Effect of nanoemulsion loading a mixture of clove essential oil and carboxymethyl chitosan‐coated ε‐polylysine on the preservation of donkey meat during refrigerated storage

Journal of Food Processing and PreservationVolume 45, Issue 9 e15733 ORIGINAL ARTICLEOpen Access Effect of nanoemulsion loading a mixture of clove essential oil and carboxymethyl chitosan-coated ε-polylysine on the preservation of donkey meat during refrigerated storage Wei Zixiang, Wei Zixiang orcid.org/0000-0002-2354-6941 Biopharmaceutical Research Institute, Liaocheng University, Liaocheng, China Contribution: Software, Writing - original draftSearch for more papers by this authorZhang Jingjing, Zhang Jingjing College of Agronomy, Shandong Engineering Technology Research Center for Efficient Breeding and Ecological Feeding of Black Donkey, Shandong Donkey Industry Technology Collaborative Innovation Center, Liaocheng University, Liaocheng, ChinaSearch for more papers by this authorZhang Huachen, Zhang Huachen College of Agronomy, Shandong Engineering Technology Research Center for Efficient Breeding and Ecological Feeding of Black Donkey, Shandong Donkey Industry Technology Collaborative Innovation Center, Liaocheng University, Liaocheng, ChinaSearch for more papers by this authorZhang Ning, Zhang Ning Biopharmaceutical Research Institute, Liaocheng University, Liaocheng, ChinaSearch for more papers by this authorZhang Ruiyan, Zhang Ruiyan Biopharmaceutical Research Institute, Liaocheng University, Liaocheng, ChinaSearch for more papers by this authorLi Lanjie, Corresponding Author Li Lanjie lilanjie@lcu.edu.cn College of Agronomy, Shandong Engineering Technology Research Center for Efficient Breeding and Ecological Feeding of Black Donkey, Shandong Donkey Industry Technology Collaborative Innovation Center, Liaocheng University, Liaocheng, China Correspondence Li Lanjie and Liu Guiqin, College of Agronomy, Shandong Engineering Technology Research Center for Efficient Breeding and Ecological Feeding of Black Donkey, Shandong Donkey Industry Technology Collaborative Innovation Center, Liaocheng University, Liaocheng 252059, Shandong Province, China. Email: lilanjie@lcu.edu.cn (L. L.) and guiqinli@lcu.edu.cn (L. G.)Search for more papers by this authorLiu Guiqin, Corresponding Author Liu Guiqin guiqinli@lcu.edu.cn College of Agronomy, Shandong Engineering Technology Research Center for Efficient Breeding and Ecological Feeding of Black Donkey, Shandong Donkey Industry Technology Collaborative Innovation Center, Liaocheng University, Liaocheng, China Correspondence Li Lanjie and Liu Guiqin, College of Agronomy, Shandong Engineering Technology Research Center for Efficient Breeding and Ecological Feeding of Black Donkey, Shandong Donkey Industry Technology Collaborative Innovation Center, Liaocheng University, Liaocheng 252059, Shandong Province, China. Email: lilanjie@lcu.edu.cn (L. L.) and guiqinli@lcu.edu.cn (L. G.)Search for more papers by this author Wei Zixiang, Wei Zixiang orcid.org/0000-0002-2354-6941 Biopharmaceutical Research Institute, Liaocheng University, Liaocheng, China Contribution: Software, Writing - original draftSearch for more papers by this authorZhang Jingjing, Zhang Jingjing College of Agronomy, Shandong Engineering Technology Research Center for Efficient Breeding and Ecological Feeding of Black Donkey, Shandong Donkey Industry Technology Collaborative Innovation Center, Liaocheng University, Liaocheng, ChinaSearch for more papers by this authorZhang Huachen, Zhang Huachen College of Agronomy, Shandong Engineering Technology Research Center for Efficient Breeding and Ecological Feeding of Black Donkey, Shandong Donkey Industry Technology Collaborative Innovation Center, Liaocheng University, Liaocheng, ChinaSearch for more papers by this authorZhang Ning, Zhang Ning Biopharmaceutical Research Institute, Liaocheng University, Liaocheng, ChinaSearch for more papers by this authorZhang Ruiyan, Zhang Ruiyan Biopharmaceutical Research Institute, Liaocheng University, Liaocheng, ChinaSearch for more papers by this authorLi Lanjie, Corresponding Author Li Lanjie lilanjie@lcu.edu.cn College of Agronomy, Shandong Engineering