Application of pectin-alginate and pectin-alginate-laurolyl arginate ethyl coatings to eliminate Salmonella enteritidis cross contamination in egg shells

Department of Life Sciences, University of Modena and Reggio Emilia, Reggio Emilia, Italy Interdepartmental Research Centre BIOGEST-SITEIA, University of Modena and Reggio Emilia, Reggio Emilia, Italy Correspondence Andrea Pulvirenti, Department of Life Sciences, University of Modena and Reggio Emilia, Via Amendola 2, 42122 Reggio Emilia, Italy. Email: andrea.pulvirenti@unimore.it Funding information Academic Found for Research of the University of Modena and Reggio Emilia, Grant/Award Number: FAR 2015; PROT.N. 81705 Abstract This study highlights the potential application of pectin-alginate blend (PA) and pectin-alginateLAE blend (PAL) coatings to eliminate Salmonella enteriditis 10,118 cross-contamination without changing the shelf-life of fresh eggs and their physico-chemical properties during storage at 7 C for 42 days. Egg shells were dipped in a solution of Salmonella enteritidis 10,118 with a concentration of 7 x 10 cfu/ml to assess Salmonella cross-contamination. PA and PAL coatings did not have a significant effect on shelf-life based on physico-chemical properties. The egg shells treated with PA and PAL coatings had a significantly lower microbial population compared to the uncoated egg shells. PA and PAL coatings effectively inhibited the growth of Salmonella after 1 and 7 days of storage, respectively. In addition, no outgrowth was observed up to 42 days.


