Functional Properties of Egg White Protein and Whey Protein in the Presence of Bioactive Chicken Trachea Hydrolysate and Sodium Chloride

Abstract

The interactions of chicken trachea collagen hydrolysate (CTH) obtained from the enzymatic hydrolysis of by-products from a chicken slaughterhouse, with two common proteins (egg white (E) and whey (W) proteins) was studied with and without sodium chloride (NaCl). The treatments with two protein types (E and W at 10% w/w) and different CTH concentrations (0, 0.25, and 0.5% w/w) and NaCl concentrations (0 and 1.5% w/w) were conducted. The addition of CTH reduced the emulsifying and foaming properties of those proteins due to their fibrillar structure, while the addition of NaCl promoted the foaming capacity. Moreover, CTH and NaCl promoted the gelling properties of those proteins, as revealed by the shift-up of the storage modulus (G′) value. The rapid shifting at 60–70 °C indicated that the heat-set gelation was observed. The strong gel strength was exhibited with the mixture containing W. The addition of 1.5% w/w NaCl improved the antioxidant and antihypertensive activities of the mixture of 0.5% w/w CTH and 10% w/w W. The best DPPH, ABTS, and FRAP radical-scavenging activities (40.00, 180.95, and 46.00 TEAC µM/mL, respectively) and the lowest IC50 value of the ACE inhibitor (30.05 mg/mL) was revealed. This mixture exhibits the highest inhibitory activity and is suited for improving the functionalities of high-protein products.

1. Introduction

Post COVID-19, consumer needs and behaviors have changed, with a high emphasis placed on health-conscious choices in lifestyle and nutrition. Functional foods that help create consumer well-being are receiving additional research interest. Understanding how functional ingredients interact is very important for designing novel products with desirable bioactive properties. Meat products are an example of foods that are deemed unhealthy due to the presence of saturated fats, and are in competition with plant-based alternatives for consumer purchases. A healthier composition is required to improve the image of meat products in the marketplace. According to a consumer insight report from Mintel^®, high protein is one of the trends that a consumer looks for [1]. In this respect, various types of protein hydrolysates and protein isolates have been added to increase the protein content of food products and improve food texture [2]. Egg white albumin has a high air absorption capacity and has been used for producing a foamy texture in food as well as its ability to form gels [3,4]. Similarly, bovine whey protein has been used for its gelling and emulsification characteristics [5,6]. These properties allow both protein types to be used as binders in meat products. Apart from these proteins, protein hydrolysate powder is also one of the main ingredients for increasing the protein level in meat products, whilst at the same time enhancing the functional and bioactive properties [7]. In addition to imparting their own properties on meat products, protein hydrolysates will also interact with the other proteins present. There are three main protein–protein interactions, including synergistic interactions, aggregation, and phase separation [8]. Several studies in the past few decades have reported that protein hydrolysates from various poultry by-product sources, including the heart, liver, gizzards, neck, head, bones, feathers, and blood, in addition to their nutritional properties, exhibited various biological functions [9]. All these tissues are high in protein that contains all the essential amino acids. Such hydrolysates provide a wide variety of health-promoting benefits including antioxidant and antihypertensive abilities [10,11]. The antioxidant properties of peptides from the extract of a chicken leg bone was revealed after hydrolysis [12]. Antihypertensive peptides isolated from a chicken bone and offal protein hydrolysate were investigated and found to inhibit the angiotensin-1-converting enzyme (ACE) effectively [13,14,15,16]. Preliminary studies have investigated chicken trachea collagen hydrolysate (CTH) produced with the commercial enzyme, Alcalase^®, and have shown these to have interesting bioactive properties [14]. Such bioactive protein hydrolysates or peptides derived from poultry by-products are safe and attractive functional ingredients in food products as nutraceuticals [17,18,19].

