Identifying the potential of ovalbumin and its’ hydrolysates to be used in a liquid food system to develop functional beverages

Abstract

Ready-to-drink protein beverages have gained attention in the functional beverage market. This study examined the possibility of using ovalbumin and its hydrolysates to develop functional protein beverages. Lyophilized ovalbumin (5%, w/v) was dissolved in distilled water (pH 10) with sugar, salt, and vanilla and pasteurized at 56.7°C for 3.5 min to prepare a basal ovalbumin beverage (OVA). The beverages were sequentially hydrolyzed using pepsin (pH 2.5, 37°C for 3 h) first, then with protease (pH 6.5, 37°C for 3 h), or protease (pH 6.5, 37°C for 3 h) followed by trypsin (pH 7.8, 37°C for 3 h) combinations. Three beverages (OVA, PrTr, and PePr) were tested for physical, functional, and quality characteristics. Complete hydrolysis by enzyme combinations was ensured using 15% SDS-PAGE. Enzymatic hydrolysis caused an increase in the solubility of ovalbumin in water. The beverages showed antioxidant activity, and hydrolyzed drinks showed antimicrobial activity. No significant difference in acidity, but a reduction in pH, an increase in TSS, and alterations in viscosity were observed among the drinks with storage. A significant difference (p < 0.05) in titratable acidity was identified between the two hydrolyzed drinks, but no difference (p > 0.05) within a treatment was observed until Day 14. In conclusion, ovalbumin and its hydrolysates have the potential for preparing functional protein beverages with a shelf-life of up to 7 days.

Introduction

Chicken egg is a readily available and affordable protein source. Eggs from most avian species may have a similar nutritional composition (Song et al., 2000). The chicken egg consists of 63% egg white, 27.5% egg yolk, and 9.5% eggshell (Kovacs-Nolan et al., 2005). Chicken eggs are composed of water (74%), proteins (12%), lipids (11%), carbohydrates (1%), and minerals (0.6%) (Belitz et al., 2009). Major egg white proteins include ovalbumin (54%), ovotransferrin (12%), ovomucoid (11%), ovoglobulins (G2 and G3, 8%), ovomucin (3.5%), and lysozyme (3.5%), while the minor egg white proteins are ovoinhibitor (1.5%), ovoglycoprotein (1%), ovoflavoprotein (0.8%), ovomacroglobulin (0.5%), avidin (0.05%), and cystatin (0.05%) (Abeyrathne et al., 2013; Kovacs-Nolan et al., 2005).

Ovalbumin is the major egg white protein (54%) utilized in various fields such as nutrition, biochemistry, and immunological studies. It is a standard protein in protein assays and is mainly used in the food industry as a protein source (Abeyrathne et al., 2014a; Geng et al., 2019; Lv et al., 2015). Ovalbumin has functional properties such as gelling, foaming, and emulsifying (Stadelman and Cotterill, 2001; Weijers et al., 2002), important in food industry applications such as meat processing, bakery, and baby food industries (Weijers et al., 2002; Zhao et al., 2016). The Possibility of utilizing ovalbumin in the food industry expands with the invention of separating techniques such as sequential separation of egg white proteins. Abeyrathne et al. (2014a) used sequential separation of egg white proteins (ovalbumin, lysozyme, ovomucin, and ovotransferrin) where ammonium sulfate and citric acid combinations have been used to yield highly purified ovalbumin (> 97% yield). The invented method was identified as a simple, non-toxic, and scalable method. The purity of ovalbumin was identified to be greater than 85% in scale-up preparations (Ji et al., 2020).

The modifications of ovalbumin, such as phosphorylation and glycation, are reported to improve the functional properties of ovalbumin (Yang et al., 2021). The enzymatic hydrolysis of ovalbumin also increased the functionality of ovalbumin significantly, which attracted the food industry's attention to using ovalbumin hydrolysate as a major functional ingredient (Abeyrathne et al., 2013; Chang et al., 2018). The activity of a particular bioactive peptide is decided by the size and amino acid sequence of the peptides (Eckert et al., 2013). Patil et al. (2020) reported that enzyme type, substrate-to-enzyme ratio, hydrolysis length, and enzyme concentration affect the functionality and sequence of peptides produced. Ovalbumin can be hydrolyzed using a single enzyme or a combination of enzymes. Various enzymes such as elastase, pepsin, trypsin, chymotrypsin, flavorzyme, neutrase, papain, and alcalase were tested to hydrolyze ovalbumin (Miguel et al., 2004; Tang et al., 2013; Wright, 1984). Peptides produced by each enzyme showed different activities, including ACE-inhibitory, antioxidant, and antimicrobial activities (Miguel et al., 2004; Tang et al., 2013). The peptides with low molecular weight showed higher antioxidant and ACE-inhibitory activities than the ones with high molecular weight (Patil et al., 2020). Peptides produced using the combinations of enzymes (pepsin, alcalase, chymotrypsin, papain, and trypsin) showed higher antioxidant, ACE-inhibitory, and metal-chelating activities than those from single enzyme hydrolysis (Abeyrathne et al., 2014b). Peptides produced by the pepsin hydrolysis of ovalbumin, such as Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu and Ser-Ala-Leu-Ala-Met, showed ACE-inhibitory and very high antioxidant activities (Miguel et al., 2004). The chymotrypsin hydrolysis of ovalbumin produced a peptide (Arg-Ala-Asp-His-Pro-Phe) with vaso-relaxing activity (Matoba et al., 1999; Miguel and Aleixandre, 2006). Fujita et al. (1995) reported that ovokinin, a vaso-relaxing octapeptide (Phe-Arg-Ala-Asp-His-Pro-Phe-Leu), was derived from ovalbumin, and the administration of the peptide (as an emulsion in 30% egg yolk) significantly lowered the systolic blood pressure of spontaneously hypertensive rats (SHR). Besides, the enzyme hydrolysis of egg white improved its texture and chewing, swallowing, and digestibility (Tavano, 2013; Garcés-Rimón et al., 2016; López-Martínez et al., 2021).