Technology Research Center for Efficient Breeding and Ecological Feeding of Black Donkey, Shandong Donkey Industry Technology Collaborative Innovation Center, Liaocheng University, Liaocheng, China Correspondence Li Lanjie and Liu Guiqin, College of Agronomy, Shandong Engineering Technology Research Center for Efficient Breeding and Ecological Feeding of Black Donkey, Shandong Donkey Industry Technology Collaborative Innovation Center, Liaocheng University, Liaocheng 252059, Shandong Province, China. Email: lilanjie@lcu.edu.cn (L. L.) and guiqinli@lcu.edu.cn (L. G.)Search for more papers by this authorLiu Guiqin, Corresponding Author Liu Guiqin guiqinli@lcu.edu.cn College of Agronomy, Shandong Engineering Technology Research Center for Efficient Breeding and Ecological Feeding of Black Donkey, Shandong Donkey Industry Technology Collaborative Innovation Center, Liaocheng University, Liaocheng, China Correspondence Li Lanjie and Liu Guiqin, College of Agronomy, Shandong Engineering Technology Research Center for Efficient Breeding and Ecological Feeding of Black Donkey, Shandong Donkey Industry Technology Collaborative Innovation Center, Liaocheng University, Liaocheng 252059, Shandong Province, China. Email: lilanjie@lcu.edu.cn (L. L.) and guiqinli@lcu.edu.cn (L. G.)Search for more papers by this author First published: 12 July 2021 https://doi.org/10.1111/jfpp.15733AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract The objective of this research was to investigate the effects of clove essential oil, the carboxymethyl chitosan-coated ε-polylysine (ε-PL) nanoemulsion (Nano-CEO-ε-PL-CMC) on the shelf-life, and the quality characteristics of donkey meat during refrigerated storage. The physicochemical, microbiota, and sensory properties of donkey meat were regularly monitored. The results indicated that Nano-CEO-ε-PL-CMC was stable with the smallest particle size. Furthermore, we found that Nano-CEO-ε-PL-CMC had a significant preservation effect on refrigerated donkey meat by maintaining the stability of pH value, inhibiting the proliferation of microorganisms (p < .05), and sharply reducing the oxidation of fat and protein (p < .05) during the storage period. Interestingly, the flesh color measurement and texture analysis revealed that the application of Nano-CEO-ε-PL-CMC could effectively reduce the color change, and delay the decline of springiness and cohesiveness of refrigerated donkey meat. Our findings suggested that Nano-CEO-ε-PL-CMC has great potential in the preservation of donkey meat during refrigerated storage. Novelty impact statement In the present study, a novel nano-dispersion system with good stability was successfully developed by incorporating the ε-PL into clove essential oil nanoemulsion. The nanoemulsion film containing clove essential oil and ε-PL (Nano-CEO-ε-PL-CMC) has a significant preservation effect on refrigerated donkey meat by inhibiting the proliferation of microorganisms and reducing the oxidation of fat and protein (p < .05). 1 INTRODUCTION The consumption of refrigerated meats has increased rapidly because of its better flavor, the improvement of meat quality, water-holding capacity, and nutrition (Zhao et al., 2019). Donkey meat contains high protein and polyunsaturated fatty acids, which makes it easy to decay and oxidative deterioration. Slaughtering, cutting, transportation, sales, and freezing may cause microbial contamination and oxidative deterioration, thereby reducing the shelf life of meat, in which the oxidative deterioration of meat could produce peculiar smell and discoloration (Verma et al., 2021). Therefore, how to prolong the shelf life of donkey meat has become a hot topic in recent years. ε-polylysine is a widely accepted food preservative produced by Streptomyces whiteflies, which has good antibacterial properties and safety (Hiraki, 2000). Additionally, ε-PL was widely used in meat coating preservation for biodegradability (Alirezalu et al., 2021; Shao et al., 2020). Clove is evergreen tree of the Myrtle family, Syzygium genus, and the flower bud of clove is one of the famous traditional imported "southern medicine" in China (Zhao et al., 