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Characterization of ethanol-induced egg white gel and transportation of active nutraceuticals
LWT - Food Science and Technology 130 (2020) 109530
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Characterization of ethanol-induced egg white gel and transportation of active nutraceuticals
T
Li Yaoa,b, Aimin Jianga,∗, Ling Chenc a
College of Food Science, South China Agricultural University, Guangzhou, 510642, China College of Food and Biotechnology, Guangdong Polytechnic of Science and Trade, Guangzhou, 510430, China c Department of Applied Engineering, Zhejiang Institute of Economics and Trade, Hangzhou, 310018, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Egg white Ethanol Curcumin Tea polyphenols Delivery system
This paper investigated the gelling characteristics of ethanol-induced egg white gel (EEWG) and also evaluated the ability of EEWG to delivery curcumin and tea polyphenols (TP). Results showed that a high concentration of ethanol induced egg white (EW) to form gel, which was attributed to the increase of zeta potential in absolute value and free sulfhydryl content. A high concentration of ethanol reduced the intrinsic fluorescence, mainly of tryptophan, and increased the viscosity. EEWG with a high concentration of protein represented greater hardness and water-holding capacity combined with the formation of abundant small bubbles. The addition of TP decreased the hardness value and water-holding capacity and reduced the storage modulus and loss modulus, determined by a rheometer, for further prevention of EW protein denaturation. The curcumin and TP stabilities were improved after adding a higher concentration of protein during storage. Adding a proper concentration of ethanol (30–40 mL/100 mL) increased the bioaccessibility of curcumin and improved the antioxidant activity and protein digestibility, but this was decreased after EEWG was loaded with TP. These results suggested that EW protein could improve the delivery of a hydrophobic active substance.
1. Introduction Active substances, such as certain plant polyphenols, vitamins, and functional oils, possess functional properties and play an important role in human health and metabolism (Endo, Kuroda, Terahara, Tsujita, & Tamura, 1976, p. 983). Active hydrophobic substances, such as β-carotene and curcumin, are unstable in water, limiting their applications and developments (Anand, Kunnumakkara, Newman, & Aggarwal, 2007). In recent years, more and more attention has been paid to researching nanoparticles supporting active hydrophobic substances, such as loading curcumin onto soybean protein, polypeptide, or egg white (EW) aggregates (Chang et al., 2019; Liu, Li, Zhang, & Tang, 2019; Zhang et al., 2018). These bioactive substances or functional factors are widely used in the research and development of various new functional foods. However, in food processing, storage, and human gastrointestinal environments, these active ingredients often are vulnerable to damage and poor stability, greatly reducing their bioavailability (McClements et al., 2015). Therefore, finding ways to improve the biological accessibility and stability of such substances is still a major challenge for the food industry. EW is rich in protein, the most abundant being ovalbumin, followed
∗
by ovotransferrin, ovomucoid, and lysozyme (Mine, 1995). EW protein is amphiphilic and possesses high digestibility, being good properties for a potential carrier material to support active substances (Selmer, Kleemann, Kulozik, Heinrich, & Smirnova, 2015). EW proteins can selfassemble under different conditions, such as heated, alkaline, acidic, high ionic strength, coenzymatic, etc (Gharbi & Labbafi, 2018). The mechanism of protein aggregation formed under these conditions is different. The gelation property is an important functional characteristic of EW, and it is often applied in the food industry, such as for preserved eggs. The formation of EW gel under specific circumstances is mainly due to protein aggregation and the self-assembly of protein. However, the common automatic coating of EW is mainly through thermal and alkali processing. So far, there are few studies on the ethanol assembly of EW gel and no studies have been conducted into the loading of active substances on ethanol-induced EW gel. Preliminary experiments found that egg whites could quickly aggregate to form gel in ethanol solution. In order to explore the formation mechanism of ethanol-induced egg white gel (EEWG), the rheological properties, the surface charge properties, and the intrinsic fluorescence and intermolecular forces of EEWG were characterized. In our previous reasearch found that TP could weaken the strength of
Corresponding author. E-mail address: [email protected] (A. Jiang).
https://doi.org/10.1016/j.lwt.2020.109530 Received 20 January 2020; Received in revised form 13 April 2020; Accepted 1 May 2020 Available online 04 June 2020 0023-6438/ © 2020 Elsevier Ltd. All rights reserved.
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from 0.1 to 100 1/s, the temperature was 25 °C, and the viscosity was the value at the sheer speed of 10 1/s.
alkali-induced gels (Ai et al., 2019). In this study, in order to further explore the effect of TP on ethanol-induced EW gels, TP were loaded onto EEWG, and the microstructure, gel strength, water-holding capacity, and rheological properties were investigated. At the same time, EEWG was utilized to load curcumin and TP, exploring the active preservation in the storage process. The bioavailability of digested curcumin and TP loaded on EEWG was studied by in vitro simulated digestion, as well as the protein digestion of EW and antioxidant activity. This study provides directions for the delivery of active substances and the expansion of the functional properties of EW gels.
2.4. Zeta potential Prior to analysis, the prepared EEWG was diluted 100-fold to determine the zeta potential. Samples were balanced for 120 s and then tested over 12 cycles. The zeta potential was measured using particle electrophoresis (Zetasizer Nano ZS-90, Malvern Instruments, Worcestershire, UK).
2. Material and methods
2.5. Free sulfhydryl content
2.1. Material
The free sulfhydryl content was adjusted according to the methods of Ai et al. (2020) under the same environment. In briefly, 3 g of samples were weighted and mixtured with 27 mL sodium phosphate buffer (0.1 mol/L pH 8.0), homogenized at 10,000 rpm for 2 min and centrifuged at 10,000 ×g for 15 min, and the supernatant was collected. The protein content in the supernatant was measured by Bradford's method. 0.2 mL of the supernatant with 2.8 mL Tris-Gly buffers solution (pH 8.0 containing 0.089 mol/L Tris, 0.09 mol/L glycine, 0.004 mol/L EDTA and 8 mol/L urea) and 0.02 mL Ellman reagent (4 mg/mL DTNB was dissolved in Tris-Gly buffer) reacted in a 40 °C water bath for 15 min before measuring the absorbance at 412 nm. The free sulfhydryl content was calculated as following: free sulfhydryl content = 73.53 × D × A412/C, where A412 is the absorbance at 412 nm, D is the dilution factor, C is the concentration of protein in mg/ mL and 73.53 is derived from 106/13,600 (13,600 is Ellman's reagent molar absorptivity).
Fresh duck eggs (1 or 2 days old) were bought from a supermarket in Guangzhou, China. TP was bought from Mengze Biological Co., Ltd. (Shanghai, China). Rhodamine b, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and trimethylaminomethane (Tris) were supplied from Sigma (St. Louis, MO, USA). Curcumin was brought from Shanghai McLean Biochemical Technology Co., Ltd (Shanghai, Chian). Pepsin (3000 u/g), trypsin (1500 u/mg) and bile extract were purchased from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, Chian). Glycine (Gly), ethanol, and ethylenediaminetetraacetic acid (EDTA) were bought from the Tianjin Fuyu Fine Chemical Co., Ltd. (Tianjin, China). All reagents were of analytical grade. 2.2. Preparation of EEWG One- or two-days old duck eggs were cleaned with tap water and were hand-broken. The EW and yolk were carefully separated from the eggshell. The EW was mixed homogeneously with a magnetic stirrer (84-1A6, Shanghai Sile Instrument Co., Ltd, Shanghai, China) at 2000 rpm for 15 min. Then, the EW protein content was diluted to 2–5 g/100g with pH 7.0 of 50 mmol/L sodium phosphate buffer solutions. Anhydrous ethanol was configured to a concentration of 0–90 mL/100 mL, and 10 mL of 3 g/100 g EW protein was mixed with 10 mL of ethanol solution to achieve the EW protein concentration of 1.5 g/ 100g, the ethanol concentrations from 5 to 45 mL/100 mL, which was quickly stirred and then rested for 1h. In addition, a series of TP ethanol (70 mL/100 mL) solutions containing 0.02, 0.06, 0.1, and 0.14 g/100g were prepared. Precisely 10 mL of the mixed solution containing TP was added with 10 mL of 3 g/100 g EW protein to achieve the EW protein concentration of 1.5 g/100g, the TP concentrations of 0.01, 0.03, 0.05 and 0.07 g/100g, and was quickly stirred and then rested for 1 h. To investigate the stability of active substances loaded on EW protein, 10 mL of EW with different protein concentrations (2–5 g/ 100g) were added with 10 mL ethanol (70 mL/100 mL) containing 3 mg/mL of curcumin and TP to achieve the EW protein concentration of 1–2.5 g/100g, the curcumin and TP concentrations of 1.5 mg/100g. These were quickly stirred and then wrapped in tin foil and placed in an incubator at 25 °C for 7 days. To explore the stability of active substances under a gastrointestinal environment, 10 mL of 3 g/100 g EW protein solution was added with 10 mL of 60–90 mL/100 mL ethanol solution containing either 3 mg/mL of curcumin or TP to achieve the protein concentration of 1.5 g/100g, the curcumin and TP concentrations of 1.5 mg/100g and the ethanol concentrations of 30–45 mL/ 100 mL, stirred quickly and then rested for 1 h.
