound by sweeping frequency auto-tracking ultrasonic (Shanghai Fangxu Co., ltd, Shanghai, China) at 500 W for 5 min to prepare nanoparticles. 2.3 Characterization of nanoparticles 2.3.1 Scanning electron microscope (SEM) observation Morphology of SDF, SPI, and SSPs with different ratio mass was evaluated by SEM (S-4700 II, Hitachi, Tokyo, Japan) as described previously with minor modification (Liu et al., 2018) . In short, after freeze-drying and preserving the samples were coated with Pt-palladium (plasma deposition method) and imaged under an accelerating voltage of 5.0 kV. 2.3.2 Particle size and polydispersity index (PDI) The mean droplet size and PDI of SSPs and control groups (0.1%, w/w) were obtained by a Zetasizer Nano ZS instrument (Malvern Panalytical Ltd, 3000HSA, UK) under 25 ℃ with a balancing time of 2 min. The refractive index and absorption index were 1.59 and 0.001, respectively. 2.3.3 Three-phase contact angle test The three-phase contact angle (θ) was measured by droplet shape analyzer DSA25 (KRüSS, Germany), static water-in-air contact angle was measured by dropping water droplets (2 µL) into particle film according to the method of Zhang et al. (2023) with minor modification. 2.3.4 Fourier transform infrared spectroscopy (FTIR) FTIR was performed in a Nicolet6700 spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Briefly, SPI, SDF, and SSPs were respectively mixed with KBr in the mass ratio of 1:100 and pressed into pellets, which were then submitted to FTIR analysis using a scanning range of 500–4000 cm − 1 . 2.4 Preparation of Pickering emulsion The oil phase (AST-O) was prepared as follows: astaxanthin was dissolved in soybean oil (0.5 mg/mL) and stirred for 30 min, and then 25 mL of oil phase was added to 25 mL aqueous samples mentioned in section 2.2 , respectively. The mixed samples were then homogenized (IKA, Germany) at a high speed of 12,000 rpm for 5 min and ultrasound by sweeping frequency auto-tracking ultrasonic (Shanghai Fangxu Co., ltd, Shanghai, China) at 500 W for 5 min. 2.5 Characterization of SSPs-stabilized Pickering emulsions 2.5.1 Mean particle and zeta-potential determination The mean particle size and zeta potential were measured using a Zetasizer Nano ZS instrument (Malvern Panalytical Ltd, 3000HSA, UK) at 25°C and the balancing time was 2 min according to the method previously described (Sun et al., 2019) . The refractive index of water (dispersed phase) was 1.59, absorption index was 0.01. 2.5.2 Rheological measurements The rheological properties were determined by a rheometer Discovery HR20 rheometer (TA Instruments, New Castle, USA) with parallel plate geometry (60 mm diameter, 1 mm gap). The temperature was set to 25°C and the emulsions were placed on the rheological plates 5 min in advance in order to reach thermal equilibrium. Regarding the dynamic oscillation experiments, the linear viscoelastic region of the Pickering emulsion was first determined using dynamic strain scanning, which were performed at a fixed strain amplitude of 1% (within the linear viscoelastic region) to collect the storage modulus (G′) and loss modulus (G″) of the emulsions in the frequency range of 0.1–100 rad/s. The dynamic frequency scanning experiments were performed at a fixed strain amplitude of 1% (within the linear viscoelastic region). Steady-state flow measurements were also performed to record the apparent viscosity and shear stress of Pickering emulsions as a function of shear rate (0.1–100 s − 1 ). 2.6. Stability of Pickering emulsions 2.6.1. Storage stability The storage stability of Pickering emulsions prepared by different SDF/SPI mass ratios were studied by recording the appearance changes of Pickering emulsion on 0, 7, 14, 21, 28 days. The Creaming Index (CI) was calculated by formula (1). CI = Hs/Ht × 100% (1) Where Hs and Ht are water layer height (mm) and emulsion height (mm), respectively. 2.6.2. Centrifugal stability 8.0 mL Pickering emulsion was added to a 10.0 mL centrifuge tube and was centrifuged at 11000 × g for 10 min. The height of the emulsion and water layer were recorded respectively and the centrifugal stability (%) was calculated after centrifugation of the emulsion according to formula (2). Centrifugal stability = 1-(Hs/Ht) × 100% (2) 2.7 Antioxidant activity 2.7.1 DPPH scavenging activity AST loaded-Pickering emulsion 1 mL was mixed with 2 mL of DPPH solution (0.1mM, m/w). The above sample was reacted in the dark for 30 min, and the absorbance was measured at 517 nm (Xu et al., 2021) . The DPPH scavenging activity of the AST loaded-Pickering emulsion was calculated by the following formula (3) DPPH scavenging activity (%) = (A DPPH -A sample )/A DPPH × 100 (3) Where A DPPH is the absorbance of the DPPH at 517 nm, and A sample is the absorbance of sample at 517 nm. 2.7.2 ABTS radical scavenging activity The ABTS scavenging activity was studied by the method of Zhang et al. (2023) with slight modifications. The ABTS radical solution was first prepared with equal volumes of 7.4 mM ABTS and 2.6 mM aqueous potassium persulphate and then left in the dark for 12 hours. After that, this solution was then diluted with methanol until the absorbance value at 734 nm reached 0.7 ± 0.1. The ABTS radical scavenging activity was analyzed by mixing 1 mL of emulsions with 2 mL of ABTS solution. The above solutions were reacted in the dark for 11h. The antioxidant activity was calculated by the following formula (4): ABTS scavenging activity (%) = (A ABTS -A sample )/A ABTS ×100 (4) Where A ABTS is the absorbance of the ABTS at 734 nm, and A sample is the absorbance of sample at 734 nm. 2.8 In vitro simulated digestion The method of in vitro simulated digestion was referred to the method of Shah et al. (2016) with little modifications. Simulated gastric fluid (SGF) was obtained by dissolving 38.0 mg NaCl and 60.8 mg pepsin in 19 mL of 0.05 M PBS buffer. The emulsion (1 mL) was mixed with 19.0mL of SGF and the pH was adjusted to 2.5. And the SGF mixture solutions were stirred at 150 rpm in the water-bath shaker for 2 h at 37°C. The simulated intestinal fluid (SIF) was obtained by dissolving 656 mg NaCl, 82.9 mg CaCl 2 , 120 mg porcine lipase, 120 mg porcine trypsin, and 378 mg bovine bile in 20.0 mL of 0.05 M PBS buffer. The pH of the SGF mixture solutions was first adjusted to 7.0 by using 1.0 M NaOH to terminate gastric digestion. And then, the above solutions were mixed with 19.0 mL of SIF and stirred at 150 rpm in the water-bath shaker for 4 h at 37°C. 2.8.1 Free fatty acid (FFA) release During intestinal digestion, the pH of the system was maintained at 7.0 by addition of 100 mM NaOH. The consumption of NaOH was recorded and used for calculating the release amount of FFA according to formula (5) (Huang et al., 2021) : FFA (%) = (V NaOH (t) × M NaOH × M WTG )/ (m TG × 2) × 100% (5) Where V NaOH (t) is the volume (mL) of NaOH required to maintain pH 7.0 at time t, M NaOH is the molar concentration (mol/L) of NaOH required for titration, M WTG is the average molecular weight (g mol − 1 ) of corn oil, and m TG is the total mass (g) of soy bean oil in the system. 2.8.2 AST bioaccessibility The bioaccessibility of AST was determined by the method of Zhang et al. (2024) with modifications. The simulated digested liquid was centrifuged at 10000 × g for 20 min. The AST was extracted by mixing 0.5 mL of the lower micelle layer after centrifugation with 4.5 mL dichloromethane/methanol mixed solution 2:1, v/v). And then the solution was centrifuged at 5550 × g for 10 min. The content of AST in micelles after in vitro digestion was calculated by the standard curve. The bioaccessibility of AST was calculated by formula (6). The chemical stability was calculated by comparing the astaxanthin content in the crude digested phase with the initial emulsion by formula (7). Bioaccessibility (%) = C Micelles /C Initial × 100 (6) Chemical stability (%) = C Emulsion /C Intitial × 100 (7) Where C Micelles is the concentration of AST in the micelle fraction, C Initial is the AST concentration calculated from the initial addition, and C emulsion is the AST concentration in the crude digestive phase. 2.9 Statistical analysis The Duncan's multiple range test with SPSS 26.0 (IBM Corporation, NY, USA) was used to statistically analyze the various treatments, different letters (a, b, c, d, e, f) represent significant differences between groups ( p < 0.05). All charts were draw by using Origin 2024 (Origin Lab, USA). Each experiment was performed in triplicate and the results were expressed as mean ± standard deviation (SD). 3. Results and discussion 3.1 Characterization of SSPs 3.1.1 The microtopography of SSPs SSPs prepared by ultrasound-assisted high-speed homogenization and dispersion were characterized by SEM at 1000 × magnification (Fig. 1 . a-e ). As shown in Fig. 1 a and 1 e, SDF and SPI showed a smooth surface lamellar stacking structure. The appearance of bands and ellipsoids can be seen in SSPs (Fig. 1 . b-d ), indicating that different degrees of cross-linking and associations between the composite particles have occurred. The nanoparticles do not have distinct boundaries between them, but are connected together to form stable particles. Compared with SSP 3:1 and SSP 1:3, SSP 1:1 showed a more ordered and plump structure.When SSPs 1:1 was observed under 10,000 × magnification ( Fig. 1 . f ), the shape of rounded and full-bodied nanoparticles can be seen as they aggregate and combine to form a stable structure. 