Fabrication egg white gel hydrolysates-stabilized oil-in-water emulsion and characterization of its stability and digestibility

Food Hydrocolloids 102 (2020) 105621

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Fabrication egg white gel hydrolysates-stabilized oil-in-water emulsion and characterization of its stability and digestibility Ziting Ling a, b, Minmin Ai a, b, 1, Quan Zhou a, b, Shanguang Guo a, b, Ledan Zhou a, b, Hong Fan a, b, Yuanyuan Cao a, b, Aimin Jiang a, b, c, * a

College of Food Science, South China Agricultural University, Guangzhou, 510642, China The National Center for Precision Machining and Safety of Livestock and Poultry Products Joint Engineering Research Center, College of Food Science, South China Agricultural University, Guangzhou, 510642, China c The Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou, 510642, China b

A R T I C L E I N F O

A B S T R A C T

Keywords: Egg white hydrolysates Rheology Emulsion stability Digestibility Antioxidant activity

This paper investigated the properties of egg white protein hydrolysate (EH) prepared from alkali-induced egg white gel as an oil-in-water emulsion stabilizer. The physicochemical properties of EH, including Fourier transform infrared (FTIR), surface hydrophobicity, particle size, zeta potential and polydispersity index were investigated. Meanwhile, the microstructure, emulsion stability, oil oxidative stability, in vitro digestive stability and antioxidant activity of emulsion prepared by EH were evaluated. Results suggested that the surface hy­ drophobicity of EH decreased due to the destruction of hydrophobic groups, and the particle size of most hy­ drolysates ranged from 124.24 to 197.7 nm, except neutrase and alcalase hydrolysates. FTIR revealed that the strength of each peak increased significantly after hydrolysis owing to the break of structure and peptide bond. Most EH-stabilized emulsions were stable against droplets aggregation as influenced by temperature, and emulsion prepared from trypsin hydrolysate exhibited better ionic strength and storage stability. Emulsion stabilized by trypsin and alcalase hydrolysates delayed the lipids oxidation during storage. Droplet aggregation occurred to emulsions after in vitro digestion due to the destruction of the emulsion structure and the accu­ mulation of proteins and lipids, and the release rate of free fatty acids ranged from 55.72% to 77.29% in the 2 h vitro intestine digestion for six different emulsions. Additionally, most digested EH-stabilized emulsion enhanced its ability to scavenge DPPH, ABTS and O2 free radical, owing to the formation of antioxidant peptides. These results suggest that EH can be as a potential stabilizer for emulsions, especially trypsin hydrolysate, and possesses antioxidant properties after digestion.

1. Introduction Bioactive substances can easily decompose and become deactivated due to the surrounding conditions, significantly affecting their digestion and absorption characteristics in the human body (Ye, Nicolas, & Cor­ delia, 2018). Therefore, an appropriate delivery method that maintains the unique functional characteristics of bioactive substances is needed. Emulsion delivery systems have attracted significant research attention, especially regarding the transfer and embedding of bioactive substances, such as β-carotene, curcumin, and resveratrol (Dickinson, 2010). Emulsions consist of a liquid (dispersed phase) dispersed as stable droplets within a second liquid (continuous phase) that is immiscible

with it (Anton, Benoit, & Saulnier, 2008). While, emulsions are sus­ ceptible to environmental influences such as pH, temperature, and ionic strength. Therefore, it is necessary to explore a kind of emulsion stabi­ lizer that performs stable under various influencing factors, such as ion or temperature. Egg whites (EWs) contain a variety of proteins, pri­ marily ovalbumin, ovotransferrin, ovomucoid, and lysozyme (Mine, 1995). Their rich protein content also provides several other functions, such as gelation, water-holding capacity, foaming, and emulsification (Tabilo & Barbosa, 2005). Thus, EWs proteins are often used to stabilize emulsion delivery systems by using pure ovalbumin, ovotransferrin fibrils, and EWs-aggregate nanoparticles (Chang et al., 2016; Xu, Tang, Liu, & Liu, 2019; Wei, Cheng, & Huang, 2019). EWs can form a kind of

* Corresponding author. College of Food Science, South China Agricultural University, Guangzhou, 510642, China. E-mail address: [email protected] (A. Jiang). 1 Co-first author. https://doi.org/10.1016/j.foodhyd.2019.105621 Received 31 August 2019; Received in revised form 12 November 2019; Accepted 23 December 2019 Available online 24 December 2019 0268-005X/© 2019 Elsevier Ltd. All rights reserved.

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Fig. 2. Hydrolyzed protein (A) particle size distribution; (B) mean particle size, zeta potential, and polydispersity index. Different letters for the same index indicate significant difference (P < 0.05).

possibilities for the application of EWs gel. Enzymatic hydrolysis is a process in which proteases are used to treat proteins to produce peptides with various characteristics, such as anti­ oxidant, antimicrobial, or anti-inflammatory properties (Feng, Ruan, Jin, Xu, & Wang, 2018). Commonly used protease include pepsin, trypsin, transaminase, flavourzyme and alcalase. These enzymes possess their own enzymatic hydrolysis characteristics and conditions, which cause variations in the characteristics of the produced hydrolysates. EWs protein contain essential amino acids necessary for daily human meta­ bolism but has the disadvantages of high viscosity, poor thermal sta­ bility, and ready foaming and sensitization, which limit its applicability in the food industry. Enzymatic hydrolysis can not only ameliorate the limitation of EWs proteins, but also improve the physiological func­ tionality in terms of antioxidant activity, anticoagulation activity, antibacterial activity, and immune regulation (Yao, Vanga, Jin, & Raghavan, 2018). However, excessive hydrolysis leads to excessive amounts of hydrolysates with small molecular weights in the emulsion system. These are more likely to be free in the aqueous phase due to their unstable adsorption at the oil-water interface (Ipsen et al., 2001). Therefore, the state of the raw materials will affect the degree of hy­ drolysis. Thereby, EWs were prepared as a type of alkali-induced gel that immobilized and embeded proteins, thereby avoiding excessive hydrolysis. This paper follows on from our previous research (Ai et al., 2019a, b). Fresh EWs were prepared as an alkali-induced gel as a raw material for further hydrolysis using six different proteases to prepare hydrolysates. The obtained hydrolysates were employed to stabilize the emulsion under alkaline conditions. Firstly, the prepared hydrolysates were characterized using Fourier transform infrared (FTIR) spectroscopy, fluorescence spectroscopy and dynamic light scattering (DLS). Secondly, the emulsion was prepared with using hydrolysates, and its morphology