2.6. Fluorescence microstructure The microstructures of the EEWG were studied with fluorescence microscopy under 10-times magnification (Axio Observer A1, Carl Zeiss, Germany). A 20 μL of 0.1 mg/mL rhodamine b solution was added into 1 mL of freshly prepared gel. A total of 10 μL of the mixed sample was absorbed onto a glass slide before gently covering with a coverslip. The fluorescence imagery was obtained under red excitation. 2.7. Hardness TPA was conducted using a TA-XT Plus texture analyzer (Stable Microsystems, Surrey, England) according to a previous study (Ai, Guo, Zhou, Wu, & Jiang, 2018) with some modifications. The gel to be tested was prepared in a 20 mL glass bottle (15 mL) and compressed to 50% of its original height fitted with a probe (P/0.5R). The pretest and test speeds were set to 1 mm/s and 1 mm/s, respectively. The hardness texture parameters were calculated with the hardness values obtained for the peak value at the time of the first compression. All samples were tested 8 times. 2.8. Water-holding capacity (WHC) The WHC of the EEWG was determined according to the method of Li et al. (2018) with slight modifications. About 3 g gel was added to a l0 mL centrifuge tube, then centrifuged at 10,000 r/min for 10 min, after which the supernatant was removed. The calculation formula of WHC is as follows:
2.3. Apparent viscosity
WHC = The apparent viscosity was measured using a rheometer (MCR301, Anton Paar, Austria). After dropping 1 mL of EEWG onto the test plane, a probe (CP50 equipped with 50 mm diameters) was pressed down before adding a paraffin seal on the probe side to prevent water loss. The shear scanning conditions were as follows: the shear rate ranged
M 2 − M1 M 3 − M1
where M1 is the weight of the centrifuge tube; M2 is the weight of the protein gel and the centrifuge tube after centrifugation, and M3 is the total weight of the protein and the centrifuge tube before centrifugation. 2
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Fig. 1. The visual appearance (A) of EW protein dispersion (3 g/100g) in the presence of increasing concentrations (0–45 mL/100 mL) of ethanol, before and after inversion. The appearance of viscosity (B), zeta potential (C), free sulfhydryl (D), and intrinsic fluorescence spectra (E) in the presence of increasing concentrations (0–45 mL/100 mL) of ethanol. Different letters for the same index indicate significant differences (P < 0.05). 0–45 represents the ethanol concentrations of 0–45 mL/100 mL.
420 nm. Similarly, 0.2 mL of the sample was added with 1 mL of Folin's phenol reagent, and 5 mL of 15 g/100g sodium carbonate solution was added after resting for 3 min. Then, after 30 min of rest, the supernatant was centrifugated at 3000 g for 5 min, and the absorbance value was determined at 760 nm (Li et al., 2015).
2.9. Rheological properties The rheological properties of EEWG were determined using a rheometer (MCR301, Anton Paar, Austria) fitted with a 50 mm steel parallel plate (CP50). The following sweep sequence was conducted: a) the strain sweep was conducted, the mixture was adsorbed and dropped onto the platform, the strain ranged from 0.1 to 1000%, and the temperature was set at 25 °C; b) the frequency sweep was determined under 1–100 rad/s at 25 °C with a 0.2% strain.
2.11. Evaluation of in vitro bioaccessibility The bioaccessibility of curcumin or TP encapsulated in the reassembled EW gel, defined as the relative amount of curcumin or TP transferred to the aqueous phase at the end of in vitro digestion (180 min), was evaluated using an in vitro simulated digestion model consisting of sequential gastric (60 min) and intestinal (120 min) digestion, according to the process described in a previous study (Chen et al., 2015) with some modifications. In brief, 10 mL of sample solution was mixed well with 10 mL of 0.1 mol/L HCl (pH 1.5) and preincubated
2.10. Curcumin and TP stability in EEWG In brief, 3 mL of ethyl acetate was added to 0.2 mL of the EEWG. The mixtures were vortexed for 60 s and left for layering, and then the supernatants (organic phase) were collected. The amount of curcumin in the obtained supernatants was determined spectrophotometrically at 3
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the protein (Ai et al., 2018). As shown in Fig. 1C, the absolute zeta potential value of EW protein increased significantly with the higher ethanol content, ranged from −11.3 to −39.46 mV, indicating that EW protein is less stable in lower concentrations of ethanol. When the ethanol concentration increased, the surface charge of EW protein also increased, resulting in greater epulsion to form stable micelles or gels (Fig. 5A).
in a shaker (at 37 °C) at a rate of 100 rpm for 10 min. Then, 8 mg of the pepsin powder was added and mixed well to start the simulated gastric digestion. After 60 min, the pH of the pepsin-digest was immediately adjusted to 7.0 with 4 mol/L NaOH, and 200 mg of bile extract was added and well dispersed in a shaker for 10 min. Lastly, 20 mg of the pancreatin powder was added to start the intestinal digestion (60–180 min). After 180 min, 500 μL of the final digest dispersion was collected and centrifuged at 6000 g for 15 min to determine the amount of curcumin or TP remaining in the supernatants. Using the standard curve of the bovine serum protein solution (0.1–1.0 mg/mL), the absorbance of supernatant after centrifugation was measured at 280 nm and the concentration of protein in the gastric fluid was obtained during digestion.
3.1.2. Conformational changes EW proteins were aggregated and combined in ethanol solutions, which may have also been related to the conformational changes in EW proteins. Therefore, Fig. 1D–E reflect the changes of free sulfhydryl content and intrinsic fluorescence spectra of EW proteins at different ethanol concentrations. However, after adding ethanol, the contents of the free sulfhydryl group significantly reduced, indicating that the sulfhydryl group on the surface of the EW after adding ethanol was embedded into the interior, and the protein structure was gradually destroyed. This may have also been responsible for the aggregation of EW protein ethanol gels in relation to the transformation of sulfhydryl and disulfide bonds. After adding ethanol, the peak protein fluorescence of EW protein is typically concentrated around 335 nm, mainly due to the fluorescence of tryptophan (Trp) residues. With increased ethanol concentration, the fluorescence intensity decreases significantly (P < 0.05; especially between 20 and 45 mL/100 mL), indicating that ethanol can interact with protein groups and cause the quenching of the intrinsic fluorescence of proteins. When the ethanol concentration is between 25 and 45 mL/100 mL, the 315 nm side peak increases significantly, which was due to the fluorescence excitation of other amino acids or the fluorescence transition of Trp. In addition, Trp and tyrosine (Tyr) in EW protein are mainly distributed on the surface of the protein, indicating that the surface groups of the EW protein are embedded within the protein structure after adding ethanol, perhaps also changing the surface hydrophobicity of protein, resulting in a change in protein conformation and causing protein aggregation. These results are consistent with the changes represented in Fig. 1A. It can be seen from Fig. S1 that the protein patterns changed under the induction of ethanol, especially between 25 and 45 mL/100 mL where the protein (especially ovalbumin and ovalbumin) degraded, indicating that the aggregated protein has been formed by the decomposed proteins interconnecting by chemical bonds, such as hydrogen bonds or van der Waals force. It is interesting to highlight that from the concentration of 25 mL/100 mL the solution becomes turbid and homogeneous along with higher zeta potential and lower fluorescence. That means that the repulsive forces increase and the Trp are no more at the surface leading to dispersion of the aggregates. For larger ethanol concentrations, this effect is not sufficient and the solutions appear turbid but heterogeneous. This would indicate that agregates at low ethanol concentrations are larger than at high ethanol ones.
