Coating Quail Eggs with a Bioactive Solution of Corn Starch and Green Propolis Extract

Abstract

Quail eggs are foods that can suffer significant losses in internal quality 14 days after laying and onwards. With the aim of minimizing early quality losses, this study investigated the effect of the combined use of corn starch and green propolis extract (CS+GPE) on the internal quality attributes of quail eggs, as well as on the bacterial level of their eggshells during storage at room temperature. The Haugh unit remained above 80.00 in eggs coated with CS+GPE at 21 days of storage, which was significantly greater than that of the eggs from the control treatment. On that same day, the bacterial load in the eggshell of the eggs coated with CS+GPE was 34.74% lower than that in eggs from the control treatment. The synergistic use of CS+GPE represents a strategy for preserving quail eggs using bioactive, green, and biodegradable materials.

1. Introduction

Food research centers focus on improving the shelf life of quail eggs. The development and widespread testing of food coatings, including those intended for quail eggs, have been suggested as a strategy to mitigate the challenges associated with post-collection handling, particularly during the storage phase [1,2,3]. This approach has directly benefited the shelf life of quail eggs by controlling adverse reactions such as the rapid loss of albumen quality, which leads to internal quality deterioration, and reducing the levels of bacterial contamination [4].

Corn starch (CS) is a raw material used in coating formulations that can enhance the water vapor permeability and oxygen barrier properties of quail eggs [5]. These properties can be further enhanced by the inclusion of bioactive compounds in the formulation, which also provide the eggs with antibacterial and antifungal barriers [5]. Propolis is a natural source with antibacterial and antifungal properties produced by bees from plants [6,7,8,9], which helps prevent undesirable physical and chemical changes in food, contributing to its preservation [10]. Its value is even greater when combined with coatings [10]. The ability of propolis to reduce the bacterial load on eggshells has been proven through eggshell-washing tests. Aygun et al. [11] demonstrated that the application of a 10% alcoholic extract of propolis significantly reduced the microbial load on the eggshells of quail eggs, covering different bacteria such as total aerobic mesophilic bacteria, coliforms, Salmonella spp., and Staphylococcus spp. Therefore, this study aims to evaluate the use of a corn-starch-based coating loaded with green propolis extract (CS5+GPE) for the preservation of quail eggs.

2. Materials and Methods

CS (Maizena, São Paulo, Brazil), GPE (Apis Flora, São Paulo, Brazil; green propolis obtained from the Brazilian native plant Baccharis dracunculifolia), and glycerol (Dinâmica, São Paulo, Brazil) were purchased from commercial suppliers. The CS-based coating solutions were prepared using a magnetic stirrer for 40 min, following the preparation methodology described by de Araújo et al. [5]. The final compositions of the obtained coatings are described in

Table 1. Composition of coatings for quail eggs.

Coating	Components
CS	5 g of corn starch 1.5 mL of glycerol 100 mL of distilled water
GPE	11 g of green propolis dry extract 100 mL of hydroalcoholic solution
CS+GPE	5 g of corn starch 1.5 mL of glycerol 10 mL of 11% green propolis extract 100 mL of distilled water

Abbreviations: CS, corn starch; GPE, green propolis extract; CS+GPE, corn starch + green propolis extract.

. The treatments were the control (no coating), CS, GPE, and CS+GPE.

Fresh quail eggs were distributed equally in each treatment (Table 1). While the control treatment eggs were not coated, the eggs in the other treatments were immersed in the coating solution for 45 s and naturally dried at room temperature. The samples were subsequently placed in plastic trays specifically designed for quail eggs. The trays were stored in the laboratory for 21 days at a temperature of 27.00 ± 2.00 °C and relative humidity of 48.00 ± 6.00%.