The study of food protein–functional peptide interactions in model systems is of interest because it can help modify and improve the textural properties of foods, which is a major factor influencing consumer acceptance. This understanding can be used for the design of new healthy or functional meat products with novel organoleptic and functional properties. Our preliminary studies have successfully produced a bioactive chicken trachea collagen hydrolysate (CTH). This was obtained from by-products from the chicken slaughterhouse by hydrolysis with the commercial enzyme Alcalase^® [20]. Another study also reported that Alcalase^® is effective at hydrolyzing chicken skin gelatin and yields a hydrolysate with high antioxidant properties [21]. In the work reported here, the effect of CTH and sodium chloride on the functional properties of two common food protein samples (egg white and whey protein) was studied. A better understanding of the synergy between CTH and food proteins and the effect on the functional properties of these mixtures will inform their application in formulated meat product systems, such as low fat-reduced sodium restructured chicken ham in a follow-on study. Building on our previous study of CTH, where the production and bioactive properties of CTH were established, [20], here, we also report on how mixing CTH with egg white or whey protein and sodium chloride influences the bioactive properties of CTH, including the antioxidant and antihypertensive properties and the rheological, foaming, and emulsifying properties. If CTH is to be used as a bioactive component in reformulated meat products, it is important to understand if the interaction with other food proteins could reduce or enhance CTH bioactivity. These findings could be used to support the application of CTH in making meat products healthier through the exploitation of the bioactivity of the hydrolysate.

2. Materials and Methods

2.1. Materials

The chicken trachea was obtained from a Thai slaughterhouse company. The commercial protease enzyme, Alcalase^®, was obtained from the Novozyme^® (Bangkok, Thailand). Egg white and whey protein were gifts from Marlow Foods (Billingham, UK) and Arla Foods (Region Midtjylland, Viby J, Denmark), respectively, both with a protein content of 80%, as determined by Kjeldahl nitrogen analysis. Sodium dodecyl-sulfate (SDS), 2, 2-diphenyl-1-picryl-hydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ), and dipeptidyl carboxypeptidase, extracted from rabbit lung for bioactivity, were from purchased from Sigma-Aldrich (St. Louis, MO, USA).

CTH was produced according to the method of Pramualkijja et al. [20] (by enzymatic hydrolysis of the trachea with Alcalase^®). The hydrolyzed chicken trachea solution was separated using centrifugation and the soluble supernatant fraction dried under vacuum using a freeze dryer at −50 °C.

2.2. Protein Mixture Preparation

Mixtures of CTH (H) with egg white (E), WPC (W), and NaCl were prepared according to the treatment shown in

Table 1. Formulations of protein mixtures containing chicken trachea hydrolysate (H), egg white protein isolates (E) or whey protein isolates (W), and sodium chloride (S). The protein concentration of E and W was fixed at 10% (w/v), with various levels of hydrolysate.

Treatment	Chicken Protein Hydrolysate (%)	Sodium Chloride (%)
E	0	0
E/0.25 H	0.25	0
E/0.50 H	0.50	0
E/1.5 S	0	1.5
E/0.25 H/1.5 S	0.25	1.5
E/0.50 H/1.5 S	0.50	1.5
W	0	0
W/0.25 H	0.25	0
W/0.50 H	0.50	0
W/1.5 S	0	1.5
W/0.25 H/1.5 S	0.25	1.5
W/0.50 H/1.5 S	0.50	1.5

. Firstly, the egg white or whey protein was dissolved in deionized water to a concentration of 10% protein (w/v). Different amounts of CTH and sodium chloride were added to the protein solution and stirred for 30 min until completely dissolved. The mixtures were covered with a watch glass, left at room temperature for 15 min, and then tested for their foaming ability, emulsifying capacity, rheological properties, antioxidant ability, and ACE inhibitory ability using the methods detailed below.

2.3. Foaming Ability of the Protein Mixtures in the System with and without Sodium Chloride

Foaming capacity and foam stability were measured according to the method of Nomana et al. [22]. A sample (50 mL) of 2% protein solution was placed in a 100 mL measuring cylinder and foam was prepared using a high-shear homogenizer (Ultra-Turrax, Ika UK.) at 20,000 rpm for 2 min. The foam volume was recorded immediately and every 1 min for 30 min. Foaming capacity and foaming stability were calculated using the equations:

$$\mathrm{Foaming} \mathrm{capacity} =\frac{\mathrm{V}2-\mathrm{V}1}{\mathrm{V}1}\times 100$$

where v1 is the volume before whipping and v2 is the volume after whipping.

$$\mathrm{foaming} \mathrm{stability} =\frac{\mathrm{foam} \mathrm{volume} \mathrm{after} 30 \min }{\mathrm{initial} \mathrm{foam} \mathrm{volume}}\times 100$$

