Investigation of the optimal production conditions for egg white hydrolysates and physicochemical characteristics

Abstract

This study aimed to investigate the potential of egg white protein hydrolysate (EWH) as a functional food by identifying the optimum production conditions for EWH with response surface methodology (the results of the sensory evaluation were considered as an essential quality indicator). At the same time, its physicochemical and biological activity was also evaluated. The optimal economic production conditions were selected: substrate concentration of 12.5%, enzyme content of 7.5%, and hydrolysis time at 100 min. The degree of hydrolysis (DH %) was 13.51%. In addition, to the better acceptance of the evaluation, it also helps to reduce the production cost of the protein hydrolysate, which is beneficial to future processing and applications. The antioxidant capacity experiments showed that EWH has good antioxidant activity, which presents a dose-dependent relationship. Hence, this study provides a theoretical basis for future research and application of EWH for processing applications, including dietary supplementation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-023-05708-0.

Introduction

Eggs are a very good source of proteins and are accessible, economical, and highly nutritious natural food in the human diet. Eggs contain essential nutrients to support human embryonic development and provide the essential amino acids necessary to maintain normal physiological functions (Moreno-Fernández et al. 2020; Lee and Paik 2019; Nasiru et al. 2022). They contain a considerable amount of lipids, minerals, vitamins, and trace elements; they are easily digestible, making them an excellent potential source of nutrients (López-Martínez et al. 2021). Therefore, egg products have been widely used in the food industry for processing applications and providing nutritional properties (Lee and Paik 2019). There has been considerable scientific interest in egg whites (in both egg albumen and yolk) since the 1990s (Zhang et al. 2020). Even though a study has concluded that introducing new nutritionally enhanced eggs offers not only potential benefits to consumers' diets but also increases new business opportunities for producers and farmers (Tian et al. 2022). However, a further increase was limited because of the lack of advanced studies on traditional egg processing (Huang and Ahn 2019).

The food industry developed functional foods for several decades to meet market demands. Functional foods are defined as foods that provide beneficial actions beyond their nutritional value, such as phenolic compounds, functional lipids, and bioactive peptides, (López-Martínez et al. 2021). Studies have shown that EWH produced by the hydrolysis of protein produces bioactive peptides with several biological activities, such as antioxidant, anti-inflammatory, anti-cancer, and antibacterial functions (Xiao et al. 2021; Moreno-Fernández et al. 2020; Kiewiet et al. 2018; Ho et al. 2021; Araiza-Calahorra et al. 2022; Jalili-Firoozinezhad et al. 2020; Bueno Gavilá et al. 2021). Interestingly, many bioactive peptides have been found in many fermented foods, such as fermented dairy products and meat products. However, most of the peptides in foods have no bioactive properties and must be released via digestion with endogenous proteases in the body or exogenous proteases during the protein hydrolysis process (Zhang et al. 2020; Taktak et al. 2021).

In vitro hydrolysis using exogenous proteases remains the most common method for peptide digestion. Compared to chemical processing, enzymatic hydrolysis is milder, and the total amino acid composition after hydrolysis is closely similar to the original composition of whole food proteins (Araiza-Calahorra et al. 2022). Additionally, the enzyme hydrolysis operating without organic solvents or toxic chemicals was suitable for the food and pharmaceutical industries (Nasri 2017; Li et al. 2021). The other reason concerning enzymatic hydrolysis deserves attention namely that some proteins can induce allergic reactions that induce immune disorders were possible (Sun et al. 2021). The necessity to exclude allergens from eggs during the production thereby highlights the need for thermal processing, and enzymatic hydrolysis helps to mitigate potential allergenicity (Zhang et al. 2020). It has been found that both positive and negative effects of the gastrointestinal tract on the IgE binding activity of egg white proteins were observed. In this case, the deletion of the carbohydrate components leads to a decrease in the immunoreactivity of the ovalbumin (Gazme et al. 2022). Therefore, it is necessary to continue efforts to increase the use of eggs and optimize their functional properties to enable new and innovative food applications (Huang and Ahn 2019). It not only serves as a non-toxic biological preservative to replace the harmful sodium nitrite, inhibits foodborne pathogens, promotes metal ion absorption, and improves the quality of meat products (Zhang et al. 2020).