2.12. Antioxidant activities The antioxidant activity of digested samples was assayed by scavenging DPPH and ABTS radical activity. The method used to determine antioxidant activity was adopted from a previous study (Nimalaratne, Bandara, & Wu, 2015). This was achieved by diluting 1 mL of digested samples into 100 mL, with the resulting DPPH concentration of 1.0 mmol/L. Solvent blanks were measured for each assay. 2.13. Statistical analysis All experiments were conducted in triplicate, except for hardness, which was performed 8 times. The results were reported as the mean ± standard deviation. The data was analyzed using SPSS V.20 software (SPSS Inc., Chicago, IL). Significant differences between means (P < 0.05) were studied through Duncan's multiple-range test by one way ANOVA using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). 3. Results and discussion 3.1. Characterization of EW-ethanol dispersion transformation into gel 3.1.1. Protein aggregation and gel formation Fig. 1A shows the visual appearance of EW proteins (1.5 g/100g, w/ w) with different concentrations of ethanol (0–45 mL/100 mL, v/v). In the 0–15 mL/100 mL concentration, the EW proteins were stacked together, but still achieved the formation of the aggregated micelle state after being shaken and rested for a period of time. When the concentration of ethanol was increased to 20 mL/100 mL, the EW solution began to appear cloudy. With the ethanol concentration increased to 25 mL/100 mL, the aggregated protein micelle disappeared, and the EW protein solution presented a cloudy state. The solution began to aggregate and became more turbid in ethanol increased to 30 mL/100 mL, but the aggregation degree was not significant and the gel that formed was easily collapsed by external forces. EW proteins formed gels in ethanol concentrations of 35–45 mL/100 mL, but as the ethanol increased, more of the ethanol solution precipitated. Ethanol usually enhances ethanol-water interactions by first disrupting the hydrogen bonds that maintain the rigid tertiary structure (oligomeric globulin is also quaternion), and then further damaging the hydrophobic regions of the protein (Brandts & Hunt, 1967; Liu et al., 2019; Romero, Lozano, Sancho, & Giraldo, 2007). As a result, proteins become associated and even aggregated. Therefore, the changes in protein structure caused by ethanol may be irreversible at higher ethanol concentrations (> 20 mL/ 100 mL). Viscosity scanning and surface charge were used to further characterize EW protein aggregation and association in ethanol solutions. With increases in the ethanol concentration, the viscosity of the whole EW solution system significantly increased (25–45 mL/100 mL; P < 0.05) but was not significant at 0–25 mL/100 mL (P > 0.05), consistent with the protein changes observable in Fig. 1A. The charge on the protein surface can reflect the aggregation state and stability of
3.2. Effects of adding TP on the gel properties of EEWG 3.2.1. Gel microstructure and appearance Our previous study revealed that the addition of TP to alkali-induced EW gels affected the EW gelling properties, for example, by reducing the textural and rheological properties but protecting the protein degradation (Ai et al., 2019). The prepared gel with different protein and TP content is shown in Fig. 2A. With an increased protein concentration, the overall transmittance of the gel decreased and the turbidity increased significantly. Interestingly, with the higher concentrations, there were a greater number of bubbles in the gel system and the actual size of the bubbles was significantly smaller. Adding 0.01–0.07 g/100 g TP led to a color change from white to reddishbrown, but the amount of small bubbles in the gel system did not change significantly. Fig. 2B shows the fluorescence micrograph with different protein and TP concentrations. As can be seen from the figure, 4
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Fig. 2. The visual appearance (A) of different concentrations of EW protein dispersion (1–2.5 g/100g) and tea polyphenols (0.01–0.07 g/100g) at a 35 g/100g ethanol concentration. (B) The fluorescence microstructure of EEWG at different concentrations of EW protein and tea polyphenols. (C and D) The hardness and WHC of prepared EEWG. Different letters for the same index indicate significant differences (P < 0.05). 1–2.5 stands for the protein concentration of 1–2.5 g/100g; 0.01–0.07 stands for the tea polyphenols contents of 0.01–0.07 g/100g. EW represents egg white; TP stands for tea polyphenols.
content, the protein ethanol gel was softer, which was related to the effect of TP on protein. EEWG are weaker than with other treatments, such as heating or adding alkali solutions (Zhao et al., 2020; Li et al., 2018), depending on the effect of the denatured agent on the protein. However, more water exists in the EEWG process, which to some extent limits the water embedding and the contact between proteins, resulting in weaker gel formation. The results of WHC of EEWG are shown in Fig. 2D. The WHC of EW protein was consistent with its textural properties, indicating that TP hindered the formation of EEWG to a certain extent. This was consistent with study results whereby TP were added to alkali-induced EW gel (Ai et al., 2019). It may be that the polar groups between TP and ethanol are responsible for possible desiccation of the protein that weakens the gel, or that the strengthening of hydrogen bonds causes other interactions to weaken, resulting in less textural properties and WHC of the prepared gel.
with the increase in protein concentration the size of bubbles grew smaller but there were more bubbles overall. The bubbles in the TP group were smaller than those in the gel without TP. Ethanol and water are infinitely miscible, but in reality, ethanol and water will still form a binary system after mixing (Zhang et al., 2004). When proteins are added to the system, they disrupt the energy transfer and stability of the system, while the energy is unstable, leading to the formation of bubbles after mixing ethanol and water (Song, Islam, & Lee, 2008). TP contain a large number of active groups that are more likely to interact with ethanol, having a certain effect on the gelling properties of the prepared ethanol gel. 3.2.2. Gel hardness and WHC Induced by a high concentration of ethanol (35 mL/100 mL), the prepared EEWG supported the texture analysis. With a higher protein concentration, the hardness value of the prepared gel increased significantly (Fig. 2C), from 4.73 g to 16.43 g, indicating that higher concentrations of protein form stronger gels. With increasing of TP
3.2.3. Rheological properties The microstructure of the formed gel was closely related to the 5
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Fig. 3. The rheological properties of EEWG at different protein and tea polyphenol concentrations. (A, B) Strain sweep results; (C, D) Frequency sweep results. A and C represents the effects of protein content (1.0, 1.5, 2.0 and 2.5 g/100g) on the rheological properties; B and D stands for the effects of tea polyphenols (0.01, 0.03, 0.05 and 0.07 g/100g) on the rheological properties.
3.3. Evaluation of EEWG delivery system
rheological properties of small deformation (Dickinson & Chen, 1999). In order to further characterize the effect of the EW protein concentration and the TP content on the rheological characteristics of the EEWG, the rheological properties of the gel were characterized (Fig. 3). As shown in Fig. 3A–B, with higher protein concentrations, the linear viscoelastic region of EEWG gradually decreased, while higher TP content did not change the linear viscoelastic region of the gel. In the linear viscoelastic region, G′ was greater than G″, indicating that the sample had the property of an elastic solid. With the increased stress, an apparent intersection occurred (G″ > G′), indicating that the network structure began to change under stress. With increasing protein concentrations, the critical stress gradually lessened, which was due to the structural changes occurring in the protein stacking under low stress. Alternatively, with increasing protein concentration, the G′ and G″ values of the EEWG also increased significantly (P < 0.05), indicating the gel that formed under high concentrations of protein were stronger. However, the G′ and G″ changes in the TP group were not significant. Fig. 3C–D demonstrate the results of frequency scanning of the gel, which can reflect the degree of dependence of the EEWG rheological response on frequency. All Gʹ and Gʹʹ values of prepared gel increase gradually with the increase in scanning frequency, indicating that Gʹ and Gʹʹ showed certain frequency dependence, and EEWG showed a kind of viscoelastic behavior in solids. With the protein concentration becoming higher, both Gʹ and Gʹʹ increased significantly (P < 0.05) in frequency scanning, revealing the gel formed had higher strength. With the increasing TP content, the Gʹ of the protein ethanol gel was much larger than Gʹʹ, demonstrating that there were more elastic crosslinking parts in the EEWG system. Further, with the higher proportions of TP, the frequency dependency also increased with lower elastic properties.