3.1.2 Mean particle size, PDI and zeta-potential test The mean particle size, PDI and zeta-potential test of SSPs prepared by different SDF/SPI ratio were shown in Fig. 2 a and 2 b, respectively. After 5 min high-speed homogenization and 500 W ultrasonication, the particle size of SSPs can be controlled in the range of 400–800 nm. The particle size can also reflect the strength of the interaction force between the polysaccharide and protein complexes (Gao et al., 2023) . As the proportion of SPI in the complex particles increased, the particle size decreased. The largest particle size 1328.33 ± 103.2 nm was observed in SDF group, while SSPs 1:3 group showed smallest particle size of 453 ± 31.6 nm. Recently, it has been shown that nanoparticles with small and uniform particle size are ideal for stabilizing Pickering emulsions. PDI reflects the degree of dispersion in the system, the smaller the value, the higher the degree of dispersion of the particles in the solution. It can be seen that SSPs 1:1 and SSPs 1:3 groups are more evenly dispersed than others in Fig. 2 a. Zeta-potential is a measure of the point bilayer around a particle in solution, with higher absolute values of zeta-potential indicating stronger electrostatic repulsion between particles (Herrman et al., 2022) . As shown in Fig. 2 b, The SDF, SPI and SSPs are negatively charged, with the SDF and SPI having the higher negative charge absolute value and the SSPs having a reduced charge value in comparison. This may be due to the fact that the electrostatic interaction between the composite particles neutralizes the charge values. And the electrostatic interactions became a key factor for stabilization. The phenomenon of negatively charged complexes of myofibrillar proteins and pectin was also studied by Chen et al. (Chen et al., 2022) . 3.1.3 Three phase contact angle Since the behavior of particles on oil-water surfaces affects the type and stability of the Pickering emulsion, the wettability of particles was also assessed by measuring the three-phase contact angle (θ) (Xiao et al., 2016) . The complex particles are hydrophilic when the contact angle is less than 90°, and the opposite means that they are lipophilic. When the contact angle reaches 90°, the stabilization barrier reaches the highest, the hydrophilicity and lipophilicity reach a balance, and the system formed is more stable (Abdullah et al., 2020) . As shown in Fig. 3a , when SDF was dispersed in the water phase, the hydrophobic groups were reunited inward, while the hydrophilic groups were exposed externally, which made the particles being in a hydrophilic state with a contact angle of 72.0 ± 0.5°. SPI was hydrophilic and the contact angle was 75.2 ± 0.4°. The three-phase contact angle raised to 81.0 ± 0.6°. The reason for the decrease of hydrophilicity of SSPs may be that the combination of SPI and SDF exposes the internal hydrophobic groups.when the ratio of SDF and SPI reached 1:1, suggesting they are amphiphilic and suitable for stabilizing Pickering emulsion. This result is consistent with the relationship that Ni et al. (2023) found between the hydrophilicity of the composite particles and the percentage of polysaccharide protein complexes. 3.1.4 FTIR FTIR is an effective method for analyzing the structure of materials, which can reflect the functional group information of samples (Wang et al., 2024) . As shown in Fig. 3b , all emulsion revealed strong absorption peaks in both the amide I region (1600–1700 cm − 1 ) and amide II region (1480–1575 cm − 1 ), which were indicative of the secondary structure of protein, and could be used to analyze the interaction between SPI and SDF ( Jiang et al., 2019 ).Compared to SDF and SPI, the peak of Osingle bondH stretching vibration in SPPs nanoparticles was shifted,which confirmed that hydrogen bonds were formed between SDF and SPI. The wavelength around 3440 cm − 1 is the peak of the O-H stretching vibration in cellulose and hemicellulose, indicating hydrogen bonding, which is closely related to water wettability. The O-H peaks of SDF and SPI were 3431.76 cm − 1 and 3442.70 cm − 1 , respectively. When the proportion of SDF in the SSPs decreased, the absorption bond was blue-shifted, which may be due to hydrogen bond breaking (Zhang et al., 2023) . The stretching vibration peak at 1630 cm − 1 – 1670 cm − 1 indicating that the combination of C = O bonds formed between SPI and SDF (Lyu et al., 2021) . The absorption peak at 950–1200 cm − 1 is considered to be the region of carbohydrates. SPI has a weaker peak there whereas the peaks of SSPs were evident. It was suggested that hydrogen bonding interactions participated in the formation and stability of SSPs [33] . 3.2 Stability of Pickering emulsion 3.2.1 Storage stability of Pickering emulsion The appearance changes of astaxanthin dissolved in oil (AST-O) and Pickering emulsion were shown in Fig. 4a-e . After 28 days, AST gradually precipitated from the oil phase in AST-O group. The color of the solution gradually changed from red to yellow, which indicated that oxidative decomposition had occurred. In contrast, the color change of Pickering emulsion embedded with astaxanthin was not significant. According to Fig. 4f , CI% of SSPs 1:3 stabilized Pickering emulsion (2.78 ± 0.48%) was much lower than that of SDF stabilized emulsion (7.72 ± 0.58%) and SSPs 3:1 (8.67 ± 0.33%) after 28 days of storage. These results indicated that SSPs 1:3 stabilized astaxanthin Pickering emulsion was the most stable. A similar study was conducted by Yang et al. (2022) to determine the storage stability of Pickering emulsion embedded with astaxanthin stabilized by composite particles of chitosan and guar gum. The stability of Pickering emulsions has also been demonstrated in the study. 3.2.2 Centrifugal stability Centrifugal stability also reflects the stability of the emulsion under centrifugal force at higher rotational speeds (Fig. 5a) . The force exerted by the centrifugal treatment causes the colloidal particles to be dislodged from the surface of the oil droplets, leading to their precipitation. The index of centrifugal stability based on the height of the water layer and emulsion separated was calculated after centrifugation. Ultimately, it was found that SSPs 1:1 and SSPs 1:3 exhibited high centrifugal stability, 67.2 ± 3.2% and 75.9 ± 3.2%, respectively. This result further demonstrated the stability of Pickering emulsions stabilized by SSPs with higher SPI fractions. 3.2.3 The mean particle size and zeta potential of emulsions The stability of the emulsions was further characterized by recording the mean particle size and zeta potential of the emulsions from 0–28 days. According to Fig. 5b , the mean particle size of the emulsions increased with the increase of storage time. This phenomenon indicates that after 28 days of storage, the Ostwald’s ripening may occur in emulsions. The force between small droplets was weakened, leading to droplet aggregation and a subsequent increase in emulsion particle size. Among them, the SSPs 1:1 group showed excellent stability and resistance to aggregation, with a particle size control of 1353.7 ± 78.9 nm at day 28, which was lower than that of 2,669.7 ± 88.7 nm in the SSPs 3:1. The zeta-potential is one of the effective indicators for evaluating electrostatic interactions (Zhai et al., 2018) . As shown in Fig. 5c , the zeta potential of SSPs 1:1 and SSPs 1:3 groups were stable at -30 mV to -35 mV after the storage period. Stronger electrostatic interactions counteracted the aggregation of the emulsions, resulting in a uniform droplet size of the emulsions. The result of zeta-potential is consistent with the particle size test described above. 3.3 Rheological measurements of Pickering emulsions The relationship between SDF/SPI mass ratio and emulsion rheology was studied. The viscosity of the emulsion decreased with an increase in shear rate ( Fig. 6a ) which suggested that emulsions stabilized by SSPs were non-Newtonian fluids. When the viscosity of the emulsion is high, the resistance to movement of the droplets increases and the collision frequency between droplets decreases, thus reducing the possibility of droplet aggregation and coalescence, which helps to improve the physical stability of the emulsion. G′ reflects the elastic solid-like character of the material, whereas G″ indicates the viscous response.According to the results of the rheological tests, the storage modulus (G′) was greater than the loss modulus (G′′) for all groups, which indicated that the an elastic-like structure was formed (Zhu et al., 2020) . The SSPs 1:1 group emulsion had the highest viscosity and both the G′ and G′′were higher compared to other groups, which demonstrated good stability in frequency scanning and oscillation ( Fig. 6b,c ). This indicates that the emulsion is most stable at this complex concentration condition. This indicator is also consistent with the storage stabilization described above. When the ration of SDF/SPI reaches 1:1, the gel strength of SSPs stabilized emulsion increased significantly, indicating that SSPs plays an important role in the formation of gel-like structures. Similar to viscosity, the increased G′ in emulsion stabilized by SSPs can be attributed to the small oil droplet size, which enhanced the interactions between oil droplets (Zou et al., 2018) . 