Ovalbumin hydrolysates are used in the food industry to improve the textures of haute cuisines. Different products, such as custard, cream, junket, cheese-like products, and functional ice cream, were developed using egg white hydrolysates (Garcés-rimón et al., 2016; López-Martínez et al., 2021). Although the beverage industry is one of the fastest growing industries, the use of ovalbumin hydrolysates in the beverage industry has not been explored in depth yet. Currently, the food industry uses egg white proteins to produce various beverages. Novel products such as high-protein egg drinks (Silva and Abeyrathne, 2014), protein beverages with milk and egg whites (Lotfian et al., 2019), and protein-enriched carbonated fruit drinks were developed (Hemanth et al., 2020). So far, the application of the above-described functional properties of ovalbumin hydrolysates and non-hydrolysate status in the beverage industry has not been reported. A reason for this can be the hydrophobicity of ovalbumin in its’ non-hydrolysate status, which makes it difficult to use as a main raw material in beverage production (Maggonage et al., 2024). However, there is a potential for the utilization of hydrolysates over the native proteins in the beverage industry because protein solubility is considerably changed with enzymatic hydrolysis. To meet the increasing demands for natural ingredients and minimize the utilization of synthetic preservatives while providing nutritional and health benefits, make ovalbumin hydrolysates a perfect alternative for the beverage industry. Hence, this study aims to investigate the ability of ovalbumin and its hydrolysates to develop a new value-added protein-rich functional drink and analyze the product's physical, chemical, functional, and quality properties of the final product.

Materials and methods

Materials

Ovalbumin was separated as a byproduct of ovotransferrin purification using ammonium sulfate and citric acid combination (Abeyrathne et al., 2014a). Freeze-dried samples were transported from Iowa State University, Ames, IA, USA. Enzymes, Pepsin from porcine gastric mucosa (≥ 3,200 U/mg protein; EC 3.4.23.1), protease from B. licheniformis (≥ 2.4 U/g solution; EC 3.4.21.64), and trypsin from bovine pancreas (≥ 7,500 BAEE U/mg solid; EC 3.4.21.4) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Butylated hydroxyanisole, thiobarbituric acid, and trichloroacetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ammonium acetate and plate count agar from HiMedia Laboratories Pvt. Ltd (Mumbai, Maharashtra, India), and iron (II) sulfate heptahydrate from Daejung Chemicals and Metals Co., Ltd (Siheung, Gyeonggi, S. Korea) were purchased.

Preparation of ovalbumin-based drinks

Lyophilized ovalbumin was dissolved in water and used for the study. Protein concentration, pH, time, and temperature combinations were tested for beverage preparation. The protein concentrations between 1% and 15% (w/v) and pH 4 and 11, along with different pasteurization temperatures and time combinations, were tested at the preliminary trials. The best percentage of ovalbumin utilized in the creation of beverages was determined using the solubility index equation. The solubility index was measured according to Yu et al. (2017) with some modifications. The ovalbumin-based drink was centrifuged (ST 40R, Thermo Scientific, Waltham, MA, USA) at 3,000 × g for 15 min at 4°C, and the supernatant was collected and freeze-dried. The following equation was used to calculate the solubility index.

$$\text{Solubility}\text{index}=\frac{\left(\text{weight}\text{of}\text{protein}\text{dissolved}\text{in}\text{Initial}\text{supernatant}\right)}{\text{weight}\text{of}\text{ovalbumin}\text{used}}\times 100\%$$

After identifying the solubility index, the preparation of ovalbumin drinks was started by randomly allocating the lyophilized ovalbumin (from Iowa states, USA) into three groups 1, 2, and 3. A randomly selected group of lyophilized ovalbumin (group 2) is used to prepare ovalbumin basal beverage (OVA). The beverage was prepared using ovalbumin, sugar, salt, and vanilla essence (Mamma Cook Pvt. Ltd., Kandana, Sri Lanka) according to a drafted recipe. The amount of sugar added to ovalbumin followed Garcia et al. (2020). The pH-adjusted beverage was pasteurized at 56.7°C for 3.5 min (Michalski et al., 2000), cooled in an ice bath for 5 min, and then stored at 4°C. The remaining two groups (groups 1 and 3) were used to prepare ovalbumin hydrolysate-based drinks. Before the hydrolysis process, the basal ovalbumin drinks were prepared separately from the remaining two lyophilized groups. The two groups were randomly chosen for the hydrolysis process described below.

Preparation of ovalbumin hydrolysate-based drinks

Pepsin from porcine gastric mucosa (Sigma-Aldrich, St. Louis, MO, USA), protease from B. licheniformis (Sigma-Aldrich, St. Louis, MO, USA), and trypsin from bovine pancreas (Sigma-Aldrich, St. Louis, MO, USA) were used. The ovalbumin-based drinks prepared from native protein were hydrolyzed using the best combinations of the two enzyme treatments from a previous study (Abeyrathne et al., 2014b). The two beverages with native proteins were hydrolyzed with pepsin (pH 2.5, 37°C for 3 h) first, then with protease (pH 6.5, 37°C for 3 h) or protease (pH 6.5, 37°C for 3 h), followed by trypsin (pH 7.8, 37°C for 3 h). The enzyme: substrate ratio used was 1:100 (w/w), and the first enzyme was inactivated by heating at 100°C for 15 min before the second enzyme treatment. At the end of the second enzyme treatment, the beverages were heated at 100°C for 15 min, cooled to room temperature, and then stored at 4°C. At the end of the process, two hydrolyzed beverages (PePr and PrTr) and a beverage with a native protein (OVA) were used for further analysis.