The internal quality of quail eggs was evaluated through weekly measurements of egg weight (EW), albumen height (AH), yolk diameter (YD), and height (YH) on a precision scale (Gehaka, São Paulo, Brazil) and a digital caliper (Mitutoyo, São Paulo, Brazil). The albumen pH (ApH) was determined using a calibrated pH meter (206–pH2, Testo, Lenzkirch, Baden-Württemberg, Germany), with measurements taken from seven eggs per treatment. The initial assessment (day 0) of internal quality was evaluated using 15 quail eggs. Different formulae were used to calculate the egg weight loss (EWL%), Haugh unit (HU), and yolk index (YI) values as follows: (1) EWL = (Initial EW − Final EW)/Initial EW × 100; (2) HU = 100 log (AH + 7.57 − 1.7 EW^0.37) [12]; and (3) YI = YH/YD [13]. In addition to the initial analysis of internal egg quality, the eggs from all treatments evaluated during the subsequent weeks were individually weighed on the first day. Later, these same eggs were reweighed during the respective week of the internal quality assessment in order to determine weight loss over time. For the calculation of the HU, the eggs were first weighed, then broken open, and the AH was measured. Regarding the YI, after measuring the albumen, a yolk separator was used to isolate the yolk, followed by the measurement of its diameter and height. All values obtained from these measurements were applied to their respective formulas to calculate the quality parameters. The analyses were conducted on a marble countertop equipped with a glass structure.

A total of 12 eggs were subjected to mesophilic bacteria count (MBC) analysis on the eggshell, with the aim of determining the bacterial load on day 0. For this purpose, the eggs were washed in sterile bags (Labplas, Sainte-Julie, QC, Canada) containing 20 mL of 0.1% peptone saline solution (PSS, Laborclin, Paraná, Brazil) (one egg per bag) for one minute. On day 21, six eggs from each treatment were subjected to the same procedure and were subsequently washed in sterile bags containing 40 mL of 0.1% PSS (two eggs per bag). On both days, the washing solutions were serially diluted, and 0.1 mL was plated onto a plate count agar (Laborclin, Paraná, Brazil). The plates were incubated at 36 °C for 48 h. The colonies were counted, and the results were expressed as log₁₀ CFU/mL.

The experiment was conducted in a completely randomized 2 × 2 design. Data analysis was performed using methods such as analysis of variance, testing principal treatments, and their interactions. The comparison of means was performed using SAS Studio 9.4 University Edition software (SAS Inst. Inc., Cary, NC, USA). Statistical significance was determined using Tukey’s test with a significance level of p < 0.05.

3. Results and Discussion

Coatings are an affordable modality for preserving quail eggs [14,15,16]. Polysaccharides are among the most commonly used natural biopolymers as key components in the formulation of sustainable food coatings [17,18,19,20,21]. Among the polysaccharides available for egg coating formulation, methylcellulose, pectin, chitosan, and starch have been investigated either individually or in combination with other compounds and are recommended for preserving the internal quality of quail and hen eggs [22,23,24,25]. To our knowledge, this is the first study to investigate the combination of CS+GPE as a coating for quail eggs.

An effect of the interaction between coating type and storage day was observed on EWL. Eggs coated with CS+GPE exhibited a weight loss rate of 4.68 ± 0.89% after 21 days of storage (

Table 2. Values obtained after evaluating the internal quality of coated or uncoated quail eggs.