| INTRODUCTION
Salmonella is one of the most common foodborne pathogens worldwide (Galiş et al., 2013;Howard, O'Bryan, Crandall, & Ricke, 2012;Whiley & Ross, 2015). Consumption of contaminated food products is one of the most prevalent sources of Salmonella infection (Hur, Kim, Choi, & Lee, 2013;Pande, Gole, McWhorter, Abraham, & Chousalkar, 2015). According to the European Food Safety Authority (EFSA) and the European Center for Disease Prevention and Control report, in 2015 a total of 94,625 salmonellosis cases were reported by 28 EU countries at a rate of 21.2 cases per 100,000 population.
Food from animal sources, especially poultry and its derivate products, including eggs, have been consistently involved in salmonellosis outbreaks (Braden, 2006;EFSA, 2016;Jin, Gurtler, & Li, 2013;Luber, 2009). Egg-related salmonellosis represents a major risk for consumers and consequently plays an important public health role (Gole et al., 2014;Patrick et al., 2004). Egg-related salmonellosis is mainly caused by the two most commonly reported Salmonella serovars: Salmonella enteritidis and Salmonella typhimurium (EFSA, 2015;Howard et al., 2012). These serotypes are regarded as unrestricted, which means that they can cause infections in animals as well as in humans (Galiş et al., 2013;Whiley & Ross, 2015). S. enteritidis outbreaks occur relatively often in EU countries. With eggs as the most common source. Salmonella typhimurium outbreaks are relatively common in Australia and New Zealand (Greig & Ravel, 2009).
Salmonella is a member of the gram-negative Enterobacteriaceae family (D'Aoust & Maurer, 2007;Galiş et al., 2013;White, Baker, & range of environmental conditions combined with a wide range of hosts, which make it difficult to control (White et al., 1997). Foods with low water activity can only stop the reproduction of Salmonella but not its survival (Al-Moghazy, Boveri, & Pulvirenti, 2014). This organism is heat-sensitive and can be readily destroyed at pasteurization temperature (Wybo et al., 2004).
Eggs can be contaminated with Salmonella by two pathways: vertical and horizontal transmission. In vertical transmission, eggs are infected from the spoiled reproductive tissues of hens during their formation in the hen's ovary and oviduct. In horizontal transmission, eggs are exposed to a contaminated environment, that is, in the presence of feces on the egg shell after being laid by the hen. Due to the humidity of the egg shell, storage at room temperature and shell damage, bacteria may be transferred through the egg shell and membranes and thus negatively impact on the egg's content (Braden, 2006;De Reu, Grijspeerdt, Messens, & Heyndrickx, 2006;Gole et al., 2014;Howard et al., 2012;Whiley & Ross, 2015). Jin et al. (2013) found that more than 90% of the cases of foodborne salmonellosis due to S. enteritidis are caused by contaminated egg shells. The moment of breaking is another delicate practice where the bacteria can be transmitted from the shell to its content, thus causing potential direct or indirect cross-contamination to other foods (Botey-Saló, Anyogu, Varnam, & Sutherland, 2012;Luber, 2009).
Egg producers employ various procedures such as dry cleaning, washing with water, chilled storage, electrolyzed oxidizing water, ozone, ultrasound, microwaves, irradiation (from high-energy gamma rays, X-rays, and accelerated electrons), gas plasma, ultraviolet light, and pulsed light technology to decontaminate egg shells and diminish the risk of salmonellosis (Galiş et al., 2013;Howard et al., 2012;Upadhyaya et al., 2016;Whiley & Ross, 2015). However, these methods have not been accepted and implemented worldwide. Just to mention a few examples, egg washing is applied in the United States, Canada, Australia, and Japan, while in the EU, it is currently banned. This is because according to the EU's egg regulation it might compromisepartially or completely-the cuticle layers, which represent an effective and natural barrier against bacteria due to their antimicrobial properties. Thus, egg washing might encourage the transfer of harmful bacteria such as Salmonella from the outside to the inside of the egg. Furthermore, with these techniques eggs can no longer be classified as fresh (EFSA, 2015;Galiş et al., 2013).
As an alternative and innovative solution, the concept of edible films and coatings has received considerable attention in the egg industry. This is because of the advantages, including the capacity to improve the shelf-life of the egg, the preservation of the egg's internal quality, the minimization of weight loss, the reduction in breakage, and increase in shell strength. Edible films and coatings act as a semipermeable barrier against oxygen, carbon dioxide and moisture, thus reducing respiration, water loss, and oxidation rates. They can also be applied on the egg shell, thus acting as carriers of substances such as natural antimicrobial compounds aimed at preventing the growth of pathogenic bacteria such as Salmonella (Ali, Maqbool, Ramachan- Edible films and coatings can be derived from several sources, such as polysaccharides, proteins, and lipids (Caner, 2005). Generally, lipids are used to reduce water transmission, proteins provide mechanical stability, while polysaccharides are used to control oxygen and other gas transmissions. Among polysaccharides, pectin, and alginate have been reported as two of the main raw materials to obtain edible films and coatings because of their natural abundance, low cost, excellent film forming properties, and renewable components (Seol, Lim, Jang, Jo, & Lee, 2009;Valdés, Burgos, Jiménez, & Garrigós, 2015). The wide use of alginate and pectin in the food industry is enhanced by their lack of toxicity and allergenicity (Solak & Dyankova, 2014).
Pectin is used as an edible film and coating because of its gelling and thickening properties and its ability to retard lipid migration and moisture loss (Moalemiyan, Ramaswamy, & Maftoonazad, 2012). It is especially suitable for low moisture foods (Dhanapal, Rajamani, Kavitha, Yazhini, & Banu, 2012). Alginate is used for edible films and coatings because of its unique colloidal properties and ability to form strong gels (Rojas-grau et al., 2008). However, it exhibits poor moisture barrier properties because of its hydrophilic nature (Dhanapal et al., 2012).
In this study, a composite coating formulation was produced by blending pectin and alginate, to achieve a synergistic effect from the  (Guerreiro, Gago, Faleiro, Miguel, & Antunes, 2015).
Egg quality includes a number of phenomena related to the shell, albumin, and yolk, which can be subdivided into external and internal quality characteristics, such as moisture loss, albumin pH, yolk index, yolk color, egg shell color, and Haugh unit (HU; Caner & Cansiz, 2007;Morsy, Sharoba, Khalaf, El-Tanahy, & Cutter, 2015). Morsy et al. (2015) studied the effects of pullulan coatings on the microbiological qualities, physical properties, and freshness parameters of fresh eggs. Pullulan coatings were shown to minimize weight loss (<1.5%) and preserved the albumen and yolk quality of eggs for 3 weeks longer than noncoated eggs at 25 C. Upadhyaya et al. (2016) demonstrated that when phytochemicals are added to pectin and arabic-gum based coatings, they were effective in reducing S. enteritidis on egg shells. In addition, coating egg shells with chitosan preserves the internal quality and extending shelf-life of eggs and acts as a protective barrier against contamination from S. enteritidis (Hur et al., 2013).
The main objective of this study was to develop an edible egg coating based on a pectin-alginate blend (PA) with LAE as an antimicrobial compound to evaluate the effects of coating on the physicochemical properties of eggs during storage at 7 C for 42 days. The effectiveness of the coating against S. enteritidis cross-contamination was also examined.

