Effects of Whey Protein, Aloe Vera, or Carnauba Wax Biofilms on the Internal Quality of Eggs from 86-Week-Old Hens

Abstract

Egg quality loss during storage is a major concern in the poultry industry, particularly for eggs from older hens, which are more susceptible to shell thinning, albumen liquefaction, and yolk weakening. This study evaluated the effect of whey protein, Aloe vera, and carnauba-wax coatings on the internal quality of eggs from 86-week-old laying hens stored at room temperature for 21 days. The experimental design consisted of four treatments (uncoated control and three coatings) and four storage times (0, 7, 14, and 21 days). Internal quality was assessed by Haugh unit (HU), yolk index (YI), albumen pH, and yolk color parameters (L*, a*, b*). The results showed that storage time significantly affected all internal quality traits (p < 0.05). Whey protein coating consistently maintained higher HU and YI values and lower albumen pH compared with the control, indicating better preservation of albumen viscosity and CO₂ retention. Aloe vera and carnauba-wax coatings had only transient effects, with values similar to the control after 14 days. Yolk color stability also declined over time, with minor protection observed only for the whey protein treatment. In conclusion, whey protein coating provided the best overall preservation of egg internal quality during storage, demonstrating superior gas barrier properties and structural stability. These findings suggest that protein-based coatings may be an effective strategy to extend the shelf life of eggs from aged laying hens.

1. Introduction

Egg quality is a key attribute for both the poultry industry and consumers, encompassing external traits such as weight and shell thickness and internal traits such as albumen height, Haugh units, yolk color, and yolk index. These parameters are influenced by genetics, nutrition, rearing conditions, storage time and temperature, and the age of the laying hens. As hens age, their eggs exhibit decreased shell quality, reduced albumen viscosity, and altered component proportions, which compromise both table use and industrial applications [1,2].

Age-related physiological changes have been extensively documented. Modifications in progesterone receptor expression and in the morphology of the magnum can impair proper albumen secretion [3,4]. In parallel, alterations in the concentration and activity of albumen antimicrobial proteins occur with hen age; for example, lysozyme concentration and enzymatic activity often increase, but these changes may interact with structural deterioration of albumen to affect resistance to contamination and storage stability [5,6,7].

Despite these limitations, flocks older than 80 weeks remain in production due to the economic importance of extending laying cycles, and their eggs are still used for both table and industrial purposes [8]. Therefore, strategies that extend shelf life and maintain egg quality are increasingly important, especially for eggs from aged hens, which are more fragile and prone to deterioration.

Coating the eggshell surface is one of the oldest methods used to reduce quality losses. Mineral oil has been widely applied since the early 20th century [9]; however, as a petroleum-derived product, its acceptance has declined among consumers seeking sustainable and residue-free alternatives. Natural edible coatings and biofilms therefore represent a promising and environmentally friendly option, acting as physical and selective barriers that reduce moisture loss and delay gas exchange [10].

Among natural coatings, carnauba wax, Aloe vera, and whey protein stand out because they represent distinct functional categories lipidic, polysaccharide, and protein-based matrices, respectively. Carnauba wax forms a hydrophobic barrier that limits water vapor transfer [11,12]. Aloe vera contains polysaccharides and bioactive compounds with antioxidant and antimicrobial activity that may slow oxidative and microbial deterioration [13]. Whey protein, in turn, can form flexible, semipermeable films that reduce CO₂ diffusion and preserve albumen viscosity [14]. The combination of these three matrices in comparative evaluation allows the identification of which type of biopolymer lipidic, polysaccharide, or protein offers the best protection for eggs from aged hens under storage conditions.

Recent reviews have confirmed the potential of edible coatings with antimicrobial and antioxidant properties to improve egg shelf life [15,16], but information about their efficacy in eggs from older hens remains limited. The present study hypothesized that natural biofilms based on whey protein, Aloe vera, or carnauba wax could mitigate the quality deterioration typically observed in eggs from aged hens stored at room temperature. Therefore, this study aimed to compare the effectiveness of these coatings on the internal quality of eggs from 86-week-old hens during 21 days of storage.

2. Materials and Methods

2.1. Study Site and Experimental Conditions

The experiment was conducted in Jaboticabal, São Paulo, Brazil (21.2437° S, 48.2995° W) in November 2024. Eggs were obtained from 86-week-old Hisex White hens raised in a conventional cage system, and no procedures were performed on live animals for the purpose of this study.