3.3.1. Stability of curcumin and TP In order to investigate the transport of TP and curcumin by EW protein, the stability of curcumin and TP stored at 25 °C was investigated. Fig. 4A and B illustrate the appearance of EEWG containing curcumin and TP prepared at different protein concentrations. It can be seen from the figures that curcumin appears red, which is related to the pH of the EW mixed with ethanol, indicating that the gel systems were alkaline. After 24 h of storage, the red color of the gel system containing curcumin became paler, indicating that the curcumin degraded or decomposed. Fig. 4B shows the appearance of TP at different protein concentrations. The EEWG prepared at the beginning was light brown, becoming deeper in color over 24 h. With increased protein concentration, the gel system presented a white gel aggregate, which was formed by the accumulation of proteins after ethanol denaturation. Fig. 4C and D reflect the stability of curcumin and TP during storage. The activity of curcumin and TP gradually lessened with the length of storage. This indicated that the increased protein concentration significantly improved (P < 0.05) the retention rate of curcumin and TP and provided a better carrier for the transport of active substances. This result is consistent with other study (Chen, Ou, & Tang, 2016). Study has shown that lipophilic components are embedded in the hydrophobic microregion of proteins, thus effectively impeding the self-assembly of thermally unstable molecules (Rahimnejad, Mokhtarian, & Ghasemi, 2010). Therefore, the encapsulation potential for curcumin of EW protein was related to the defolding and reaggregation behavior of proteins, which determine the formation of hydrophobic microregions of proteins (Chang et al., 2019). As a substance that can be dissolved in 6
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Fig. 4. The visual appearance of different concentrations (1–2.5 g/100g) of protein at 35 g/100g ethanol concentration with 1.5 mg/mL curcumin (A) and tea polyphenols (B). The storage stability of curcumin (C) and tea polyphenol-loaded (D) EW gel at 25 °C for 7 days. ( ) 1.0 g/100g of protein, ( ) 1.5 g/100g of protein, ( ) 2.0 g/100g of protein, ( ) 2.5 g/100g of protein.
less than 30%. Liu et al. (2019) previously reported that the digestibility of soybean-loaded curcumin increased significantly with increased ethanol content, which was related to the interaction and complexation of protein and curcumin. In our experiment, at an ethanol concentration of 45 mL/100 mL, the availability of curcumin decreased, which may have been because the protease activity was affected, and more curcumin became embedded in the EW protein and could not be released. In contrast, the accessibility of TP was poor, which was related to the interaction between TP and EW proteins. Abundant active groups and hydrophilic groups in TP are more likely to combine with the hydrophilic parts during interaction with EW proteins, making them more prone to decomposition and degradation during the digestion process and significantly reducing the accessibility of the TP. The antioxidant activity of the curcumin initially increased but then decreased. At an ethanol concentration of 40 mL/100 mL, the DPPH and ABTS radical
both water and ethanol, the degradation rate of TP in ethanol is much lower than that in water (Puligundla, Mok, Ko, Liang, & Recharla, 2017). As the protein content increased, the percentage of preserved TP also increased, suggesting that the protein aggregates provided better shelter for TP. 3.3.2. Digestive properties of supported active nutraceuticals To evaluate the activity and bioavailability of curcumin and TP after digestion, the antioxidant activity (DPPH and ABTS) and accessibility of curcumin and TP in the digested system were determined (Table 1). At a protein concentration of 1.5 g/100 g EW, the accessibility of curcumin and TP changed in opposite ways at different ethanol concentrations. The curcumin firstly became more accessible, but then became less so with increased ethanol concentration, while the accessibility of TP decreased significantly (P < 0.05). The accessibility of all groups was 7
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Fig. 5. Schematic illustration for EEWG formed (A) and the formation of curcumin and tea polyphenols-loaded reassembled EEWG (B).
The digestive characteristics of EW protein after digestion were characterized (Table 1). The digestibility of protein loaded with curcumin firstly increased and then decreased, while protein digestion loaded with TP was significantly reduced. This is consistent with the accessibility changes of curcumin and TP, proving that proteins loaded with active substances of different properties significantly affect the digestion properties of proteins and the stability of active substances, and also indicated that protease acted more on the hydrophilic part during digestion. TP are rich in hydrophilic groups and when added they compete with EW proteins to interact with proteases. Obviously, TP bind better with protease at high concentrations of ethanol, thereby reducing the digestibility of protein (Fig. 5B). From the above results, it can be seen that EEWG can not only improve the accessibility of hydrophobic active substances but also improve the antioxidant properties and protein digestibility after digestion, offering greater possibilities for the development of functional properties of egg-white gel and the transfer of hydrophobic active substances.
Table 1 The bioaccessibility of curcumin and tea polyphenols-loaded on egg white protein, antioxidant activity and protein digestibility after digestion. Sample
Bioaccessibility of CU or TP (%)
CU-30 CU-35 CU-40 CU-45 TP-30 TP-35 TP-40 TP-45
19.46 28.52 29.49 28.31 23.97 18.67 18.42 17.32
± ± ± ± ± ± ± ±
0.74d 0.58b 0.82a 0.70b 0.68c 0.59e 0.34e 0.42f
DPPH scavenging rate (%) 39.83 43.99 49.21 47.39 52.28 45.11 41.44 38.72
± ± ± ± ± ± ± ±
0.44f 0.71d 0.62b 0.58c 0.53a 0.77d 0.59e 0.82g
ATBS scavenging rate (%) 45.88 51.05 55.21 53.15 49.52 43.21 41.24 36.21
± ± ± ± ± ± ± ±
0.62e 0.45c 0.56b 0.50a 0.62d 0.73f 0.65g 0.55h
Protein digestibility (%)
55.02 62.93 71.24 66.80 76.06 61.58 59.46 60.23
± ± ± ± ± ± ± ±
0.54h 0.75d 0.65b 0.73c 0.91a 0.52e 0.44g 0.78f
*Different letters indicate significant difference (p < 0.05). CU stands for curcumin; TP stands for tea polyphenols.
scavenging ability were strongest, at 49.21% and 55.21%, respectively. While for the gel containing TP, the antioxidant activity significantly declined (P < 0.05). This was consistent with the accessibility changes of curcumin and TP after digestion, indicating that curcumin and TP have a greater influence on the antioxidant properties of the loading system. Some researchers have reported that the main degradation products of curcumin contain phenolic groups with antioxidant activity (Schneider, 2019; Tsuda, 2018). In conclusion, the EEWG system improves the transport capacity of hydrophobic active substances.
4. Conclusion This study firstly reported and characterized the physicochemical properties of EEWG and prepared the EEWG to act as the delivery carrier. EW protein aggregated under the influence of high concentrations of ethanol due to the improvement of molecule forces reflected by the increase of the absolute value of zeta potential and the decrease of free sulfhydryl content and the intrinsic fluorescence intensity. While 8
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the addition of TP reduced the hardness and WHC of EEWG, increasing protein content improved the WHC, hardness and rheological properties of EEWG. The increasing protein content improved the stability of curcumin and TP loaded on EEWG, and the appropriate ethanol content increased the bioaccessibility of curcumin, the digestibility of protein and antioxidant activity. However, as the ethanol concentration increased, the addition of TP into EEWG decreased the bioaccessibility of TP, the digestibility of protein and antioxidant activity. Therefore, EW protein can successfully be used as the carrier of hydrophobic active substances, providing a reference for the development of a hydrophobic active substance delivery system.
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CRediT authorship contribution statement Li Yao: Conceptualization, Methodology, Writing - original draft, Writing - review & editing, Software. Aimin Jiang: Visualization, Investigation. Ling Chen: Supervision. 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: The National Center for Precision Machining and Safety of Livestock and Poultry Products Joint Engineering Research Center, China. Acknowledgement The authors thanked all those involved in the design and operation of the experiment and thanked the laboratory for the financial support: The National Center for Precision Machining and Safety of Livestock and Poultry Products Joint Engineering Research Center ((2016)2203), China. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lwt.2020.109530. References 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., 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. Ai, M., Zhou, Q., Xiao, N., Guo, S., Cao, Y., Fan, H., et al. (2020). Enhancement of gel characteristics of NaOH-induced duck egg white gel by adding Ca (OH)2 with/ without heating. Food Hydrocolloids, 103, 105654. Anand, P., Kunnumakkara, A. B., Newman, R. A., & Aggarwal, B. B. (2007).