Currently, protein hydrolysates derived from natural foods such as milk, eggs, fish, shellfish, and microalgae are known to have immunomodulatory physiological functions that protect the body from pathogens. Therefore, regulating immune function by protein hydrolysates is a potential approach (Chalamaiah et al. 2018; Kiewiet et al. 2018; Moreno-Fernández et al. 2020). There are many reported pieces of evidence that protein hydrolysates have antioxidant physiological functions (Kiewiet et al. 2018; Mendis et al. 2005; Moraes et al. 2022), indicating that it has the potential to be developed as a healthy food material (Araiza-Calahorra et al. 2022). Hence, this study aimed to evaluate the optimal egg white hydrolysis production by substrate concentration, enzyme content, and reaction time, which the EWH obtained. In addition, the physical and chemical evaluation was performed.

Materials and methods

Materials

Egg whites were supplied by Fuji Frozen Food Co., Ltd (Kaohsiung, Taiwan). Protease N (152 U/mg) was purchased from Ho Jun Biotechnology Co., Ltd. (Taoyuan, Taiwan). Papain W- 40 (from Carica papaya) (30 U/mg) was purchased from Champion Co., Ltd. (Taipei, Taiwan). Bovine serum albumin (BSA) and Reagents A, B, and S were purchased from Bio-Rad, represented by Genmall Biotechnology Co., Ltd. (Taipei, Taiwan). Serine and HPLC reagents (HPLC grade) were purchased from Sigma-Aldrich® (Merck KGaA, Darmstadt, Germany). Sodium tetraborate decahydrate (borax) was purchased from Showa Chemical Industry Co., Ltd. (Tokyo, Japan). Ethanol (95%), sodium dodecyl sulfate (SDS), and ortho-phthalaldehyde (OPA) were purchased from Alfa Aesar, represented by Echo Chemical Co., Ltd. (Taipei, Taiwan). Dithiothreitol (DTT) was purchased from R&D Systems, represented by Union Biomed Inc. Co., Ltd. (Taipei, Taiwan). The other reagents were of analytical grade.

Experimental design of RSM for the EWH

The study used Design-Expert software version 10 (Statease Inc., Minneapolis, MN) for experimental design with further statistical analysis and regression analysis using response surface methodology (RSM). According to the three variables-three levels responses surface analysis proposed by Box and Behnken (1960) was used to investigate the optimal conditions for the hydrolysis of EWH (Table 1). Following the substrate concentration (%), enzyme content (%), and reaction time (min) a better quality (highly DH) of EWH is obtained. Additionally, the concentration of substrate was 5, 12.5, and 20%; the enzyme content was 3, 7.5, and 12% (obtained by mixing the above two enzymes in a 1:1 ratio). The reaction time was 20, 60, and 100 min (Table 1), which were used as the independent variables to determine the optimum processing conditions for producing EWH.

Table 1.

Three-factor crossover design and RSM optimization experiment results

Level	Substrate (%)	Enzyme (%)	Time (min)	Hydrolysis rate (%)
− 1	5	3	20	–
0	12.5	7.5	60	–
1	20	12	100	–
Exp No
1	5	3	20	1.31
2	20	3	20	1.91
3	5	12	20	5.68
4	20	12	20	9.36
5	5	3	100	0.42
6	20	3	100	9.88
7	5	12	100	12.21
8	20	12	100	15.28
9	5	7.5	60	3.97
10	20	7.5	60	13.24
11	12.5	3	60	0.79
12	12.5	12	60	12.44
13	12.5	7.5	20	7.55
14	12.5	7.5	100	13.51
15	12.5	7.5	60	9.97
16	12.5	7.5	60	9.65
17	12.5	7.5	60	11.02
The optimal conditions	20	12	100	15.28
The optimal economic conditions	12.5	7.5	100	13.31

In general, the EWH formulation process consists of homogenizing the fresh egg whites for 30 s at 3000 rpm, mixing with distilled water to the proper consistency (5–20%), and then stirring in a heated mixer (maintain the temp. at 50 °C). Then, Protease N and Papain (3–12%) were added for hydrolysis (20–100 min), followed by secondary hydrolysis in a 70 °C water bath for 120 min, followed by a boiling water bath for 5 min to end the hydrolysis reaction, which resulted in EWH by centrifugation and sterilization. Finally, the hydrolysate was freeze-dried into powder and stored until use.

Sensory evaluation

Before the sensory evaluation of this research, we provided informed consent (supplement material “Informed consent”) to the participants and confirmed that these experiments were conducted according to established ethical guidelines, and informed consent was obtained from the participants, then gave them the Egg white hydrolysates evaluation and questionnaire (supplement material “Egg white hydrolysates evaluation and questionnaire”).