Fig. 1. FTIR curves (A) and surface hydrophobicity (B) of hydrolyzed egg white protein. Table 1 The secondary structure changes of hydrolyzed egg white protein. Sample

β-sheet (%)

Random structure (%)

α-helical (%)

β-turn (%)

EHA

23.46 � 0.79d 17.73 � 0.11e 30.47 � 0.65c 34.36 � 0.92b 33.41 � 0.73b 36.72 � 0.58a

12.32 � 0.76b

26.98 � 0.83b 27.67 � 0.88a 25.18 � 0.92c

37.24 0.48b 43.87 1.47a 32.17 0.58c 29.76 0.45d 30.00 0.57d 36.87 0.39b

EHN EHF EHT EHPe EHPa

10.73 � 0.58

c

12.18 � 0.47b 12.07 � 1.05b 12.35 � 0.96

b

13.02 � 0.49

a

23.81 � 0.57d 24.24 � 0.66d 13.39 � 0.50e

� � � � � �

a Different letters represent significant difference between different samples in the same column.

gel via adding NaOH and tea polyphenol in the previous study (Ai et al., 2019b), but the EWs gel formed was limited by its state to be applied to stabilize the emulsion system. Therefore, it is necessary to use some efficient and economical techniques to treat the gel formed, so that the EWs gel can be used in stabilizing the emulsion. Enzymatic hydrolysis, as an efficient and rapid method, has been widely used in the treatment of various proteins, and can well open the cross-linked proteins of EWs gel, causing the proteins to be re-uncoiled and released for the preparation of EWs hydrolysates (EH). Thus, th proteine released can stabilize the emulsion after homogenization under high pressure, which adds new 2

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Fig. 3. Size distribution and fluorescence-observed microstructural changes in freshly-prepared emulsions stabilized with EHA, EHN, EHF, EHT, EHPe, and EHPa. (A) particle size distribution; (B) microstructural changes under red excitation; and (C) microstructural changes under green excitation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3

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Fig. 4. Loss modulus (G00 ) and complex viscosity of EH-prepared fresh emulsion. (A) G00 ; (B) complex viscosity; (C) G00 at 10 rad/s; (D) complex viscosity at 10 rad/s.

was characterized using a fluorescence microscope microscopy. The ionic, temperature, and storage stabilities of the emulsion were also evaluated. Meanwhile, the oxidation characteristics of the emulsion’s lipids were investigated along with its characteristics and antioxidant properties during a simulated in vitro digestion process. The aim of this study was to filtrate a suitable protease to obtain EH and stabilize an emulsion system.

Germany). Protease was added to the sample to a concentration of 2000 u/g EWs protein and appropriate pHs (8.0, 2.0, 9.0, 6.8, 6.2, and 7.5 for trypsin, pepsin, alcalase, neutrase, flavourzyme, and papain, respec­ tively) and temperatures (55 � C, 37 � C, 55 � C, 55 � C, 50 � C, and 50 � C, respectively) were selected. The pH was regulated every 20 min using 1 moL/L NaOH or HCl over the entire enzymatic hydrolysis process, which lasted 4 h. The obtained hydrolysates were placed in a boiling water bath for 10 min to deactivate the protease activity. The hydrolysates were leached using a 0.45 μm filter when the temperature dropped to 25 � C and were stored at 4 � C. A portion of the prepared enzymatic solution was freeze-dried and stored at 18 � C before testing. The hydrolysates prepared by using trypsin, pepsin, alcalase, neutrase, flavourzyme and papain were defined as EHT, EHPe, EHA, EHN, EHF and EHPa, respectively.

2. Materials and methods 2.1. Materials Fresh duck egg (1 or 2 days old) and corn oil were purchased from a supermarket in Guangzhou, China. Rhodamine b, 2,20 -azino-bis-(3-eth­ ylbenzthiazoline-6-sulfonic acid) (ABTS), 8-aniline-1-naphthalenesul­ fonic acid (ANS), 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) and proteases (flavourzyme (50,000 u/g), neutrase (102,786 u/g), alcalase (217,434 u/g), papain (71,461 u/g), pepsin (3000 u/g) and trypsin (3500 u/g) were purchased from the Sigma Chemical Company (St. Louis MO, U.S.A.). Cholate and lipase were obtained from Shanghai Yuanye Biotechnology Company (Shanghai, China). Ammonium thio­ cyanate, cumene hydroperoxide, and 1, 1, 3, 3-tetraethoxypropane were purchased from Aladdin Reagent Corporation (Shanghai, China). All other chemicals were of analytical grade and used without further purification.

2.3. Characterization of EH The surface hydrophobicity, FTIR spectra, particle size, poly­ dispersity index (PDI) and zeta potential were determined as described in our previous study under the same experimental conditions (Ai et al., 2019a). 2.4. Emulsion preparation Freeze-dried hydrolysates were dissolved in distilled water to obtain a protein content of 1% (w/v) and the pH was adjusted to 12.0 using 1 moL/L NaOH. The mixture was placed into a 4 � C refrigerator overnight to complete hydration. An 80 mL protein solution and 20 mL corn oil were mixed and precut at a speed of 10,000 rpm for 1 min, then passed three times through a homogenizer at 80 MPa (AH-BASIC, ATS Engi­ neering Co Ltd, Jiangsu, China). To prevent microbial growth, 0.02% of iterative sodium was added to the emulsion. The obtained emulsion prepared by different EH was defined as EHTE, EHPeE, EHAE, EHNE, EHFE and EHPaE, respectively.

2.2. EH preparation To prepare EH, whole, fresh duck eggs were selected randomly before carefully collecting their EWs by hand. The EWs were prepared as an alkali-induced gel by adding 0.57% NaOH and 0.04% tea polyphenol, as described in our previous study (Ai et al., 2019b). The enzymes and experimental conditions used were the same as in previous work (Ai et al., 2019a). Briefly, six groups of 10 g of fractured EWs gel and 90 mL deionized water were homogenized (T25 easy clean digital, IKA, 4

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Fig. 5. Ionic stability of EH-prepared fresh emulsion. (A) Mean particle size; (B) zeta potential; (C) fluorescent microstructure; and (D) photographs of emul­ sion samples.