2.4. Emulsifying Capacity of the Protein Mixtures in the System with and without Sodium Chloride

The emulsifying activity index (EAI) and the emulsion stability index (ESI) were determined using the method previously described by Noman et al. [22], with some modifications. A total of 30 mL of the various protein solutions were mixed with 10 mL of sunflower oil and homogenized at 20,000 rpm for a minute using a high-shear mixer (Ultra-Turrax, Ika UK) to create the emulsion. Then, 50 μL of the emulsion was taken at the position of 1 cm above the bottom of the container and diluted with 5 mL of 0.1% sodium dodecyl sulfate solution. The absorbance of the solutions was measured at 500 nm at times of 0 and 10 min after the end of homogenization using a spectrophotometer (Thermo Scientific, Waltham, MA, USA, Genesys10 s UV-Vis spectrophotometer). All EAI and ESI values were reported as the mean of at least five measurements. The EAI is calculated using the equation:

$$\mathrm{EAI} \left({\mathrm{m}}^2/\mathrm{g}\right)=\frac{2\times \mathrm{Con}\times \mathrm{dil} \times \mathrm{A}}{\mathrm{C} \times \mathsf{\theta } \times 10,000\times \mathsf{\varphi }}$$

where Con is the constant of 2.303, dil is the dilution factor (200), A is the absorbance at 500 nm, C is the protein concentration (g/mL), θ is the disperse phase volume fraction (0.25), and ϕ is the optical path (0.01 m). In this equation, the absorbance is converted to a turbidity using the equation,

$$\mathrm{T}=\frac{\mathrm{Con}\times \mathrm{A}\times \mathrm{dilution}}{\mathrm{light} \mathrm{path} \mathrm{length}}$$

The ESI is calculated as,

$$\mathrm{ESI} \left(\min \right)=\frac{{\mathrm{A}}_0\times \mathsf{\Delta }\mathrm{t}}{\mathsf{\Delta }\mathrm{A}}$$

where ΔA = A0–A10 and Δt = 10 min.

2.5. Rheological Properties of the Protein Mixtures in the System with and without Sodium Chloride

All rheological properties were determined using a Malvern Kinexus controlled stress rheometer (Kinexus, NETZSCH GmbH, Benton Harbor, MI, USA), fitted with a 4°/40 mm cone and plate-measuring geometry with a 140 μm gap. The temperature was maintained at 4 °C. The linear viscoelastic region (LVER) was determined by an amplitude sweep test with strain increased from 0.1 to 100% at a constant frequency of 1 Hz. Temperature sweeps were carried out between 4 and 90 °C at a heating rate of 2 °C/min. The expansion during temperature sweep test was monitored and there was no evidence of the cone touching the plate. Moisture loss from the sample was reduced by using a water trap and surrounding the plate with low viscosity silicone oil.

2.6. Antioxidant Ability of the Protein Mixtures in the System with and without Sodium Chloride

Protein solutions were centrifuged at 12,000 rpm for 10 min using a high-speed microcentrifuge (Benchmark, MC-12™, Tempe, AZ, USA) and the supernatant was collected and analyzed for antioxidant activity using three tests.

2.6.1. DPPH Assay

The scavenging effects of protein solutions or Trolox (as a control) for the DPPH radical were measured using a method given by Chakka et al. [23]. First, 500 µL of test sample was mixed with 500 µL of 0.1 mM DPPH in 95% ethanol, vortex mixed for 1 min, and left at room temperature for 30 min in the dark. The absorbance was read at 517 nm with a UV/Vis spectrophotometer (Thermo Scientific, Genesys10 s). The DPPH radical scavenging ability was calculated using the equation:

$$\%\mathrm{DPPH} \mathrm{scavenging} \mathrm{activity} =1-\frac{{\mathrm{A}}_{\mathrm{sample}}-{\mathrm{A}}_{\mathrm{blank}}}{{\mathrm{A}}_{\mathrm{control}}}\times 100$$

where A_control is the absorbance of DPPH solution without sample, A_sample is the absorbance of the DPPH solution plus test sample, and A_blank is the absorbance of phosphate buffer with DPPH solution (without sample).

Trolox in the range of 5–50 µM was used to make a standard curve. The DPPH activity of CTH was expressed as µmol Trolox equivalents (TE)/mg CTH.