2006). Many researches indicate that clove essential oil (CEO) can effectively inhibit foodborne spoilage and pathogenic bacteria, such as Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus (Anacarso et al., 2019; Cui et al., 2018; Zhang et al., 2017). The study found that CEO could effectively protect the texture of fish fillets and delay the lipid oxidation of sutchi catfish fillets in the process of refrigeration (Binsi et al., 2017). Although CEO with good antibacterial and antioxidant activities, its volatile, low solubility, and aromatic properties limit the application in food preservation. In order to reduce the volatilization and hydrophobicity of essential oils, nanoemulsions were applied in the encapsulation and protection of essential oils (Prakash et al., 2018). Nanoemulsions are submicron oil-in-water (O/W) transparent or translucent emulsions with the diameter of the droplet between 100 and 600 nm. In addition, an antibacterial agent coated with oil droplets promotes the ability to penetrate bacterial cell membranes (Salvia-Trujillo et al., 2017). Therefore, the nanoemulsion formed by embedding essential oil may have a great prospect in food preservation. In the application of meat preservation, the nanoemulsion is usually loaded on the edible coating (Huang et al., 2020). To our knowledge, there was no related research for the CEO-ε-PL-CMC coating on donkey meat preservation. CEO-ε-PL-CMC coating solution was prepared as a composite film for refrigerated donkey meat to investigate its antioxidant, antibacterial activities, and shelf-life effectiveness. 2 MATERIALS AND METHODS 2.1 Materials Donkey hind leg meat was provided by Tianlong Food Co. LTD (Liaocheng, China). ε-polylysine hydrochloride was purchased from Silver-Elephant Biological Engineering Co. LTD (Zhejiang, China). CEO was purchased from Xue Mailon Food Spices Co. LTD (Zhengzhou, China). Lecithin and carboxymethyl chitosan (CMC) were purchased from Qilu Biotechnology Co. LTD (Liaocheng, China). WPI was provided by Agropur LTD (Canada). The remaining reagents and consumables were provided by Sinopec Chemical Reagents Co. LTD (Shanghai, China). 2.2 Preparation of composite coating solution The preparation of nanoemulsion and coarse emulsion was modified according to the methods of Liu and Neves (Liu et al., 2020; Neves et al., 2016). Initially, an emulsifier aqueous solution containing 1.50% WPI (w/v) and 0.05% lecithin (w/v) was prepared, and 0.125% (wt) polylysine was added at the same time. Then 0.50% (wt) of clove essential oil phase and 99.60% (wt) of water were mixed. After that, the coarse emulsion (ε-polylysine 0.125%, clove essential oil 0.50%, WPI 1.50%, lecithin 0.05%) was formed by shearing with high-speed dispersion at 5,000 rpm for 10 min. Nanoemulsions were prepared with 100 MPa through high-pressure homogenization (NS1001L 2K; GEA Niro, Italy) five times. CMC was dissolved in ultra-pure water and stirred to form a clarifies the transparent coating solution (3.00% w/v) (Xiong et al., 2020). Then, CEO-ε-PL coarse emulsion, CEO-ε-PL nanoemulsion, and ultra-pure water were mixed and stirred with CMC coating solution with 25% volume fraction and 75% volume fraction, respectively. Finally, four types of composite coating solution were formed (Table 1). TABLE 1. Description of sample composition Treatment Sample composition CON Ultra-pure water CMC 2.25% CMC CMC-CEO 2.25% CMC+0.5% CEO Emul-CEO-ε-PL-CMC 2.25% CMC+0.5% CEO+0.125% ε-PL Nano-CEO-ε-PL-CMC 2.25% CMC+0.5% CEO+0.125% ε-PL Abbreviations: ε-PL, ε-polylysine; CEO, clove essential oil; CMC, carboxymethyl chitosan; CON, control. 2.3 Sample treatment The chilled sour donkey meat was transported to the laboratory by the cold chain. After removing the fascia and fat, the donkey meat was divided into 5 × 5 × 4 cm (length × width × height) rectangular pieces. Simultaneously selected 3 samples as the 0th day for index determination. Then, a total of 75 meat samples were randomly divided into five groups with three parallel samples in each group for 5-time points and stored at 4℃ in sterile crisper sealed. The following indexes were evaluated. 