| Experimental design
Conventional "AA" class, medium size eggs (53-63 g) were purchased from a local shop (Reggio Emilia, Italy). The eggs had been laid 8 days before being purchased. The eggs were individually marked and randomly assigned to each experiment. Two treatments were evaluated: a coating with PA and a coating with pectin-alginate-LAE blend (PAL).
Uncoated eggs were used as controls (C). The first experiment monitored the physical and chemical features and the microbiological charge of the eggs every 7 days for up to 42 days (1, 7, 14, 21, 28, 35, and 42;Caner & Yüceer, 2015). In the second experiment, the egg shells were inoculated with S. enteriditis 10,118 (Zooprophylactic Institute of Palermo) before the coating treatments (Jin et al., 2013). The microbial charge was examined on the same days as the other analysis. Nine eggs per treatment were analyzed three times at each storage interval during the 42 days of analysis.

| Coating formulation
Distilled water was used as the solvent for preparing the film solutions. The pectin-alginate coating (PA) was formulated as follows: pectin 15 g/L, sodium alginate 10 g/L, glycerol 6.75 ml/L, sodium bicarbonate 2 g/L. Sodium bicarbonate was used to neutralize the pH of the coating and to prevent negative effects on the calcium carbonate of the egg shells. The compounds were mixed with constant stirring (750 rpm) at 40 C until the polymers had been completely dissolved. The final coating solution was degassed under vacuum for 15 min. The same procedure was applied for the PAL, substituting glycerol with 7.5 ml/L of MIRENAT-G.

| Coating application
Each egg was uniformly sprayed with the coating formulation, and any excess coating solution was drained off. The eggs were then reticulated with anhydrous CaCl 2 solution (50 g/L). Each coated egg was picked up with beeswax-coated tweezers and placed on a petri dish with a diameter of 140 mm, covered with beeswax to avoid gel adhesion to the bottom of the petri dish. After drying at 25 C for 1 hr under ventilation (30 m/s), the eggs were stored for 1 day at 7 C before the analysis. Each egg was weighed before storage using a laboratory scale (BL 2002 XS BALANCE, China).

| Physical and chemical analysis of the eggs
On each day of the analysis (1, 7, 14, 21, 28, 35, and 42), the following parameters were measured.

| Moisture loss
The loss of water and the consequent weight loss were calculated by subtracting the final weight from the initial weight of the eggs divided by the initial weight for each day of the analysis. The percentage moisture loss was calculated by multiplying the moisture loss by 100.
A European 1,700 technical scale was used for this measurement (Gibertini, Novate Milanese, Milan, Italy).

| Shell and yolk color
The egg shell and yolk color were measured with a Minolta Chroma Meter Model CR-400 (Minolta Co., Ltd., Osaka, Japan). Three egg shells and yolks were analyzed in three different points and the measurements were averaged. The results were expressed as L value (Lightness), a value (redness), and b value (yellowness). ΔEab indicated the size of the color differences compared with the control and was calculated by the following equation (Caner, 2005):

| Haugh unit
The HU was measured before separating the yolk from the albumen.
A digital caliper (CDJAAB 15, Borletti, Antegnate, Bergamo, Italy) was used to measure the height of the albumen placed on a glass surface.
The height was the mean of three measurements in three different points of the albumen (Yüceer, Aday, & Caner, 2016). The HU was calculated with the following formula: where H is the height of the albumen (mm) and G is the weight of the eggs (g).

| Albumen pH
After the eggs had been broken, the albumen was separated from the yolk with a glass pipette (50 ml) and the small volumes of albumen were homogenized for 20 s in a blender. The pH of homogenized albumen was measured with a pH meter (VWR, pH110, Milan, Italy).