2.2. Experimental Design

A completely randomized 4 × 4 factorial design was used, consisting of four coating treatments (control, whey protein, Aloe vera, and carnauba wax) and four storage periods (0, 7, 14, and 21 days). Each treatment–time combination was replicated 15 times, with one egg per replicate, totaling 240 eggs. Eggs analyzed on day 0 represented the baseline control.

The factorial design allows evaluation of the main effects of coating and storage period as well as their interaction (coating × time). Data were analyzed according to Equation (1):

$$Y_{ijk}=\mu + C_i+T_j+(C \times T{)}_{ij} + e_{ijk}$$

(1)

where $Y_{ijk}$ is the observed value, $\mu$ is the overall mean, $C_i$ is the effect of coating, $T_j$ is the effect of storage time, $(C \times T{)}_{ij}$ is the interaction, and $e_{ijk}$ is the experimental error.

2.3. Preparation of Biofilms

All reagents and materials were obtained from commercial suppliers. Whey protein concentrate (100% Whey Concentrate, Profit Labs^®, 840 g, Rio de Janeiro, Brazil) and carnauba wax (10% solution, composed of 60% wax and 40% vegetable resin; Cera Aruá BR18, Aruá Comércio e Serviços Ltd., São Paulo, Brazil) were formulated using glycerol (Glicerina USP bidestilada 100% pura, Quimivida^®, São Paulo, Brazil) as the plasticizer in both the whey-based and the carnauba-wax coatings.

The coating solutions were prepared according to the literature protocols, with adaptations for this study. The whey protein solution contained 8% whey protein and 4% glycerol [17]. Carnauba wax, which was commercially diluted in vegetable resin (60% carnauba wax and 40% resin), was used at 10% with 4% glycerol. Aloe vera gel was used in its natural form without dilution or heating.

Glycerol was added as a plasticizer to improve film flexibility and reduce brittleness, as hydrophilic plasticizers are commonly incorporated into protein and lipid matrices [18]. Since wax-based coatings tend to form rigid but less homogeneous barriers [19], additional glycerol was included to increase flexibility while maintaining protective properties.

Solutions (with the exception of Aloe vera) were heated in a water bath using beakers placed in an aluminum pan over a tripod with asbestos mesh and a refractory disk to avoid direct flame contact. The whey protein mixture was maintained at 40–50 °C for 10 min, while the carnauba wax mixture was heated to 60–80 °C for 10 min. The temperature was monitored via a digital penetration thermometer (SA9791 PLUS M9; Incoterm^®, Porto Alegre, Brazil). After heating, the solutions were cooled and homogenized on a magnetic stirrer (Fisatom^®, São Paulo, Brazil). The pH was adjusted following the methods of Alleoni and Antunes [20] to ensure stability and uniformity.

2.4. Preparation of Aloe Vera Gel

Fresh Aloe vera leaves were purchased from a local market (Jaboticabal, São Paulo, Brazil). Leaves were washed under running water and longitudinally cut, and the mucilaginous parenchyma was manually extracted. The gel was homogenized in a blender until a uniform gel was obtained and used directly for egg coating.

2.5. Application of Coatings

Eggs were immersed in the coating solutions for 1 min in 500 mL beakers. The eggs were then placed on metal grids and air-dried for 1 h. After drying, the eggs were stored in cellulose pulp trays under room conditions, simulating common commercial practices. The temperature and relative humidity were monitored with a digital thermohygrometer throughout the experiment. The average maximum temperature was 31.3 ± 0.6 °C, with a minimum of 20.7 ± 0.6 °C, and the relative humidity ranged from 29.4 ± 10.0% (minimum) to 75.2 ± 10.2% (maximum).

2.6. Quality Assessment

On day 0, eggshell thickness was measured to characterize initial quality. Throughout storage, egg weight, albumen height, yolk diameter and height, albumen pH, and yolk color were determined.

Eggs were weighed on a precision balance (Bel Engineering^®, S2202H, Monza, Italy) and then broken onto a marble surface. The albumen height was measured via a tripod micrometer (AMES^®, model Egg Tester IV, Waltham, MA, USA). Yolks were separated, and their diameter and height were measured with a digital caliper (Mitutoyo, São Paulo, Brazil). Yolk color was evaluated via a Minolta CR-400 colorimeter (Konica Minolta Sensing, Inc., Osaka, Japan) in the CIELab system: L* (lightness), a* (redness), and b* (yellowness) with SpectraMagic NX software, version 2.7.