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Characterization of ethanol-induced egg white gel and transportation of active nutraceuticals
T
Li Yaoa,b, Aimin Jianga,∗, Ling Chenc a
College of Food Science, South China Agricultural University, Guangzhou, 510642, China College of Food and Biotechnology, Guangdong Polytechnic of Science and Trade, Guangzhou, 510430, China c Department of Applied Engineering, Zhejiang Institute of Economics and Trade, Hangzhou, 310018, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Egg white Ethanol Curcumin Tea polyphenols Delivery system
This paper investigated the gelling characteristics of ethanol-induced egg white gel (EEWG) and also evaluated the ability of EEWG to delivery curcumin and tea polyphenols (TP). Results showed that a high concentration of ethanol induced egg white (EW) to form gel, which was attributed to the increase of zeta potential in absolute value and free sulfhydryl content. A high concentration of ethanol reduced the intrinsic fluorescence, mainly of tryptophan, and increased the viscosity. EEWG with a high concentration of protein represented greater hardness and water-holding capacity combined with the formation of abundant small bubbles. The addition of TP decreased the hardness value and water-holding capacity and reduced the storage modulus and loss modulus, determined by a rheometer, for further prevention of EW protein denaturation. The curcumin and TP stabilities were improved after adding a higher concentration of protein during storage. Adding a proper concentration of ethanol (30–40 mL/100 mL) increased the bioaccessibility of curcumin and improved the antioxidant activity and protein digestibility, but this was decreased after EEWG was loaded with TP. These results suggested that EW protein could improve the delivery of a hydrophobic active substance.
1. Introduction Active substances, such as certain plant polyphenols, vitamins, and functional oils, possess functional properties and play an important role in human health and metabolism (Endo, Kuroda, Terahara, Tsujita, & Tamura, 1976, p. 983). Active hydrophobic substances, such as β-carotene and curcumin, are unstable in water, limiting their applications and developments (Anand, Kunnumakkara, Newman, & Aggarwal, 2007). In recent years, more and more attention has been paid to researching nanoparticles supporting active hydrophobic substances, such as loading curcumin onto soybean protein, polypeptide, or egg white (EW) aggregates (Chang et al., 2019; Liu, Li, Zhang, & Tang, 2019; Zhang et al., 2018). These bioactive substances or functional factors are widely used in the research and development of various new functional foods. However, in food processing, storage, and human gastrointestinal environments, these active ingredients often are vulnerable to damage and poor stability, greatly reducing their bioavailability (McClements et al., 2015). Therefore, finding ways to improve the biological accessibility and stability of such substances is still a major challenge for the food industry. EW is rich in protein, the most abundant being ovalbumin, followed
∗
by ovotransferrin, ovomucoid, and lysozyme (Mine, 1995). EW protein is amphiphilic and possesses high digestibility, being good properties for a potential carrier material to support active substances (Selmer, Kleemann, Kulozik, Heinrich, & Smirnova, 2015). EW proteins can selfassemble under different conditions, such as heated, alkaline, acidic, high ionic strength, coenzymatic, etc (Gharbi & Labbafi, 2018). The mechanism of protein aggregation formed under these conditions is different. The gelation property is an important functional characteristic of EW, and it is often applied in the food industry, such as for preserved eggs. The formation of EW gel under specific circumstances is mainly due to protein aggregation and the self-assembly of protein. However, the common automatic coating of EW is mainly through thermal and alkali processing. So far, there are few studies on the ethanol assembly of EW gel and no studies have been conducted into the loading of active substances on ethanol-induced EW gel. Preliminary experiments found that egg whites could quickly aggregate to form gel in ethanol solution. In order to explore the formation mechanism of ethanol-induced egg white gel (EEWG), the rheological properties, the surface charge properties, and the intrinsic fluorescence and intermolecular forces of EEWG were characterized. In our previous reasearch found that TP could weaken the strength of
Corresponding author. E-mail address: [email protected] (A. Jiang).
https://doi.org/10.1016/j.lwt.2020.109530 Received 20 January 2020; Received in revised form 13 April 2020; Accepted 1 May 2020 Available online 04 June 2020 0023-6438/ © 2020 Elsevier Ltd. All rights reserved.
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from 0.1 to 100 1/s, the temperature was 25 °C, and the viscosity was the value at the sheer speed of 10 1/s.
alkali-induced gels (Ai et al., 2019). In this study, in order to further explore the effect of TP on ethanol-induced EW gels, TP were loaded onto EEWG, and the microstructure, gel strength, water-holding capacity, and rheological properties were investigated. At the same time, EEWG was utilized to load curcumin and TP, exploring the active preservation in the storage process. The bioavailability of digested curcumin and TP loaded on EEWG was studied by in vitro simulated digestion, as well as the protein digestion of EW and antioxidant activity. This study provides directions for the delivery of active substances and the expansion of the functional properties of EW gels.
2.4. Zeta potential Prior to analysis, the prepared EEWG was diluted 100-fold to determine the zeta potential. Samples were balanced for 120 s and then tested over 12 cycles. The zeta potential was measured using particle electrophoresis (Zetasizer Nano ZS-90, Malvern Instruments, Worcestershire, UK).
2. Material and methods
2.5. Free sulfhydryl content
2.1. Material
The free sulfhydryl content was adjusted according to the methods of Ai et al. (2020) under the same environment. In briefly, 3 g of samples were weighted and mixtured with 27 mL sodium phosphate buffer (0.1 mol/L pH 8.0), homogenized at 10,000 rpm for 2 min and centrifuged at 10,000 ×g for 15 min, and the supernatant was collected. The protein content in the supernatant was measured by Bradford's method. 0.2 mL of the supernatant with 2.8 mL Tris-Gly buffers solution (pH 8.0 containing 0.089 mol/L Tris, 0.09 mol/L glycine, 0.004 mol/L EDTA and 8 mol/L urea) and 0.02 mL Ellman reagent (4 mg/mL DTNB was dissolved in Tris-Gly buffer) reacted in a 40 °C water bath for 15 min before measuring the absorbance at 412 nm. The free sulfhydryl content was calculated as following: free sulfhydryl content = 73.53 × D × A412/C, where A412 is the absorbance at 412 nm, D is the dilution factor, C is the concentration of protein in mg/ mL and 73.53 is derived from 106/13,600 (13,600 is Ellman's reagent molar absorptivity).
Fresh duck eggs (1 or 2 days old) were bought from a supermarket in Guangzhou, China. TP was bought from Mengze Biological Co., Ltd. (Shanghai, China). Rhodamine b, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and trimethylaminomethane (Tris) were supplied from Sigma (St. Louis, MO, USA). Curcumin was brought from Shanghai McLean Biochemical Technology Co., Ltd (Shanghai, Chian). Pepsin (3000 u/g), trypsin (1500 u/mg) and bile extract were purchased from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, Chian). Glycine (Gly), ethanol, and ethylenediaminetetraacetic acid (EDTA) were bought from the Tianjin Fuyu Fine Chemical Co., Ltd. (Tianjin, China). All reagents were of analytical grade. 2.2. Preparation of EEWG One- or two-days old duck eggs were cleaned with tap water and were hand-broken. The EW and yolk were carefully separated from the eggshell. The EW was mixed homogeneously with a magnetic stirrer (84-1A6, Shanghai Sile Instrument Co., Ltd, Shanghai, China) at 2000 rpm for 15 min. Then, the EW protein content was diluted to 2–5 g/100g with pH 7.0 of 50 mmol/L sodium phosphate buffer solutions. Anhydrous ethanol was configured to a concentration of 0–90 mL/100 mL, and 10 mL of 3 g/100 g EW protein was mixed with 10 mL of ethanol solution to achieve the EW protein concentration of 1.5 g/ 100g, the ethanol concentrations from 5 to 45 mL/100 mL, which was quickly stirred and then rested for 1h. In addition, a series of TP ethanol (70 mL/100 mL) solutions containing 0.02, 0.06, 0.1, and 0.14 g/100g were prepared. Precisely 10 mL of the mixed solution containing TP was added with 10 mL of 3 g/100 g EW protein to achieve the EW protein concentration of 1.5 g/100g, the TP concentrations of 0.01, 0.03, 0.05 and 0.07 g/100g, and was quickly stirred and then rested for 1 h. To investigate the stability of active substances loaded on EW protein, 10 mL of EW with different protein concentrations (2–5 g/ 100g) were added with 10 mL ethanol (70 mL/100 mL) containing 3 mg/mL of curcumin and TP to achieve the EW protein concentration of 1–2.5 g/100g, the curcumin and TP concentrations of 1.5 mg/100g. These were quickly stirred and then wrapped in tin foil and placed in an incubator at 25 °C for 7 days. To explore the stability of active substances under a gastrointestinal environment, 10 mL of 3 g/100 g EW protein solution was added with 10 mL of 60–90 mL/100 mL ethanol solution containing either 3 mg/mL of curcumin or TP to achieve the protein concentration of 1.5 g/100g, the curcumin and TP concentrations of 1.5 mg/100g and the ethanol concentrations of 30–45 mL/ 100 mL, stirred quickly and then rested for 1 h.