2.5. Particle size, zeta potential and PDI Prior to analysis, the prepared emulsions were diluted 400-fold to determine the zeta potential and 200-fold to determine the PDI, and particle size, respectively. The samples were balanced for 120 s and then tested over 12 cycles. The particle size distribution and zeta potential of the emulsions were measured using particle electrophoresis (Zetasizer Nano ZS-90, Malvern Instruments, Worcestershire, UK). Each sample was measured in triplicate.

was carried out by a rheometer (MCR301, Anton Parr, Austria). A 50 mm stainless steel parallel plate geometry with a 0.1 mm gap was used for the determination. The fresh prepared emulsion was placed on the platform (approximately 1.5 mL), and the probe was pressed down before adding the paraffin seal to prevent water loss. The frequency scanning conditions were as follows: angular frequency ranged from 1 to 100 rad/s and temperature 25 � C, and the amplitude gamma was set to 2%. Therefore, the loss modulus (G00 ) and complex viscosity of the sample were obtained with 31 points.

2.6. Fluorescence microscopy

2.8. Emulsion stability

The microstructures of the emulsion samples were studied with fluorescence microscopy under 40-times magnification (Axio Observer A1, Carl Zeiss, Germany). A 20 μL sample of 0.1 mg/mL rhodamine b solution was added into 1 mL of freshly prepared emulsion. A total of 10 μL of the mixed emulsion was absorbed onto a glass slide before gently with a coverslip. The microstructure of the emulsion was observed under a bright light field and fluorescence imagery was obtained under red and green excitation.

The prepared emulsion was tested for its ionic, temperature, and storage stabilities. Temperature stability was determined by placing samples in 30, 50, 70, and 90 � C water baths for 30 min, then imme­ diately cooling the heated samples with flowing tap water. Ionic stability was probed by adding different amounts of NaCl into the emulsions to obtain ionic strengths of 10, 50, 100, 150, and 200 mmoL/L. Storage stability was determined after the samples were stored at a constant temperature of 25 � C for 30 days. The average particle size, zeta po­ tential, and optical microstructure of all treated samples were determined.

2.7. Rheological property Rheological property of different hydrolysates prepared emulsion 5

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Fig. 6. Thermal stability of EH-prepared fresh emulsion. (A) Mean particle size; (B) zeta potential; (C) fluorescent microstructure; and (D) photographs of emul­ sion samples.

2.9. Lipid oxidation

each emulsion was mixed in test tubes with 2.0 mL of TBA reagent (15% w/v trichloroacetic acid and 0.375% w/v trichloroacetic acid in 0.25 moL/L HCl). The resultant mixtures were then heated in a boiling water bath for 15 min and cooled to room temperature in air for approximately 10 min. The heated mixtures were filtered using a 1.5 mm microporous membrane (0.22 μm). The absorbances of the filtrates were recorded at 532 nm and the TBARS concentrations (μmol/L of emulsion) were determined using the standard curve of 1, 1, 3, 3-tetraethoxypropane.

2.9.1. Lipid hydroperoxide The emulsions were placed in test tubes and allowed to oxidize at 25 � C for 30 days. The lipid peroxide formation was evaluated using the method of Shantha and Decker (1994) with some modifications. Briefly, 0.3 mL of each emulsion was mixed with 1.5 mL isooctane/2-propanol (3:1, v/v) by vertexing with each sample three times for approxi­ mately 10 s. The organic solvent phase of the mixtures was collected through centrifugation at 1000g for 2 min. The organic solvent phase (200 μL) was added to 2.8 mL of the methanol:1-butanol mixture (2:1, v/v), followed by addition of 15 μL of 3.94 moL/L ammonium thiocy­ anate and 15 μL of a ferrous iron solution (prepared by mixing 0.132 moL/L BaCl2 and 0.144 moL/L FeSO4). The absorbances of the resultant solutions were measured at 510 nm for 20 min after addition of the iron solution. The lipid hydroperoxide concentrations were determined using a standard curve based on cumene hydroperoxide.

2.10. Gastrointestinal tract model The freshly-prepared emulsions were passed through a gastrointes­ tinal tract (GIT) model that simulated the mouth, stomach, and small intestine. This method was developed based on a previous study (Zhang, Zhang, Zhang, Decker, & McClements, 2015); therefore, only a brief summary is given here. First, both the sample and simulated saliva were incubated at 37 � C. Second, 15 mL of sample was mixed with 15 mL simulated saliva (containing 0.03 g/mL mucin) and the pH adjusted to 6.8. Third, the mixture was incubated in an incubator shaker for 10 min at 37 � C to mimic agitation in the mouth. Fourth, 30 mL of the mouth-phase emulsion was mixed with 30 mL of simulated gastric fluid containing 0.096 g pepsin, then preheated to 37 � C and the pH adjusted

2.9.2. Thiobarbituric acid-reactive substances The thiobarbituric acid-reactive substance (TBARS) content in the emulsions upon storage was determined using the process described by Mcdonald and Hultin (1987) with some modifications. Briefly, 0.3 mL of 6

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Fig. 7. Storage stability of EH-prepared fresh emulsion during storage at room temperature for 1 month. (A) Mean particle size; (B) zeta potential; (C) fluorescent microstructure; and (D) photographs of emulsion samples.

to 2.5. The mixture was incubated in the incubator shaker for 2 h at 37 � C to mimic stomach conditions. Fifth, small intestine conditions were modeled through the addition of simulated intestinal fluids (3 mL), lipase (5 mL), and bile salts (7 mL) and the mixture adjusted to pH 7.0. The pH was maintained at 7.0 throughout the small intestine phase by titrating 0.25 mmoL/L NaOH into the mixture at 37 � C throughout the 2 h incubation period. The average particle size, surface charge, and fluorescence-observed microstructure of the emulsions were character­ ized at each stage of digestion. The volume of NaOH required to neutralize the released free fatty acids (FFAs) was recorded over time and used to calculate the percentage of FFAs released during the digestion process using formula (1): FFA ​ ð%Þ ¼

VNaOH � CNaOH � Mlipid � 100 Wlipid � 2

2.11. Antioxidant activity The antioxidant activity of the emulsion at each stage was assayed by scavenging DPPH, ABTS, and O2 radical activity. The method used to determine antioxidant activity was adopted from a previous study (Nimalaratne, Bandara, & Wu, 2015). The EWs protein in the simulation samples was diluted to a concentration of 1 mg/mL, and the resulting DPPH concentration was 7.4 � 10 2 mmoL/L. Solvent blanks were measured for each assay and each sample was run in triplicate. 2.12. Statistical analysis The experiments were conducted in triplicate using freshly-prepared samples. Results are expressed as the mean � standard deviation of the triplicate measurements. The data were analyzed using SPSS statistical software (version 16.0, Chicago, IL, USA).