The physical properties, such as hydrolysate yields and total soluble solids (TSS) in the beverages, were determined. The yields of hydrolysates were measured for the beverages made with ovalbumin hydrolysates. The samples were centrifuged (ST 40R, Thermo Scientific) at 3,000 × g for 15 min at 4°C after the completion of the hydrolysis process. The supernatant was freeze-dried to calculate hydrolysate yields.

$$\text{Yield}(\%)=\frac{\begin{array}{c}\text{Final}\text{weight}\text{of}\text{hydrolysates}\text{dissolved}\text{in}\text{the}\text{supernatant}\\ \text{after}\text{two}\text{enzyme}\text{treatments}\end{array}}{\text{Initial}\text{weight}\text{of}\text{ovalbumin}\text{used}}\times 100\%$$

The total soluble solids of the three beverage samples were measured using a hand-held refractometer (2312 Master-T, ATAGO Co., Ltd, Fukaya, Japan). The changes in total soluble solids were calculated periodically on days 1, 3, 5, 8, 10, and 14. Total soluble solids were given in Brix values (Hemanth et al., 2020).

The hydrolysis of the ovalbumin in the drinks was confirmed using 15% SDS-PAGE gels (Green and Sambrook, 2014). The SDS-PAGE was conducted using a Mini-Protean® Tetra System (Bio-Rad, Bio-Rad Laboratories, Hercules, California, USA) and stained with Coomassie Brilliant Blue G-250 (HiMedia Laboratories Pvt. Ltd, Mumbai, Maharashtra, India).

Analyses of the functional property of developed beverages

TBAR Assay. The 2-thiobarbituric acid reactive substance (TBARS) assay was used to determine antioxidant activity (Abeyrathne et al., 2014b). An oil-in-water emulsion was prepared by homogenizing 1 g of olive oil (Aceites Agro Sevilla, Agrosevilla, Sevilla, Spain) and 100 µL of tween-20 (Research Lab Fine Chem, India) with 100 mL of distilled water using a polytron homogenizer (D-500, Scilogex, Rocky Hill, CT, USA) for 2 min in an ice bath at full power. Samples were prepared by mixing 8 mL of oil emulsion, 0.5 mL distilled water, 0.5 mL of 200 ppm Fe²⁺ (Iron (II) sulfate heptahydrate, Daejung, Siheung, Gyeonggi, S. Korea), and 1 mL sample from the drink. Then samples were incubated at 37°C for 16 hours in a water bath (YCW-010E, GEMMY INDUSTRIAL CORP., Taipei, Taiwan). After incubation, 2 ml of 20 mM TBA/15% TCA; w/v and 20 µL of 10% butylated hydroxyanisole (Sigma-Aldrich, St. Louis, MO, USA) in 90% ethanol were added to 1 mL of sample and then vortex mixed. The prepared solution was incubated at 90°C in a water bath for 15 min, cooled in an ice bath for 10 min, and centrifuged (ST 40R, Thermo Scientific) at 3,000 × g for 15 min at 5°C. The sample's absorbance was measured using a UV-Vis spectrophotometer (Spectrophotometer UV-2005, J. P. Selecta, Barcelona, Spain) at 532 nm against a blank prepared with 1 mL of distilled water and 2 mL of TBA/TCA solution. A control sample was prepared using 1 mL emulsion with 2 mL TBA/TCA solution. The TBARS values of the drinks were expressed as milligrams of malondialdehyde per liter of emulsion.

DPPH (1, 1-diphenyl-2-picrylhydrazyl) Radical Scavenging Activity. DPPH radical scavenging activity assesses an antioxidant molecule's free radical scavenging potential (Mishra et al., 2012). DPPH-radical scavenging activity was determined using the method of Blois (1958) with some modifications. Two mL of beverage sample and 18 mL of distilled water were added to 50 mL Falcon tubes, homogenized using a polytron homogenizer (D-500, Scilogex) for 10 seconds, and centrifuged (ST 40R, Thermo Scientific) at 3,000 × g for 10 min. The sample (0.4 mL) was transferred to a 15 mL Falcon tube, added with 1.6 mL distilled water and 2 mL DPPH (Sisco Research Laboratories Pvt. Ltd., Mumbai, India, 0.2 mM in methanol), and then vortex mixed. After storing the samples in a dark place for 60 min, the absorbance was measured at 517 nm using a UV-Vis Spectrophotometer (Spectrophotometer UV-2005, J. P. Selecta) against a blank. Blank samples were prepared by adding 2 mL distilled water and 2 mL methanol. Ascorbic acid samples were prepared by adding 2 mL of 10% ascorbic acid solution and 2 mL DPPH solution. Control samples were prepared by mixing 2 mL distilled water and 2 mL DPPH solution. The following equation was used to calculate the DPPH-radical scavenging activity.

$$\text{DPPH}-\text{radical}\text{scavenging}\text{activity}(\%)=\frac{(\text{Absorbance}\text{of}\text{control}-\text{Absorbance}\text{of}\text{the}\text{sample})}{\text{Absorbance}\text{of}\text{control}}\times 100\%$$