3.4 Antioxidant activity DPPH free radical scavenging and ABTS free radical scavenging are the most common methods for evaluation in vitro antioxidant activity (Zhang et al., 2022) . As shown in Fig. 6d , DPPH scavenging activity of SSPs 3:1 and SSPs 1:1 groups were 65.5% and 52.6%, respectively. ABTS scavenging activity of SSPs 3:1 and SSPs 1:1 groups were 97.6% and 96.25% respectively, which were higher than free AST in AST-O (33.7% and 40.3%, respectively), which is consistent with the stability of emulsions. The SSPs stabilized Pickering emulsions encapsulated AST, which can delay the contact of AST with the external environment and slow down the rate of oxidative deterioration (Foti, 2015) . In addition, the excellent DPPH and ABTS radical scavenging rates suggest that the synergistic encapsulation of SDF and SPI facilitates the optimal antioxidant effect of astaxanthin in Pickering emulsion systems. And this property also offers the possibility of subsequent application of the emulsions in the development of functional foods. 3.5 In vitro simulated digestion 3.5.1 Digestive Stability In vitro simulation of gastrointestinal digestion has been widely used as a tool to study the biochemical changes that occur in food and drug compounds during their passage through the gastrointestinal tract. After the intestinal digestion, the droplet size of the SSPs 1:1 and SSPs 1:3 groups were smaller than that of the separate SPI and SDF particle-stabilized Pickering emulsion. This phenomenon was verified in the average particle size determination after the intestinal digestion in Fig. 7a , where the mean particle sizes of the SSPs 3:1 and SSPs 1:1 groups determined at 492.8 nm and 548.1 nm, respectively. The particle sizes above were lower than 3,237 nm in the SDF group and 1425.2 nm in the SPI group. Regarding the capacity to maintain low droplet sizes, this could be because of the molecular interactions and hydrogen bonding that create a repulsive effect on the merging aggregation among droplets among combinations of SSPs composite particles. The microstructural changes of the Pickering emulsion during in vitro digestion are shown in Fig. 7c . The trend of emulsion droplet size increased due to breakage and droplet aggregation in an acidic environment during the gastric digestion stage. Smaller droplets had a higher specific surface area, which could better interact with enzymes in the digestive juice ( Chen et al., 2019 ). Additionally, Guo et al. (2022) suggested that that large oil droplets may reduce the effectiveness of contact and reaction with digestive enzymes. Zeta-potential was tested to determine the electrostatic force between emulsions and to verify one of the driving forces that keep emulsions stable. When emulsions went through the gastric digestion phase, the aboslute of zeta-potential decreased because of the acidic environment in stomach ( Fig. 7.b ).Finally, at the completion of the intestinal digestion phase,emulsions return to pH 7, and the zeta-potential reverts to between − 30 to -50 mV. The emulsion droplets are held in the lower range by electrostatic forces. This test verified the reason for the emulsion size change in the particle size test described above as well as in the microscopic observations. Additionally, an earlier investigation showed that the chitosan-caseinophosphopeptide complex's irreversible adsorption at the oil-in-water interface created a densely packed, stiff layer that surrounded the oil droplets and stabilized the emulsions' anti-coalescing action (Huang et al., 2019) . 3.5.2 FFA release FFA is a surfactant that facilitates the conversion of emulsion-loaded bioactive compounds into micelles. The surface structure and structural properties of the emulsion droplets are the main factors affecting lipid digestion. During simulated intestinal digestion, the hydrophobic core of AST was exposed due to structural destruction of the emulsion in simulated intestinal fluid (Yang et al., 2021) . The emulsions were digested faster during the first 60 min, then the rate of lipolysis gradually decreased and reached a maximum at 240 min, indicating that the soybean oil emulsion loaded with AST was broken down by the digestive solution. According to the Fig. 8a , the highest FFA release rate (78.3. ± 3.65%) was observed in SSPs 1:1 group. The release of FFA indicates the degree of lipid digestion in the emulsion, and the higher the FFA release rate, the more the emulsion was digested and broken down during digestion in the small intestine ( Zhang et al., 2021 ). 3.5.3 Bioaccessibility and chemical stability The bioaccessibility of AST in emulsions was evaluated ( Fig. 8b ). The SSPs 1:1 group had the highest bioaccessibility and chemical stability of all the groups, 62.5 ± 2.3% and 44.9 ± 0.4%, respectively. The reasons for this may be as follows: (i) the decrease in droplet size and the increase in specific surface area at SPI/SDF ratios close to 1:1 may facilitate the digestion of the oil droplets and the AST transferred from the droplets to the digestive solution; (ii) the three-dimensional network structure of the SSPs inhibits the coalescence and flocculation of the lipid droplets, thus increasing the chance of lipase and bile salts entering the surface of the oil droplets and increasing the amount of astaxanthin transferred from the oil phase to absorbable micelles [45] . 4. Conclusion In summary, a strategy for stabilization of Pickering emulsion delivery of AST by SPI/SDF composite nanoparticles is proposed by constructing composite nanoparticles with different mass ratios. The results showed that the composite nanoparticles of SSPs 1:1 and SSPs 1:3 groups have better water-oil amphiphilicity and stronger hydrogen bonding force compared to SPI or SDF nanoparticles. Furthermore, SSPs 1:1 and SSPs 1:3 groups maintained better stability in storage period and centrifugal stability experiments. After the in vitro digestion, the maximum bioaccessibility and chemical stability of AST in the SSPs 1:1 group were 62.5 ± 2.3% and 44.9 ± 0.4%, respectively. Synthesizing the results, it was concluded that the stability of the Pickering emulsion and the encapsulated delivery AST were best when the ratio of SDF and SPI in the SSPs was 1:1. These results provided a reference for the preparation of Pickering emulsions with natural nutrient delivery systems for vegetarians. Declarations CRediT authorship contribution statement Yue Qiu: Writing-review & editing, Conceptualization, Supervision; Feifan Fu: Investigation, Writing-original draft, Writing-review & editing; Jiajun Cheng: Methodology; Aodong Yue: Conceptualization;Tingting Yang: Conceptualization, Methodology; Ligen Zou: Supervision, Writing-review & editing; Fei Lu: Supervision; Jianyou Zhang: Supervision, Project administration, Conceptualization, Methodology, Visualization, Funding acquisition Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work was supported by the Hangzhou Science and Technology Development Plan (20241203A09). Author Contribution Q: Writing-review & editing, Conceptualization, Supervision; F: Investigation, Writing-original draft, Writing-review & editing; C: Methodology; Yue: Conceptualization; Yang: Conceptualization, Methodology; Zou: Supervision, Writing-review & editing; L: Supervision;Zhang: Supervision, Project administration, Conceptualization, Methodology, Visualization, Funding acquisition Acknowledgements The authors thank the help and support received from the College of Food Science and Technology, Zhejiang University of Technology and Hangzhou Science and Technology Development Plan. References Abdullah, Weiss, J., Ahmad, T., Zhang, C., & Zhang, H. (2020). A review of recent progress on high internal-phase Pickering emulsions in food science. Trends in Food Science & Technology 106:91-103.https://doi.org/10.1016/j.tifs.2020.10.016 Chen, H.-H., Wu, J.-X., Huang, R., Dai, J.-L., Liang, M.-H., & Jiang, J.-G. (2024). 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Modification of pea dietary fibre by superfine grinding assisted enzymatic modification: Structural, physicochemical, and functional properties. International Journal of Biological Macromolecules 267:https://doi.org/10.1016/j.ijbiomac.2024.131408 Yi, J., Gan, C., Wen, Z., Fan, Y., & Wu, X. (2021). Development of pea protein and high methoxyl pectin colloidal particles stabilized high internal phase pickering emulsions for β-carotene protection and delivery. Food Hydrocolloids 113:https://doi.org/10.1016/j.foodhyd.2020.106497 Zhai, X., Lin, D., Liu, D., & Yang, X. (2018). Emulsions stabilized by nanofibers from bacterial cellulose: New potential food-grade Pickering emulsions. Food Research International 103:12-20.https://doi.org/10.1016/j.foodres.2017.10.030 Zhang, G., Hao, M., He, Y., Ahmad, I., Ding, Y., & Lyu, F. (2023). Structural, physicochemical, and functional properties of insoluble dietary fiber derived from okara by Viscozyme®L. 