2.6.2. ABTS Assay

The method of Binsan et al. [24] was used to measure ABTS radical scavenging activity of the protein mixture solution or Trolox. First, 10 mL of 7 mM 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was mixed with 5 mL of 2.45 mM potassium persulfate and incubated for 15 h at 37 °C. Then, 900 µl of this ABTS solution was added to 100 µL of protein solution and allowed to stand for 6 min at room temperature. For the control, 100 µL of phosphate buffer was used instead of the protein solution. A sample blank was prepared by adding buffer instead of ABTS. Absorbance of the reaction mixture was measured at 734 nm (Thermo Scientific, Genesys10 s) and scavenging activity was calculated with the equation below:

$$\%\mathrm{ABTS} \mathrm{scavengineg} \mathrm{activity} =1-\frac{{\mathrm{A}}_{\mathrm{sample}}-{ \mathrm{A}}_{\mathrm{blank}}}{{\mathrm{A}}_{\mathrm{control}}}\times 100$$

Trolox in the range of 20–200 µM was used to prepare a standard curve. ABTS scavenging activity was expressed as µmol Trolox equivalents (TE)/mg sample.

2.6.3. FRAP Assay

The ferric reducing ability (FRAP) method, as described by Benzie and Strain [25], was applied to measure the ferric ion reducing capacity of the protein solutions. In this assay, a complex is formed between the ferric ions of FeCl₃ and tripiridiltriazine (TPTZ). In the presence of reducing agents, the Fe(III) ions are reduced to Fe(II), resulting in a blue-colored complex that adsorbs at 595 nm. FRAP solution was prepared by mixing acetate buffer (300 mM, pH 3.6), TPTZ (10 mM in 40 mM HCl), and FeCl₃.6H₂O (20 mM) in the volume ratio 10:1:1. Then, 300 μL of FRAP solution was added to 10 μL of protein solution and 30μL of distilled water, all at 37 °C. Absorbance was measured 30 min after mixing at 595 nm in a UV/Vis spectrophotometer (Thermo Scientific, Genesys10 s). Ferrous sulfate was used to make a standard curve, which relates the concentration of this compound to the absorbance at 595 nm. Results were expressed as Trolox equivalents (TE)/mg sample.

2.7. ACE Inhibitor Ability of the Protein Mixtures in the System with and without Sodium Chloride

The protein mixture which had the best antioxidant properties for each protein was measured for its ACE inhibitory activity. CTH, egg protein, and whey protein were used as controls. ACE inhibition was monitored by the method proposed by Cheng et al. [26], with slight modifications. The protein mixture was diluted using distilled water at ratios of 100:0, 75:25, 50:50, and 25:75 (v/v). The samples and enzyme were dissolved in 50 mM HEPES-NaOH/300 mM NaCl (pH 8.3) buffer. The, 50 µL of ACE solution (8 mU/50 mL) was added to 30 μL of protein in HEPES buffer and held at 37 °C for 5 min. To this mixture, 50 μL of hippuryl-L-histidyl-L-leucine (HHL, 6 mg/mL) solution was added and held in a water bath at 37 °C for 15 min. The enzyme in the ACE solution hydrolyzed the HHL, releasing hippuric acid. After 15 min, the enzyme reaction was terminated by adding 380 μL of 1 M HCl. Then, 1.5 mL of ethyl acetate was added to extract the hippuric acid after vortex mixing for 1 min. The mixture was centrifuged at 12,000 rpm for 10 min using a high-speed microcentrifuge (Benchmark, MC-12™). Next, 1 mL of the supernatant was removed and the ethyl acetate evaporated by heating in boiling water. After that, 1 mL of water was added to dissolve the remaining hippuric acid and the absorbance read at 228 nm. The ACE-inhibitory activity (%) was calculated as:

$$\%\mathrm{ACE} \mathrm{inhibitor} \mathrm{activity} =1-\frac{{\mathrm{A}}_{\mathrm{sample}}-{ \mathrm{A}}_{\mathrm{sample} \mathrm{blank}}}{{\mathrm{A}}_{\mathrm{control}}-{ \mathrm{A}}_{\mathrm{control} \mathrm{blank}}}\times 100$$

where A_control is the absorbance of 50 mM HEPES-NaOH/300 mM NaCl buffer without sample, A_sample is the absorbance of 50 mM HEPES-NaOH/300 mM NaCl buffer with sample, and A_{control blank} and A_{sample blank} are ACE solutions added to the reaction without termination of the reaction with 1 M HCl.