(1)

where VNaOH is the volume of added NaOH (mL), CNaOH is the molarity of the NaOH solution (0.25 mmoL/L), Mlipid is the average molecular weight of corn oil, and Wlipid is the mass of the oil in the small intestine phase.

3. Results and discussion 3.1. FTIR The FTIR spectrum for the EH is shown in Fig. 1A. The 3200–3400 7

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from several coordinate displacements; Haris & Severcan, 1999; Jackson & Mantsch, 2008). However, the amide band I of the EHN was weaker than that of the other hydrolysates. The absorption peak near 1076 cm 1 – O bonds (Jiang et al., 2011). is attributed to the absorption of –C– Different action sites for each type of protease led to different FTIR spectra, especially for the EHN. Amide I band (1700–1600 cm 1) represents the major characteristics of the secondary structure of the proteins, as caused by changes in the hydrogen-bonding and dipole-dipole interactions of the polypeptide backbones (Surewicz, Mantsch, & Chapman, 1993). Based on previous reports (Surewicz et al., 1993), the peaks within 1600–1700 cm 1 were assessed as follows: 1600–1640 cm 1 for β-sheets, 1640–1650 cm 1 for random structures, 1650–1660 cm 1 for α-helixes and 1660–1700 cm 1 for β-turns. Fresh EW protein contains 42.25% β-sheets, 15.32% random structures, 23.85% α-helixes, and 18.58% β-turns (Ji et al., 2013), while alkali-induced EW protein (0.57% NaOH) comprises 18.26% β-sheets, 23.39% random structures, 23.21% α-helixes, and 35.14% β-turns (Ai et al., 2019b). For the enzymatic proteins, the β-sheet and random structure decreased content decreased while the α-helix (except EHPa), and β-turn contents increased relative to those of fresh EW protein (shown in Table 1). However, compared with the alkali-induced EW gel, the β-sheet (except EHN) and α-helix contents increased while the random structure content declined, and β-turn of different hydrolysates content changed variously. Previous study showed that the secondary structures of the hydrolysates prepared with preserved EWs gel using the same proteases were significantly different (Ai et al., 2019a). This could be related to the treatment method and processing aids used to treat the preserved eggs (CuSO4, black tea, NaOH, NaCl). Each component of the secondary structures in EHN (especially the β-sheets, random structures, and β-turns) varied from those of the other proteases due to the specific characteristics of the neutrase used to hydrolyze the protein. This may affect the other properties of the hydrolysate, such as its emulsifying capacity or antioxidant activity. 3.2. Surface hydrophobicity

Fig. 8. Lipid oxidation of EH-prepared fresh emulsions during storage at room temperature for 1 month. (A) Lipid hydroperoxide; (B) TBARS.

Surface hydrophobicity is closely related to the functional properties of proteins, such as their solubility, rheology, and emulsify capacity (Li et al., 2018). Therefore, monitoring surface hydrophobicity is an important method to reflect the functional characteristics of proteins. The hydrophobicity of the proteins was mainly caused by repulsion of water molecules from aggregated non-polar groups rather than from van der Waals forces between non-polar groups (Young, Jernigan, & Covell, 2010). And ANS can be used as a probe to reflect the level of hydro­ phobicity in proteins based on bonding with this anionic protein (Smith, Galazka, Wellner, & Sumner, 2010). Surface hydrophobicity of the EH is illustrated in Fig. 1B. EHPa possessed the highest surface hydrophobicity except control group, which is consistent with our previous research (Ai et al., 2019a). This was followed by EHF and EHPe. The surface hy­ drophobicity of EHT and EHPa showed an insignificant difference in surface hydrophobicity (P < 0.05), while the EHN sample had the lowest surface hydrophobicity. Neutrase is an endonuclease, which may destroy more hydrophobic groups embedded in the interior molecule, showing the lowest surface hydrophobicity. Our previous study found that the surface hydrophobicity of trypsin-treated preserved EWs pro­ tein was the lowest, which might be caused by enzymatic inactivation from the addition of Cu2þ into the pickling solution (Ai et al., 2019a). At the same time, the surface hydrophobicity of control group was signif­ icantly higher than hydrolyzed proteins (P < 0.05). This illustrates that the hydrophobic groups of the EWs protein were destroyed significantly after enzymatic hydrolysis. Hydrophobic groups are often embedded in the interior structure of proteins (Croguennec, Renault, Beaufils, Dubois, & Pezennec, 2007). The protease decomposed part of the protein to produce hydrolysates or amino acids, which destroyed additional hy­ drophobic groups and led to differences in surface hydrophobicity.

cm 1 band is considered the stretching vibration mode of –OH, and the 2800–3000 cm 1 band is considered the stretching vibration mode of �nchez-Gonza �lez, Desobry, Chiralt, & Tehrany, 2014). –CH (Jim� enez, Sa In the 3200–3400 cm 1 band, the peak was at 3286.64 cm 1, of which the strength was enhanced after hydrolysis, and the same results occurred to other peaks. However, the peak values of EWs treated with different enzymes varied significantly within this band. And EHN had the strongest peak, while EHPe had the weakest, which might be related to the enzymatic hydrolysis characteristics of different proteases, resulting in prominent difference in electronegativity between the two ends of the bond. The neutrase had the greatest effect on the protein of EWs gel, which might also affect the stability of emulsion prepared by using EHN. Furthermore, the peak width of EHN in the 3200–3400 cm 1 region was lower than that of the other samples, illustrating that there were strong hydrogen bonds between the hydrolysates induced by neutrase, causing the EHN to interact with water molecules after hy­ dration, and proteins at the interface of oil to become easier to disperse into the water during emulsion preparation, which led to instability of the emulsion. The peak at 3070.28 cm 1 could be due to Ar–H stretching vibration mode (Haris & Severcan, 1999) in Fig. 1A. Two peaks (EHN) or three peaks occurred in the 2800–3000 cm 1 range, which might be caused by the stretching vibration modes of the –CH3 group in different parts of the material (Haris & Severcan, 1999). The amide bands of the EWs protein were mainly composed of band I (1700–1600 cm 1, 80% – O stretching with a minor contribution from the C–N stretching C– mode), band II (1550–1530 cm 1, 60% N–H bending and 40% C–N stretching) and band III (1310–1250 cm 1, a complex band resulting 8

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Fig. 9. In vitro digestibility of EH-prepared fresh emulsion in various parts of a simulated gastrointestinal tract. (A) Particle size distributions; (B) zeta potential; (C) fluorescence microscope images (2.5D denotes 2.5-dimensional); and (D) FFAs released by emulsion.