Treatment	EWL (%)
	0 weeks	1 weeks	2 weeks	3 weeks
Control	0.00 ± 0.00 aA	2.20 ± 0.43 aB	5.31 ± 1.04 aC	7.76 ± 2.66 aD
CS	0.00 ± 0.00 aA	2.13 ± 0.52 aB	3.70 ± 0.46 aB	6.87 ± 1.02 abC
GPE	0.00 ± 0.00 aA	1.58 ± 0.19 aAB	3.35 ± 0.93 aB	7.18 ± 4.32 aC
CS+GPE	0.00 ± 0.00 aA	1.50 ± 0.37 aAB	2.98 ± 0.47 aBC	4.68 ± 0.89 bC
p value
Treatment	<0.0001
Day	<0.0001
Treatment x day	0.0038
Treatment	HU
	0 weeks	1 weeks	2 weeks	3 weeks
Control	89.56 ± 2.63 aA	84.02 ± 5.31 aB	76.98 ± 2.97 aC	74.13 ± 5.29 bC
CS	89.56 ± 2.63 aA	84.55 ± 3.24 aAB	80.72 ± 3.92 aBC	78.06 ± 2.64 abC
GPE	89.56 ± 2.63 aA	83.08 ± 2.63 aB	78.68 ± 4.10 aBC	76.65 ± 2.47 abC
CS+GPE	89.56 ± 2.63 aA	85.92 ± 4.77 aAB	82.52 ± 3.51 aB	80.28 ± 1.03 aB
p value
Treatment	0.0003
Day	<0.0001
Treatment x day	0.1449
Treatment	YI
	0 weeks	1 weeks	2 weeks	3 weeks
Control	0.34 ± 0.03 aA	0.23± 0.04 aB	0.15 ± 0.03 bC	0.14 ± 0.02 bC
CS	0.34 ± 0.03 aA	0.23± 0.02 aB	0.15 ± 0.01 bC	0.15 ± 0.03 bC
GPE	0.34 ± 0.03 aA	0.22± 0.04 aB	0.16 ± 0.01 bC	0.15 ± 0.01 bC
CS+GPE	0.34 ± 0.03 aA	0.27 ± 0.04 aB	0.22 ± 0.03 aBC	0.20 ± 0.02 aC
p value
Treatment	<0.0001
Day	<0.0001
Treatment x day	0.0014
Treatment	ApH
	0 weeks	1 weeks	2 weeks	3 weeks
Control	8.94 ± 0.04 aA	9.37 ± 0.06 aB	9.52 ± 0.03 aBC	9.69 ± 0.21 aC
CS	8.94 ± 0.04 aA	9.26 ± 0.27 aB	9.35 ± 0.08 aB	9.51 ± 0.22 abB
GPE	8.94 ± 0.04 aA	9.34 ± 0.10 aB	9.37 ± 0.20 aB	9.45 ± 0.13 abB
CS+ GPE	8.94 ± 0.04 aA	9.28 ± 0.04 aB	9.27 ± 0.14 aB	9.28 ± 0.14 bB
p value
Treatment	<0.0001
Day	<0.0001
Treatment x day	0.0175

^{A–C; a,b} Different uppercase (row) or lowercase (column) letters indicate significant differences among means (p < 0.05). Abbreviations: CS, corn starch; GPE, green propolis extract; CS+GPE, corn starch + green propolis extract; EWL, egg weight loss; HU, Haugh unit; YI, yolk index, ApH, albumen pH.

), which was 39.69% lower than that observed in the uncoated control group. This reduction can possibly be attributed to the sealing properties of the coating, which effectively control the uncontrolled or premature passage of water and gas (CO₂). This behavior aligns with the findings of Pires et al. [26], who, by coating eggs with rice protein and propolis and storing them for 42 days at 20 °C, reported similar results.

The HU started at 89.56 ± 2.63, and on the 21st day of storage, a value of 74.13 ± 5.29 was observed for eggs from the control treatment; a value of 78.06 ± 2.64 was observed for eggs coated with CS; a value of 76.65 ± 2.47 was observed for eggs coated with GPE; and a value of 80.28 ± 1.03 was observed for eggs coated with CS+GPE (Table 2). The HU remained above 80.00 only for the eggs coated with CS+GPE, which was significantly greater than that of the eggs from the control treatment (p  < 0.05). When CS and GPE were applied separately, there was no significant difference (p  > 0.05) in HU in relation to eggs from the control treatment. The association between hydrophilic and hydrophobic substances in coatings makes the role of coatings even more efficient [27], as in the case of the CS5+GPE, which may have minimized protein alterations that could lead to earlier albumen liquefaction [28]. For example, Akpinar et al. [29] demonstrated that eggs coated with a 10/15% ethanolic propolis solution exhibited a significantly higher albumen index compared to uncoated eggs. In this study, all eggs were considered to be of excellent quality from the beginning to the end of storage [30].

An effect of the interaction between the coating type and storage day was observed on the YI. After 21 days of storage, eggs coated with CS+GPE (0.20 ± 0.02) presented a significantly higher yolk index (p < 0.05) compared to the other treatments (0.15 ± 0.02) (Table 2), demonstrating their potential ability to control issues related to elasticity, fragility, and vitelline membrane rupture, which compromise yolk quality [31]. CS and GPE applied alone were also unable to minimize the loss of yolk quality observed in the control treatment.