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Trends in Food Science & Technology 109:169-196.https://doi.org/10.1016/j.tifs.2021.01.026 Zhang, W., Gu, X., Liu, X., & Wang, Z. (2021). Fabrication of Pickering emulsion based on particles combining pectin and zein: Effects of pectin methylation. Carbohydrate Polymers 256:https://doi.org/10.1016/j.carbpol.2020.117515 Zhang, W., Huan, Y., Ren, P., Li, J., Wei, Z., Xu, J., & Tang, Q. (2024). Zein/hyaluronic acid nanoparticle stabilized Pickering emulsion for astaxanthin encapsulation. International Journal of Biological Macromolecules 255:https://doi.org/10.1016/j.ijbiomac.2023.127992 Zhang, X., Zhao, X., Tie, S., Li, J., Su, W., & Tan, M. (2022). A smart cauliflower-like carrier for astaxanthin delivery to relieve colon inflammation. Journal of Controlled Release 342:372-387.https://doi.org/10.1016/j.jconrel.2022.01.014 Zhang, X., Zhao, X., Tie, S., Wang, H., & Tan, M. (2021). Ultrasonic Self-Emulsification Nanocarriers for Cellular Enhanced Astaxanthin Delivery. Journal of Agricultural and Food Chemistry 69(9):2719-2728.https://doi.org/10.1021/acs.jafc.0c05983 Zhang, Y., Wang, H., Hou, Y., Song, J., Shang, W., Zhang, P., Hou, S., & Tan, M. (2023). Pickering emulsion stabilized by gliadin nanoparticles for astaxanthin delivery. Journal of Food Engineering 345:https://doi.org/10.1016/j.jfoodeng.2023.111417 Zhou, Q., Yang, L., Xu, J., Qiao, X., Li, Z., Wang, Y., & Xue, C. (2018). Evaluation of the physicochemical stability and digestibility of microencapsulated esterified astaxanthins using in vitro and in vivo models. Food Chemistry 260:73-81.https://doi.org/10.1016/j.foodchem.2018.03.046 Zhu, Y., Huan, S., Bai, L., Ketola, A., Shi, X., Zhang, X., Ketoja, J. A., & Rojas, O. J. (2020). High Internal Phase Oil-in-Water Pickering Emulsions Stabilized by Chitin Nanofibrils: 3D Structuring and Solid Foam . ACS Applied Materials & Interfaces 12(9):11240-11251.https://doi.org/10.1021/acsami.9b23430 Zou, Y., Yang, X., & Scholten, E. (2018). Rheological behavior of emulsion gels stabilized by zein/tannic acid complex particles. Food Hydrocolloids 77:363-371.https://doi.org/10.1016/j.foodhyd.2017.10.013 Additional Declarations No competing interests reported. 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2","display":"","copyAsset":false,"role":"figure","size":88298,"visible":true,"origin":"","legend":"\u003cp\u003eMean particle size and PDI (a) and Zeta-potential (b) of SSPs with different SDF/SPI mass ratio\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6808307/v1/1791f58537eb2ba917754064.png"},{"id":84814108,"identity":"77faa646-1038-459b-88c4-6fc57db10de7","added_by":"auto","created_at":"2025-06-17 15:20:36","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1522990,"visible":true,"origin":"","legend":"\u003cp\u003eThree-phase contact angle(a) and FTIR (b) of SSPs with different ratios of SDF/SPI\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6808307/v1/c154ddde5b34dc73c3519dfd.jpeg"},{"id":84814111,"identity":"5843c8d6-a9fd-4a8c-9a4c-f4f2b849316a","added_by":"auto","created_at":"2025-06-17 15:20:36","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":834800,"visible":true,"origin":"","legend":"\u003cp\u003eAppearance of emulsions stabilized by SSPs with different SDF/SPI ratios at 0 day (a); 7 day (b); 14 day (c); 21 day (d); 28 day (e) and Creaming index (f).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6808307/v1/68177e66dd795ab78f1bbd0b.jpeg"},{"id":84815170,"identity":"e7b16f0f-67d1-4ccc-a938-174ed5559437","added_by":"auto","created_at":"2025-06-17 15:28:36","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1416750,"visible":true,"origin":"","legend":"\u003cp\u003eCentrifugal stability of Pickering emulsions (a); Particle size (b) and zeta-potential (c) of SSPs with different SDF/SPI ratio mass during the storage time\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6808307/v1/d956b291180bdf922da85359.jpeg"},{"id":84815775,"identity":"79ce5e08-f6a7-4a91-852c-664556cbd9e2","added_by":"auto","created_at":"2025-06-17 15:36:36","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":198258,"visible":true,"origin":"","legend":"\u003cp\u003eRheological properties of Pickering emulsions stabilized by SSPs with different SDF/SPI ratios. Viscosity (a); Storage modulus (G′) (b) and loss modulus (G″) (c); DPPH scavenging activity (%) and ABTS scavenging activity (%) of Pickeringemulsions(d)\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6808307/v1/cd6bf54ee4cb7cabd22cbaa4.jpg"},{"id":84816584,"identity":"bb152de0-5159-48b4-8469-2079f3b708bb","added_by":"auto","created_at":"2025-06-17 15:44:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5673398,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6808307/v1/a6ebcc4e-9150-4be2-9d86-8584a25966d9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Soy dietary fiber/soy protein isolates nanoparticles to stabilize Pickering emulsion for astaxanthin delivery","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAstaxanthin (AST), a kind of β-carotene with strong antioxidant properties. It also possesses biological activities such as anti-inflammatory and anti-cancer and widely used in food and medicine \u003cb\u003e(Chen et al., 2024)\u003c/b\u003e. Astaxanthin has been widely extracted from red algae, shrimp and crab shells. However, AST application is restricted due to low bioavailability, poor water solubility and chemical instability \u003cb\u003e(\u003c/b\u003eZhou et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Currently, many methods were being adopted to improve the utilization of astaxanthin, including micro-encapsulation (Zhang et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), liposomes \u003cb\u003e(Pan et al., 2018)\u003c/b\u003e, nanoparticles \u003cb\u003e(\u003c/b\u003eZhang et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and emulsions. Emulsions are now widely used because of easy production, low cost and high stability.\u003c/p\u003e \u003cp\u003ePickering emulsion is a new type of emulsion that replace traditional emulsifiers with solid, insoluble colloidal particles. Additionally, Pickering emulsion is freeze-thaw stable, resistant to oxidation, and resistant to Ostwald Ripening \u003cb\u003e(\u003c/b\u003eZhang et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Since Pickering emulsion is made of combination of compact nanoparticle materials, the gap between them facilitates slow-release of encapsulated actives and allow for precise delivery. By dispersing and adsorbing nanoparticles at the interface of the immiscible two-phase interface and inhibiting droplet aggregation, a stable emulsion system is successfully formed for the encapsulation and delivery of biologically active substances \u003cb\u003e(Zhang et al., 2022)\u003c/b\u003e. Pickering emulsion has been used successfully to encapsulate polyphenols and β-carotenoids. Du used cinnamic acid-modified acid-ethanol hydrolyzed starch-stabilized Pickering emulsion to encapsulate curcumin with 96.2% encapsulation rate, and better bioavailability and antioxidant activity \u003cb\u003e(\u003c/b\u003eDu et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Ge et al. \u003cb\u003e(2022)\u003c/b\u003e prepared Pickering emulsion stabilized by zein/Adzuki bean seed coat polyphenol nanoparticles to enhance the stability and bioaccessibility of astaxanthin.\u003c/p\u003e \u003cp\u003eNowadays, more green edible materials such as polysaccharides and proteins are being used to stabilize Pickering emulsions. Polysaccharides can act as a barrier against enzymatic degradation, while proteins can provide affinity for binding bioactive compounds with strong emulsifying activity \u003cb\u003e(Hou et al., 2022)\u003c/b\u003e. Soy dietary fiber (SDF) is one kind of polysaccharide mainly extracted from plants that cannot be digested and absorbed by the body \u003cb\u003e(Liu et al., 2024)\u003c/b\u003e. SDF, extracted from soybean processing by-products, contains hydroxyl and carboxyl groups, providing excellent gelling and emulsifying properties. These features enable SDF to enhance food texture and stability, making it highly promising for food industry applications \u003cb\u003e(Yang et al., 2024)\u003c/b\u003e. Soy protein isolate (SPI) is one of the most frequent and widely used proteins in food because of its low cost and relatively balanced nutritional profile \u003cb\u003e(\u003c/b\u003ePinho et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). At the same time, SPI is an organic polymer consisting of a series of peptide-bonded amino acids that rely on their amphiphilicity and thermally induced self-assembly properties to act as novel Pickering emulsion stabilizers \u003cb\u003e(Ezo et al., 2022)\u003c/b\u003e. To the best of our knowledge, the application of soy dietary fiber (SDF) and soy isolate protein (SPI) complexes as solid particle emulsifiers for encapsulated delivery of AST has not been explored.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to use the SDF-SPI nanocomplex as a stabilizer to prepare Pickering emulsions that encapsulated and delivered AST. The microstructure, chemical bonding, and hydrophilicity of SDF/SPI with varying complex particle ratios were characterized to explain the effect of composite particles on emulsion stability. Additionally, the storage stability and centrifugal stability of Pickering emulsions was determined. Furthermore, after simulating \u003cem\u003ein vitro\u003c/em\u003e digestion, the bioavailability and chemical stability of AST was determined. This study could facilitate the design of a novel delivery system based on Pickering emulsions to encapsulate and protect bioactive for various applications.