2.6. Fluorescence microstructure The microstructures of the EEWG were studied with fluorescence microscopy under 10-times magnification (Axio Observer A1, Carl Zeiss, Germany). A 20 μL of 0.1 mg/mL rhodamine b solution was added into 1 mL of freshly prepared gel. A total of 10 μL of the mixed sample was absorbed onto a glass slide before gently covering with a coverslip. The fluorescence imagery was obtained under red excitation. 2.7. Hardness TPA was conducted using a TA-XT Plus texture analyzer (Stable Microsystems, Surrey, England) according to a previous study (Ai, Guo, Zhou, Wu, & Jiang, 2018) with some modifications. The gel to be tested was prepared in a 20 mL glass bottle (15 mL) and compressed to 50% of its original height fitted with a probe (P/0.5R). The pretest and test speeds were set to 1 mm/s and 1 mm/s, respectively. The hardness texture parameters were calculated with the hardness values obtained for the peak value at the time of the first compression. All samples were tested 8 times. 2.8. Water-holding capacity (WHC) The WHC of the EEWG was determined according to the method of Li et al. (2018) with slight modifications. About 3 g gel was added to a l0 mL centrifuge tube, then centrifuged at 10,000 r/min for 10 min, after which the supernatant was removed. The calculation formula of WHC is as follows:
2.3. Apparent viscosity
WHC = The apparent viscosity was measured using a rheometer (MCR301, Anton Paar, Austria). After dropping 1 mL of EEWG onto the test plane, a probe (CP50 equipped with 50 mm diameters) was pressed down before adding a paraffin seal on the probe side to prevent water loss. The shear scanning conditions were as follows: the shear rate ranged
M 2 − M1 M 3 − M1
where M1 is the weight of the centrifuge tube; M2 is the weight of the protein gel and the centrifuge tube after centrifugation, and M3 is the total weight of the protein and the centrifuge tube before centrifugation. 2
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Fig. 1. The visual appearance (A) of EW protein dispersion (3 g/100g) in the presence of increasing concentrations (0–45 mL/100 mL) of ethanol, before and after inversion. The appearance of viscosity (B), zeta potential (C), free sulfhydryl (D), and intrinsic fluorescence spectra (E) in the presence of increasing concentrations (0–45 mL/100 mL) of ethanol. Different letters for the same index indicate significant differences (P < 0.05). 0–45 represents the ethanol concentrations of 0–45 mL/100 mL.
420 nm. Similarly, 0.2 mL of the sample was added with 1 mL of Folin's phenol reagent, and 5 mL of 15 g/100g sodium carbonate solution was added after resting for 3 min. Then, after 30 min of rest, the supernatant was centrifugated at 3000 g for 5 min, and the absorbance value was determined at 760 nm (Li et al., 2015).
2.9. Rheological properties The rheological properties of EEWG were determined using a rheometer (MCR301, Anton Paar, Austria) fitted with a 50 mm steel parallel plate (CP50). The following sweep sequence was conducted: a) the strain sweep was conducted, the mixture was adsorbed and dropped onto the platform, the strain ranged from 0.1 to 1000%, and the temperature was set at 25 °C; b) the frequency sweep was determined under 1–100 rad/s at 25 °C with a 0.2% strain.
2.11. Evaluation of in vitro bioaccessibility The bioaccessibility of curcumin or TP encapsulated in the reassembled EW gel, defined as the relative amount of curcumin or TP transferred to the aqueous phase at the end of in vitro digestion (180 min), was evaluated using an in vitro simulated digestion model consisting of sequential gastric (60 min) and intestinal (120 min) digestion, according to the process described in a previous study (Chen et al., 2015) with some modifications. In brief, 10 mL of sample solution was mixed well with 10 mL of 0.1 mol/L HCl (pH 1.5) and preincubated
2.10. Curcumin and TP stability in EEWG In brief, 3 mL of ethyl acetate was added to 0.2 mL of the EEWG. The mixtures were vortexed for 60 s and left for layering, and then the supernatants (organic phase) were collected. The amount of curcumin in the obtained supernatants was determined spectrophotometrically at 3
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the protein (Ai et al., 2018). As shown in Fig. 1C, the absolute zeta potential value of EW protein increased significantly with the higher ethanol content, ranged from −11.3 to −39.46 mV, indicating that EW protein is less stable in lower concentrations of ethanol. When the ethanol concentration increased, the surface charge of EW protein also increased, resulting in greater epulsion to form stable micelles or gels (Fig. 5A).
in a shaker (at 37 °C) at a rate of 100 rpm for 10 min. Then, 8 mg of the pepsin powder was added and mixed well to start the simulated gastric digestion. After 60 min, the pH of the pepsin-digest was immediately adjusted to 7.0 with 4 mol/L NaOH, and 200 mg of bile extract was added and well dispersed in a shaker for 10 min. Lastly, 20 mg of the pancreatin powder was added to start the intestinal digestion (60–180 min). After 180 min, 500 μL of the final digest dispersion was collected and centrifuged at 6000 g for 15 min to determine the amount of curcumin or TP remaining in the supernatants. Using the standard curve of the bovine serum protein solution (0.1–1.0 mg/mL), the absorbance of supernatant after centrifugation was measured at 280 nm and the concentration of protein in the gastric fluid was obtained during digestion.
3.1.2. Conformational changes EW proteins were aggregated and combined in ethanol solutions, which may have also been related to the conformational changes in EW proteins. Therefore, Fig. 1D–E reflect the changes of free sulfhydryl content and intrinsic fluorescence spectra of EW proteins at different ethanol concentrations. However, after adding ethanol, the contents of the free sulfhydryl group significantly reduced, indicating that the sulfhydryl group on the surface of the EW after adding ethanol was embedded into the interior, and the protein structure was gradually destroyed. This may have also been responsible for the aggregation of EW protein ethanol gels in relation to the transformation of sulfhydryl and disulfide bonds. After adding ethanol, the peak protein fluorescence of EW protein is typically concentrated around 335 nm, mainly due to the fluorescence of tryptophan (Trp) residues. With increased ethanol concentration, the fluorescence intensity decreases significantly (P < 0.05; especially between 20 and 45 mL/100 mL), indicating that ethanol can interact with protein groups and cause the quenching of the intrinsic fluorescence of proteins. When the ethanol concentration is between 25 and 45 mL/100 mL, the 315 nm side peak increases significantly, which was due to the fluorescence excitation of other amino acids or the fluorescence transition of Trp. In addition, Trp and tyrosine (Tyr) in EW protein are mainly distributed on the surface of the protein, indicating that the surface groups of the EW protein are embedded within the protein structure after adding ethanol, perhaps also changing the surface hydrophobicity of protein, resulting in a change in protein conformation and causing protein aggregation. These results are consistent with the changes represented in Fig. 1A. It can be seen from Fig. S1 that the protein patterns changed under the induction of ethanol, especially between 25 and 45 mL/100 mL where the protein (especially ovalbumin and ovalbumin) degraded, indicating that the aggregated protein has been formed by the decomposed proteins interconnecting by chemical bonds, such as hydrogen bonds or van der Waals force. It is interesting to highlight that from the concentration of 25 mL/100 mL the solution becomes turbid and homogeneous along with higher zeta potential and lower fluorescence. That means that the repulsive forces increase and the Trp are no more at the surface leading to dispersion of the aggregates. For larger ethanol concentrations, this effect is not sufficient and the solutions appear turbid but heterogeneous. This would indicate that agregates at low ethanol concentrations are larger than at high ethanol ones.