3.3. Particle size, zeta potential and PDI

had the smallest particle sizes, which were still larger than those of most other hydrolysates obtained from fresh EWs preparations in our present study. This suggests that the hydrolysate accumulations obtained from the hydrolysis of fresh EWs are significantly smaller than those in pre­ served EWs, which is related to the characteristics of the raw material itself. Each hydrolysate showed a different PDI, which was related to the charged and hydrophilic nature of the surface of each enzymatic polypeptide. The zeta potential can be used to indicate the stability of a colloid or solution, where higher values indicate greater stability and smaller molecules or particles (Ai, Guo, Zhou, Wu, & Jiang, 2018). The zeta potentials of the EH varied from 36.07 to 16.93 mV, with EHN having the lowest and EHPa the highest. During our experiments, the

The particle size of a protein or complex sample has an important effect on its emulsion stability. An appropriate particle size can lead to the formation of a stable emulsion and prevent aggregation (Chen et al., 2018). The average particle size of the EHA sample was the largest at 824.67 nm, followed by EHN at 755.83 nm, EHPe at 197.70 nm, EHF at 152.33 nm, EHPa at 159.33 nm, and EHT at 124.24 nm (Fig. 2A and B). Each protease has different pathways by which it hydrolyzes EWs pro­ tein, which results in the hydrolysate having different particle sizes. The results of a previous study showed that the protein hydrolysates ob­ tained from the flavourzyme treatment of preserved EWs aggregated at a size of 8316.58 nm (Ai et al., 2019a), while neutrase-treated samples 9

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Table 2 The changes in DPPH, ABTS, and O2 radical scavenging rate of emulsion during in vitro digestion. EHAM DPPH radical scavenging rate (%)

ABTS radical scavenging rate (%)

O2 radical scavenging rate (%)

Initial Mouth Stomach Small intestine Initial Mouth Stomach Small intestine Initial Mouth Stomach Small intestine

EHNM c

65.93 � 0.52 99.34 � 0.74a 87.25 � 0.35b 89.45 � 0.41b 45.31 � 0.29c 69.57 � 0.83a 52.82 � 0.44b 53.62 � 0.18b 85.86 � 0.35a 49.53 � 0.29c 56.10 � 0.31b 83.93 � 0.43a

82.86 � 87.03 � 41.76 � 50.33 � 71.18 � 81.64 � 40.35 � 22.65 � 54.62 � 51.79 � 66.67 � 80.58 �

EHFM b

0.48 0.35a 0.51c 0.26c 0.41b 0.39a 0.30c 0.28d 0.35c 0.27c 0.46b 0.34a

82.20 � 95.38 � 99.56 � 92.31 � 19.71 � 68.77 � 36.19 � 85.25 � 70.17 � 55.74 � 36.47 � 81.88 �

EHTM c

0.52 0.59b 0.85a 0.42b 0.19d 0.72b 0.47c 0.66a 0.38b 0.59c 0.25d 0.83a

92.31 � 85.27 � 78.47 � 71.21 � 31.64 � 75.20 � 52.82 � 77.48 � 29.87 � 40.00 � 43.16 � 78.74 �

EHPeM a

0.77 0.69b 0.45c 0.53d 0.49c 0.52a 0.38b 0.81a 0.55c 0.53b 0.34b 0.49a

46.59 91.87 72.53 81.76 53.22 69.57 53.22 87.53 16.92 53.04 40.66 80.00

EHPaM d

� 0.31 � 1.04a � 0.58c � 0.72b � 0.48c � 0.45b � 0.50c � 0.39a � 0.33d � 0.58b � 0.74c � 0.83a

47.47 81.98 90.33 65.49 46.78 68.77 53.62 86.60 21.74 42.55 74.77 61.15

� 0.25d � 0.99b � 0.85a � 0.91c � 0.30d � 0.51b � 0.49c � 0.58a � 0.74d � 0.51c � 0.52a � 0.67b

a

Different letter in the same index as the column indicate significant differences (P < 0.05). The concentrations of hydrolyzed protein content during digestion to determine antioxidant activity were set to 1 mg/mL. The concentration of DPPH was 7.4 � 10 2 mmoL/L. b

freeze-dried enzymatic EWs gel showed stronger dispersibility in water in comparison. However, if prolonged standing is performed during reconstitution, some of the protease hydrolysis solution may precipitate, such as EHPa. That might be related to the hydrolysate zeta potential and the rearrangement and aggregation of peptides caused by in­ teractions between them.

(Fig. 4A). It has been reported that non-covalent cross-links in the emulsion system could reflect frequency dependence degree, while more chemical crosslinking of network reduced frequency dependence degree (Liu & Tang, 2014; Tang & Liu, 2013). This indicated that chemical crosslinking was enhanced after enzymatic hydrolysis. The complex viscosity of emulsions decreased significantly with the increase of angular frequency (Fig. 4B), among which EHNE and EHAE were significantly much higher than that of other emulsions, which man­ ifested better flow behavior. In order to compare the rheological prop­ erty of these emulsions in a clearer way, G00 -value and complex viscosity at the frequency of 10 rad/s were selected and demonstrated in Fig. 4C and D. EHNE represented the largest value, followed by EHAE, while EHPaE showed the smallest value. EHNE would exhibit creaming after being placed for 2 h, which might be linked to its viscosity. That result was similar to the particle size of hydrolysates, indicating that larger particle size stable emulsions tend to droplets aggregation and increase complex viscosity due to molecule interaction enhancement. Through the above analyses, the internal structure of the tested emulsion was illustrated, and it was verified that hydrolysis weakened the non-covalent cross-links between proteins. Meanwhile, the emulsion prepared by the protein hydrolysate with larger particle size showed higher viscosity, which lead to the instability of the emulsion.