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eSoy dietary fiber was obtained from Henan Wanbang Industrial Company, Zhengzhou, China. Soy protein isolate was provided by Rhawn, shanghai, China. Soy oil was purchased from Yihaikerry Co., ltd, Shanghai, China. Astaxanthin was obtained from Shanghai Macklin Biochemical Co., ltd, Shanghai, China. All other chemical reagents used were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of SDF/SPI complex particles (SSPs)\u003c/h2\u003e \u003cp\u003eMixed with different SDF:SPI (m/m) ratios of 3:1, 1:1, and 1:3, 4g mixed particles were selected and named as SSPs 3:1, SSPs 1:1, and SSPs 1:3, respectively.The 4 g separate SDF and SPI were prepared as controls. And then each of the five sets of samples was added to 100 mL of ultrapure water. The aqueous samples were then homogenized (IKA, Germany) at a high speed of 12,000 rpm for 5 min and ultrasound by sweeping frequency auto-tracking ultrasonic (Shanghai Fangxu Co., ltd, Shanghai, China) at 500 W for 5 min to prepare nanoparticles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Characterization of nanoparticles\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Scanning electron microscope (SEM) observation\u003c/h2\u003e \u003cp\u003eMorphology of SDF, SPI, and SSPs with different ratio mass was evaluated by SEM (S-4700 II, Hitachi, Tokyo, Japan) as described previously with minor modification \u003cb\u003e(Liu et al., 2018)\u003c/b\u003e. In short, after freeze-drying and preserving the samples were coated with Pt-palladium (plasma deposition method) and imaged under an accelerating voltage of 5.0 kV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Particle size and polydispersity index (PDI)\u003c/h2\u003e \u003cp\u003eThe mean droplet size and PDI of SSPs and control groups (0.1%, w/w) were obtained by a Zetasizer Nano ZS instrument (Malvern Panalytical Ltd, 3000HSA, UK) under 25 ℃ with a balancing time of 2 min. The refractive index and absorption index were 1.59 and 0.001, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 Three-phase contact angle test\u003c/h2\u003e \u003cp\u003eThe three-phase contact angle (θ) was measured by droplet shape analyzer DSA25 (KR\u0026uuml;SS, Germany), static water-in-air contact angle was measured by dropping water droplets (2 \u0026micro;L) into particle film according to the method of Zhang et al. \u003cb\u003e(2023)\u003c/b\u003e with minor modification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4 Fourier transform infrared spectroscopy (FTIR)\u003c/h2\u003e \u003cp\u003eFTIR was performed in a Nicolet6700 spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Briefly, SPI, SDF, and SSPs were respectively mixed with KBr in the mass ratio of 1:100 and pressed into pellets, which were then submitted to FTIR analysis using a scanning range of 500\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Preparation of Pickering emulsion\u003c/h2\u003e \u003cp\u003eThe oil phase (AST-O) was prepared as follows: astaxanthin was dissolved in soybean oil (0.5 mg/mL) and stirred for 30 min, and then 25 mL of oil phase was added to 25 mL aqueous samples mentioned in section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e, respectively. The mixed samples were then homogenized (IKA, Germany) at a high speed of 12,000 rpm for 5 min and ultrasound by sweeping frequency auto-tracking ultrasonic (Shanghai Fangxu Co., ltd, Shanghai, China) at 500 W for 5 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Characterization of SSPs-stabilized Pickering emulsions\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Mean particle and zeta-potential determination\u003c/h2\u003e \u003cp\u003eThe mean particle size and zeta potential were measured using a Zetasizer Nano ZS instrument (Malvern Panalytical Ltd, 3000HSA, UK) at 25\u0026deg;C and the balancing time was 2 min according to the method previously described \u003cb\u003e(Sun et al., 2019)\u003c/b\u003e. The refractive index of water (dispersed phase) was 1.59, absorption index was 0.01.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Rheological measurements\u003c/h2\u003e \u003cp\u003eThe rheological properties were determined by a rheometer Discovery HR20 rheometer (TA Instruments, New Castle, USA) with parallel plate geometry (60 mm diameter, 1 mm gap). The temperature was set to 25\u0026deg;C and the emulsions were placed on the rheological plates 5 min in advance in order to reach thermal equilibrium. Regarding the dynamic oscillation experiments, the linear viscoelastic region of the Pickering emulsion was first determined using dynamic strain scanning, which were performed at a fixed strain amplitude of 1% (within the linear viscoelastic region) to collect the storage modulus (G\u0026prime;) and loss modulus (G\u0026Prime;) of the emulsions in the frequency range of 0.1\u0026ndash;100 rad/s. The dynamic frequency scanning experiments were performed at a fixed strain amplitude of 1% (within the linear viscoelastic region). Steady-state flow measurements were also performed to record the apparent viscosity and shear stress of Pickering emulsions as a function of shear rate (0.1\u0026ndash;100 s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Stability of Pickering emulsions\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. Storage stability\u003c/h2\u003e \u003cp\u003eThe storage stability of Pickering emulsions prepared by different SDF/SPI mass ratios were studied by recording the appearance changes of Pickering emulsion on 0, 7, 14, 21, 28 days. The Creaming Index (CI) was calculated by formula (1).\u003c/p\u003e \u003cp\u003eCI\u0026thinsp;=\u0026thinsp;Hs/Ht \u0026times; 100% (1)\u003c/p\u003e \u003cp\u003eWhere Hs and Ht are water layer height (mm) and emulsion height (mm), respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. Centrifugal stability\u003c/h2\u003e \u003cp\u003e8.0 mL Pickering emulsion was added to a 10.0 mL centrifuge tube and was centrifuged at 11000 \u0026times; g for 10 min. The height of the emulsion and water layer were recorded respectively and the centrifugal stability (%) was calculated after centrifugation of the emulsion according to formula (2).\u003c/p\u003e \u003cp\u003eCentrifugal stability\u0026thinsp;=\u0026thinsp;1-(Hs/Ht) \u0026times; 100% (2)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Antioxidant activity\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1 DPPH scavenging activity\u003c/h2\u003e \u003cp\u003eAST loaded-Pickering emulsion 1 mL was mixed with 2 mL of DPPH solution (0.1mM, m/w). The above sample was reacted in the dark for 30 min, and the absorbance was measured at 517 nm \u003cb\u003e(Xu et al., 2021)\u003c/b\u003e. The DPPH scavenging activity of the AST loaded-Pickering emulsion was calculated by the following formula (3)\u003c/p\u003e \u003cp\u003eDPPH scavenging activity (%) = (A\u003csub\u003eDPPH\u003c/sub\u003e-A\u003csub\u003esample\u003c/sub\u003e)/A\u003csub\u003eDPPH\u003c/sub\u003e \u0026times; 100 (3)\u003c/p\u003e \u003cp\u003eWhere A\u003csub\u003eDPPH\u003c/sub\u003e is the absorbance of the DPPH at 517 nm, and A\u003csub\u003esample\u003c/sub\u003e is the absorbance of sample at 517 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2 ABTS radical scavenging activity\u003c/h2\u003e \u003cp\u003eThe ABTS scavenging activity was studied by the method of \u003cb\u003eZhang et al. (2023)\u003c/b\u003e with slight modifications. The ABTS radical solution was first prepared with equal volumes of 7.4 mM ABTS and 2.6 mM aqueous potassium persulphate and then left in the dark for 12 hours. After that, this solution was then diluted with methanol until the absorbance value at 734 nm reached 0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1.\u003c/p\u003e \u003cp\u003eThe ABTS radical scavenging activity was analyzed by mixing 1 mL of emulsions with 2 mL of ABTS solution. The above solutions were reacted in the dark for 11h. The antioxidant activity was calculated by the following formula (4):\u003c/p\u003e \u003cp\u003eABTS scavenging activity (%) = (A\u003csub\u003eABTS\u003c/sub\u003e-A\u003csub\u003esample\u003c/sub\u003e)/A\u003csub\u003eABTS\u003c/sub\u003e\u0026times;100 (4)\u003c/p\u003e \u003cp\u003eWhere A\u003csub\u003eABTS\u003c/sub\u003e is the absorbance of the ABTS at 734 nm, and A\u003csub\u003esample\u003c/sub\u003e is the absorbance of sample at 734 nm.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.8 In vitro simulated digestion\u003c/h2\u003e \u003cp\u003eThe method of \u003cem\u003ein vitro\u003c/em\u003e simulated digestion was referred to the method of \u003cb\u003eShah et al. (2016)\u003c/b\u003e with little modifications. Simulated gastric fluid (SGF) was obtained by dissolving 38.0 mg NaCl and 60.8 mg pepsin in 19 mL of 0.05 M PBS buffer. The emulsion (1 mL) was mixed with 19.0mL of SGF and the pH was adjusted to 2.5. And the SGF mixture solutions were stirred at 150 rpm in the water-bath shaker for 2 h at 37\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe simulated intestinal fluid (SIF) was obtained by dissolving 656 mg NaCl, 82.9 mg CaCl\u003csub\u003e2\u003c/sub\u003e, 120 mg porcine lipase, 120 mg porcine trypsin, and 378 mg bovine bile in 20.0 mL of 0.05 M PBS buffer. The pH of the SGF mixture solutions was first adjusted to 7.0 by using 1.0 M NaOH to terminate gastric digestion. And then, the above solutions were mixed with 19.0 mL of SIF and stirred at 150 rpm in the water-bath shaker for 4 h at 37\u0026deg;C.