The IC50 value was defined as the concentration of inhibitor required to inhibit 50% of the ACE activity.

2.8. Statistical Analysis

The statistical analysis was performed using computational program. Statistical significance at 95% significance interval was analyzed using an independent sample T-test and one-way analysis of variance (ANOVA), and the significance level was tested using Duncan’s multiple range test (p ≤ 0.05) located in the SPSS software package (12.0 for Windows, SPSS Inc., Bangkok, Thailand).

3. Results and Discussion

3.1. CTH Properties

In a previous paper [20], we reported the properties of bioactive CTH solutions. We noted that the main amino acids in CTH were glutamic acid (Glu, 11.80%), glycine (Gly, 8.65%), and proline (Pro, 5.82%). Furthermore, CTH showed significant antioxidant potential with a DPPH of 4.42 TEAC mM/mL, ABTS of 134.29 TEAC mM/mL, and FRAP of 22.48 TEAC mM/mL. Additionally, the ACE inhibitory test showed an IC50 of 0.422 mg/mL (p ≤ 0.05).

3.2. Foaming Ability of the Protein Mixtures in the System with and without Sodium Chloride

The effect of CTH and NaCl on the foaming capacity and foam stability of egg white and whey protein are presented in Figure 1. The mixture of egg white mostly had a higher foaming ability than the mixture with whey protein (p < 0.05), and all egg white protein treatments gave a higher foam stability than those made with whey protein (Figure 1B). The increasing CTH concentration gave a trend of a decreasing foaming capacity for egg white, although this was only statistically significant for the foam with 0.5% CTH added compared to the egg white foam with no added CTH. The addition of CTH to the WPC system did not give a significant difference in all the added concentrations (p > 0.05). The addition of CTH only gave a significant decrease in foam stability for the E/0.5 H treatment, whilst there was no effect observed on the stability of the WPC mixture. The addition of NaCl to the protein and CTH mixture markedly improved the foaming ability for both the egg white and WPC foams, while foam stability improvement was found in the mixture with egg white. The effect of salt on the WPC and CTH mixture was a marginal increase with a non-significant effect (p > 0.05). The foaming properties of food proteins are controlled by the diffusion, penetration, and rearrangement of molecules at the air–water interface. Hydrophobic regions largely determine the adsorption of molecules at the air–water interface, whilst the extent of protein–protein interactions at the interface affects the nature of film formation and foam stability [27]. Presumably, CTH and NaCl interfere with these interactions and alter the foaming properties of egg white and WPC.

Raikos et al. [27] reported that increasing the NaCl concentration, heating temperature, and whipping time enhanced egg white protein foam formation but did not affect foam stability, results that are broadly in line with our own. They speculated that this was due to the screening of the protein charge by ions from the NaCl which allowed a greater adsorption of protein at the air–water interface. The same effect was observed by others [28]. Ovalbumin is the most abundant protein in egg white, an ingredient widely used to promote foam formation properties in food systems. The excellent foaming property of whipped egg white is the result of interactions between conalbumin, ovomucin, and ovoglobulin [29]. As well as promoting protein adsorption, it is possible that NaCl also alters the interactions between the proteins in egg white. The reduction of foam stability by CTH could be explained by competitive adsorption between the CTH peptides and egg white proteins. This is a phenomenon where different proteins compete for space at interfaces based on their relative surface activity [30]. If CTH outcompetes intact proteins at the air–water foam interface, and if the CTH is a poorer foaming agent, then the hydrolysate will reduce the foaming ability and stability.

3.3. Emulsifying Capacity of the Protein Mixtures in the System with and without Sodium Chloride

The emulsifying activity index of the different protein mixture is shown in Figure 2A. Emulsions containing whey protein had higher emulsifying activity index values than those containing egg protein. The addition of CTH and NaCl had a small but statistically insignificant impact (p < 0.05) on emulsion formation by the whey protein solution. The addition of CTH decreased the emulsifying ability of the egg protein solution, which slightly improved when NaCl was added (p > 0.05). On the other hand, the addition of CTH and salt to the egg protein solution significantly improved the emulsion stability (p < 0.05), whereas this was not observed for the whey protein solution (Figure 2B), where a reduction in stability occurred in the presence of CTH and/or salt.