3.4. Characteristics of fresh prepared emulsions The particle size distributions of the freshly-prepared emulsions using different EHs are shown in Fig. 3. Various particle sizes were ob­ tained for the emulsions containing different hydrolysates. The particle size distributions for the EHTE, EHAE, EHFE, EHPeE, and EHPaE were smooth and contained two peaks, while that of the EHNE had three peaks, indicating that nonuniform aggregation occurred between the emulsion droplets due to electrostatic interactions or the rearrangement of polypeptide segments. The average particle size of each emulsion was 359.4 nm for the EHAE, 1040.0 nm for EHNE, 454.3 nm for EHFE, 409.3 nm for EHTE, 425.3 nm for EHPeE, and 495.7 nm for EHPaE. The par­ ticle size of EHNE was 2–2.9 times that of the other emulsions. Emulsion ability index (EAI) and emulsion stability index (ESI) found that the EHN possessed the lowest EAI but that the ESI was the best (supplemental material, Fig. S1). However, the EHNE using high-pressure homogeni­ zation had the largest emulsion particle size and lowest stability, which was unexpected. At a high pH, the Hþ concentration was low and the -COOH- and –NHþ 3 groups were protonated, resulting in a negative static charge (Adjonu, Doran, Torley, & Agboola, 2014). However, the nega­ tive charge of the fresh emulsion prepared with EHN ( 58.8 mV, Fig. 9 B) was greater than that of the others, which caused it to be more prone to electrostatic repulsion. This resulted in the dissociation and rear­ rangement of small peptides and aggregation of emulsion droplets. However, this phenomenon is also related to internal chemical bonding, such as van der Waals forces, covalent bonding, or hydrogen bonding. The microstructures of the freshly-prepared emulsions are shown in Fig. 3. Fluorescent microscopy found tight contacts between the droplets of the EHNE, with some of them being aggregated. There was lower dispersion in the EHAE and EHFE than that in EHTE, with the uniform dispersion of EHTE and EHPeE droplets.

3.6. Emulsion stability The environmental stability of an emulsion, such as its ionic, ther­ mal, pH, and storage stability, is of great significance to its utilization and development. Thus, an environmental stability study was performed on the emulsions described above. 3.6.1. Ionic strength Ionic stability is based on the particle size, zeta potential, visual appearance, and microstructure of emulsions with different ionic strengths, as shown in Fig. 5. With increases in NaCl concentration, the average particle size of each emulsion increased significantly (P < 0.05), indicating that ionic strength greatly affects aggregation. At low ionic concentrations (10 mmoL/L), the EHNE was stratified, while the other EH-prepared emulsions were more stable. At high ionic concentrations (200 mmoL/L), the emulsions (except for EHTE and EHPeE) showed different degrees of stratification, with EHAE and EHNE being the most stratified. The EHFE and EHPaE showed limited amounts of layering. The instability of a protein emulsion at high ionic strengths is caused by electrostatic shielding between droplets. The absolute value of the zeta potential tended to decrease gradually because the additional Naþ group interacted with the –COO– group on the surface of the droplets and reduced the static charge via electrostatic shielding (Salminen & Weiss, 2014). At high ionic strength, the attraction between droplets was greater than the electrostatic repulsion, leading to droplet aggregation

3.5. Rheological property The rheological property of food emulsions is important for the storage and application of emulsions, so the dynamic viscoelastic behavior of the fresh prepared emulsions was measured with using a rheometer to reflect the relation between the overall composition and flow behavior. The G00 and complex viscosity are represented in Fig. 4. The G00 -value of all emulsion showed low in the early scanning range, while the frequency dependence appeared in the later scanning period 10

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Food Hydrocolloids 102 (2020) 105621

(Qian, Decker, Xiao, & Mcclements, 2012). Changes in the microstruc­ ture indicate that the effects of NaCl concentrations on the emulsions were different, EHFE and EHPeE showed droplets aggregation under the influence of 200 mmoL/L NaCl. In general, the emulsions prepared with EHTE had more stable ionic strengths.

Usually, lipid hydroperoxide oxidizes and decomposes into alde­ hydes, ketones, and other species. Aldehydes and other products pose health risks and are sources of bad flavors. The TBARS values of all emulsions were observed to increase significantly (P < 0.05) after storage for up to 30 days. The TBARS value of the EHFE reached 26.209 moL/kg oil, which is 2.67 times that of the EHTE at 9.781 mol/kg oil, while that of EHAE remained low. These results demonstrate that the EHAE had better inhibition of lipid oxidation. The cross-linked interfa­ cial protein layer may act as a physical barrier to the transmission of oxygen and, consequently, limit lipid oxidation (Phoon, Paul, Burgner, San Martin-Gonzalez, & Narsimhan, 2014). The analysis above showed that the proteins were distributed on the exteriors of emulsion droplets, and that the hydrolyzed polypeptide had certain antioxidant properties that resisted lipid oxidation.

3.6.2. Temperature The thermal stability of protein emulsions treated at different tem­ peratures (30–90 � C) is shown in Fig. 6. The average particle sizes of the EHAE, EHNE, and EHPaE increased significantly with increases in treatment temperature. The average particle size of EHAE increased from 447.7 to 667.8 nm, that of EHNE increased from 1040.0 to 1619.0 nm, and that of papain increased from 330.9 to 603.9 nm. At 30 � C, only the EHNE showed the phenomenon of layered. However, when the temperature was increased to 90 � C, the EHAE also showed this prop­ erty. Based on visual appearance, the other emulsions (EHTE, EHFE, EHPeE, and EHPaE) were more stable. The emulsion microstructure showed that the EHN-prepared emulsion was aggregated, which is consistent with the variations in its size and visual appearance. The absolute zeta potential value for the EHAE and EHNE increased, which could be caused by transfer or rearrangement of the protein hydrolysates covering the surface of the droplets due to heating. The heating process also altered the surface hydrophobicity, leading to flocculation of the emulsions (Zhang, Wu, Yang, He, & Wang, 2012).

