Effect of combined chlorogenic acid and chitosan coating on antioxidant, antimicrobial, and sensory properties of snakehead fish in cold storage

Abstract Degradation of meat quality has always been a burning issue in fish preservation. To maintain the quality, a novel combination of chlorogenic acid (CGA) and chitosan (CS) coating was applied to snakehead fish fillets. Fish fillets were soaked into 2% chitosan (2CS), 0.2% CGA in 2% chitosan (0.2CGA/2CS), 0.5% CGA in 2% chitosan (0.5CGA/2CS), or 1.0% CGA in 2% chitosan (1.0CGA/2CS) solution; and then, coated samples were vacuum‐packaged and stored at 2 ± 0.5°C. pH values, color values, microbial loads, hardness, sensory qualities, and oxidization of lipids and proteins of stored fish fillets were investigated for 5 months. Antimicrobial activity was found to be nonsignificant (p ≤ .05) among different coated fish fillets, while color, antioxidant, and pH values were significantly (p ≤ .05) different. Lipid oxidation and protein oxidation were found to be inhibited in 2CS‐, 0.5CGA/2CS‐ and 1.0CGA/2CS‐coated fish fillet. All CGA/CS coating delayed increase in pH (p ≤ .05) and resulted brown color. However, only CS coating resulted in higher sensory scores (p ≤ .05) and controlled browning. Considering antioxidant properties and other quality parameters, CGA/CS coating might be applied commercially in fish preservation.

Fish processing and preservation have developed rapidly to provide new exciting knowledge for addressing industry requirements.
The application of edible coating with bioactive compounds in preservation has been successfully studied. The characteristics of edible coatings and their physicochemical nature have been given great interest (Fang, Lin, Warner, & Ha, 2018;Hassannejad, Nouri, Soltani, & Molavi, 2019). Macromolecules of protein, starch, modified starch, and polysaccharides have been applied in edible coating for preservation (Abdulkareem, Abdalsalam, & Bohan, 2019;Cardoso et al., 2019;Hassannejad et al., 2019). Chitosan (CS) coating is a nontoxic, attractive, and natural coating agent used in the food industry for inhibiting microorganism proliferation and lipid oxidization (Abdulkareem et al., 2019;Bharathi, Ranjithkumar, Chandarshekar, & Bhuvaneshwari, 2019;Reesha, Panda, Bindu, & Varghese, 2015). Use of additives in edible coating further enhances its activity in preservation by releasing antioxidants and antimicrobial substances (Ao et al., 2019;Cardoso et al., 2019). Thus, incorporation of chlorogenic acid (CGA) with chitosan coatings would exhibit oxygen barrier properties, since CGA has been known for its antioxidant activity (Gokoglu et al., 2012;Jiao, Wang, Yin, Xia, & Mei, 2018;Liu & Park, 2010). Regarding CGA incorporation, it is important to know the consequences of CGA/CS coating on qualities of snakehead fish fillets during cold storage. As the following exploration, CGA/CS coating is expected to maintain high quality of cool storage fish than CS coating.
Little research has been reported on CGA/CS coating in the preservation of fresh fish; thus, less information is available on the characteristics of CGA/CS-coated fish. Therefore, a study was designed and carried out to evaluate the sensory qualities, texture, and color and to investigate oxidation of proteins and lipids of CGA/ CS-coated snakehead fish under vacuum package and stored at refrigeration temperature. This research will contribute to preserving fish and unveil the effects of CGA/CS edible coating on the product quality of fresh fish fillets during cold storage.

| Material and coating
Snakehead fish (15 cm long, 1.5 cm diameter, growth of 12 months) were purchased from Guangzhou Zhengyuan Food Technology Company Limited. Snakehead fish were cut into 3-mm-thick fillet (axial cutting). Chlorogenic acid was bought from Luye company in China; chitosan and other chemicals used were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
After fish fillets were soaked into the coating solutions for 30 s, the soaked samples were air-dried at 40°C for 40 min under 1.8 m/s air velocity. A comparison had been performed by soaking in stilled water cosolvent of 1% citric acid for 30 s. Dried samples were placed on a glass tray (5 cm × 5 cm × 4 cm) with absorbing paper covered the bottom of the tray. Then, glass tray was packed at 0.7 MPa vacuum; the packed glass trays were stored at 2 ± 0.5°C in a refrigerator. Sampling and assay intervals were performed in every month for 5 months.