2.12. Antioxidant activities The antioxidant activity of digested samples was assayed by scavenging DPPH and ABTS radical activity. The method used to determine antioxidant activity was adopted from a previous study (Nimalaratne, Bandara, & Wu, 2015). This was achieved by diluting 1 mL of digested samples into 100 mL, with the resulting DPPH concentration of 1.0 mmol/L. Solvent blanks were measured for each assay. 2.13. Statistical analysis All experiments were conducted in triplicate, except for hardness, which was performed 8 times. The results were reported as the mean ± standard deviation. The data was analyzed using SPSS V.20 software (SPSS Inc., Chicago, IL). Significant differences between means (P < 0.05) were studied through Duncan's multiple-range test by one way ANOVA using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). 3. Results and discussion 3.1. Characterization of EW-ethanol dispersion transformation into gel 3.1.1. Protein aggregation and gel formation Fig. 1A shows the visual appearance of EW proteins (1.5 g/100g, w/ w) with different concentrations of ethanol (0–45 mL/100 mL, v/v). In the 0–15 mL/100 mL concentration, the EW proteins were stacked together, but still achieved the formation of the aggregated micelle state after being shaken and rested for a period of time. When the concentration of ethanol was increased to 20 mL/100 mL, the EW solution began to appear cloudy. With the ethanol concentration increased to 25 mL/100 mL, the aggregated protein micelle disappeared, and the EW protein solution presented a cloudy state. The solution began to aggregate and became more turbid in ethanol increased to 30 mL/100 mL, but the aggregation degree was not significant and the gel that formed was easily collapsed by external forces. EW proteins formed gels in ethanol concentrations of 35–45 mL/100 mL, but as the ethanol increased, more of the ethanol solution precipitated. Ethanol usually enhances ethanol-water interactions by first disrupting the hydrogen bonds that maintain the rigid tertiary structure (oligomeric globulin is also quaternion), and then further damaging the hydrophobic regions of the protein (Brandts & Hunt, 1967; Liu et al., 2019; Romero, Lozano, Sancho, & Giraldo, 2007). As a result, proteins become associated and even aggregated. Therefore, the changes in protein structure caused by ethanol may be irreversible at higher ethanol concentrations (> 20 mL/ 100 mL). Viscosity scanning and surface charge were used to further characterize EW protein aggregation and association in ethanol solutions. With increases in the ethanol concentration, the viscosity of the whole EW solution system significantly increased (25–45 mL/100 mL; P < 0.05) but was not significant at 0–25 mL/100 mL (P > 0.05), consistent with the protein changes observable in Fig. 1A. The charge on the protein surface can reflect the aggregation state and stability of
3.2. Effects of adding TP on the gel properties of EEWG 3.2.1. Gel microstructure and appearance Our previous study revealed that the addition of TP to alkali-induced EW gels affected the EW gelling properties, for example, by reducing the textural and rheological properties but protecting the protein degradation (Ai et al., 2019). The prepared gel with different protein and TP content is shown in Fig. 2A. With an increased protein concentration, the overall transmittance of the gel decreased and the turbidity increased significantly. Interestingly, with the higher concentrations, there were a greater number of bubbles in the gel system and the actual size of the bubbles was significantly smaller. Adding 0.01–0.07 g/100 g TP led to a color change from white to reddishbrown, but the amount of small bubbles in the gel system did not change significantly. Fig. 2B shows the fluorescence micrograph with different protein and TP concentrations. As can be seen from the figure, 4
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Fig. 2. The visual appearance (A) of different concentrations of EW protein dispersion (1–2.5 g/100g) and tea polyphenols (0.01–0.07 g/100g) at a 35 g/100g ethanol concentration. (B) The fluorescence microstructure of EEWG at different concentrations of EW protein and tea polyphenols. (C and D) The hardness and WHC of prepared EEWG. Different letters for the same index indicate significant differences (P < 0.05). 1–2.5 stands for the protein concentration of 1–2.5 g/100g; 0.01–0.07 stands for the tea polyphenols contents of 0.01–0.07 g/100g. EW represents egg white; TP stands for tea polyphenols.
content, the protein ethanol gel was softer, which was related to the effect of TP on protein. EEWG are weaker than with other treatments, such as heating or adding alkali solutions (Zhao et al., 2020; Li et al., 2018), depending on the effect of the denatured agent on the protein. However, more water exists in the EEWG process, which to some extent limits the water embedding and the contact between proteins, resulting in weaker gel formation. The results of WHC of EEWG are shown in Fig. 2D. The WHC of EW protein was consistent with its textural properties, indicating that TP hindered the formation of EEWG to a certain extent. This was consistent with study results whereby TP were added to alkali-induced EW gel (Ai et al., 2019). It may be that the polar groups between TP and ethanol are responsible for possible desiccation of the protein that weakens the gel, or that the strengthening of hydrogen bonds causes other interactions to weaken, resulting in less textural properties and WHC of the prepared gel.
with the increase in protein concentration the size of bubbles grew smaller but there were more bubbles overall. The bubbles in the TP group were smaller than those in the gel without TP. Ethanol and water are infinitely miscible, but in reality, ethanol and water will still form a binary system after mixing (Zhang et al., 2004). When proteins are added to the system, they disrupt the energy transfer and stability of the system, while the energy is unstable, leading to the formation of bubbles after mixing ethanol and water (Song, Islam, & Lee, 2008). TP contain a large number of active groups that are more likely to interact with ethanol, having a certain effect on the gelling properties of the prepared ethanol gel. 3.2.2. Gel hardness and WHC Induced by a high concentration of ethanol (35 mL/100 mL), the prepared EEWG supported the texture analysis. With a higher protein concentration, the hardness value of the prepared gel increased significantly (Fig. 2C), from 4.73 g to 16.43 g, indicating that higher concentrations of protein form stronger gels. With increasing of TP
3.2.3. Rheological properties The microstructure of the formed gel was closely related to the 5
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Fig. 3. The rheological properties of EEWG at different protein and tea polyphenol concentrations. (A, B) Strain sweep results; (C, D) Frequency sweep results. A and C represents the effects of protein content (1.0, 1.5, 2.0 and 2.5 g/100g) on the rheological properties; B and D stands for the effects of tea polyphenols (0.01, 0.03, 0.05 and 0.07 g/100g) on the rheological properties.
3.3. Evaluation of EEWG delivery system
rheological properties of small deformation (Dickinson & Chen, 1999). In order to further characterize the effect of the EW protein concentration and the TP content on the rheological characteristics of the EEWG, the rheological properties of the gel were characterized (Fig. 3). As shown in Fig. 3A–B, with higher protein concentrations, the linear viscoelastic region of EEWG gradually decreased, while higher TP content did not change the linear viscoelastic region of the gel. In the linear viscoelastic region, G′ was greater than G″, indicating that the sample had the property of an elastic solid. With the increased stress, an apparent intersection occurred (G″ > G′), indicating that the network structure began to change under stress. With increasing protein concentrations, the critical stress gradually lessened, which was due to the structural changes occurring in the protein stacking under low stress. Alternatively, with increasing protein concentration, the G′ and G″ values of the EEWG also increased significantly (P < 0.05), indicating the gel that formed under high concentrations of protein were stronger. However, the G′ and G″ changes in the TP group were not significant. Fig. 3C–D demonstrate the results of frequency scanning of the gel, which can reflect the degree of dependence of the EEWG rheological response on frequency. All Gʹ and Gʹʹ values of prepared gel increase gradually with the increase in scanning frequency, indicating that Gʹ and Gʹʹ showed certain frequency dependence, and EEWG showed a kind of viscoelastic behavior in solids. With the protein concentration becoming higher, both Gʹ and Gʹʹ increased significantly (P < 0.05) in frequency scanning, revealing the gel formed had higher strength. With the increasing TP content, the Gʹ of the protein ethanol gel was much larger than Gʹʹ, demonstrating that there were more elastic crosslinking parts in the EEWG system. Further, with the higher proportions of TP, the frequency dependency also increased with lower elastic properties.