The method described by López-Martínez et al. (2021) was modified as follows to the specified operations. For the development of sensory characteristics, the group with more than 9% hydrolysis in the RSM design was selected and evaluated in terms of bitterness, spiciness, burnt taste, fishy egg taste, and odor. Each element was represented by + and −. +: slightly, ++: moderate, +++: strong, −: weak, ––: none. Moreover, the overall acceptability was assessed by the ordinal scale. The room temperature was always maintained at 25 ± 2 °C. 30 trained panelists then assessed the sensory evaluation. The panelists tasted each sample and rinsed their mouths with drinking water twice in between.

Degree of hydrolysis (DH %)

The method was performed according to Ho et al. (2021). In brief, the serine equivalent of 100 ppm standard and 0.08% protein content of EWH were formulated, respectively. Next, the OPA reagent protocol was as follows: 7.620 g of Borax and 200 mg of SDS dissolved in 150 mL of deionized water. Add 160 mg of OPA solution dissolved in 4 mL of 95% ethanol followed by 176 mg of DTT and madeup to 200 mL. The absorbance was measured at 340 nm (BioTek ELISA Reader, Agilent Technologies, Inc. Santa Clara, CA, USA) following the reaction of 20 μL/well of the EWH sample and 150 μL/well of the OPA reagent. The DH was determined using the following formula:

$$\text{DH}(\%)=[({\text{OD}}_{\text{Sample}}-{\text{OD}}_{\text{Blank}})/({\text{OD}}_{\text{Standard}}-{\text{OD}}_{\text{Sample}})\times \left(0.9516\times 0.1\right)/\left(\text{X}\times \text{P}\right)-\beta ]/(\alpha \times {\text{h}}_{\text{tot}})\times 100$$ 1

OD_Standard: absorbance of standard solution (Serine); OD_Sample: absorbance of sample solution; OD_Blank: absorbance of blank; X: volume of 1 mL 0.08% protein content sample (mL); P: protein content of sample (%); α, β, and h_tot: 1.0, 0.4, and 8.0, respectively.

Amino acids analysis

The determination of the amino acid compositions of the samples was performed with some modifications following the method described by Su et al. (2021). EWH 50 mg was dissolved in 1 mL of 0.1% Formic acid (containing 5% Acetonitrile), filtered through a 0.45 μm filter membrane, and analyzed by high-performance liquid chromatography (HPLC)(Hitachi, Ltd., Tokyo, Japan). The HPLC conditions were as follows: a Capcell Pak C-18MGII 100A (250 nm × 4.6 mm) column with an injection volume of 30 μL, a flow rate of 1 mL/min, in UV wavelength of 214 nm. The solutions used for the linear gradient were A (50:40:10 volume ratio mixture of acetic acid buffer, methanol, and acetonitrile) and B (50:50 volume ratio mixture of acetic acid buffer, methanol, and acetonitrile).

The hydrolysate was separated by HPLC to provide 8 mL of each product and then analyzed by RP-HPLC/MS/MS, followed by TurboSEQUEST (Therom Finning, San Jose, CA, USA) software for protein identification. When the Xcorr value of three peptides in the same protein was greater than 2.5, the protein was considered trustworthy. Additionally, the Xcorr is a comparison score in TurboSEQUEST software, which compares the identified peptides with the peptide database, whenever the peptide sequence is more similar to the database, the Xcorr value will be higher, as long as the Xcorr value is greater than 2.5, the result is reliable.

Determination of molecular weight

The methodology was modified according to the Su et al. (2021) reported and used. EWH was filtered by 0.45 μm PVDF membrane, and molecular weight distribution was measured by HPLC-Chromatograph system with BioSep-SEC-S2000 (600–7.8 mm). The eluate solution comprised 45% (v/v) ethanol and 1% trichloroacetic acid buffer solution at a 1 mL/min flow rate. An injection volume of 25 μL was measured at wavelength 214 nm.

Determination of chromaticity

The EWH samples were measured using a colorimeter (Nippon Denshoku, SA-4000, Tokyo, Japan) for each sample in three replicates, and the results were expressed as mean ± SD values, evaluated according to the colorimetric system established by the International Commission on Illumination (CIE) as follows: The L*, a*, and b* values represent the lightness-darkness, red-green, and yellow-blue components of the samples, respectively.

