**1. Introduction**

Eggs are produced and consumed daily all over the world on a large scale, as excellent and inexpensive sources of high quality protein, certain minerals, and vitamins [1]. Nowadays, they have also been considered as ideal vehicles of bioactive components to enhance human nutrition [2]. Although the eggshells have been regarded as natural protective barriers of eggs, quantities of tiny pores presenting on the eggshells for gas exchange lead to the moisture and CO2 loss of eggs, as well as the penetration of microbial, which would then cause the quality deterioration of eggs [3,4]. Moreover, eggshells are too fragile to retain their integrity during transport. Even a tiny crack in an eggshell would largely increase the risk of microbial contamination, particularly when eggs are stored at room temperature.

Attracted by the huge economic benefit, many strategies have been studied for egg preservation, which can be generally classified into two types based on the mechanism. The first one is to deactivate the microorganisms on the eggshells, including techniques such as ultrasonic treatment [5], ozone treatment [6], application of AgNPs-doped paper egg trays [7], and vacuum packaging [8]. The other one is to seal the pores on eggshells by coating, which serves as good barrier layers towards water vapor, gases, and microorganisms. The most widely studied coating materials are oil [9,10], proteins [11], biopolymers [3], etc. Biopolymers composed of biomass or derived polymers are intensively studied as edible coatings [12,13]. Among them, chitosan has attracted much attention of researchers due to its extraordinary performance, such as the excellent film forming property, low gas permeability, satisfied biocompatibility [14], and strong antimicrobial activities [15]. Therefore, coating of chitosan on eggshells can protect egg from both physicochemical changes and microbial contamination, leading to promising effects on quality preservation of eggs. Owing to the low cost, wide availability of chitosan and the operational feasibility of coating process, chitosan coating is considered an efficient and practical way to preserve eggs at room temperature. The influencing factors on the efficiency of chitosan coating such as the molecular weight [16], plasticizer types [17], sources of chitosan [18], and crosslinking agents [19] have been widely investigated. Combination coatings of chitosan with natural antimicrobial agents [20], pullulan [3], oil [21], and montmorillonite [22] have also been applied to eggs and showed improved performance.

However, although the coating materials have been intensively studied, the structure of the chitosan coating layer and their effects on egg preservation has not been carefully investigated so far. Suresh et al. [23] have coated eggs by chitosan a single time and three times, respectively. The coating layer on the eggshell with three-time coating was 20 μm thicker than that with single time coating, but no significant differences were found in weight loss, Haugh unit and albumin index of eggs between these two groups at 22 ± 1 ◦C. The preservation effects of chitosan coatings should be mainly dependent on their barrier properties and antimicrobial activities, which are highly related to the thickness and structures of coating layers on the eggshells.

Therefore, in this study, eggs were coated with chitosan one, two, and three times, separately, to observe their differences in coating structure and structural stability during storage in order to study the relationship between the coating structure and preservation effects. The quality of coated eggs were compared with those of the untreated, water washed and acetic acid treated eggs.

#### **2. Materials and Methods**

#### *2.1. Materials and Reagents*

The freshly laid eggs were provided by a local farm of Chongqing, China. Chitosan with molecular weight of 180 kDa and deacetylation degree of 90% was supplied by Weifang Haizhiyuan Biological Products Co., Ltd. (Weifang, China). Other chemicals were of analytical grade and were used as received.

#### *2.2. Treatment of Eggs*

Chitosan coating solution with a concentration of 0.5% (*w*/*w*) was prepared by dissolving certain amount of chitosan granules in 1 vol % acetic acid and stirring for 24 h. The freshly laid eggs were divided into 6 groups with 50 eggs in each. There were three coated groups, which were coated by chitosan solution for one, two and three times, separately, and were designated as CS1, CS2, and CS3, correspondingly. Each coating process was carried out as immersing eggs in the prepared coating solution for 2 min and drying under ambient conditions overnight with their small-ends down. The other three groups were set as control, including the untreated eggs (CK1), water washed eggs (CK2), and eggs immersed in 1 vol % acetic acid for 2 min (CK3). After the above treatments, all the eggs were placed in egg trays with their small-ends down, and were stored at 25 ◦C and 80% relative humidity (RH). Five marked eggs of each group were taken out to weigh every 5 days, and were returned back right after the measurement. Another five eggs of each group were picked up randomly to measure the Haugh unit, yolk index and pH of albumen every 5 days.

#### *2.3. Characterizations of Eggs*

#### 2.3.1. Weight Loss

Weight loss (%) of a whole egg was calculated as the weight difference in percentage of an egg during storage compared to its whole weight at day 0 [24]. The weight of five marked eggs in each groups was measured using a balance every 5 days and given as average value ± standard deviation.