The albumen pH was measured via a handheld penetration pH meter (Testo 205), which was previously calibrated. The albumen and yolk were separated into labeled containers, and the pH was recorded individually.

The yolk index (YI) was calculated as Equation (2); the Haugh unit (HU) was calculated using the standard Equation [3]:

$$\mathrm{Y}\mathrm{I}={\displaystyle \frac{\mathrm{Y}\mathrm{o}\mathrm{l}\mathrm{k} \mathrm{h}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}}{\mathrm{Y}\mathrm{o}\mathrm{l}\mathrm{k} \mathrm{d}\mathrm{i}\mathrm{a}\mathrm{m}\mathrm{e}\mathrm{t}\mathrm{e}\mathrm{r}}}$$

(2)

$$\mathrm{H}\mathrm{U}=100\times \mathrm{l}\mathrm{o}\mathrm{g}(\mathrm{H}+7.57-1.7\times {\mathrm{W}}^{0.37})$$

(3)

where H is albumen height (mm) and W is egg weight (g).

2.7. Statistical Analysis

Data were subjected to analysis of variance (ANOVA) using SAS^® OnDemand for Academics, version 9.4 (SAS Institute Inc., Cary, NC, USA), and figures were created in Microsoft Excel, version 365 (Microsoft Corporation, Redmond, WA, USA). Means were compared via Tukey’s test at the 5% significance level. Polynomial orthogonal contrasts (linear and quadratic) were applied to evaluate storage time effects.

3. Results

The internal quality parameters of the eggs were significantly affected by both storage time and coating treatment (p < 0.05), with an interaction effect (time × coating) observed for most traits. The results are summarized in

Table 1. Internal quality of eggs from 86-week-old hens coated with whey protein, Aloe vera, or carnauba wax and stored at room temperature for 21 days.

Storage Times (Days)	Treatments	Haugh Unit (HU)	Yolk Index (YI)	Albumen pH	Yolk Color b*
0 (Fresh)		90	0.4	7.98	42.11
7	Control	76.90 aA	0.32 bA	8.87 aB	47.27 aB
	Whey	78.35 aA	0.35 abA	8.33 bB	46.59 abA
	Wax	79.65 aA	0.37 aA	8.13 bB	44.40 abB
	Aloe vera	80.38 aA	0.37 aA	8.22 bB	42.82 bB
14	Control	60.35 bB	0.28 bB	9.31 aA	51.95 aA
	Whey	70.81 aA	0.32 aAB	8.39 bB	47.63 bA
	Wax	62.42 abB	0.29 abB	9.17 bA	50.59 abA
	Aloe vera	62.53 abB	0.30 abB	9.06 bA	49.54 abA
21	Control	44.38 bC	0.20 bC	9.33 aA	54.91 aA
	Whey	70.46 aA	0.31 aB	8.97 aA	48.62 bA
	Wax	44.71 bC	0.20 bC	9.23 aA	54.17 aA
	Aloe vera	49.08 bC	0.20 bC	9.09 aA	49.97 bA
p value
	Storage (S)	<0.001	<0.001	<0.001	<0.001
	Coating (C)	<0.001	<0.001	<0.001	<0.001
	S × C	0.014	<0.001	0.014	0.014
	Linear (L)	<0.001	<0.001	<0.001	<0.001
	Quadratic (Q)	<0.001	<0.001	<0.001	<0.001
	SEM	2.38	0.01	0.11	1.09

SEM = standard error of the mean. Means followed by the same uppercase letter in the row and the same lowercase letter in the column for the same variable do not differ according to the Tukey test (p < 0.05).

.

SEM = standard error of the mean. Means followed by the same uppercase letter in the row and the same lowercase letter in the column for the same variable do not differ according to the Tukey test (p < 0.05).

Fresh eggs had an average HU of 90.00. A significant decrease was observed with storage time (p < 0.001).

At 7 days, whey protein-coated eggs (78.35) and Aloe vera-coated eggs (80.38) maintained higher HU values than the control (76.90).

However, by day 14 and day 21, only whey protein maintained significantly higher HU values (70.81 and 70.46, respectively), whereas Aloe vera and carnauba wax showed similar values to the control (p > 0.05).