Iron-Binding Capability. The iron-binding capability of hydrolysates was tested using the Ferrozine method with some modifications (Abeyrathne et al., 2014b). One hundred microliters of beverage sample, 0.9 mL of distilled water, and 1 mL of 10 mg/kg Fe²⁺ (iron (II) sulfate heptahydrate, Daejung Chemicals and Metals Co., Ltd, S. Korea) were vortex-mixed in a 15 mL Falcon tube and incubated for 5 min at room temperature. Nine hundred microliters of 11.3% TCA (Sigma-Aldrich, St. Louis, MO, USA) were added to the sample mixture and centrifuged at 2,500 × g for 10 min at 5°C. One milliliter of the supernatant was transferred to a disposable culture tube, 1 mL of distilled water, 800 µL of 10% ammonium acetate (HiMedia Laboratories Pvt. Ltd, Mumbai, Maharashtra, India), and ferroin color indicator (75 mg of ferrozine, 75 mg of neocuprion, and 1 drop of 6 N HCl in 25 mL of distilled water) and vortex mixed. After 5 min of incubation at room temperature, the absorbance was measured at 562 nm using a UV spectrophotometer (Spectrophotometer UV-2005, J. P. Selecta).

The Fe-chelating activity was calculated using the following equation.

$$\text{Fe}2+\text{chelating}\text{activity}(\%)=\left[1-\frac{\text{Sample}\text{absorbance}}{\text{Absorbance}\text{of}\text{control}}\right]\times 100\%$$

Antimicrobial Activity. The total plate count method described by Moon et al. (2012) was utilized to determine the antimicrobial activity. Culture media and equipment were autoclaved at 121°C for 20 min before use. A 20-25 mL of total plate agar (HiMedia Laboratories Pvt. Ltd, Mumbai, Maharashtra, India) was poured into each sterilized petri dish, solidified for 1 hour, and then the agar surface was streaked with a sterilized cotton swab that was contained bacteria. Wells were aseptically punched on the agar surface after 30 min, and 100 microliters of each beverage were pipetted into it. The Minimum Inhibition Concentration of the beverages for the bacteria was determined using a series of solutions containing 20,000, 10,000, 5,000, 2,500, 1,250, and 625 ppm of ovalbumin or ovalbumin hydrolysates. Complete diffusion was facilitated by allowing about 30 min and incubating at 37°C for 48 hours. The bacterial inhibition zones of the beverages were measured. Augmentin (GlaxoSmithKline, Brentford, UK) was used as the positive control, and distilled water was used as the negative control. The beverage's antimicrobial activity was calculated using the following equation.

$$\text{Antimicrobial}\text{index}=\frac{\text{Inhibition}\text{zone}\text{of}\text{the}\text{sample}}{\text{Inhibition}\text{zone}\text{of}\text{the}\text{control}}\times 100\%$$

Quality analyses of the developed beverages

The quality parameters analyzed were pH, titratable acidity, total soluble solids, and dynamic viscosity. The viscosity of the drinks was measured using a viscometer (Lotfian et al., 2019). The pH of the beverages was measured using a digital pH meter (PL-700PV, EZDO, Taipei City, Taiwan) on days 1, 3, 5, 8, 10, and 14. The beverages' acidity was calculated using titratable acidity (Hemanth et al., 2019). Two milliliters of the sample were titrated with 0.1N NaOH using phenolphthalein as an indicator. The acidity of the samples was calculated using the following equation.

$$\text{Titratable}\text{Acidity}=\frac{\mathrm{N}\times \mathrm{V}\times \mathrm{W}}{\mathrm{M}\times 100}$$

Where N = Normality of NaOH, V= Volume of titrant, W = Equivalent weight of acid, M = Mass of the sample.

Statistical analysis

Data were analyzed with MINITAB18.0 statistical software (Minitab Ltd., Coventry, UK). Three replicates were carried out for each experiment. One-way and Two-way ANOVA were used. Tukey test was performed to compare means at a 95% confidence interval.