2.4 Meat quality index determination 2.4.1 pH determination Donkey meat samples (10.0 g) were cut and placed in a beaker containing 90.0 ml of distilled water for homogenization (Zhang et al., 2016). The pH value was measured by a digital pH meter (FE28; METTLER TOLEDO, Zurich, Switzerland). 2.4.2 Total viable aerobic bacteria (TVC) determination The microorganisms were cultured on Days 0, 2, 4, 6, 8, and 10. Refer to Zhang's methods for sample processing and microbial count (Souza et al., 2019; Zhang et al., 2020). Donkey meat sample (25.0 g) and sterile physiological saline (225.0 ml) were fully homogenized by a high-speed homogenizer (XHF-DY; SCIENTZ, Ningbo, China), and then the homogenized samples were diluted in a 1:10 ratio. The samples (1.0 ml) were added to the Petri dish. Total viable counts (TVC) were determined by nutrient agar plate after incubated at 37℃ for 48 hr. The results were expressed in lg (CFU/g). 2.4.3 TBARS determination of lipid oxidation TBARS determination refers to the method of Souza (Souza et al., 2019), 10 g of minced donkey meat was mixed with 7.5% trichloroacetic acid (20.0 ml) and shook for 1 hr, and collected the supernatant after filtration. Five milliliters of the supernatant were combined well with 0.02 mol/L of 2-thiobarbituric acid. After heating in a water bath (95℃) for 30 min, the absorbance of the sample was measured at 530 nm. The standard curve was drawn with the known concentration of malondialdehyde (MDA), and the result was expressed in mg MDA/kg. 2.4.4 Carbonylation determination of protein oxidation Carbonylation determinations (nmol/mg prot) were measured by Protein Carbonyl assay kit (NJJCBIO, Nanjing, China). Prior to the protein carbonylation assay, the total protein concentrations (mg/ml) were measured by the total protein quantitative assay kit (NJJCBIO, Nanjing, China). Each step of the operation was carried out in strict accordance with the manufacturer's instructions. 2.4.5 Color determination The color of refrigerated donkey meat was measured by a color sensor (CR-10 Plus, Minolta Inc, Osaka, Japan) without removing the coating (Cao et al., 2019). The Colorimeter had an 8 mm-diameter aperture with D65 illumination. And the lightness (L*) redness (a*) and yellowness (b*) values were measured directly three times on each sample surface in a 10° observer angle set. 2.4.6 Textural Properties determination Donkey meat was formed into a sample of 5 × 3 × 3 cm (length × width × height). The determination method was based on Wang's method to determine the hardness, springiness, cohesiveness, and chewiness of the sample (Wang et al., 2020). Measurements were conducted by the texture analyzer (TA.TOUCH, BosinTech, Shanghai, China) mode at a speed of 1 mm/s and within a 5 mm distance. 2.5 Statistical analysis All data were expressed as means ± standard deviation (SD). Analysis of Duncan's test was performed on SPSS Statistics 23.0 to evaluate the significance of differences among mean values. 3 RESULTS AND DISCUSSION 3.1 Nanoemulsion characterization The changes in particle size and polydispersity index (PDI) of nanoemulsions and coarse emulsions were obtained by high-pressure homogenization within 10 days. The minimum particle sizes of nanoemulsion by high-pressure homogenization and coarse emulsions were 110.30 ± 0.45 and 203.40 ± 0.77 mm (Table 2). Compared with the previous reports on CEO nanoemulsion, the nanoemulsion of this study has a smaller particle size (Majeed et al., 2016; Sharma et al., 2017; Wan et al., 2019). The particle size of nanoemulsion was significantly smaller than the coarse emulsion at all time points (p < .05). With the extension of storage time, the particle sizes of the two groups of emulsions increased gradually, which was due to Ostwald Ripening (Wooster et al., 2008). Briefly, it is the dissolution of small particles of oil droplets in the emulsion, which is deposited on the larger particles to form larger particles. TABLE 2. Emulsion particle size and dispersion at 4℃ for 10 days Storage time (d) Nano-CEO-ε-PL-CMC Emul-CEO-ε-PL-CMC Particle size (nm) PDI Particle size (nm) PDI 0 110.3 ± 0.45b 0.20 ± 0.00 203.4 ± 0.77a 0.24 ± 0.01 1 142.6 ± 0.33b 0.19 ± 0.01 222.1 ± 0.56a 0.23 ± 0.02 3 155.7 ± 0.49b 0.18 ± 0.01 243.2 ± 0.61a 0.27 ± 0.02 5 171.9 ± 0.37b 0.20 ± 0.02 310.2 ± 0.91a 0.32 ± 0.03 7 190.8 ± 0.85b 0.20 ± 0.02 370.4 ± 1.26a 0.38 ± 0.04 10 257.7 ± 0.79b 0.22 ± 0.03 459.9 ± 1.72a 0.46 ± 0.03 Note Lowercase letters represent significant differences on the same day p < .05, data are expressed as mean ± SD (n = 3). PDI represents the uniformity of particle size distribution, the smaller value of PDI represents the more uniform distribution of nanoemulsion (Liew et al., 2020). It is generally believed that emulsions with PDI < 0.25 are stable. The PDI of nanoemulsion with 0.18–0.22 is more stable than coarse emulsion (Table 2). During 10 days of storage, the nanoemulsion did not delaminate. 3.2 pH values The pH value is one of the important indicators of refrigerated meat quality. In the present study, the initial pH value of donkey meat was 5.84 (Figure 1). However, after 10 days stroage, the final pH values of donkey meat in each treatment group were significant differences (p < .05). Among them, the coating group without essential oils and preservatives revealed the worst effect on inhibiting the increase of pH value. Interestingly, the Nano-CEO-ε-PL-CMC group presented the best inhibitory effect, and the pH value increased by 0.36 units during storage. The Emul-CEO-ε-PL-CMC and CMC-CEO group increased by 0.38 and 0.47 units, respectively. The results indicated that donkey meat was prone to spoilage without any treatment during refrigerated storage, while preservation treatment could greatly slow down its spoilage rate. Moreover, our study suggested that the pH value of donkey meat could be more effectively maintained by the emulsion preservation with nano-particle size (for example, Nano-CEO-ε-PL-CMC). These findings were similar to previous reports (Amiri et al., 2019; Ghani et al., 2018; Milani et al., 2020). The pH value of the donkey meat in different treatment groups gradually increased with the extension of the storage period. The possible reason could be attributed to the increase of microorganisms during the storage, which decomposed the proteins and gradually accumulated alkaline substances in the meat (Verma et al., 2019). FIGURE 1Open in figure viewerPowerPoint Effects of different coating treatments on pH value of donkey meat during storage at 4℃ (different lowercase letters represent significant differences on the same day p < .05) 3.3 Microbial measurement The TVC of different samples during the storage period of 10 days are presented in Figure 2. The initial bacteria count of fresh donkey meat was 4.01 lg CFU/g, which was lower than the microbial limit of spoiled meat of 7.00 lg CFU/g (Karabagias et al., 2011), and this indicated the good meat quality (Figure 2). The TVC of meat samples in each treatment group increased with the extension of the storage period. The increase rate of TVC in the control group was fastest, which exceeded the microbial limit on the 6th day, and reached 8.35 lg CFU/g on the 10th day. Besides, the TVC in the CEO-CMC group increased slowly in the early stage of storage, then it was significantly different from the Nano-CEO-ε-PL-CMC group and the Emul-CEO-ε-PL-CMC group in the later stage of storage. The clove essential oil is volatile, so its antibacterial effect is far worse than that of Nano-CEO-ε-PL-CMC and Emul-CEO-ε-PL-CMC group. These results suggested that the combination of CEO and ε-PL with the coating agent could effectively slow down the growth of microorganisms. Because the eugenol in CEO is an effective antibacterial agent, and ε-PL also has a strong inhibitory effect on bacteria (Bentayeb et al., 2014; Chheda & Vernekar, 2015). Remarkably, the TVC of donkey meat in the Nano-CEO-ε-PL-CMC treatment group showed the least increase, and it was significantly different from other treatment groups during the storage period (p < .05). Even though, on the 10th day, the TVC of donkey meet in Nano-CEO-ε-PL-CMC treatment group was 6.71 lg cfu/g, which was lower than the standard for spoiled meat. This can be interpreted that nano-encapsulation could increase the release time of antimicrobial agents. Additionally, the