3.8. Influence of GIT conditions on emulsion stability When food enters the human GIT, its structural characteristics change due to various physicochemical and physiological processes (Mcclements, Decker, & Park, 2008). The average particle size, zeta potential, microstructure, and FFAs release of each emulsion during digestion are shown in Fig. 9. Average particle sizes increased signifi­ cantly in the oral phase. Fluorescence microscopy revealed that, except for the uniform distributions of EHFE and EHTE droplets, the other emulsions showed various degrees of aggregation due to interactions of their droplets with the mucin present in the simulated saliva. Under simulated oral conditions, the mucin caused the protein-prepared emulsions to produce bridging or flocculation (Vingerhoeds, Silletti, Groot, Schipper, & Aken, 2009). The charge characteristics of all emulsions also changed (EHAE, EHFE, EHTE, and EHPeE increased, while EHNE and EHPaE decreased), which is primarily related to the electrostatic shielding effect of the anionic mucin molecules or the polyvalent equilibrium ions adsorbed onto the droplet surfaces and mineral ions (Mao & Mcclements, 2012). After entering the gastric phase, the average particle size of the emulsions increased significantly to >2000 nm. The microstructures exhibited serious droplet aggregation, because the lipid droplets were dispersed in a simulated gastric solution that caused the pH, ionic strength, and enzyme activity to change. A relatively low pH in the stomach leads to high positive charges in the droplets. This causes strong electrostatic attraction between the cationic lipid droplets and the anionic mucin molecules, which facilitates bridging flocculation. The relatively high ionic strength in the simulated gastric solution would weaken the electrostatic interactions in the system, thereby promoting droplet aggregation by reducing the electrostatic repulsion between droplets. The relatively low pH in the stomach is expected to produce droplets with large positive charges, while a negative droplet charge could be attributed to charge shifts caused by the adsorption of anionic mineral ions or mucin molecules from the saliva to the surface of the cationic lipid droplets (Gumus, Davidov-Pardo, & Mcclements, 2016). The average particle size of the emulsions was significantly lower in the small intestine phase than in the stomach phase. The microstructures exhibited protein aggregation, especially for the EHNE, EHPeE, and EHPaE. A decrease in particle size may be caused by the enzymatic breakdown of proteins and fats during digestion. After exposure to simulated digestion, all the emulsions obtained negative charges. This is attributed to the neutral pH conditions of the small intestine causing the proteins to have relatively high negative charges. During intestinal digestion, the emulsion structures were destroyed generally, and the mean particle size of each emulsion decreased, which might be due to the decomposition effect of protease during digestion and the influence of pH and ionic strength. The significant increase in the absolute value of zeta potential was attributed to the production of various types of anionic colloidal particles in the mixture. The fluores­ cence microstructure showed the formation of protein aggregates, possibly small aggregates of digested mixtures. Emulsified digestible

3.6.3. Storage stability Storage stability was related to the average particle size, zeta po­ tential, microstructure, and apparent morphology of emulsions stored at room temperature (25 � C) for 30 days, as shown in Fig. 7. The particle sizes of each protein hydrolysate emulsion increased with time. The average particle size of the EHAE increased from 497.2 to 862.1 nm, that of EHNE increased from 1205.0 to 2143.0 nm, that of EHPaE increased from 546.3 to 876.2 nm, and those of EHPeE, EHTE, and EHFE remained stable after 20 days’ storage. However, the particle sizes of the EHTE and EHPeE began to increase significantly at 20 and 25 days, respectively. Increases in particle size may be related to droplet flocculation (Qiu, Zhao, & Mcclements, 2015). Morphological observations revealed that the EHN-prepared emulsion precipitated on the first day of storage. Over the 30 days of storage, the emulsions showed different degrees of stratification. The EHTE had better storage stability than the other emulsions. At the same time, it can be seen from the changes in micro­ structure that each emulsion aggregated inordinately at 30 days, form­ ing larger particles of emulsion droplets. The absolute zeta potential for each emulsion increased significantly in the middle and later periods of storage (15–25 days) and decreased significantly during the final period (especially EHFE and EHNE). It might be the lipid oxidation products that affected the charge behavior of the emulsions. These results sug­ gested that EHTE was stable to ionic, thermal and storage influences. 3.7. Emulsion oxidative stability Lipid oxidation is the main cause of decreases in the quality of food demulsification (Coupland & Mcclements, 1996). The oxidative stability of the different hydrolysate-prepared emulsions was determined by monitoring both lipid hydroperoxide and TBARS formation during storage, as displayed in Fig. 8A and B. The hydroperoxide content of EHAE was the lowest, with a range of 0.208–0.496 mmoL/kg oil. The hydroperoxide content of EHPaE was the lowest, with a range of 0.177–1.234 mmoL/kg oil. Variations in the hydroperoxide content of the other emulsions ranged from 0.209 to 0.919 mmoL/kg oil. The dif­ ference in hydroperoxide content between EHTE and EHPeE was insignificant, while the content in EHFE was greater than in EHNE. Kong and Xiong (2006) demonstrated that alcalase-hydrolyzed zein provided strong antioxidant activity in a liposomal model system, which was related to the composition of the specific amino acids/peptides present in the zein hydrolysates, which is consistent with our results. 11

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triacylglycerol was hydrolyzed into monoglycerol and FFAs under the competitive adsorption of lipase at the oil-water interface after entering the small intestine phase (Zhang et al., 2016). Fig. 9D shows the kinetics of the FFAs released during the digestion of the EH-stabilized emulsions in the small intestine. The samples were digested rapidly by pancreatin within 30 min. The FFAs release rate for the EHNE was significantly greater than those of the other emulsions. There were no obvious dif­ ferences in the rate and extent of FFAs released by the EHAE and EHTE. The FFAs release rate after digestion for 2 h was 77.29% for EHNE, 68.76% for EHPaE, 67.82% for EHPeE, 64.56% for EHFE, 56.46% for EHAE, and 55.72% for EHTE. The release of FFAs is related to the contact between lipase and oil droplets through proteins, and the vol­ ume in the small intestine is large, which extends the distance between the spread of biopolymer and the surrounding solution and lipase con­ tact (Ma, Tu, Wang, Zhang, & Mcclements, 2018), and EHTE and EHAE may delay the contact between lipase and oil.

as an emulsion stabilizer, improving the emulsion stability and func­ tional properties, giving them great potential for application in the food industries. Declaration of competing interest There are no conflicts of interest to declare. The author thanked all those involved in the design and operation of the experiment and thanked the laboratory for the financial support: Guangdong province livestock and poultry products processing technology engineering research center construction (2014B090904075); The National Center for Precision Machining and Safety of Livestock and Poultry Products Joint Engineering Research Center, China. Acknowledgements The author thanks all those involved in the design and operation of the experiments and thanks the following laboratories for their financial support: the Guangdong Province Livestock and Poultry Products Pro­ cessing Technology Engineering Research Center Construction (2014B090904075), The National Center for Precision Machining, and the Safety of Livestock and Poultry Products Joint Engineering Research Center, China. And the author thanked my best friend Wenkai Zhou for his help in grammar and spell checking.