Results and discussion

Conditions for the preparation of ovalbumin beverage

Ovalbumin sedimentation was initially observed when it was dissolved (1-15%, w/v range) in water. It is known that half of the amino acids in ovalbumin are hydrophobic, and factors such as pH, ionic strength, temperature, and surface charge of proteins and solvents affect the solubility (Geng et al., 2019; Li and Yan, 2017). In this study, a significant increase in sedimentation (p < 0.05) was observed as the percentage of ovalbumin increased (1 to 15%, w/v)). When the ovalbumin concentration was reduced to 1-5% (w/v), the 5% solution had significantly higher sedimentation (p < 0.05) than 1%. However, no significant difference was found between 3%, 4%, and 5% ovalbumin at pH 6.3. The sedimentation of insoluble protein and layer separation were visible at an acidic pH. This may be due to the iso-electric point of ovalbumin, pI = 4.9 (Huntington and Stein, 2001). This result is consistent with the observations of other studies, where minimal solubility was identified around pH 5.0 (Xu et al., 2020). After several preliminary trials, the optimum pH value for beverage production was selected as pH 10.0. However, no significant difference was observed in sedimentation percentages between pH 7, 9, and 10 (p < 0.05, Table 1), and a homogenous texture was manifested at higher pH levels (pH > 7.0). The recommended pasteurization temperature for egg white is 56.7°C for 3.5 min (Michalski et al., 2000), and heat denaturation of ovalbumin is not expected at this condition because ovalbumin heat denaturation temperature is 84°C (Li et al., 2013). Thus, pasteurizing a 5% (w/v) ovalbumin (pH 10) solution at 56.7°C for 3.5 min was acceptable for the albumin beverage preparation. A milky or dairy-like texture was observed following pH adjustment and heat treatment. The drafting of a recipe was done with a minimum amount of ingredients. A recipe was prepared with the minimum amount of ingredients since the protein is an isolated and novel protein. On the other hand, the protein behavior within the food system can be easily identified when there are lesser amounts of ingredients. It was necessary to identify whether the individual protein and its’ hydrolysates showed antioxidant and antimicrobial activities when they were alone in the medium of water. Therefore, the recipe was formulated as follows. Sugar (10%, w/v) was added to the ovalbumin solution following a recipe for yogurt, as mentioned in Garcia et al. (2020). Vanilla essence (0.04%, w/v) and salt (0.05%, w/v) were added as final ingredients in the recipe to increase organoleptic qualities. The evaluation of physical and functional properties was conducted and discussed in this study. However, the final ingredient percentages of the recipe will be developed with further studies on sensory evaluation not conducted in the current study. The primary intention of developing a temporary or drafted recipe was to identify how protein and hydrolysates behave within a food system as a component of others such as sugar, vanilla, and salt. Moreover, sensory evaluation is the most crucial step to be followed in future extensions of the current study. The developed beverage using the above recipe (OVA) was stored at refrigerated conditions (4°C). The same recipe was also used in the preparation of ovalbumin hydrolysate beverages.

Table 1.

Average sedimentation of 5% ovalbumin in water under pH 7, 8, 9, 10 and 11.

PH	AVERAGE SEDIMENTATION (%)
7	73.63±0.38 b,c
8	72.45±1.75 c
9	73.67±0.26 b,c
10	76.52±0.65 b
11	80.45±1.55 a

^a-cValues with different letters differ significantly (p < 0.05).

Values are means ±SE.

Solubility index of ovalbumin beverage (OVA)

The solubility index of proteins is important in liquid foods and beverages because it can affect the functionality of proteins (Bao et al., 2017). The separation of insoluble proteins was the main hindrance in producing a homogeneous texture in this study. Previous studies on preparing beverages from whey protein concentrates have also shown sedimentation of insolubilized proteins (Lotfian et al., 2019). The solubility index of beverages with native ovalbumin was 27.18±1.18%. The possible reason for insolubility is that half of the amino acids in the ovalbumin sequence are hydrophobic (such as valine, leucine, and tryptophan) (Guha et al., 2019). During processing steps (mixing and pasteurization), the unfolding of the hydrophobic core of the ovalbumin can occur because ovalbumin readily denatures and coagulates when exposed to new surfaces (Stadelman and Cortterill, 2001). Another reason for insolubility would be alkaline pH (10), where ovalbumin can undergo unfolding (Zhao et al., 2016). Strong alkalis can completely denature ovalbumin molecules by exposing their hydrophobic core. The major deficiency identified in OVA-based drinks in this study was low solubility. However, the insolubility of ovalbumin is identified as a limitation or a challenge of its’ potential use in large-scale preparations. This challenge was expected to be resolved after hydrolysis.

Hydrolysis of ovalbumin drinks using two-enzyme combinations

The hydrolysis procedure was carried out under the predetermined conditions where a combination of Protease-Trypsin and Pepsin-Protease showed the highest Fe²⁺-chelating and antioxidant activity (Abeyrathne et al., 2014b). After the addition of the first enzyme during hydrolysis, a homogeneous texture was observed, but it changed during heat inactivation of the enzyme (100°C for 15 min). The beverages lost the homogeneous milky texture observed in the OVA. The beverages were slightly brownish translucent liquid. Small aggregates were visible as a small top layer in hydrolysates at the end of two enzyme hydrolysis. The formation of layers can be due to impurities present in ovalbumin (the purity level of ovalbumin used was > 85%). Bao et al. (2017) also recorded the formation of a few aggregates in the hydrolysates of egg yolk. Previous studies showed that egg white subjected to enzymatic hydrolysis (Aminopeptidase from Aspergillus oryzae, pH 5.5, 50°C, and 120 min) formed gels upon heat inactivation (Garcés-Rimón et al., 2016). However, the gelation of ovalbumin hydrolysates was not observed during the heat inactivation process of the beverages. Therefore, enzymatically treated ovalbumin under given conditions could be utilized to produce beverages.

The yield of hydrolysates of PePr and PrTr enzyme combination

The percentage yield of hydrolysates (%) dissolved in the beverage was calculated separately for the two hydrolyzed beverages. The PePr combination showed a higher yield (53.38±1.71%) than the PrTr combination (38.95±0.13%) (Fig. 1). A similar result was observed in a previous study that used egg white powder with a peptide yield of 77.5±0.3% (Li et al., 2018). As mentioned above, the solubility index of OVA is lower than the hydrolysates. Therefore, enzymatic hydrolysis has increased the solubility of ovalbumin. Chen et al. (2012) also reported that the protease hydrolysate of egg white was more water-soluble than the egg white. Therefore, hydrolysates of ovalbumin are more favorable for beverage production than the native ovalbumin. As discussed above, the total solubility of ovalbumin was not observed even after enzymatic hydrolysis, which caused a separation of insoluble ovalbumin as a layer with time. This was identified as a limitation in preparing a protein beverage on a commercial scale.

Fig. 1.

Graphical expression of the yield of hydrolysates in PePr and PrTr drinks.