reduction of particle size extends the contact area between the antibacterial substances and the bacterial cell membrane, which can enhance its antiseptic and antibacterial effect (Donsì & Ferrari, 2016). In summary, Nano-CEO-ε-PL-CMC treatment is an effective way to slow down the microbial proliferation during donkey meat cold storage. FIGURE 2Open in figure viewerPowerPoint Effects of different coating treatments on TVC of donkey meat during storage at 4℃ (different lowercase letters represent significant differences on the same day p < .05) 3.4 Lipid oxidation In the present study, we found that the TBA values of refrigerated donkey meat in the treatment group and the control group gradually increased. There was no significant difference among the treatment groups during the storage period from Day 0 to 4, and the TBA value of the CEO-CMC group showed the slowest increase (Figure 3). On the 6th day of the storage, the TBA value of the CON group and the CMC group reached 3.91 mg MDA/kg and 3.21 mg MDA/kg, respectively, which were significantly different from that of the other three groups (p < .05). The results indicated that different treatments of essential oils could effectively inhibit the oxidative decomposition of fat in donkey meat. On the 8th day, the TBA value of the CON group was about 5.33 mg MDA/kg. For the CMC group, the TBA value was about 5.26 mg MDA/kg on the 10th day. These TBA values of meat are equal to or greater than 5 mg MDA/kg, which are considered as the threshold for detecting off-odors and off-taste for humans (Insausti et al., 2001). It may be due to the coating treatment effectively blocked oxygen and slowed down the oxidation of fat. Pabast reported that the coarse emulsion and nanoemulsion with Satureja plant essential oil dramatically reduced TBARS of lamb, which was confirmed by our findings (Pabast et al., 2018). Furthermore, this study also found that the combination of essential oils and ε-PL can effectively inhibit fat oxidation. The TBARS value of the Emul-CEO-ε-PL-CMC group was lower than that of the Nano-CEO-ε-PL-CMC group during storage, but there was no significant difference (p > .05). It means that reducing the particle size has no obvious effect on improving the antioxidant capacity of the composite preservative. FIGURE 3Open in figure viewerPowerPoint Effects of different coating treatments on TBARS of donkey meat during storage at 4℃ (different lowercase letters represent significant differences on the same day p < .05) 3.5 Protein oxidation Protein oxidation is an important factor of quality deterioration in meat, and an important index of protein oxidation is protein carbonyl content (Stadtman, 1992). As presented in Figure 4, from the 1st day to the 9th day of storage, the carbonyl content of samples in each group continuously increased, which indicated that oxidation promoted the side chain carbonylation of protein. The carbonyl content of the CON group was higher than that of other treatment groups (p < .05) during the storage period of day 2 to day 10. This suggests that the coating treatment can reduce the degree of protein oxidation. We found that the meats in the groups with essential oils treatment had lower carbonyl values compared to the group without essential oils treatment. Several reports had also obtained similar results (Al-Hijazeen et al., 2018; Fourati et al., 2020; Jokanović et al., 2020). Utrera considered that the hydroxyl group in essential oil phenols had a scavenging effect on free radicals, thereby reducing protein oxidation (Utrera & Estévez, 2013). Although there was no significant difference between the Emul-CEO-ε-PL-CMC group and the Nano-CEO-ε-PL-CMC group during the entire storage process (p > .05), the carbonyl value of the Nano-CEO-ε-PL-CMC group is lower in 8–10 days storage. It indicated that reducing the particle size of these emulsions can further improve their ability to resist protein oxidation. FIGURE 4Open in figure viewerPowerPoint Effects of different coating treatments on the carbonyl content of donkey meat during storage at 4℃ (different lowercase letters represent significant differences on the same