Dynamic rheological analysis

Dynamic rheological analysis was followed the reported by Liu et al. (2022) and Ping-Hsiu Huang et al. (2022), in short, the measurements of egg white and EWH were obtained with a rheometer (Solids Dynamic Analyzer II, Rheometric Scientific Ltd., New Castle, DE, USA). The measurement condition was performed at shear rates ranging from 0.1 to 300 (S⁻¹), all measured at room temperature of 25 °C and in units of (mPa s).

Determination of viscosity

According to the method described by Monkos (2015), with some modifications to measure the viscosity of the samples. The samples were measured with a digital viscometer (DV-E, AMETEK Brookfield, Inc., Middleboro, MA), and the probe was rotated at 100 rpm/15 s while the value displayed was recorded with the unit of cP.

DPPH radical scavenging activity assay

The method described by Ping-Hsiu Huang et al. (2011) with modified appropriately for DPPH radical scavenging ability determination in samples. Various concentrations of EWHP and 100 μL of Vitamin C solution (1 mg/mL, as positive control) were mixed with 25 μM DPPH solution 500 μL and PBS 400 μL, then the reaction for 20 min at room temperature and protected from light, followed by the measurement of absorbance at 517 nm. The DPPH radical scavenging activity equation was calculated as follows:

$$\text{DPPH}\text{radical}\text{scavenging}\text{activity}(\%)=(1-\text{Abs}\text{sample}/\text{Abs}\text{control})\times 100$$ 2

Determination of ABTS^·+ radical scavenging

The reaction was carried out by the method of Pv (2022) 100 μL each of EWH and 1 mg/mL Vitamin C solution (as a control group) were added to 2 mM ABTS^·+ and 70 mM Potassium persulfate solution, respectively, with 16 h incubation. The absorbance values were diluted with PBS to 0.800 ± 0.03 before usage. Take 10 μL of different concentrations of EWH, add 990 μL of diluted ABTS^·+ solution to the reaction for 6 min, and measure the absorbance value at 734 nm. The ABTS^·+ radical scavenging was calculated as follows:

$${\text{ABTS}}^{·+}\text{radical}\text{scavenging}(\%)=\left[\left(\text{Abs}\text{control}-\text{Abs}\text{sample}\right)/\text{Abs}\text{control}\right]\times 100$$ 3

Determination of reducing power

The method was slightly modified as described by Bueno Gavilá et al. (2021) and Ping-Hsiu Huang et al. (2011). 150 μL of EWH and Vitamin C solutions (1 mg/mL as a control group) mixed with each 150 μL of 0.2 M pH 6.6 PBS and 0.1% erythrosine, respectively, followed by a water bath at 50 °C for 20 min. Next, the reaction was cooled rapidly with 10% Trichloroacetic acid and centrifuged at 860 × g for 10 min. Then, 500 μL of the supernatant was added to 600 μL of distilled water and 120 μL of 0.1% Ferricchloride during 10 min of standing reaction. The absorbance value was measured at 700 nm, and the reduction capacity was calculated as follows:

$$\text{Reducing}\text{power}(\%)=\left(\text{Abs}\text{sample}-\text{Abs}\text{control}\right)\times 100$$ 4

Ferrous ion chelating activity assay

Ferrous ion chelating activity assay was performed as described by Ping-Hsiu Huang et al. (2011) with minor modifications. In brief, either 500 μL of different concentrations of EWH and the control group 1 mg/mL of ethylenediaminetetraacetic acid (EDTA) solution, added 1.85 mL of methanol and 2 mM Iron (II) chloride 50 μL, stood for 30 s. Next, added 5 mM Ferrozine 50 μL, and the reaction was allowed to stand for 10 min. Finally, measured the absorbance value at 562 nm, and the ability to chelate ferrous iron was calculated as follows:

$$\text{Chelating}\text{effects}(\%)=\left[1-(\text{Abs}\text{sample}/\text{Abs}\text{control})\right]\times 100$$ 5

Statistical analysis

All the data obtained from the experiments were compared within groups using the One-way ANOVA program in IBM® SPSS® Statistics software Version 12.0 (Armonk, NY, USA). In addition, Duncan's multiple range tests or T-tests were used to evaluate whether the experimental data were significantly different from each other, and the significance of the difference was P < 0.05.