PePr = Ovalbumin beverage hydrolyzed with Pepsin and Protease, PrTr = Ovalbumin beverage hydrolyzed with Protease and Trypsin. Values with different letters (a-b) differ significantly (P < 0.05).

Total soluble solids (TSS) of ovalbumin-based beverage and hydrolyzed drinks

The TSS content in beverages can vary under different conditions. Temperature can be the main factor that influences TSS (Lotfian et al., 2019), but the TSS content of OVA and OVA hydrolysates varied with storage time: on day 1, the TSS content of OVA was 11.2±0.2% Brix, while the hydrolyzed ovalbumin was 13.93±0.23 and 14.80±0.20 (PePr and PrTr, respectively). On the 10^th day, the TSS contents in all three beverages were significantly higher than those at day 1 (OVA=12.20±0.2%, PePr=15.9±0.14%, PrTr=16.2±0.01%) (Fig. 2). In a similar study, protein beverages with milk and egg white (14%(v/v) and 16% (v/v)) under sterilized and pasteurized conditions did not show any significant difference in total soluble solids content (Lotfian et al., 2019). The herbal apple-whey-based ready-to-serve beverages showed a slight increase in TSS with storage due to the presence of an acid that might have caused solubilizing insoluble carbohydrates (Sharma et al., 2019). The increase of TSS in this study could be due to the storage conditions, such as temperature (4°C) and pH changes.

Fig. 2.

Changes of total soluble solids (TSS) content in OVA, PePr, and PrTr drinks during 14 days of storage.

OVA = Ovalbumin-based beverage; PePr = Ovalbumin beverage hydrolyzed by Pepsin and Protease; PrTr = Ovalbumin beverage hydrolyzed with Protease and Trypsin. Values with different letters (a-b), (x-z), and (q-s) differ significantly (P < 0.05).

The viscosity of ovalbumin-based beverage (OVA)

A significant difference in the viscosity of an ovalbumin-based drink was identified between the 1%(w/v) and 5% (w/v) treatments (5% OVA beverage; 7.5±0.71 MPa•s, 1% OVA beverage; 5±0.01 mPa•s on day 1). No significant difference in dynamic viscosity (MPa•s) was identified in 1% OVA until Day 7 but a significant difference on Day 10 (5±0.01 on Day 1 to 7.5±2.12 on Day 10). Similarly, a significant difference (P < 0.05) in dynamic viscosity (MPa•s) was identified on Day 10 of the 5% OVA drink (7.5±0.71 on day 1 to 11±1.41 on Day 10) (Fig. 3). Previous studies showed the beverages with higher protein content had higher viscosity.

Fig. 3.

The changes of dynamic viscosity in 5% (w/v) and 1% (w/v) OVA. OVA = Ovalbumin based drink.

Values with different letters (A-B) differ significantly (P < 0.05) between treatments (1% and 5%) during storage. Values with different letters (a-b and x-y) differ significantly (P < 0.05) within 1% and 5%, respectively (p < 0.05).

Lotfian et al. (2019) reported that increasing egg white protein percentages in the protein beverages developed with milk and egg proteins showed decreasing viscosity, while lowering egg white protein percentages caused increasing viscosity during storage. Higher than 14% of egg white caused a reduction in viscosity. Increasing TSS in the OVA beverage during storage could be the reason for increasing viscosity with time. Therefore, like OVA, an increase in the viscosity of hydrolyzed beverages was also expected and observed. Viscosity change with time was calculated only for OVA (unhydrolyzed drink) due to the limitation of resources such as enzymes. Although ovalbumin peptides are reported to have good functional activities, using them on a large scale is challenging, especially in the food industry, because of higher raw material costs than ovalbumin. Therefore, in terms of cost, OVA drink is in the upper hand.

Sodium dodecyl sulfate poly acrylamide gel electrophoresis of the beverages produced

Fig. 4 shows the complete hydrolysis of ovalbumin in the drink by PePr and PrTr treatments. A thick band was observed at near 45 kDa in the OVA beverage, confirming the presence of ovalbumin in the native state. However, no 45 kDa band was identified in the hydrolyzed drinks. The SDS results of the hydrolyzed drinks were similar to those of Abeyrathne et al. (2014b), who used the same two enzyme combinations to hydrolyze ovalbumin. However, Photchanachai et al. (2002) reported that ovalbumin had aggregated and polymerized when heated at > 88°C, which was not observed in the current study after hydrolysis. Most peptides formed by the two-enzyme hydrolysis had a low molecular weight (< 2 kDa), and most of the peptide bands were not seen in 15% SDS-PAGE (Abeyrathne et al., 2014b). No bands in the PePr and PrTr lanes confirmed a high degree of hydrolysis with the two-enzyme combinations.

Fig. 4.

Sodium dodecyl sulfate-PAGE of ovalbumin-based drinks and hydrolyzed drinks. M = Marker, OVA= Ovalbumin-based beverage, PePr = Ovalbumin beverage hydrolyzed by Pepsin and Protease, PrTr = Ovalbumin beverage hydrolyzed with Protease and Trypsin.