CTH and NaCl significantly increased the stability of the egg protein emulsions but decreased the stability of the whey protein emulsions (Figure 2A,B). The emulsifying activity index reflects the ability of the protein to be rapidly adsorbed at the water/oil interface during the formation of the emulsion [31]. The addition of salt is known to promote protein-stabilized emulsion droplet aggregation, with an increased salt concentration giving greater instability [32]. Clearly, CTH and salt affect the emulsifying ability of egg white and whey proteins, but in different ways. CTH contains abundant hydrophobic amino acid residues that can interact with hydrophobic patches on the surface of proteins. This effect on emulsion stability may be due to differences in the way that the two proteins interact with CTH.

3.4. Rheological Properties of the Protein Mixtures in the System with and without Sodium Chloride

The rheological properties of the solutions of egg white and whey protein with added CTH were characterized. The amplitude sweep is shown in Figure 3 for different protein and CTH combinations. Adding CTH to a 10% egg white solution significantly increased the Gʹ value in the LVER, while the addition of CTH to 10% whey protein produced only a modest increase in Gʹ. Adding salt had a similar effect, with the egg white and egg white + CTH solutions producing a further significant increase in Gʹ in the LVER, while the increase for the whey protein solutions was again modest. There was no apparent effect on the yield strain of the solution. In Figure 3, strains above about 0.25% led to a flow in the whey protein solution, as the structure was broken by the increased strain [33]. Egg white solutions with and without CTH were more resistant to the increasing strain; a strain above about 0.5% is required for the solutions to flow. Consequently, a strain of 0.1% (within the linear viscoelastic region) was selected for the heating tests, to remain within the LVER.

The results from the temperature sweep test are shown in Figure 4 as Gʹ versus temperature. The Gʹ rheograms of the protein solution showed that all egg protein solutions (with and without CTH or salt) had lower initial Gʹ values than those from whey protein solutions. There were higher Gʹ values for egg + CTH and/or salt and whey protein + CTH and/or salt solutions than solutions of the proteins alone, confirming the data in the amplitude sweep. All protein solutions had similar temperature profiles, where Gʹ initially decreased as the temperature increased to about 28 °C. Being a mixture of proteins, both egg white and whey protein (to a lesser degree) will show a complex heat denaturation behavior. In Figure 4, above about 40 °C, the viscosity started to increase as proteins started to denature and aggregate. Above 60 °C, there was a more rapid increase in Gʹ, corresponding to the denaturation and gelation temperature of the egg white [34]. While the CTH and salt addition increased the G′ value of the protein solutions, it did not change the denaturation and gelation temperature of the proteins, which remained at 60 °C for all combinations of protein, CTH, and salt. Notably, from Figure 4, the final gel strength (G′ at 90 °C) increased for egg white as more CTH/salt was added; however, there was no effect of CTH or salt on the final G′ values for the whey protein solutions. This suggests a synergistic interaction between CTH and egg white that is largely absent with whey protein.

The rheological properties of protein solutions depend on a number of factors (composition, molecular weight, size, shape, flexibility, degree of hydration, and intermolecular interactions). These are, in turn, influenced by extrinsic factors such as concentration, temperature, pH, ionic strength, and previous processing treatments [35]. The effect of NaCl on G′ can be explained by the enhanced formation of larger-size aggregates and the rate of aggregation, as observed by others for β-lactoglobulin or albumin with increasing NaCl concentrations [36,37].

3.5. Antioxidant Ability and ACE Inhibitory Ability of the Protein Mixtures in the System with and without Sodium Chloride

The antioxidant activity of the protein mixture solution was evaluated using free radical-scavenging activity assays, which relate to the scavenging activity of the Trolox concentration. The whey protein solution mixed with 0.5% CTH and 1.5% NaCl had the highest antioxidant activity (Figure 5) with DPPH, ABTS, and FRAP radical-scavenging activities of 40.00, 180.95, and 46.00 TEAC µM/mL, respectively. Figure 5 also shows that increasing the CTH concentration increased the antioxidant activity of both protein mixtures. Increasing the NaCl concentration, however, had a small effect of increasing the antioxidant activity of CTH in the presence of whey protein, but had no significant effect on CTH + egg white.