3.9. Assessment of emulsions’ antioxidant activities during digestion The antioxidant properties of the various emulsions during digestion, including the amounts of scavenging DPPH radicals, ABTS radicals, and O2 radicals, were characterized (Table 2). In terms of the DPPH free radical scavenging ability, the antioxidant activities of the EHAE improved to 65.93–89.45%, that of EHFE to 82.20–92.31%, EHPeE to 46.59–81.76%, and EHPaE to 47.47–65.49%. However, the antioxidant activities of EHNE decreased to 82.86–50.33% and those of EHTE decreased to 92.31–71.21%. The ABTS free radical scavenging ability of the EHNE was significantly reduced to 71.18–22.65%, while those of the other emulsions were increased greatly, especially in the EHFE, EHTE, EHPeE, and EHPaE. The O2 free radical scavenging abilities showed significant increases, especially for the EHPeE and EHPaE, while that of the EHAE slightly decreased (from 85.86 to 83.93%, P > 0.05). Some of the emulsions exhibited volatility antioxidant activities during diges­ tion. For example, in EHPaE, DPPH free radicals were removed in the mouth but increased significantly in the stomach phase. These free radicals then declined greatly in the small intestine phase. The EHAE and EHFE showed higher removal capacity of O2 free radicals in the fresh and intestinal phases, which represents reduced in the mouth and stomach phases. We have three hypotheses for the above changes. 1) The protease used in digestion caused the polypeptide segments to decom­ pose or aggregate. 2) An increase in the ionic concentration during digestion changed the protein aggregation effect and altered the ex­ pressions of active sites. 3) The amino acid composition of the enzymatic hydrolysate was also closely related to the free radical scavenging ac­ tivity. The tyrosine, methionine, histidine, lysine and tryptophan have been proven to have remarkable antioxidant activities (You, Zhao, Regenstein, & Ren, 2010). The results above indicate that most of the emulsions prepared using hydrolysates had higher antioxidant proper­ ties after digestion, especially in terms of the scavenging capacities of ABTS and O2 free radicals.

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.foodhyd.2019.105621. Notes The authors declare no competing financial interests. References Adjonu, R., Doran, G., Torley, P., & Agboola, S. (2014). Whey protein peptides as components of nanoemulsions: A review of emulsifying and biological functionalities. Journal of Food Engineering, 122(1), 15–27. Ai, M., Guo, S., Zhou, Q., Wu, W., & Jiang, A. (2018). The investigation of the changes in physicochemical, texture and rheological characteristics of salted duck egg yolk during salting. LWT- Food Science and Technology, 88, 119–125. Ai, M., Tang, T., Zhou, L., Ling, Z., Guo, S., & Jiang, A. (2019). Effects of different proteases on the emulsifying capacity, rheological and structure characteristics of preserved egg white hydrolysates. Food Hydrocolloids, 87, 933–942. Ai, M., Zhou, Q., Guo, S., Ling, Z., Zhou, L., Fan, H., et al. (2019). Effects of tea polyphenol and Ca(OH)2 on the intermolecular forces and mechanical, rheological, and microstructural characteristics of duck egg white gel. Food Hydrocolloids, 94, 11–19. Anton, N., Benoit, J. P., & Saulnier, P. (2008). Design and production of nanoparticles formulated from nano-emulsion templates-A review. Journal of Controlled Release, 128(3), 185–199. Chang, C., Niu, F., Gu, L., Xin, L., Hao, Y., Bei, Z., et al. (2016). Formation of fibrous or granular egg white protein microparticles and properties of the integrated emulsions. Food Hydrocolloids, 61, 477–486. Chen, Y. B., Zhu, X. F., Liu, T. X., Lin, W. F., Tang, C. H., & Liu, R. H. (2018). Improving freeze-thaw stability of soy nanoparticle-stabilized emulsions through increasing particle size and surface hydrophobicity. Food Hydrocolloids, 87, 404–412. Coupland, J. N., & Mcclements, D. J. (1996). Lipid oxidation in food emulsions. Trends in Food Science & Technology, 7(3), 0-91. Croguennec, T., Renault, A., Beaufils, S., Dubois, J. J., & Pezennec, S. (2007). Interfacial properties of heat-treated ovalbumin. Journal of Colloid and Interface Science, 315(2), 627–636. Dickinson, E. (2010). Food emulsions and foams: Stabilization by particles. Current Opinion in Colloid & Interface Science, 15(1–2), 40–49. Feng, Y. X., Ruan, G. R., Jin, F., Xu, J., & Wang, F. J. (2018). Purification, identification, and synthesis of five novel antioxidant peptides from Chinese chestnut (Castanea mollissima Blume) protein hydrolysates. LWT- Food Science and Technology, 92, 42–46. Gumus, C. E., Davidov-Pardo, G., & Mcclements, D. J. (2016). Lutein-enriched emulsionbased delivery systems: Impact of Maillard conjugation on physicochemical stability and gastrointestinal fate. Food Hydrocolloids, 60, 38–49. Haris, P. I., & Severcan, F. (1999). FTIR spectroscopic characterization of protein structure in aqueous and non-aqueous media. Journal of Molecular Catalysis B: Enzymatic, 7(1–4), 207–221.

4. Conclusion In summary, EWs gel was separated into small particles and six types of enzymes were employed for proteolysis. The obtained nano­ polypeptides were then prepared with corn oil as an emulsion delivery system. Most hydrolysates performed the small particle size and surface hydrophobicity decreased attributed to the embedding or destruction of hydrophobic groups. The prepared emulsion by EH represented different particle size distribution and microstructural properties. The EHTprepared emulsion had better ionic, thermal, and storage stabilities and inhibited lipid oxidation. The antioxidant activities of the emulsions improved during digestion, especially in terms of the scavenging of DPPH, ABTS, and O2 radicals. The release rate of FFAs for EHTE and EHAE was lowest and reached 55.72% and 56.46% respectively. This study provides a basic approach to the development of EWs gel proteins 12

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