\u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e2.8.1 Free fatty acid (FFA) release\u003c/h2\u003e \u003cp\u003eDuring intestinal digestion, the pH of the system was maintained at 7.0 by addition of 100 mM NaOH. The consumption of NaOH was recorded and used for calculating the release amount of FFA according to formula (5) \u003cb\u003e(Huang et al., 2021)\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eFFA (%) = (V\u003csub\u003eNaOH (t)\u003c/sub\u003e \u0026times; M\u003csub\u003eNaOH\u003c/sub\u003e \u0026times; M\u003csub\u003eWTG\u003c/sub\u003e)/ (m\u003csub\u003eTG\u003c/sub\u003e \u0026times; 2) \u0026times; 100% (5)\u003c/p\u003e \u003cp\u003eWhere V\u003csub\u003eNaOH (t)\u003c/sub\u003e is the volume (mL) of NaOH required to maintain pH 7.0 at time t, M\u003csub\u003eNaOH\u003c/sub\u003e is the molar concentration (mol/L) of NaOH required for titration, M\u003csub\u003eWTG\u003c/sub\u003e is the average molecular weight (g mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of corn oil, and m\u003csub\u003eTG\u003c/sub\u003e is the total mass (g) of soy bean oil in the system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e2.8.2 AST bioaccessibility\u003c/h2\u003e \u003cp\u003eThe bioaccessibility of AST was determined by the method of Zhang et al. \u003cb\u003e(2024)\u003c/b\u003e with modifications. The simulated digested liquid was centrifuged at 10000 \u0026times; g for 20 min. The AST was extracted by mixing 0.5 mL of the lower micelle layer after centrifugation with 4.5 mL dichloromethane/methanol mixed solution 2:1, v/v). And then the solution was centrifuged at 5550 \u0026times; g for 10 min. The content of AST in micelles after \u003cem\u003ein vitro\u003c/em\u003e digestion was calculated by the standard curve. The bioaccessibility of AST was calculated by formula (6). The chemical stability was calculated by comparing the astaxanthin content in the crude digested phase with the initial emulsion by formula (7).\u003c/p\u003e \u003cp\u003eBioaccessibility (%)\u0026thinsp;=\u0026thinsp;C \u003csub\u003eMicelles\u003c/sub\u003e/C\u003csub\u003eInitial\u003c/sub\u003e \u0026times; 100 (6)\u003c/p\u003e \u003cp\u003eChemical stability (%) \u003csub\u003e=\u003c/sub\u003e C\u003csub\u003eEmulsion\u003c/sub\u003e/C\u003csub\u003eIntitial\u003c/sub\u003e \u0026times; 100 (7)\u003c/p\u003e \u003cp\u003eWhere C\u003csub\u003eMicelles\u003c/sub\u003e is the concentration of AST in the micelle fraction, C\u003csub\u003eInitial\u003c/sub\u003e is the AST concentration calculated from the initial addition, and C\u003csub\u003eemulsion\u003c/sub\u003e is the AST concentration in the crude digestive phase.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe Duncan's multiple range test with SPSS 26.0 (IBM Corporation, NY, USA) was used to statistically analyze the various treatments, different letters (a, b, c, d, e, f) represent significant differences between groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). All charts were draw by using Origin 2024 (Origin Lab, USA). Each experiment was performed in triplicate and the results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Characterization of SSPs\u003c/h2\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 The microtopography of SSPs\u003c/h2\u003e \u003cp\u003eSSPs prepared by ultrasound-assisted high-speed homogenization and dispersion were characterized by SEM at 1000 \u0026times; magnification (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003cb\u003ea-e\u003c/b\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, SDF and SPI showed a smooth surface lamellar stacking structure. The appearance of bands and ellipsoids can be seen in SSPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003cb\u003eb-d\u003c/b\u003e), indicating that different degrees of cross-linking and associations between the composite particles have occurred. The nanoparticles do not have distinct boundaries between them, but are connected together to form stable particles. Compared with SSP 3:1 and SSP 1:3, SSP 1:1 showed a more ordered and plump structure.When SSPs 1:1 was observed under 10,000 \u0026times; magnification \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003cb\u003ef\u003c/b\u003e), the shape of rounded and full-bodied nanoparticles can be seen as they aggregate and combine to form a stable structure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Mean particle size, PDI and zeta-potential test\u003c/h2\u003e \u003cp\u003eThe mean particle size, PDI and zeta-potential test of SSPs prepared by different SDF/SPI ratio were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, respectively. After 5 min high-speed homogenization and 500 W ultrasonication, the particle size of SSPs can be controlled in the range of 400\u0026ndash;800 nm. The particle size can also reflect the strength of the interaction force between the polysaccharide and protein complexes \u003cb\u003e(Gao et al., 2023)\u003c/b\u003e. As the proportion of SPI in the complex particles increased, the particle size decreased. The largest particle size 1328.33\u0026thinsp;\u0026plusmn;\u0026thinsp;103.2 nm was observed in SDF group, while SSPs 1:3 group showed smallest particle size of 453\u0026thinsp;\u0026plusmn;\u0026thinsp;31.6 nm. Recently, it has been shown that nanoparticles with small and uniform particle size are ideal for stabilizing Pickering emulsions. PDI reflects the degree of dispersion in the system, the smaller the value, the higher the degree of dispersion of the particles in the solution. It can be seen that SSPs 1:1 and SSPs 1:3 groups are more evenly dispersed than others in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. Zeta-potential is a measure of the point bilayer around a particle in solution, with higher absolute values of zeta-potential indicating stronger electrostatic repulsion between particles \u003cb\u003e(Herrman et al., 2022)\u003c/b\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, The SDF, SPI and SSPs are negatively charged, with the SDF and SPI having the higher negative charge absolute value and the SSPs having a reduced charge value in comparison. This may be due to the fact that the electrostatic interaction between the composite particles neutralizes the charge values. And the electrostatic interactions became a key factor for stabilization. The phenomenon of negatively charged complexes of myofibrillar proteins and pectin was also studied by Chen et al. \u003cb\u003e(Chen et al., 2022)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3 Three phase contact angle\u003c/h2\u003e \u003cp\u003eSince the behavior of particles on oil-water surfaces affects the type and stability of the Pickering emulsion, the wettability of particles was also assessed by measuring the three-phase contact angle (θ) \u003cb\u003e(Xiao et al., 2016)\u003c/b\u003e. The complex particles are hydrophilic when the contact angle is less than 90\u0026deg;, and the opposite means that they are lipophilic. When the contact angle reaches 90\u0026deg;, the stabilization barrier reaches the highest, the hydrophilicity and lipophilicity reach a balance, and the system formed is more stable \u003cb\u003e(Abdullah et al., 2020)\u003c/b\u003e. As shown in \u003cb\u003eFig.\u0026nbsp;3a\u003c/b\u003e, when SDF was dispersed in the water phase, the hydrophobic groups were reunited inward, while the hydrophilic groups were exposed externally, which made the particles being in a hydrophilic state with a contact angle of 72.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;. SPI was hydrophilic and the contact angle was 75.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u0026deg;. The three-phase contact angle raised to 81.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6\u0026deg;. The reason for the decrease of hydrophilicity of SSPs may be that the combination of SPI and SDF exposes the internal hydrophobic groups.when the ratio of SDF and SPI reached 1:1, suggesting they are amphiphilic and suitable for stabilizing Pickering emulsion. This result is consistent with the relationship that Ni et al. \u003cb\u003e(2023)\u003c/b\u003e found between the hydrophilicity of the composite particles and the percentage of polysaccharide protein complexes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e3.1.4 FTIR\u003c/h2\u003e \u003cp\u003eFTIR is an effective method for analyzing the structure of materials, which can reflect the functional group information of samples \u003cb\u003e(Wang et al., 2024)\u003c/b\u003e. As shown in \u003cb\u003eFig.\u0026nbsp;3b\u003c/b\u003e, all emulsion revealed strong absorption peaks in both the amide I region (1600\u0026ndash;1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and amide II region (1480\u0026ndash;1575 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), which were indicative of the secondary structure of protein, and could be used to analyze the interaction between SPI and SDF \u003cb\u003e(\u003c/b\u003eJiang et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).Compared to SDF and SPI, the peak of Osingle bondH stretching vibration in SPPs nanoparticles was shifted,which confirmed that hydrogen bonds were formed between SDF and SPI. The wavelength around 3440 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is the peak of the O-H stretching vibration in cellulose and hemicellulose, indicating hydrogen bonding, which is closely related to water wettability. The O-H peaks of SDF and SPI were 3431.76 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 3442.70 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. When the proportion of SDF in the SSPs decreased, the absorption bond was blue-shifted, which may be due to hydrogen bond breaking \u003cb\u003e(Zhang et al., 2023)\u003c/b\u003e. The stretching vibration peak at 1630 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026ndash; 1670 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003eindicating that the combination of C\u0026thinsp;=\u0026thinsp;O bonds formed between SPI and SDF \u003cb\u003e(Lyu et al., 2021)\u003c/b\u003e. The absorption peak at 950\u0026ndash;1200 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is considered to be the region of carbohydrates. SPI has a weaker peak there whereas the peaks of SSPs were evident. It was suggested that hydrogen bonding interactions participated in the formation and stability of SSPs \u003csup\u003e\u003cb\u003e[33]\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Stability of Pickering emulsion\u003c/h2\u003e \u003cdiv id=\"Sec31\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Storage stability of Pickering emulsion\u003c/h2\u003e \u003cp\u003eThe appearance changes of astaxanthin dissolved in oil (AST-O) and Pickering emulsion were shown in \u003cb\u003eFig.