All egg white protein mixture solutions had lower antioxidant activity levels than the equivalent whey protein solution (p > 0.05). The results indicated that the DPPH, ABTS, and FRAP radical-scavenging activities of the protein mixture solution depended on the CTH concentration, the protein type, and the addition of NaCl.

The antihypertensive activity is defined as the concentration of the solution (based on the total protein content) required to inhibit 50% of the ACE activity, with the results presented in Figure 6. The CTH solution (0.5%) showed 50% inhibition of the reaction at 0.84 mg/mL, which was much lower than the IC50 for all mixtures of CTH with egg or whey protein. Of the protein + CTH mixtures, the whey protein solution mixed with 0.5% CTH and 1.5% NaCl had the lowest IC50 (30.05 mg/mL, highest antihypertensive activity), although this was not significantly (p < 0.05) different to the whey protein solution without NaCl (31.84 mg/mL). The addition of CTH and NaCl into the protein mixtures slightly enhanced antihypertensive activity. It is clear from these results that egg protein and whey protein interfere with the bioactive properties of CTH, with egg interfering more than whey protein.

It has been reported that ovalbumin and β-lactoglobulin, which are the main proteins found in egg white and whey protein, respectively, exhibit antioxidant properties through their multiple-binding sites and free radical-trapping properties [38,39]. Several studies have indicated the relationship between the antioxidative and antihypertensive properties of protein hydrolysates. Ambigaipalan et al. (2015) prepared date seed protein hydrolysates using commercial protease and reported that it had the lowest reducing power and ABTS radical scavenging activity, but a higher ACE inhibition and hydroxyl radical scavenging [40]. Yousr and Howell (2015) purified bioactive peptide from egg yolk protein using gel filtration and reported that it had a high ACE inhibitory activity (69%) and a high IC50 value (3.35 mg/mL) [41]. Chalamaiah et al. (2016) prepared protein hydrolysate from Rohu egg using Alcalase and reported that the maximum ACE inhibitory activity was 45% at the concentration of 1 mg/mL [42]. CTH is a protein hydrolysate from chicken trachea consisting of small peptides and amino acid residues. Hydrophobic amino acids in hydrolysates have been suggested to have antioxidant and antihypertensive properties [43], while some peptides are well-known to enhance antioxidant action [44]. Our results showed that the addition of egg protein or whey protein to CTH promoted the antioxidative, but reduced the antihypertensive properties of CTH. The effect of NaCl action on the antioxidant activity of protein systems was demonstrated by Tunieva and Kotenkova [45]. Furthermore, NaCl encourages increased protein solubility [46] which in the protein mixtures would increase the concentration of free amino acids from the hydrolysate in solution that may partly explain its effect on bioactive properties. It has been reported that the antioxidant and antihypertensive activity of hydrolyzed proteins were enhanced with the increasing free amino acid concentration [47,48].

4. Conclusions

In conclusion, the investigation of interactions between the selected two protein types (egg white and whey protein) with CTH in the system with and without NaCl was revealed and expressed in terms of bioactive and functional properties. The protein–salt mixture exhibited the most prominent properties suited for further application in real meat model systems. This mixture composed of 10% w/w whey protein, 0.5% w/w CTH, and 1.5% w/w NaCl. Moreover, CTH did not affect the emulsifying and foaming ability of whey protein, but reduced those for egg white protein. CTH had a positive effect on the rheological properties of both proteins. The mixture with whey protein exhibited a stronger gel strength than that of the egg white protein mixture. The addition of CTH and NaCl promoted the gelling properties of both proteins. The antioxidant and antihypertensive properties of the protein solutions were not influenced significantly by NaCl and had a higher activity when mixed with CTH, especially for the whey protein system. These results show that CTH has potential as a functional ingredient. However, when mixed with the common food proteins, egg white and whey protein, the antihypertensive effect of CTH is greatly reduced. If hydrolysates such as CTH are to be formulated into functional food meat products with health claims, it is important to understand the interactions between the bioactive components and other ingredients, and the trade-offs that occur when bioactive ingredients with properties that have been proven in simple model systems are incorporated into more complex food systems.