These results indicate that the protective effect of Aloe vera was transient, while whey protein coatings consistently preserved albumen height and viscosity throughout the 21 days (Figure 1).

The YI followed a similar trend to that of HU. Fresh eggs averaged 0.40, which declined over time (p < 0.001).

Whey protein coatings maintained the highest YI values at 14 and 21 days (0.32 and 0.31), whereas the other treatments declined to 0.20–0.29. Thus, whey protein delayed vitelline membrane weakening, while Aloe vera and carnauba wax were ineffective after 14 days. (Table 1).

Albumen pH increased progressively with storage (p < 0.001). Whey protein and Aloe vera coatings initially limited the increase (8.33 and 8.22 vs. 8.87 for control at day 7).

However, after 14 and 21 days, the Aloe vera treatment showed pH values close to the control, whereas whey protein remained significantly lower (8.39–8.97).

This confirms the superior barrier properties of protein-based coatings in reducing CO₂ loss and delaying albumen alkalinization (Figure 2).

Lightness (L*) and redness (a*) were not significantly affected by storage or coating (p > 0.05). However, yellowness (b*) increased in all groups (p < 0.05), reflecting pigment oxidation during storage.

At 21 days, control and wax-coated eggs exhibited the highest b* values (54.9 and 54.2), whereas whey protein and Aloe vera treatments had smaller increases (48.6 and 50.0).

Overall, the application of natural biofilms, particularly those based on whey protein and Aloe vera, effectively preserved albumen viscosity and yolk structure, as evidenced by higher HU and YI values and lower albumen pH during storage. These results confirm the potential of protein and polysaccharide-based coatings to extend the shelf-life of eggs from aged hens under room temperature conditions.

4. Discussion

The internal quality of eggs declined progressively during storage, as expected due to the migration of CO₂ and moisture through the shell pores and the consequent degradation of albumen proteins [8,21]. These alterations are associated with the breakdown of the carbonic acid equilibrium and the weakening of the thick albumen structure [22]. However, the magnitude of such changes varied according to the coating applied. Among the tested biofilms, whey protein coating showed the most consistent protective effect on albumen quality, while Aloe vera and carnauba-wax coatings presented similar results to the uncoated control after 14 days of storage.

The preservation of Haugh unit (HU) values in whey protein-coated eggs throughout the 21-day period indicates that this coating provided an effective physical barrier against gas exchange and water loss [14,23]. Protein-based coatings form cohesive and dense matrices due to intermolecular interactions, which reduce permeability to oxygen and carbon dioxide [15,20]. This barrier delays CO₂ diffusion from the albumen, maintaining internal viscosity and slowing albumen thinning [10]. Consequently, the slower decrease in HU values observed in whey protein-coated eggs agrees with the reduced rate of quality loss reported for protein films [10].

In contrast, the Aloe vera and carnauba-wax coatings exhibited only transient effects on HU and YI during the first week of storage, with no significant differences compared with the control at 14 and 21 days. Although Aloe vera gel contains polysaccharides and organic acids that can temporarily reduce gas permeability [13], the integrity of this coating may decrease during prolonged storage due to dehydration and shrinkage [24]. Similarly, the hydrophobic carnauba-wax layer forms a less flexible film that can develop microcracks during drying or handling, facilitating gas diffusion [11]. These structural limitations likely explain the limited efficacy of both coatings after the initial storage period.

Changes in albumen pH followed a pattern consistent with gas exchange dynamics. The pH increased with storage time in all treatments, but the lowest values were maintained in eggs coated with whey protein, indicating reduced CO₂ loss [10,14,23]. The maintenance of lower albumen pH is a reliable indicator of better internal preservation, as alkalinization leads to the unfolding of albumen proteins, reduced viscosity, and increased susceptibility to microbial growth [16]. In the present study, the pH of Aloe vera-coated eggs did not remain stable over time and became comparable to the control after 14 days, confirming that its barrier effect was not sustained throughout storage [13]. These findings demonstrate that only the whey protein coating effectively limited both the increase in albumen pH and the decrease in HU and YI, maintaining higher internal quality compared with uncoated eggs and those treated with Aloe vera or carnauba wax.