Results and discussion

The optimal economic production conditions

The rate of hydrolysis has been determined in Section “Degree of hydrolysis (DH %)” above and calculated by the formula. In general, EWH was prepared with a controlled constant temperature for hydrolysis of the commercial enzymatic at its optimum reaction temperature. After 2 steps of hydrolysis, the residual protein content will be approximately 6%. The result was similar to previous studies, where the commercial enzymatic hydrolysis (Proteinase A and Papain) hydrolyzed egg albumen from chicken at 6.19% (Ho et al. 2021). Therefore, the substrate concentration, enzyme content, and reaction time will influence hydrolysate production under different hydrolysis conditions.

Table 1 shows the results of 17 experimental combinations and the DH by expanding the variation conditions through RSM, among which groups 15, 16, and 17 represent the centroid groups in the RSM conditions, and EWH performed according to these design conditions. The optimized hydrolysis conditions and DH results were as follows: The optimum was group 8, with 20% substrate, 12% enzyme content, and 100 min hydrolysis time, whose DH reached 15.28%. However, as shown in Fig. 1A, the reaction effect has yet to reach the maximum. This phenomenon might be related to the reaction of the enzyme substrate. Moreover, continuously raising the enzyme content will yield more products in the enzyme hydrolysis reaction, yet the cost of commercial enzymatic hydrolysis enzyme input will increase. In this study, the DH rose from 13.24% (group 10) to 15.28% (group 8) when the enzyme content was increased from 7.5% to 12%. Thus, the slight increase in DH (2.04%) was not cost-effective. In case of poor sensory evaluation (e.g., spiciness, fishy egg taste, burnt taste, etc.) or high viscosity, the derivative processing application will lead to difficulties in formulation preparation, requiring the use of more food additives (e.g., spices, etc.) to possibly alleviate the unpleasant taste problem, which will be contrary to the purposes of this study to produce EWH as a functional food (Johny et al. 2022). From a sensory and technical functional point of view, whole hydrolysates represent another focus for developing new functional foods (Moreno-Fernández et al. 2020). Thus, in this study, the palatability of EWH was evaluated sensory, followed by further determination of the optimal conditions for the subsequent production.

Fig. 1.

A Response surface of the hydrolysis conditions of the protein hydrolysate. B Rheological analysis of egg white hydrolysate (EWH) and egg white

Synthesizing the results of the sensory evaluation, the results of the EWH were shown in Table 2. The DH was 15.28% at 20% substrate concentration, 12% enzyme content, and 100 min hydrolysis time, yet significant spiciness, fishy egg taste, and odor were observed in the sensory evaluation under this hydrolysis condition. However, previous studies showed that the DH could increase to 25–67.57% (Ho et al. 2021; Horimoto and Lim 2017; Su et al. 2021; Yap and Gan 2020). Intriguingly, the sensory evaluation of group 15 showed the same results as group 14. Group 14 contained a 12.5% substrate concentration, with 7.5% enzyme content, at 100 min reaction, showed a protein DH of 13.51%, which was not much different from group 15 (difference of 1.77% only). In Brief, group 14 has significantly improved the acceptability of sensory evaluation, such as less spicy taste, fishy egg taste, and odor. Simultaneously, it can reduce the issues of poor sensory evaluation. Moreover, it has the benefit of reducing the use of substances and commercial enzyme hydrolysis, which will significantly reduce the production cost of EWS and contribute to future mass production and promotional applications.

Table 2.

Sensory evaluation of EWH

Groupa	Substrate/Enzyme	Hydrolysis time (min) at 50/70 °C	Sensory evaluationb
			Bitterness	Spiciness (tongue feel)	Burnt taste	Fishy egg
						taste	odor
4	20/12	20/120	+	+++	−	++	+++
6	20/3	100/120	+	–	−	+−	+
7	5/12	100/120	+	++	−	+	++
8	20/7.5	100/120	+	+++	−	++	+++
10	20/7.5	60/120	+	++	−	+	++
12	12.5/12	60/120	+	+	++	+−	+
14	12.5/7.5	100/120	+	+	+	+−	+
15	12.5/7.5	60/120	+	−	+	+−	+

^aSensory evaluation of groups with hydrolysis rates above 9%

^bEach element was represented by + and −. +: slightly, ++: moderate, +++: strong, −: weak, ––: none