| Yolk index
The yolk index was measured after separating the yolk from the albumen, with a digital caliper (CDJAAB 15, Borletti, Antegnate, Bergamo, Italy) to estimate the height and width of the yolk placed on a glass surface. The percentage yolk index was calculated with the following formula: where H is the height (mm) and W is the width of the yolk (mm).

| Pore number and dimension
An optical microscope (CHK model, Olympus, Japan) with 32x magnification was used to determine the number and dimension of the pores of the egg shells. Before the analysis, the egg shells were treated with nitric acid using a modified protocol (Tyler, 1953

| Moisture loss
The weight loss of the control (C) and coated eggs (PA and PAL) during 42 days of storage at 7 C is shown in Figure 1. The weight of the eggs decreased until 35 days in C, PA, and PAL groups. The highest weight loss was obtained for the C after 35 days of storage with 1.67%. The weight loss continued to decrease until 42 days in PA and PAL with 1.6 and 1.5%, respectively. No significant differences were found among C, PA, and PAL eggs during 42 days of storage at 7 C. Kim, No, and Prinyawiwatkul (2008) reported no significant differences in weight loss among chitosan-coated eggs with different plasticizer types after 5 weeks of storage. Jin et al. (2013) reported that during storage at either 7 or 4 C eggs coated with chitosan lost approximately 4% of the moisture, while uncoated eggs lost approximately 6% of their weight, and all the coated eggs had significantly less weight loss than the uncoated eggs. The weight loss of the eggs during storage was caused by the evaporation of water and the loss of carbon dioxide from the albumen through the shells. This parameter can be used as an index for egg quality, and prevention of weight loss is important for maintaining egg quality (Caner, 2005;Jin et al., 2013). Differences in weight loss among studies may be due to the storage conditions, temperature, egg size, or shell porosity (Caner, 2005; Jo, Ahn, Liu, Kim, & Nam, 2011). This study showed that eggs coated with pectin-alginate did not negatively affect the evaporation process, and coated eggs showed similar trends as the controls after 42 days of storage.

| Shell color
The ΔE between the control color at Day 1 and all the treatments (control included) in the subsequent analysis times are shown in

| Haugh unit
The HU measures the egg protein quality and is often measured based on the height of the albumen and the egg weight. A fresh and good quality egg has a HU index of around 80 which decreases physiologically with the aging of the egg (Caner & Yüceer, 2015). The initial value of HU represents the main marker to evaluate the egg protein quality, and its expression provides an indication of the egg shelf-life as well as the storage conditions (Figueiredo et al., 2014). Changes in the HU of the C, PA, and PAL groups are shown in

| Albumen pH
Beside the moisture loss and HU, albumen pH can also be used as an indicator of egg quality (Caner & Yüceer, 2015;Kim, Daeschel, et al., 2008;Morsy et al., 2015;Nongtaodum et al., 2013). Changes in albumen pH of the C, PA, and PAL groups during 42 days of storage at 7 C are shown in Table 3. After 42 days of storage, the albumen pH of the control eggs increased from an initial value of 8.48-9.61, while those of the PA and PAL coated eggs increased from 8.55 and 8.58 to 10.02 and 9.93, respectively. No significant differences in pH values among C, PA, and PAL were observed throughout the 42 days of storage. Morsy et al. (2015) observed that the albumen pH of noncoated eggs increased after 5 weeks of storage at room temperature from an initial value of 8.02-8.48, while those of coated eggs with pullulan and pullulan containing nisin increased to 8.15 and 8.14, respectively. Caner and Yüceer (2015) reported that a protein-based coating using whey protein isolate (WPI), whey protein concentrate (WPC), zein, and shellac had a significant effect on albumen pH. The albumen pH for uncoated eggs ranged from initially 7.50-9.50 at the end of 5 weeks of storage at 24 C, while for coated eggs, albumen pH values reached 9.33 (WPC), 9.31 (WPI), 8.90 (Zein), and 8.83 (shellac).
During the shelf-life of eggs, CO 2 is released from the albumen to the external environment through the egg shell pores. This CO 2 loss increases the albumen pH during storage. The carbon dioxide loss from the breakdown of carbonic acid in albumen results in changes in the bicarbonate buffer system; which consequently causes an increase in the albumen pH (Biladeau & Keener, 2009;Nongtaodum et al., 2013;Yüceer et al., 2016). Integrated with other parameters such as HU, YI, the numbers and dimension of the pores, the pH value can be used to evaluate the shelf-life of eggs. Means AE SD of 3 measurements on 3 eggs. Different letters within a column indicate significant differences (p < .05). Means AE SD of 3 measurements on 3 eggs. Different letters within a column indicate significant differences (p < .05).
The results of this study indicated that coating eggs with PA and PAL blends did not influence the carbon dioxide release through the shell, thus providing evidence that the egg quality and consequently the egg shelf-life did not change after coating. Table 4 shows the ΔE calculated for each day of analysis between the treatments and the control. The table shows that a large variation is time dependent and ΔE increased with time in all the treatments, due to the aging of the eggs (Figueiredo et al., 2014). Conversely, the color variation was not significant among the different treatments. The values shown in Table 4 did not display a decreasing linear trend, and the differences among C, PA, and PAL were not statistically significant. A significant difference was evident at Day 42, due to the end of the shelf-life period, in line with the pH trend described in Section 3.4.