\u0026nbsp;4a-e\u003c/b\u003e. After 28 days, AST gradually precipitated from the oil phase in AST-O group. The color of the solution gradually changed from red to yellow, which indicated that oxidative decomposition had occurred. In contrast, the color change of Pickering emulsion embedded with astaxanthin was not significant. According to \u003cb\u003eFig.\u0026nbsp;4f\u003c/b\u003e, CI% of SSPs 1:3 stabilized Pickering emulsion (2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48%) was much lower than that of SDF stabilized emulsion (7.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58%) and SSPs 3:1 (8.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33%) after 28 days of storage. These results indicated that SSPs 1:3 stabilized astaxanthin Pickering emulsion was the most stable. A similar study was conducted by Yang et al. (2022) to determine the storage stability of Pickering emulsion embedded with astaxanthin stabilized by composite particles of chitosan and guar gum. The stability of Pickering emulsions has also been demonstrated in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Centrifugal stability\u003c/h2\u003e \u003cp\u003eCentrifugal stability also reflects the stability of the emulsion under centrifugal force at higher rotational speeds \u003cb\u003e(Fig.\u0026nbsp;5a)\u003c/b\u003e. The force exerted by the centrifugal treatment causes the colloidal particles to be dislodged from the surface of the oil droplets, leading to their precipitation. The index of centrifugal stability based on the height of the water layer and emulsion separated was calculated after centrifugation. Ultimately, it was found that SSPs 1:1 and SSPs 1:3 exhibited high centrifugal stability, 67.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2% and 75.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2%, respectively. This result further demonstrated the stability of Pickering emulsions stabilized by SSPs with higher SPI fractions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 The mean particle size and zeta potential of emulsions\u003c/h2\u003e \u003cp\u003eThe stability of the emulsions was further characterized by recording the mean particle size and zeta potential of the emulsions from 0\u0026ndash;28 days. According to \u003cb\u003eFig.\u0026nbsp;5b\u003c/b\u003e, the mean particle size of the emulsions increased with the increase of storage time. This phenomenon indicates that after 28 days of storage, the Ostwald\u0026rsquo;s ripening may occur in emulsions. The force between small droplets was weakened, leading to droplet aggregation and a subsequent increase in emulsion particle size. Among them, the SSPs 1:1 group showed excellent stability and resistance to aggregation, with a particle size control of 1353.7\u0026thinsp;\u0026plusmn;\u0026thinsp;78.9 nm at day 28, which was lower than that of 2,669.7\u0026thinsp;\u0026plusmn;\u0026thinsp;88.7 nm in the SSPs 3:1. The zeta-potential is one of the effective indicators for evaluating electrostatic interactions \u003cb\u003e(Zhai et al., 2018)\u003c/b\u003e. As shown in \u003cb\u003eFig.\u0026nbsp;5c\u003c/b\u003e, the zeta potential of SSPs 1:1 and SSPs 1:3 groups were stable at -30 mV to -35 mV after the storage period. Stronger electrostatic interactions counteracted the aggregation of the emulsions, resulting in a uniform droplet size of the emulsions. The result of zeta-potential is consistent with the particle size test described above.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Rheological measurements of Pickering emulsions\u003c/h2\u003e \u003cp\u003eThe relationship between SDF/SPI mass ratio and emulsion rheology was studied. The viscosity of the emulsion decreased with an increase in shear rate (\u003cb\u003eFig.\u0026nbsp;6a\u003c/b\u003e) which suggested that emulsions stabilized by SSPs were non-Newtonian fluids. When the viscosity of the emulsion is high, the resistance to movement of the droplets increases and the collision frequency between droplets decreases, thus reducing the possibility of droplet aggregation and coalescence, which helps to improve the physical stability of the emulsion. G\u0026prime; reflects the elastic solid-like character of the material, whereas G\u0026Prime; indicates the viscous response.According to the results of the rheological tests, the storage modulus (G\u0026prime;) was greater than the loss modulus (G\u0026prime;\u0026prime;) for all groups, which indicated that the an elastic-like structure was formed \u003cb\u003e(Zhu et al., 2020)\u003c/b\u003e. The SSPs 1:1 group emulsion had the highest viscosity and both the G\u0026prime; and G\u0026prime;\u0026prime;were higher compared to other groups, which demonstrated good stability in frequency scanning and oscillation (\u003cb\u003eFig.\u0026nbsp;6b,c\u003c/b\u003e). This indicates that the emulsion is most stable at this complex concentration condition. This indicator is also consistent with the storage stabilization described above. When the ration of SDF/SPI reaches 1:1, the gel strength of SSPs stabilized emulsion increased significantly, indicating that SSPs plays an important role in the formation of gel-like structures. Similar to viscosity, the increased G\u0026prime; in emulsion stabilized by SSPs can be attributed to the small oil droplet size, which enhanced the interactions between oil droplets \u003cb\u003e(Zou et al., 2018)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Antioxidant activity\u003c/h2\u003e \u003cp\u003eDPPH free radical scavenging and ABTS free radical scavenging are the most common methods for evaluation \u003cem\u003ein vitro\u003c/em\u003e antioxidant activity \u003cb\u003e(Zhang et al., 2022)\u003c/b\u003e. As shown in \u003cb\u003eFig.\u0026nbsp;6d\u003c/b\u003e, DPPH scavenging activity of SSPs 3:1 and SSPs 1:1 groups were 65.5% and 52.6%, respectively. ABTS scavenging activity of SSPs 3:1 and SSPs 1:1 groups were 97.6% and 96.25% respectively, which were higher than free AST in AST-O (33.7% and 40.3%, respectively), which is consistent with the stability of emulsions. The SSPs stabilized Pickering emulsions encapsulated AST, which can delay the contact of AST with the external environment and slow down the rate of oxidative deterioration \u003cb\u003e(Foti, 2015)\u003c/b\u003e. In addition, the excellent DPPH and ABTS radical scavenging rates suggest that the synergistic encapsulation of SDF and SPI facilitates the optimal antioxidant effect of astaxanthin in Pickering emulsion systems. And this property also offers the possibility of subsequent application of the emulsions in the development of functional foods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec36\" class=\"Section2\"\u003e \u003ch2\u003e3.5 \u003cem\u003eIn vitro\u003c/em\u003e simulated digestion\u003c/h2\u003e \u003cdiv id=\"Sec37\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1 Digestive Stability\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e simulation of gastrointestinal digestion has been widely used as a tool to study the biochemical changes that occur in food and drug compounds during their passage through the gastrointestinal tract. After the intestinal digestion, the droplet size of the SSPs 1:1 and SSPs 1:3 groups were smaller than that of the separate SPI and SDF particle-stabilized Pickering emulsion. This phenomenon was verified in the average particle size determination after the intestinal digestion in \u003cb\u003eFig.\u0026nbsp;7a\u003c/b\u003e, where the mean particle sizes of the SSPs 3:1 and SSPs 1:1 groups determined at 492.8 nm and 548.1 nm, respectively. The particle sizes above were lower than 3,237 nm in the SDF group and 1425.2 nm in the SPI group. Regarding the capacity to maintain low droplet sizes, this could be because of the molecular interactions and hydrogen bonding that create a repulsive effect on the merging aggregation among droplets among combinations of SSPs composite particles. The microstructural changes of the Pickering emulsion during in vitro digestion are shown in \u003cb\u003eFig.\u0026nbsp;7c\u003c/b\u003e. The trend of emulsion droplet size increased due to breakage and droplet aggregation in an acidic environment during the gastric digestion stage. Smaller droplets had a higher specific surface area, which could better interact with enzymes in the digestive juice \u003cb\u003e(\u003c/b\u003eChen et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, Guo et al. (2022) suggested that that large oil droplets may reduce the effectiveness of contact and reaction with digestive enzymes.\u003c/p\u003e \u003cp\u003eZeta-potential was tested to determine the electrostatic force between emulsions and to verify one of the driving forces that keep emulsions stable. When emulsions went through the gastric digestion phase, the aboslute of zeta-potential decreased because of the acidic environment in stomach (\u003cb\u003eFig.