Thus, it was apparent in the results of sensory evaluation, that a high DH cannot increase the performance of sensory evaluation. Notably, the previously reported bioactive hydrolysates DPP-IV and ACE were typically hydrolyzed using whole egg whites for 3–90 h (Moreno-Fernández et al. 2020; Johny et al. 2022; Moraes et al. 2022; Su et al. 2021; Bueno Gavilá et al. 2021); while the protein hydrolysate extracted from duck egg white required 19.24 h (Pv 2022). Therefore, it showed that COX-2, Ang II, ACE, and oxidative stress were responsible for the beneficial effects of EWH, possibly through its antioxidant and anti-inflammatory properties, which improved the toxic effects of Cd (Moraes et al. 2022). Meanwhile, considering the economic efficiency of cost, the minimum substrate concentration and commercial enzymatic content should be considered for the same reaction time, yielding a product with a suitable DH (Fig. 1A). While sensory results obtained were appropriate, the components have not always conformed to health profiles; weaknesses such as stability, texture, nutrition, and sensory still need to be overcome (López-Martínez et al. 2021; Nasiru et al. 2022). According to the theoretical basis of enzyme reaction kinetics, the reaction products will continue to increase when the substrate and enzyme contents rise. However, in practice, when the substrate (egg white) concentration increases, the viscosity rises, which means that the reaction time is required to increase; thus, a more costly input of commercial enzymatic hydrolysis is needed.

In this study, the DH reached up to 15.28% with the two different commercial enzymatic hydrolysis treatments. Pepsin (Protease N) and papain were most widely used in the plastering reaction (Sun et al. 2021). The hydrolysis was carried out in a boiling water bath at 95–98 °C for 10 min to prevent the foaming of the hydrolysate and to inactivate the two commercial enzymatic reactions. Then, it was centrifuged and filtered to obtain the supernatant, which was sterilized and packaged as the EWH product and then stored at refrigeration below 7 °C for follow-up analysis (Ho et al. 2021).

Relationship between EWH after enzyme treatment and its characteristics

Eggs provide the best source of protein supplementation, with the enzymatic hydrolysis transforming the protein from large molecules to small molecules, which facilitates the digestion and absorption of egg protein (Jiang et al. 2021). Therefore, this study investigated the composition of amino acids in the EWH. The results showed that EWH contained 17 amino acids, which were aspartic acid, glutamic acid, serine, histamine, glycine, threonine, arginine, propionic acid, tyramine, cystine, valine, methyl thiamine, phenyl propionic acid, isoleucine, leucine, lysine, and proline (Table 5). The total hydrolyzed amino acid content was 1,444 mg/100 g, in which glutamic acid was the highest, followed by asparagine, and cystine, whose contents were 204, 141, and 125 mg/100 g, respectively. Notably, the content of histidine, threonine, valine, isoleucine, leucine, and lysine was higher than the WHO recommended daily intake for adults (WHO 2007); thus, the EWH application can be diluted for future use. It reported that methionine combined with two-fold the amount of choline is higher than the cysteine level, which combination of these two amino acids can alleviate fatty liver; otherwise, it can promote fatty liver; yet EWH showed good performance in improving fatty liver either as their amino acid efficiency was possibly higher or because they were digested to produce peptides rather than they had overall amino acid composition (Jiang et al. 2021).

Table 5.

Amino acid composition of egg white hydrolysate (EWH)

Amino acid	Results (mg/100 g)	WHO R.D.I.a for adults (mg/Kg)
Aspargine	141	–
Glutamic acid	204	–
Serine	93.6	–
Histidine	45.7	10
Glycine	45	–
Threonine	54.4	15
Arginie	85	–
Alanine	82	–
Tyrosine	48.8	–
Cystine	125	–
Valine	85.3	26
Methionine	19.9	–
Phenylalanine	94	–
Isoleucine	69.5	20
Leucine	113	39
Lysine	85.3	30
Proline	52	–
Total hydrolyzed amino acids	1444	–

^aRecommended daily intake

The results of the molecular weight of EWH showed in Fig. 2. The molecular weight of EWH from egg albumen hydrolyzed by two commercials enzymatic (Protease N and Papain) was calculated by size exclusion chromatography (SEC) of HPLC, and the molecular weight of EWH fell in the range of 4,075—5,396 Da. However, the average molecular weight of commercialized EWH was 700 Da as a functional ingredient (Jiang et al. 2021). It was mentioned that these components were primarily smaller peptides (200 to 500 Da) (Su et al. 2021).

Fig. 2.