An effect of the interaction between coating type and storage day was observed on the ApH. The quail eggs exhibited an increase (p < 0.05) in ApH, ranging from 9.28 ± 0.14 to 9.69 ± 0.21 over the 21-day storage period (Table 2). On the last day, eggs coated with CS+GPE presented a significantly lower pH (p < 0.05) compared to those in the control group. Similarly, on the 42nd day of storage at 20 °C, a reduction in ApH was observed in eggs coated with rice protein plus green propolis [32]. These results suggest that coatings containing propolis can help preserve albumen quality by controlling carbonic acid dissociation and CO₂ release [33].

An effect of the interaction between the coating type and storage day was observed on the MBC. The MBC of the quail eggshells increased (p < 0.05) between days 0 and 21 of storage only in the control treatment and in eggs coated with CS (

Table 3. Values obtained after the bacterial counting of coated or uncoated eggshells.

Treatment	MBC
	Before Treatment (Day 0)	After Treatment (Day 21)
Control	2.81 ± 0.21 aB	4.03 ± 0.16 aA
CS	2.81 ± 0.21 aB	3.98 ± 0.23 aA
GPE	2.81 ± 0.21 aA	2.16 ± 0.62 bB
CS+GPE	2.81 ± 0.21 aA	2.63 ± 0.26 bA
p value
Treatment	<0.0001
Day	<0.0001
Treatment x day	<0.0001

^{A,B; a,b} Different uppercase (row) or lowercase (column) letters indicate significant differences among means (p < 0.05). Abbreviations: CS, corn starch; GPE, green propolis extract; CS+GPE, corn starch + green propolis extract; MBC, mesophilic bacteria count.

). On the other hand, eggs coated with GPE presented a significant reduction in colony count (p < 0.05), whereas those treated with CS+GPE maintained a stable bacterial load with no significant changes (p > 0.05). It is evident that the bacterial load on the eggshell surface tends to increase progressively during storage at room temperature. In this study, the increase in MBCs on the eggshell surface without the use of a coating over the 21-day period was 43.42%. However, GPE exhibited a significant residual antibacterial effect, effectively limiting bacterial growth over time. In contrast, the CS-based coating alone did not show any antibacterial activity (an increase of 41.64% in MBCs was observed from day 0 to day 21). Nonetheless, the addition of GPE led to the stabilization of MBCs, indicating that its incorporation conferred effective antibacterial activity to the CS-based coating. Reis et al. [34] reported that the GPE at 21% showed, in vitro, inhibition halos against Bacillus subtilis, Salmonella Typhimurium, Staphylococcus aureus, Escherichia coli, and Shigella spp. In vivo, Vilela et al. [35] demonstrated that hens’ eggs sanitized with 2400 µg, 240 µg, or 24 µg/mL of GPE at 24% presented significantly lower MBCs compared to untreated eggs. These findings support and reinforce the results observed in the present study, highlighting the antimicrobial effectiveness of GPE under different experimental conditions. On the 21st day of storage, quail eggs coated with GPE, as well as those with the CS+GPE, exhibited significantly lower MBCs (p < 0.05) than those of the other treatments. For eggs coated with GPE, the reduction was 46.40%, whereas for those coated with CS+GPE, the reduction amounted to 34.74%. Once again, these results demonstrate that coatings involving either the isolated or combined use of propolis inhibited bacterial growth. This is because propolis can disrupt the cell wall and bacterial membrane, causing the leakage of nucleic acids essential for bacterial survival [36]. Ezazi et al. [37] reported that eggs coated with chitosan and propolis did not exhibit Salmonella Enteritidis on the shell after 14 days of storage at room temperature.

4. Conclusions

The combined application of CS+GPE effectively maintained the internal quality of quail eggs during storage at room temperature and reduced the MBCs of the eggshells. The use of these compounds alone did not yield the same benefits for the internal quality of the eggs. In general, the isolated application of GPE did not show significantly negative effects on the internal quality of the eggs or on the bacterial control of the shell, nor did the isolated use of CS show negative effects in terms of internal quality. However, more satisfactory results were observed when the compounds were combined, possibly due to the interaction between the hydrophilic characteristics of CS and the hydrophobic properties of GPE, which may have favored the formation of a coating with improved mechanical and barrier properties. Additionally, the presence of GPE conferred antibacterial properties to the coating, which is attributed to its broad-spectrum activity against various bacteria.