| pH measurement
Snakehead fish were unwrapped, and pH was determined by a pH meter (SevenCompact S220-Micro, Mettler Toledo Company). The pH of the samples was measured by inserting pH sensor into the fillet. When pH value reached maximum and was stable, it was documented with a precision of 0.01 (Cihlar, Drdlik, Cihlarova, & Hadraba, 2013).

| Color measurement
CR400 colorimeter (Konica Minolta) was calibrated twice with a white board. After cutting the coating of samples, the sensor was placed on the surface of the sample and values of L, a, and b were measured and recorded. L, a, and b represent lightness, redness, and yellowness, respectively. ΔE was calculated using Equation (1) (Islam, Zhang, Adhikari, Xinfeng, & Xu, 2014).
In Equation (1), L 0 , a 0 , b 0 and L 1 , a 1 , b 1 represent the values of the fresh samples and stored samples, respectively.

| Microbiological array
Total viable microbes were measured by incubation method (Fadıloğlu & Emir Çoban, 2018;Öz, 2018). Sample (20 g) was shifted into a sterilized stomacher bag (180 ml peptone water of 0.1 g/100 ml) and stomached in 2 min under 25°C. Concentration of samples was serially diluted in 10-fold by injecting peptone solution of 0.1 g/100 ml, and diluted solutions (1 μl) inoculated and were spread on plate with MS medium (Murashige & Skoog, 1962). Inoculated plates were incubated at 37°C for 48 hr, and then, count of viable microbes was arrayed. Total viable microbes were calculated by multiplying dilution factor (fold) in log CFU/g.
After agitation (800 rpm, 60 s), the liquid supernatant with 2-thiobarbituric acid was incubated in 30 min under 90°C. The 532 nm absorbance was quantified by a spectrophotometer. A comparison was carried out using a blank sample. Blank solution consisted of 10% TCA and of 20 mM TBA (two solution, w/w = 1:1).

| Texture analysis
Samples texture was analyzed by texture analyzer (TMS-PRO,

Food Technology Corporation). Test program was set as compress-
ibility method (Peh, Khan, & Ch'Ng, 1999). Cylindrical probe (2 mm diameter) was used to penetrate through the fish fillets (thickness 3 mm). Pretest speed was 0.5 mm/s, test speed was 1 mm/s, and penetrate depth was 3 mm. Standard weight of 1.0 kg was used for calibration. Texture values were recorded, and the mean value was calculated.

| Sensory evaluation
After removing package, samples (20 g) were fried in 170°C oil with some salt in 60 s. 20 trained panelists (10 men and 10 women, between 30 and 50 years) were recruited for sensory evaluation according to the earlier method (Xu, Song, et al., 2018). Fried samples (2 g) were randomly delivered to each panelist for evaluation.
Evaluation scores were collected in different aspects of food. The score was analyzed by serial rank of 5, excellent; 4, good; 3, acceptable; 2, fair; and 1, unacceptable. Evaluation was operated in a panel test room at 25°C temperature under natural light.

| Data analysis
Data were analyzed using analysis of variance (ANOVA), and mean comparisons were done using Duncan's multiple range test (DMRT) with a confidence level (p ≤ .05) of 95% using SPSS software (SPSS 20.0, IBM). All tests were carried out in triplicate unless stated. Data were presented as mean values with significant letters. TA B L E 1 pH trend of snakehead fish fillets subjected to different chlorogenic acid (CGA) chitosan coatings during storage Table 1 shows an upward trend in pH during storage of snakehead fish fillets. In noncoated fish fillets, the pH increased significantly from about 5.1 to 7.2 during storage, which significantly differed from the treated group (Table 1). The reason of high pH over the storage in control samples might be that volatile base nitrogen (TVB-N) is formed by enzymatic hydrolysis of fish proteins (Chauhan et al., 2019;Trabelsi et al., 2019;Xu, Song, et al., 2018). This finding supported the fact that fresh fish is viable to decay. Higher pH values present higher content of TVB-N formed by bacterial metabolites. In Table 1