The molecular weight of egg white hydrolysate (EWH)

For the future derivative processing of EWH, it was preferred to be used as beverage or tablet products, either as material (semi-product). Therefore, it is necessary to evaluate the color change of the hydrolyzed solution to meet the requirements for processing food. The color change of EWH during the 4-month storage period showed in Table 3. The pH values and brightness (L* value) decreased from 0 to 4 months during the storage period. The product's brightness was 72.40 ± 0.01 at the initial storage period, which significantly reduced to 46.77 ± 0.03 at 4 months, with a significant difference (P < 0.05). The pH value was 8.39 ± 0.07 at the beginning of the storage period, yet it decreased to 7.53 ± 0.01 at 4 months, which was a significant difference in comparison (P < 0.05); hence, it needs to be modified to low acidity (pH < 4.6) for evaluation in the future. In addition, there was no significant difference in the green–red (a* value) at the end of 4 months of storage. The blue-yellow value (b* value) showed a significant decrease compared to the initial storage period (P < 0.05). The previous case reported that L* and a* values decreased significantly with EWH concentration, while the b* value had no effect (Ho et al. 2021).

Table 3.

Physical changes of egg white hydrolysate (EWH) during 4 months of storage

Month	L*	a*	b*	pH
0	72.40 ± 0.01a	3.10 ± 0.02b	11.37 ± 0.06a	8.39 ± 0.07a
1	71.79 ± 0.26b	2.65 ± 0.09c	9.48 ± 0.27b	8.02 ± 0.01b
2	71.56 ± 0.49b	1.98 ± 0.08d	7.68 ± 0.11c	7.98 ± 0.01b
3	69.09 ± 0.39c	1.95 ± 0.06d	6.99 ± 0.19d	7.56 ± 0.29c
4	46.77 ± 0.03d	3.55 ± 0.01a	7.54 ± 0.05c	7.53 ± 0.01c

Data are expressed as mean ± SD (n = 3). Different alphabet letters mean that the values differ significantly from the others at P < 0.05 (Duncan’s multiple range tests)

In rheological analysis, rheological properties indicate the deformation of the fluid when shear was applied to it or the stretching resistance, which suggests the properties of the sample, such as viscosity or friction within the fluid. The egg albumen (as the control group) and EWH samples were analyzed, respectively, which showed that the egg albumen viscosity was 1666.70 mPa s, while EWH was 98.17 mPa s at a re-shear rate of 0.1 (S⁻¹) (Fig. 1B), and it declined with increasing shear rate. The viscosity was an important indicator that affected the mouthfeel of the liquid food, thus influencing the processability, formability, and flowability in food development. As known, egg albumen has a thick consistency with high viscosity (4256.33 ± 101.02), while EWH is thin and fluid with low viscosity (2320.00 ± 87.01) (Table 6). Following these characteristics, the EWH product development requirements include the lower viscosity apart from the application product development mentioned above in this study.

Table 6.

Viscosity analysis of egg white and egg white hydrolysate (EWH)

Sample	Viscosity (cP)
Egg white	4256.33 ± 101.02a
Egg white hydrolysates (EWH)	2320.00 ± 87.01b

Data are expressed as mean ± SD (n = 3). Different alphabet letter means that the values significantly differ with the others at P < 0.05 (Duncan’s multiple range tests)

Bioactive-related properties of EWH

Many studies have confirmed that excessive production of ROS will damage human cell membranes, DNA, and biomolecules, causing the development of chronic diseases (Kurutas 2016; Villalpando-Rodriguez and Gibson 2021; Pizzino et al. 2017; Srinivas et al. 2019). Moreover, food quality decreases due to lipid oxidation caused by free radicals during processing and storage. Hence, it is important to regulate the damage caused by free radicals and prevent food degradation by natural substances with antioxidant functions (Elsayed Azab et al. 2019).