day p < .05) 3.6 Color change The L* value, b* value, and a* value of refrigerated donkey meat of different treatments are summarized in Table 3. The increase in meat brightness is related to high-myofibril degeneration and internal water exudation (Faustman & Suman, 2017; Petracci & Cavani, 2012). The L* value of meat in each group increased with the prolongation of storage time. During the storge, the L* value of Nano-CEO-ε-PL-CMC group, Emul-CEO-ε-PL-CMC group and CMC-CEO group increased by 28.3%, 24.9% and 26.9%, respectively, which were lower than 30.9% in the CMC group. It indicated that using CEO and ε-PL coating to treat donkey meat can effectively prevent the donkey meat from turning pale. TABLE 3. Changes in the color attributes of refrigerated donkey samples stored at 4℃ for 10 days Attributes Storage days Treatments 0d 2d 4d 6d 8d 10d L* CON 34.77 ± 0.69BCa 35.43 ± 1.06BCa 39.20 ± 2.24Ba 45.37 ± 1.2Aa 46.53 ± 2.76Aa 48.03 ± 1.64Aa Nano-CEO-ε-PL-CMC 34.77 ± 0.69Da 35.03 ± 0.50 Da 38.60 ± 0.70Ca 41.87 ± 2.25Bab 41.63 ± 1.25Bab 44.63 ± 0.61Aab Emul-CEO-ε-PL-CMC 34.77 ± 0.69Ca 35.70 ± 0.41BCa 37.53 ± 0.45Ba 41.36 ± 1.43Aab 42.07 ± 1.18Ab 43.43 ± 1.54Ab CMC-CEO 34.77 ± 0.69Da 36.23 ± 1.13CDa 36.97 ± 0.48BCa 38.33 ± 0.31Bbc 42.46 ± 1.30Ab 44.13 ± 0.86Ab CMC 34.77 ± 0.69Da 35.23 ± 0.40Da 38.67 ± 1.03Ca 42.47 ± 1.68Bc 43.90 ± 1.18ABb 45.50 ± 1.34Ab a* CON 16.87 ± 0.40ABa 17.83 ± 0.53Aa 16.77 ± 0.21ABa 15.27 ± 1.58BCa 14.63 ± 1.31BCb 13.73 ± 0.78Cb Nano-CEO-ε-PL-CMC 16.87 ± 0.40ABa 17.09 ± 1.07ABab 17.47 ± 1.08Aa 17.17 ± 0.68ABa 17.03 ± 1.03ABa 15.53 ± 0.62Ba Emul-CEO-ε-PL-CMC 16.87 ± 0.40ABa 16.93±054Aab 17.23 ± 0.52Aa 16.83 ± 0.99ABa 16.20 ± 0.59ABab 15.27 ± 1.32Ba CMC-CEO 16.87 ± 0.40Aa 16.17 ± 1.02Ab 16.47 ± 0.83Aa 16.57 ± 0.34Aa 15.43 ± 0.94ABb 15.07 ± 0.25Ba CMC 16.87 ± 0.40Aa 17.33 ± 0.45Aab 17.10 ± 1.49Aa 16.40 ± 0.36Aa 15.67 ± 0.34ABab 14.90 ± 0.51Ba b* CON 11.17 ± 0.56Aa 11.80 ± 0.49Aab 12.23 ± 0.37ABa 12.33 ± 0.05BCa 12.87 ± 0.49CDa 13.73 ± 0.24Da Nano-CEO-ε-PL-CMC 11.17 ± 0.56Aa 12.27 ± 0.26Aa 12.10 ± 0.71Aa 12.67 ± 0.42Aa 12.53 ± 0.54Aab 12.67 ± 0.17Bb Emul-CEO-ε-PL-CMC 11.17 ± 0.56Aa 10.77 ± 0.98Ab 11.43 ± 0.69Aa 11.67 ± 0.58Aa 11.87 ± 0.21Ab 12.57 ± 0.71Ab CMC-CEO 11.17 ± 0.56ABa 11.87 ± 0.12Aab 11.96 ± 0.74ABa 12.07 ± 0.21ABa 12.47 ± 0.33ABab 12.97 ± 0.37Aab CMC 11.17 ± 0.56Aa 12.13 ± 0.53Aa 11.53 ± 0.73ABa 11.93 ± 0.61ABa 12.57 ± 0.37BCab 13.40 ± 0.36Cab Note Different lowercase letters indicate significant differences between different samples on the same day; different capital letters indicate significant differences in the same sample at different times (p < .05). Data are expressed as mean ± SD (n = 3). The protein denaturation and fat oxidation would cause the increase of b* value in meat, so the b* value of the five groups showed an upward trend (Akamittath et al., 1990). The b* value of the Nano-CEO-ε-PL-CMC group and Emul-CEO-ε-PL-CMC group increased by 13.4% and 12.5%, respectively, which was lower than that of the other three groups (16.1%, 20.0%, and 22.9%). It suggested that the preservative activity of the combination of CEO and ε-PL coating is better than that of the CEO. The use of ε-PL may inhibit bacterial proliferation and reduce the degree of protein and fat oxidation. The a* value decreased with time during the entire storage period. After storage to the 10th day, the a* value of the control group was significantly lower than that of the other three treatment groups with essential oils (p < .05). The results indicated that the CEO hindered the oxidation of myoglobin. The donkey meat in the Nano-CEO-ε-PL-CMC group and Emul-CEO-ε-PL-CMC group had higher redness value than that of the CMC-CEO group during the storage, which may be attributed to the protective effect of essential oils emulsion or nano-emulsion. The encapsulating essential oils in microsomes could improve the antioxidant capacity of essential oils (Bakkali et al., 2008). In general, combining the results of fat oxidation and protein oxidation, we can conclude that the Nano-CEO-ε-PL-CMC and Emul-CEO-ε-PL-CMC can effectively alleviate the color change and extend the shelf life of donkey meat in cold storage.