Antioxidant capacity of the developed beverage

2-Thiobarbituric Acid Reactive Substance (TBARS) Assay. Native ovalbumin does not have an antioxidant activity (Lechevalier et al., 2007). However, all three beverages showed lower TBARS values than the control (Fig. 5). The PePr sample has the highest antioxidant activity (6.2±0.33) and was significantly different from that of the control (7.85±0.49). Abeyrathne et al. (2014b) also observed the lowest TBARS value when a protease and trypsin combination was used to hydrolyze ovalbumin. The hydrolysis of ovalbumin with pepsin (1%, 37°C, 3 h), α-chymotrypsin (1%, 37°C, 3 h), and combinations of pepsin-alcalase (1% pepsin, 37°C for 3 h followed by 1% alcalase, 37°C for 3 h), alcalase-trypsin (1% alcalase, 37°C for 3 h followed by 1% trypsin, 37 °C for 3 h), and pepsin-papain (1% pepsin, 37°C for 3 h followed by 1% papain, 37°C for 3 h) all showed antioxidant activity and no significant difference was identified between the enzyme treatments (Abeyrathne et al., 2014b). Heating ovalbumin to more than 80°C caused an increase in antioxidant and superoxide radical scavenging activities, which might be due to the exposure of amino acids like cysteine and histidine (Yamamoto et al., 1996). Ovalbumin-based beverages heated at 56.7°C can also have an impact from heat. However, a significant difference (p < 0.05) was not identified between the PePr and OVA, where PePr was subjected to heat inactivation (100°C, 15 min). Although native ovalbumin has no antioxidant activity (Lechevalier et al., 2007), some antioxidant activity was observed in the OVA drink, probably due to alterations of molecular structure during the heat process.

Fig. 5.

The TBARS values (mg of MDA/L) of beverages.

OVA = Ovalbumin-based beverage, PePr = Ovalbumin beverage hydrolyzed by Pepsin and Protease, PrTr = Ovalbumin beverage hydrolyzed with Protease and Trypsin, oil = Corn oil. Values with different letters (a-b) differ significantly (P < 0.05).

DPPH (1,1-diphenyl-2-picrylhydrazyl) Radical Scavenging Activity. DPPH assay is used in the food industry to measure antioxidant activity by measuring the ability of a compound to act as a hydrogen donor (Kedare and Singh, 2011). In the current study, OVA showed the highest DPPH radical scavenging activity (21.85±0.97%), but the three beverages (OVA, PePr, and PrTr) did not show significant difference (p < 0.05) (Fig. 6) compared to the positive control (ascorbic acid) even though the PePr showed the lowest DPPH radical scavenging activity (14.04±4.78%). The PrTr beverage manifested 20.97±5.23% DPPH radical scavenging activity. According to Chen et al. (2012) both factors, type of enzyme and time, impacted hydrolysis. In the current study, the time and ratio of enzyme to substrate are constants. Therefore, the activity difference can be due to the type of enzyme used. Ultimately, all three beverages have manifested DPPH radical scavenging activity. Therefore, within a food system, protein and peptides can depict radical scavenging activity.

Fig. 6.

The DPPH radical scavenging activity (%) of beverages.

OVA = Ovalbumin-based beverage, PePr = Ovalbumin beverage hydrolyzed by Pepsin and Protease, PrTr = Ovalbumin beverage hydrolyzed with Protease and Trypsin, Ascorbic acid = Positive control. Values with different letters (a-b) differ significantly (P < 0.05).

Fe ²⁺-Chelating Activity. The chelation of transition metal ions can also measure the antioxidant activity of peptides. Transition metals promote lipid oxidation (Bueno-Gavilá et al., 2021; Goddard et al., 2012). Abeyrathne et al. (2014b) reported that the ovalbumin hydrolysates of protease-trypsin (90%), pepsin-papain, and pepsin-alcalase (40-60%) combinations showed Fe²⁺-chelating activity, but protease-trypsin combination had the highest activity. The current study also showed that the PrTr combination has the highest Fe²⁺-chelating activity. Ovalbumin-based beverages did not manifest Fe²⁺-chelating activity, while hydrolyzed drinks (OVA; -0.94±0.58%, PePr; 12.57±0.22%, PrTr; 13.37±0.47%) have shown positive results on the test (Fig. 7). Nisbet et al. (1981) reported that natural ovalbumin does not have metal-binding capacity, similar to the current studies. Oxidative stress is caused by the imbalances between reactive oxygen species and antioxidants in the body and can cause various diseases (Antolovich et al., 2002). Natural antioxidants can minimize the adverse effects of free radicals within the body (Mishra et al., 2012). The hydrolyzed ovalbumin drinks are superior to the natural ovalbumin-based beverages in metal-chelating activity, and thus, they can help lower oxidative stress in the body. Therefore, using hydrolyzed ovalbumin would be more beneficial than using natural ovalbumin in making beverages.

Fig. 7.

The Fe²⁺-chelating activity (%) of beverages.

OVA = Ovalbumin-based beverage, PePr = Ovalbumin beverage hydrolyzed by Pepsin and Protease, PrTr = Ovalbumin beverage hydrolyzed with Protease and Trypsin. Values with different letters (a-b) differ significantly (P < 0.05).