3.3.1. Stability of curcumin and TP In order to investigate the transport of TP and curcumin by EW protein, the stability of curcumin and TP stored at 25 °C was investigated. Fig. 4A and B illustrate the appearance of EEWG containing curcumin and TP prepared at different protein concentrations. It can be seen from the figures that curcumin appears red, which is related to the pH of the EW mixed with ethanol, indicating that the gel systems were alkaline. After 24 h of storage, the red color of the gel system containing curcumin became paler, indicating that the curcumin degraded or decomposed. Fig. 4B shows the appearance of TP at different protein concentrations. The EEWG prepared at the beginning was light brown, becoming deeper in color over 24 h. With increased protein concentration, the gel system presented a white gel aggregate, which was formed by the accumulation of proteins after ethanol denaturation. Fig. 4C and D reflect the stability of curcumin and TP during storage. The activity of curcumin and TP gradually lessened with the length of storage. This indicated that the increased protein concentration significantly improved (P < 0.05) the retention rate of curcumin and TP and provided a better carrier for the transport of active substances. This result is consistent with other study (Chen, Ou, & Tang, 2016). Study has shown that lipophilic components are embedded in the hydrophobic microregion of proteins, thus effectively impeding the self-assembly of thermally unstable molecules (Rahimnejad, Mokhtarian, & Ghasemi, 2010). Therefore, the encapsulation potential for curcumin of EW protein was related to the defolding and reaggregation behavior of proteins, which determine the formation of hydrophobic microregions of proteins (Chang et al., 2019). As a substance that can be dissolved in 6
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Fig. 4. The visual appearance of different concentrations (1–2.5 g/100g) of protein at 35 g/100g ethanol concentration with 1.5 mg/mL curcumin (A) and tea polyphenols (B). The storage stability of curcumin (C) and tea polyphenol-loaded (D) EW gel at 25 °C for 7 days. ( ) 1.0 g/100g of protein, ( ) 1.5 g/100g of protein, ( ) 2.0 g/100g of protein, ( ) 2.5 g/100g of protein.
less than 30%. Liu et al. (2019) previously reported that the digestibility of soybean-loaded curcumin increased significantly with increased ethanol content, which was related to the interaction and complexation of protein and curcumin. In our experiment, at an ethanol concentration of 45 mL/100 mL, the availability of curcumin decreased, which may have been because the protease activity was affected, and more curcumin became embedded in the EW protein and could not be released. In contrast, the accessibility of TP was poor, which was related to the interaction between TP and EW proteins. Abundant active groups and hydrophilic groups in TP are more likely to combine with the hydrophilic parts during interaction with EW proteins, making them more prone to decomposition and degradation during the digestion process and significantly reducing the accessibility of the TP. The antioxidant activity of the curcumin initially increased but then decreased. At an ethanol concentration of 40 mL/100 mL, the DPPH and ABTS radical
both water and ethanol, the degradation rate of TP in ethanol is much lower than that in water (Puligundla, Mok, Ko, Liang, & Recharla, 2017). As the protein content increased, the percentage of preserved TP also increased, suggesting that the protein aggregates provided better shelter for TP. 3.3.2. Digestive properties of supported active nutraceuticals To evaluate the activity and bioavailability of curcumin and TP after digestion, the antioxidant activity (DPPH and ABTS) and accessibility of curcumin and TP in the digested system were determined (Table 1). At a protein concentration of 1.5 g/100 g EW, the accessibility of curcumin and TP changed in opposite ways at different ethanol concentrations. The curcumin firstly became more accessible, but then became less so with increased ethanol concentration, while the accessibility of TP decreased significantly (P < 0.05). The accessibility of all groups was 7
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Fig. 5. Schematic illustration for EEWG formed (A) and the formation of curcumin and tea polyphenols-loaded reassembled EEWG (B).
The digestive characteristics of EW protein after digestion were characterized (Table 1). The digestibility of protein loaded with curcumin firstly increased and then decreased, while protein digestion loaded with TP was significantly reduced. This is consistent with the accessibility changes of curcumin and TP, proving that proteins loaded with active substances of different properties significantly affect the digestion properties of proteins and the stability of active substances, and also indicated that protease acted more on the hydrophilic part during digestion. TP are rich in hydrophilic groups and when added they compete with EW proteins to interact with proteases. Obviously, TP bind better with protease at high concentrations of ethanol, thereby reducing the digestibility of protein (Fig. 5B). From the above results, it can be seen that EEWG can not only improve the accessibility of hydrophobic active substances but also improve the antioxidant properties and protein digestibility after digestion, offering greater possibilities for the development of functional properties of egg-white gel and the transfer of hydrophobic active substances.
Table 1 The bioaccessibility of curcumin and tea polyphenols-loaded on egg white protein, antioxidant activity and protein digestibility after digestion. Sample
Bioaccessibility of CU or TP (%)
CU-30 CU-35 CU-40 CU-45 TP-30 TP-35 TP-40 TP-45
19.46 28.52 29.49 28.31 23.97 18.67 18.42 17.32
± ± ± ± ± ± ± ±
0.74d 0.58b 0.82a 0.70b 0.68c 0.59e 0.34e 0.42f
DPPH scavenging rate (%) 39.83 43.99 49.21 47.39 52.28 45.11 41.44 38.72
± ± ± ± ± ± ± ±
0.44f 0.71d 0.62b 0.58c 0.53a 0.77d 0.59e 0.82g
ATBS scavenging rate (%) 45.88 51.05 55.21 53.15 49.52 43.21 41.24 36.21
± ± ± ± ± ± ± ±
0.62e 0.45c 0.56b 0.50a 0.62d 0.73f 0.65g 0.55h
Protein digestibility (%)
55.02 62.93 71.24 66.80 76.06 61.58 59.46 60.23
± ± ± ± ± ± ± ±
0.54h 0.75d 0.65b 0.73c 0.91a 0.52e 0.44g 0.78f
*Different letters indicate significant difference (p < 0.05). CU stands for curcumin; TP stands for tea polyphenols.
scavenging ability were strongest, at 49.21% and 55.21%, respectively. While for the gel containing TP, the antioxidant activity significantly declined (P < 0.05). This was consistent with the accessibility changes of curcumin and TP after digestion, indicating that curcumin and TP have a greater influence on the antioxidant properties of the loading system. Some researchers have reported that the main degradation products of curcumin contain phenolic groups with antioxidant activity (Schneider, 2019; Tsuda, 2018). In conclusion, the EEWG system improves the transport capacity of hydrophobic active substances.
4. Conclusion This study firstly reported and characterized the physicochemical properties of EEWG and prepared the EEWG to act as the delivery carrier. EW protein aggregated under the influence of high concentrations of ethanol due to the improvement of molecule forces reflected by the increase of the absolute value of zeta potential and the decrease of free sulfhydryl content and the intrinsic fluorescence intensity. While 8
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the addition of TP reduced the hardness and WHC of EEWG, increasing protein content improved the WHC, hardness and rheological properties of EEWG. The increasing protein content improved the stability of curcumin and TP loaded on EEWG, and the appropriate ethanol content increased the bioaccessibility of curcumin, the digestibility of protein and antioxidant activity. However, as the ethanol concentration increased, the addition of TP into EEWG decreased the bioaccessibility of TP, the digestibility of protein and antioxidant activity. Therefore, EW protein can successfully be used as the carrier of hydrophobic active substances, providing a reference for the development of a hydrophobic active substance delivery system.
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CRediT authorship contribution statement Li Yao: Conceptualization, Methodology, Writing - original draft, Writing - review & editing, Software. Aimin Jiang: Visualization, Investigation. Ling Chen: Supervision. 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: The National Center for Precision Machining and Safety of Livestock and Poultry Products Joint Engineering Research Center, China. Acknowledgement The authors thanked all those involved in the design and operation of the experiment and thanked the laboratory for the financial support: The National Center for Precision Machining and Safety of Livestock and Poultry Products Joint Engineering Research Center ((2016)2203), China. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.lwt.2020.109530. References 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., 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. Ai, M., Zhou, Q., Xiao, N., Guo, S., Cao, Y., Fan, H., et al. (2020). Enhancement of gel characteristics of NaOH-induced duck egg white gel by adding Ca (OH)2 with/ without heating. Food Hydrocolloids, 103, 105654. Anand, P., Kunnumakkara, A. B., Newman, R. A., & Aggarwal, B. B. (2007).
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