| Total viable count
Total viable count value of snakehead fish fillet was subjected to the coating treatments during storage at 2°C (Table 3). After 5 months, increasing TVC values of about 6.5, 5.2, 5.3, 5.4, and 5.5 log CFU/g were responsible to noncoating sample and samples coated in 0.2CGA/2CS, 0.5CGA/2CS, and 1.0CGA/2CS. Naturally, 7 log CFU/g is the limit of microbiological safety in fresh fish fillets (Fadıloğlu & Emir Çoban, 2018;Fang et al., 2018;Olawuyi et al., 2019;Öz, 2018). In this study, coated samples were below 5.5 log CFU/g during storage at 2°C. In the absence of vacuum packaging, the shelf life of coated samples was within a week in refrigerator. Coating profiles combined with vacuum package met the demand of preservation of fresh fish fillets. It was implied that chitosan coating and vacuum package can inhibit the microbial growth (Table 3). Before 4 months, there was no difference between different chitosan-treated profiles. These results suggest that CGA did  (Table 3).  (Feng et al., 2017a;Rui et al., 2017). Moreover, it has been reported that chitosan film with additional 1.5% cinnamon oil deters lipid oxidization in fish fillets (Ojagh, Rezaei, Razavi, & Hosseini, 2010). Here, this result shows that 0.5CGA/2CSand 1.0CGA/2CS-treated fish fillets implied low lipid oxidization from 2 to 5 months of storage (Table 4). Thus, the results of this experiment suggest that 0.5%-1.0% CGA could be useful in the chitosan coating formula in preservation of snakehead fish.  The free thiol group values in 2CS-and 0.2CGA/2CS-coated samples were lowered significantly (p ≤ .05) from third month to fourth month, while no significant changes were observed between fourth month and fifth month. This phenomenon represents that chitosan coating significantly lowered protein oxidization, and addition of 0.5% CGA or less did not affect much in free thiol group values (Jiao et al., 2018;Liu & Park, 2010), while higher amount of CGA did not improve the antioxidant properties of chitosan coating in snakehead fish during storage (Table 5).

| Protein oxidization
The main reason of reduced protein oxidation is the application of chitosan coating and vacuum package which hindered product exposure to oxygen gas for responsible degradation (Gokoglu et al., 2012;Li et al., 2019;Özalp Özen et al., 2011). Hence, the antioxidant agent decreased oxygen gas to interact with fish fillets which inhibited the oxidization. Earlier studies have shown similar results that protein and lipid oxidization were delayed by adding extracts of plants (Gokoglu et al., 2012;Öz, 2018;Özalp Özen et al., 2011).  (Hassannejad et al., 2019;Jongberg, Terkelsen, Miklos, & Lund, 2014;Underwood et al., 2010). The other reason is that hardening of protein enhanced to high value (Table 6). This result is in accordance with the earlier finding (Fang et al., 2018).

| Texture analysis
Different concentration of CGA also demonstrated similar hardness of all treated samples from 2 to 5 months. This meant additional CGA is not related to hardness. Synthetically, chitosan coating not only hindered the lipid oxidization but also impeded protein oxidization during fish fillet storage.

| CON CLUS IONS
Chitosan coating possesses antioxidant and antimicrobial properties in coating fish fillets; the additional CGA further enhanced antioxidant properties but not influence hardness of snakehead fish fillets in preservation. This work implied that CGA/CS coating will enhance the food safety and quality in preservation of fresh fish. and Zhanjiang Technology Program (2019A01033).

CO N FLI C T O F I NTE R E S T
The author(s) declared no potential conflicts of interest with the research, authorship, and publication of this article.

Xiaohuang
TA B L E 7 Sensory scores of the fried samples subjected to different chitosan coating after 5 months of storage