Antimicrobial index of ovalbumin-based beverage and hydrolyzed drinks

The antimicrobial index of the hydrolyzed drinks relative to the control was calculated. The antimicrobial index of the PePr-hydrolyzed drink was 33.34±0.001%, and that of the PrTr was 27.77±7.86% at 2% (w/v) protein concentration. However, OVA did not show any antimicrobial activity up at 2% (w/v) concentration. A single enzyme treatment with pepsin (2.5 kU/g, pH 2, 37°C for 4 h) showed the highest antibacterial activity over other enzymes such as flavorzyme (5 kU/g, pH 7, 50°C), alcalase (2 kU/g, pH 9, 60°C), trypsin (2 kU/g, pH 7.5, 37°C), neutrase (1.5 kU/g, pH 7, 50°C), or papain (5 kU/g, pH 6.5, 50°C) where ovalbumin was hydrolyzed for 1, 2, 3, 4, and 5 h (Tang et al., 2013). Pellegrini et al. (2004) digested ovalbumin with trypsin and chymotrypsin and found five peptides (Ser-Ala-Leu-Ala-Met, Ser-Ala-Leu-Met-Val-Tyr, and Tyr-Pro-Ile-Leu-Pro-Glu-Tyr-Leu-Gln), and three peptides with chymotrypsin (Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu-Pro-Glu-Tyr-Leu, Gly-Ile-Ile-Arg-Asn, Tyr-Ser-Ser-Asn-Val-Met-Glu-Glu-Arg) with strong bactericidal properties against B. subtilis. The amino acid sequences of peptides have a strong impact on their functional properties, and the enzymes determine the peptides produced (Chang et al., 2018).

Variation of pH and titratable acidity of ovalbumin-based beverages and hydrolyzed drinks with time

Under refrigerated conditions, the pH changes of the beverages were measured to observe their shelf life. A significant difference in pH was identified in the beverages on the 10^th day (OVA; 9.94±0.02, PePr; 5.81±0.12, PrTr; 7.21±0.08 on Day 1 reduced to 9.44±0.07, 4.79±0.15, 6.86±0.15 on Day 10) onwards (p < 0.05) (Table 2). Significant pH variations in high-protein egg-based drinks are not common, but the whole egg-incorporated drink showed a reduction in pH on Day 10 (Silva and Abeyrathne, 2014). According to the USDA (2019), the storage time for liquid egg products in refrigerated condition (4°C) without an expiration date is seven days. Furthermore, odor and visual property changes were identified in the beverages on the 10^th day. Consistency changes were identified in all three beverages. Therefore, it can be considered that liquid egg-based products are perishable and can go for about seven days of maximum shelf-life when considering the standards and current study.

Table 2.

The pH changes of beverages with storage time.

DAY	OVA	PEPR	PRTR
1	9.94±0.02 a	5.82±0.12 q	7.21±0.08 y
3	9.97±0.04 a	5.76±0.05 q	7.42±0.05 y
8	9.86±0.06 a	5.61±0.08 q	7.51±0.04 x,y
10	9.44±0.07 b	4.79±0.15 r	6.86±0.15 z
14	9.49±0.03 b	4.87±0.04 r	6.67±0.10 z

OVA = Ovalbumin-based beverage, PEPR = Ovalbumin beverage hydrolyzed by Pepsin and Protease, PRTR = Ovalbumin beverage hydrolyzed with Protease and Trypsin.

^a-bValues,

^q-rValues, and

^x-zValues with different letters differ significantly (p < 0.05).

Values are means ±SE.

Although a significant difference (p < 0.05) in titratable acidity was identified between the two hydrolyzed drinks, no significant difference (p > 0.05) within a treatment was observed until Day 14 (PePr; 2.01±0.56% and PrTr; 0.79±0.11% in Day 1 changed to PePr; 1.7±0.11% and PrTr; 0.91±0.001% in Day 14) (Fig. 8). In a previous study, egg albumin was used to fortify a carbonated guava drink, and titratable acidity increased from 0.495% to 0.563% (Hemanth et al., 2019). In the current study, although there was no significant difference in acidity with storage, a reduction in pH, an increase in TSS, and alterations in viscosity were observed. A study on whey-based pineapple beverages also showed similar results where pH was reduced while acidity and TSS increased with storage (Kumar et al., 2017). According to the references and current results, the maximum shelf-life of the beverage can be seven days under refrigerated conditions (4°C).

Fig. 8.

The changes of titratable acidity in PePr and PrTr drinks during 14 days of storage. PePr = Ovalbumin beverage hydrolyzed by Pepsin and Protease, PrTr = Ovalbumin beverage hydrolyzed with Protease and Trypsin.

Values with letters A-B indicate a significant difference (P < 0.05) between treatments within a storage day. Letters a-b and x-y show the significant difference during storage within a treatment.

Although titratable acidity did not significantly change during the experiment, a reduction in pH, an increase in TSS, and alterations in viscosity were observed. A study on whey-based pineapple beverages also showed similar results where pH was reduced while acidity and TSS increased with storage (Kumar et al., 2017). According to the USDA (2019), the storage time for liquid egg products in refrigerated conditions (4°C) without an expiration date is seven days. Furthermore, odor and visual property changes were identified in the beverages on the 10^th day. Consistency changes were identified in all three beverages. Therefore, it can be considered that liquid egg-based products are perishable and can go for a maximum of about seven days of shelf-life.

Conclusions

The current study was developed to explore the possibility of utilizing lyophilized ovalbumin (a protein extracted from egg white) to produce a simple beverage and to investigate the potential of improving such a developed product by enzymatic hydrolysis. The beverage with native protein resulted dairy like appearance, which was affected during the enzyme hydrolysis. However, enzymatic hydrolysis resulted in higher solubility of the protein and the incorporation of other additional benefits such as antioxidant, antimicrobial, and metal-chelating activities. In conclusion, it was identified that ovalbumin and its hydrolysates could be utilized in beverage production with a shelf life of 7 days under refrigerated conditions (4°C) and can benefit a wider community. However, several areas were identified that require further improvement, which includes enhancing the texture of the hydrolyzed products. Therefore, future research studies will focus on adding value to the beverages to get better organoleptic qualities while conducting sensory evaluations and shelf-life studies related to the microbial population.

Declaration of competing interest

The authors have no conflict of interest to declare.