| Yolk index
The yolk index is an indicator of freshness, obtained by the measurements of the yolk height and width. A yolk index decrease indicates a gradual deterioration of the vitelline membrane and liquefaction of the yolk, caused by water diffusion from the albumen (Yüceer & Caner, 2014). Table 5 shows the changes in the yolk index of C, PA, and PAL during 42 days of storage at 7 C. The yolk index of the control eggs was lower than PA and PAL during storage. After 42 days of storage, the yolk index of the C decreased from 39.96 to 30.99%. The yolk index of PA and PAL decreased from 38.45% and 39.06% to 33.2% and 32.72%, respectively. No significant differences were observed among C, PA, and PAL. However, the yolk index value of coated eggs was higher than the control eggs. Nongtaodum et al. (2013)    Means AE SD of 3 measurements on 3 eggs. Different letters within a column indicate significant differences (p < .05). Means AE SD of 3 measurements on 3 eggs. Different letters within a column indicate significant differences (p < .05). 3.9 | Salmonella enteritidis challenge test

| Determination of mesophilic aerobic charge
As shown in Table 8, colonies on eggs from the C group were two orders of magnitude higher than PA and PAL. The S. enteritidis load decreased during 42 days of storage in the control and coated eggs in all the groups. After 42 days, 107 colonies were still found in the control group. In the C group, cross-contamination occurred for up to 42 days, when 107 colonies migrated from the egg shells to the Petri dishes. In the PA group, cross-contamination was blocked at Day 14.
Finally, with PAL from Day 7 no colonies were detected on the Petri dishes. Thus, PAL blocks cross-contamination earlier than the other two treatments.
It is, therefore, possible to confirm that the coating does not allow Salmonella cells to arrive from the eggs to the surfaces; in general, the coating blocks any cross-contamination. In the formulation with LAE, this blocking is enhanced due to the strong antimicrobial activity of LAE (Jin et al., 2013).

| CONCLUSIONS
The aim of this study was to develop an edible egg coating to protect against Salmonella cross-contamination without changing the shelf-life of the fresh egg and its properties. This work demonstrated that the coatings with the PA did not negatively affect the quality parameters or the shelf-life of the eggs. In Europe, any treatment related to the food safety of eggs (apart from brushing) is strictly forbidden, due to the potential damage to the shell structure that will affect all the other Means AE SD of 3 measurements on 3 eggs. Different letters within a column indicate significant differences (p < .05).
FIGURE 2 Development of total mesophilic aerobic bacteria population during the storage at 7 C for 42 days parameters. However, brushing does not guarantee protection against Salmonella contamination. The development of technologies such as an antimicrobial coating is, therefore, an effective alternative for the food sector to ensure the quality and safety of the food product. The polymer structure and pH of the coatings described in this study did not damage the structure of the shell. Regarding the microbial charge, the coatings significantly reduced the total aerobic mesophilic population, thus providing a higher level of safety for the consumer. Finally, the cross-contamination test showed positive results in the control of S. enteritidis, as it drastically reduced cross-contamination, which is one of the main causes of salmonellosis in Europe. Emilia.