The results of the antioxidant capacity analysis (Table 4) showed that the reducing power of EWH freeze-dried powder (EWHP) at 100 mg/mL was significantly higher than that of 10 mg/mL, which was equivalent to 2.7 and 22.7 folds of 1 mg/mL of Vitamin C, respectively, when compared with the control group. In the ferrous ion chelating activity assay, EWHPs (10 and 100 mg/mL) were significantly higher than EDTA (1 mg/mL) and 1.2 and 2.2 folds higher than EDTA, respectively (P < 0.05), particularly the best metal ion chelating ability of EWHP at 100 mg/mL. The scavenging capacity of DPPH radicals was significantly higher for EWHP at 100 than 10 mg/mL, whereby 10 and 100 mg/mL were equivalent to 4.7 and 30.9%, respectively, of Vitamin C. It has been reported that high DH above 50% of the protein provides significant free radical scavenging effects with a positive correlation, which might be attributed to high DH implicating more peptide content (Yap and Gan 2020). The time and cost of producing EWS in this study were more economical. Nonetheless, a study on duck egg albumen found that papain hydrolysate showed the DH of hydrolysis and the greatest immunomodulatory activity (He et al. 2021). The concentration of 100 mg/mL of EWHP was also significantly higher than 10 mg/mL in terms of ABTS^·+ free radical scavenging ability. It was reported that similar observations were made for producing antioxidant protein hydrolysates from different foods (Pv 2022; Moraes et al. 2022; Wu et al. 2017; Wen et al. 2020). The above results indicate that EWHP exhibited a certain antioxidant capacity, where 100 mg/mL concentration performed better. Previous studies have shown that the hydrolysis of scallops, fish, and egg white proteins by proteolytic enzymes showed significant biological activity (Wang et al. 2021; Rabiei et al. 2022; Bueno Gavilá et al. 2021). Thus, it means that enzyme hydrolysis technology applied to protein hydrolysis transforms protein from large to small molecules, which increases the absorption and utilization by human organisms and enhances the bioactivity performance.

Table 4.

Changes in Bioactive-related properties of egg white hydrolysis freeze-dried products (EWHP)

Sample (mg/mL)	Reducing power	Ferrous ion chelating activity	DPPH free radical scavenging activity	ABTS·+ free radical scavenging activity
	%
Vitamin C 1	93.60 ± 0.94a	–	83.62 ± 0.4a	–
EDTA 1	–	40.72 ± 7.32c	–	–
EWHP 10	2.56 ± 0.22c	50.14 ± 6.34b	3.89 ± 1.31c	48.93 ± 1.27b
EWHP 100	21.27 ± 1.42b	90.35 ± 0.87a	25.83 ± 2.76b	94.18 ± 0.28a

Data are expressed as mean ± SD (n = 3)

Different alphabet letters mean that the values differ significantly from the others at P < 0.05 (Duncan’s multiple range tests)

Conclusions

This study showed that the optimal production conditions for EWH were selected to be implemented under the best economic conditions after considering the evaluated sensory's maximum hydrolysis rate and palatability, contributing to the cost-effectiveness after mass production. Desirable physicochemical properties were obtained for the group 14 EWH obtained from the optimized hydrolysis conditions, even at the expense of biological activity (especially antioxidant capacity). Moreover, the fluidity and low viscosity of the EWH match the requirements of the future products (beverages/tablets) developed in this study. Therefore, the antioxidant mechanisms involved in this study and the identification of bioactive components with worthy directions for future research. The study will provide valuable insights and suggest ways within the EWH might develop healthier commercialized products.

Electronic supplementary material

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Abbreviations

RSM

Response surface methodology

EWH

Egg white hydrolysate

DH

Degree of hydrolysis

ABS

Absorbance

EDTA

Ethylenediaminetetraacetic acid

Appendix

See Fig. 2 and Tables 5, 6.

Author contributions

Conceptualization, C-YH and SHH; methodology, S-LH, C-WH and J-YC; software, C-KC, and S-LH; investigation, C-CH, and C-WT; resources, C-YH; data curation, SHH, C-CH, and C-YH; writing—original draft preparation, P-HH, and SHH; writing—review and editing, SHH, C-YH, and P-HH; visualization, C-WH, and M-KS, and M-HC; supervision, C-YH, and S-LH; project administration, J-YC, C-WH, and M-KS.; funding acquisition, C-YH.

Funding

This research was funded by the Ministry of Science and Technology, the Republic of China (Grant No. 110-2320-B-992 -001-MY3, 111-2622-E-992 -002—and 111-2221-E-328 -001 -MY3).

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

Consent to participate

Before the sensory evaluation of this research, we provided informed consent (supplement material “Informed consent”) to the participants and confirmed that these experiments were conducted according to established ethical guidelines, and informed consent was obtained from the participants, then give them the evaluation and questionnaire (supplement material “Egg white hydrolysates evaluation and questionnaire”).

Consent for publication

All authors have read and agreed to the published version of the manuscript.