\u0026nbsp;7.b\u003c/b\u003e).Finally, at the completion of the intestinal digestion phase,emulsions return to pH 7, and the zeta-potential reverts to between \u0026minus;\u0026thinsp;30 to -50 mV. The emulsion droplets are held in the lower range by electrostatic forces. This test verified the reason for the emulsion size change in the particle size test described above as well as in the microscopic observations. Additionally, an earlier investigation showed that the chitosan-caseinophosphopeptide complex's irreversible adsorption at the oil-in-water interface created a densely packed, stiff layer that surrounded the oil droplets and stabilized the emulsions' anti-coalescing action \u003cb\u003e(Huang et al., 2019)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003e3.5.2 FFA release\u003c/h2\u003e \u003cp\u003eFFA is a surfactant that facilitates the conversion of emulsion-loaded bioactive compounds into micelles. The surface structure and structural properties of the emulsion droplets are the main factors affecting lipid digestion. During simulated intestinal digestion, the hydrophobic core of AST was exposed due to structural destruction of the emulsion in simulated intestinal fluid \u003cb\u003e(Yang et al., 2021)\u003c/b\u003e. The emulsions were digested faster during the first 60 min, then the rate of lipolysis gradually decreased and reached a maximum at 240 min, indicating that the soybean oil emulsion loaded with AST was broken down by the digestive solution. According to the \u003cb\u003eFig.\u0026nbsp;8a\u003c/b\u003e, the highest FFA release rate (78.3. \u0026plusmn; 3.65%) was observed in SSPs 1:1 group. The release of FFA indicates the degree of lipid digestion in the emulsion, and the higher the FFA release rate, the more the emulsion was digested and broken down during digestion in the small intestine \u003cb\u003e(\u003c/b\u003eZhang et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section3\"\u003e \u003ch2\u003e3.5.3 Bioaccessibility and chemical stability\u003c/h2\u003e \u003cp\u003eThe bioaccessibility of AST in emulsions was evaluated (\u003cb\u003eFig.\u0026nbsp;8b\u003c/b\u003e). The SSPs 1:1 group had the highest bioaccessibility and chemical stability of all the groups, 62.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3% and 44.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4%, respectively. The reasons for this may be as follows: (i) the decrease in droplet size and the increase in specific surface area at SPI/SDF ratios close to 1:1 may facilitate the digestion of the oil droplets and the AST transferred from the droplets to the digestive solution; (ii) the three-dimensional network structure of the SSPs inhibits the coalescence and flocculation of the lipid droplets, thus increasing the chance of lipase and bile salts entering the surface of the oil droplets and increasing the amount of astaxanthin transferred from the oil phase to absorbable micelles \u003csup\u003e\u003cb\u003e[45]\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn summary, a strategy for stabilization of Pickering emulsion delivery of AST by SPI/SDF composite nanoparticles is proposed by constructing composite nanoparticles with different mass ratios. The results showed that the composite nanoparticles of SSPs 1:1 and SSPs 1:3 groups have better water-oil amphiphilicity and stronger hydrogen bonding force compared to SPI or SDF nanoparticles. Furthermore, SSPs 1:1 and SSPs 1:3 groups maintained better stability in storage period and centrifugal stability experiments. After the \u003cem\u003ein vitro\u003c/em\u003e digestion, the maximum bioaccessibility and chemical stability of AST in the SSPs 1:1 group were 62.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3% and 44.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4%, respectively. Synthesizing the results, it was concluded that the stability of the Pickering emulsion and the encapsulated delivery AST were best when the ratio of SDF and SPI in the SSPs was 1:1. These results provided a reference for the preparation of Pickering emulsions with natural nutrient delivery systems for vegetarians.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003cb\u003eCRediT authorship contribution statement\u003c/b\u003e \u003c/p\u003e \u003cp\u003eYue Qiu: Writing-review \u0026amp; editing, Conceptualization, Supervision; Feifan Fu: Investigation, Writing-original draft, Writing-review \u0026amp; editing; Jiajun Cheng: Methodology; Aodong Yue: Conceptualization;Tingting Yang: Conceptualization, Methodology; Ligen Zou: Supervision, Writing-review \u0026amp; editing; Fei Lu: Supervision; Jianyou Zhang: Supervision, Project administration, Conceptualization, Methodology, Visualization, Funding acquisition\u003c/p\u003e\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Hangzhou Science and Technology Development Plan (20241203A09).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eQ: Writing-review \u0026amp; editing, Conceptualization, Supervision; F: Investigation, Writing-original draft, Writing-review \u0026amp; editing; C: Methodology; Yue: Conceptualization; Yang: Conceptualization, Methodology; Zou: Supervision, Writing-review \u0026amp; editing; L: Supervision;Zhang: Supervision, Project administration, Conceptualization, Methodology, Visualization, Funding acquisition\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors thank the help and support received from the College of Food Science and Technology, Zhejiang University of Technology and Hangzhou Science and Technology Development Plan.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdullah, Weiss, J., Ahmad, T., Zhang, C., \u0026amp; Zhang, H. 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Rheological behavior of emulsion gels stabilized by zein/tannic acid complex particles. \u003cem\u003eFood Hydrocolloids\u003c/em\u003e 77:363-371.https://doi.org/10.1016/j.foodhyd.2017.10.013\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pickering emulsion, Astaxanthin, Anti-oxidation","lastPublishedDoi":"10.21203/rs.3.rs-6808307/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6808307/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAstaxanthin (AST), a powerful antioxidant, faces challenges due to its susceptibility to decomposition, poor solubility and low bioavailability. In this study, green edible plant-Pickering emulsion was prepared by soy dietary fiber (SDF) and soy protein isolate (SPI) complex nanoparticles (SSPs) and used for AST delivery. The SSPs characterized by their negatively charged fibrous rod-like structures, are primarily held together by hydrogen bonding. When the SDF/SPI mass ratio of SSPs is closed to 1:1, it exhibit good dispersibility and water-oil amphiphilicity. The storage stability test and rheological measurement showed that Pickering emulsions stabilized by SSPs have high viscosity and fluid stability, with a higher creaming index than the emulsions by separate SDF/SPI material. DPPH and ABTS free radical scavenging of AST in the 1:1 ratio of SDF and SPI stabilized Pickering emulsion, were higher( 65.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7% and 97.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5% respectively). In vitro digestion indicated that the bioaccessibility and chemical stability of AST in Pickering emulsion was 20.56% and 22.34% higher than free astaxanthin in oil (AST-O) group. Thus, Pickering emulsion stabilized by SDF/SPI complex particles is a potential delivery system for AST in the food industry.\u003c/p\u003e","manuscriptTitle":"Soy dietary fiber/soy protein isolates nanoparticles to stabilize Pickering emulsion for astaxanthin delivery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 15:20:31","doi":"10.21203/rs.3.rs-6808307/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-10T11:19:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-07T03:22:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-03T18:44:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-01T12:23:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-28T13:33:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-24T11:19:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"96751424950429443374538282676674402660","date":"2025-06-23T16:11:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136802763043164611567114510477315103433","date":"2025-06-21T16:20:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180037430671936537234064152835422383813","date":"2025-06-17T23:20:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24470381529100665756102395703557968478","date":"2025-06-16T10:46:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216798417506125105202898438575141728331","date":"2025-06-16T09:15:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182338549262643910125630859293400171668","date":"2025-06-16T05:08:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224994346491275139805716412168151286268","date":"2025-06-16T02:30:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"331225171500053093335797978835357142643","date":"2025-06-16T02:15:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-15T23:04:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-10T12:02:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-10T12:02:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Food Research and Technology","date":"2025-06-03T07:31:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"71c52c0b-c3f7-4189-82af-ae3b98d5c6be","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-11T22:23:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-17 15:20:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6808307","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6808307","identity":"rs-6808307","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}