Yolk color parameters were also affected by storage time, mainly for the b* coordinate (yellowness), which increased progressively in all treatments [18]. This increase is generally associated with pigment oxidation and water migration from the albumen to the yolk, leading to concentration of carotenoids [19]. In the present study, the increase in b* was slightly lower in eggs coated with whey protein, suggesting a moderate protective effect against pigment oxidation. Conversely, Aloe vera and carnauba-wax coatings showed color changes similar to those observed in uncoated eggs. These results differ from the initial assumption that Aloe vera would stabilize yolk color, as no significant difference was maintained after 14 days of storage. Therefore, any protective effect of Aloe vera on yolk color was temporary and probably related to moisture retention rather than to a stable barrier effect [13].

Lightness (L*) and redness (a*) values were not significantly influenced by coating type or storage time [18]. The stability of these parameters indicates that lipid oxidation, although initiated, did not progress enough to cause perceptible discoloration during 21 days of storage. The slower increase in b* values for whey protein-coated eggs may be associated with the lower pH and reduced oxygen diffusion promoted by the protein matrix, delaying oxidative reactions in yolk lipids [18].

Shell thickness (0.36 ± 0.02 mm) was measured only at the beginning of the experiment to characterize the eggs used. This value is slightly lower than that reported for younger layers (0.38–0.44 mm) and reflects the natural thinning of eggshells with advancing hen age [25]. Thinner shells have higher gas permeability and thus accelerate internal quality loss [26]. Therefore, the application of coatings is particularly relevant in eggs from aged hens, as the external film can partially compensate for the reduced natural barrier of the shell. This helps explain the greater relative benefit observed for the whey protein coating, which effectively maintained albumen quality despite the intrinsic fragility of eggs from older hens.

The recent literature further supports our findings by demonstrating that bio-based edible coatings composed of different biopolymer matrices can significantly preserve the internal quality of table eggs during storage. Protein-based films, particularly those formulated with whey or casein, have been reported to provide superior gas-barrier properties compared with polysaccharide- or lipid-based coatings, thereby maintaining higher Haugh unit (HU) and yolk index (YI) values for longer periods under room-temperature conditions [15,27]. In contrast, lipid matrices such as waxes mainly reduce water-vapor transmission but show a more modest effect on gas permeability and CO₂ retention [28]. This behavior is consistent with our observations, where the whey protein coating limited albumen pH elevation and HU decline more effectively than the carnauba-wax coating. Polysaccharide-based coatings such as Aloe vera gels have shown variable or transient protective effects, maintaining egg quality only in the early stages of storage [27,29]. Other studies combining protein and polysaccharide matrices (e.g., casein–chitosan blends) reinforced the role of the protein phase in preserving HU and YI, while the polysaccharide or lipid components mainly contributed to moisture control [27,29]. The enhanced performance of whey protein films observed in the present study is therefore coherent with previous reports demonstrating that the semipermeable nature of protein networks slows gas diffusion and moisture loss, extending the internal quality of eggs during ambient storage [30].

Overall, the comparative performance of coatings demonstrates that film composition plays a decisive role in maintaining egg quality. Protein-based films create cohesive matrices with strong intermolecular interactions that reduce porosity and gas permeability [20]. In contrast, polysaccharide- and wax-based coatings may undergo cracking or shrinkage during storage, losing their barrier function over time [19]. These differences confirm that the effectiveness of a coating depends not only on its initial physical properties but also on its mechanical stability throughout storage. Future studies should focus on combining biopolymers or adding plasticizers and crosslinking agents to improve the long-term integrity of Aloe vera and wax-based coatings.

5. Conclusions

Storage time had a marked impact on the internal quality of eggs from 86-week-old hens, leading to progressive reductions in Haugh unit and yolk index values and an increase in albumen pH. Among the tested coatings, whey protein provided the most effective preservation of internal quality throughout 21 days of storage at room temperature. Its protein matrix formed a dense and cohesive film that reduced gas exchange and moisture loss, thereby maintaining albumen viscosity, lower pH, and better yolk firmness.

In contrast, Aloe vera and carnauba-wax coatings exhibited only temporary or limited effects, showing quality losses comparable to the uncoated control after 14 days. Yolk color stability followed the same pattern, with minor improvement observed only in whey protein-coated eggs.

These findings confirm that protein-based coatings offer superior barrier properties and mechanical stability compared with polysaccharide- or wax-based films. Considering the thinner eggshells typical of aged hens, applying whey protein coatings can be an effective strategy to extend egg shelf life and reduce post-production losses. Future studies should investigate polymer blends or crosslinking agents to improve the durability of Aloe vera and wax-based coatings.