Improving the quality of the chicken fillet using chitosan, gelatin, and starch coatings incorporated with bitter orange peel extract during refrigeration

Abstract The preserving potential of biopolymer coatings can be improved by adding natural antimicrobial and antioxidant compounds. The objective of this study was to evaluate the effect of natural coatings (gelatin (Gel), chitosan (Ch), and modified starch (MS)) incorporated with bitter orange peel extract (BOE) on the quality of the chicken fillets during cold. BOE had a high amount of phenolic compounds (145.28 mgGAE/g). Coating the fillets with pure BOE exerted a higher inhibitory effect against bacterial growth compared to composite coatings without extract. Lower microbial count (2–3 log CFU/g on days 9 and 12 of storage) was observed in the samples coated with composite biopolymers incorporated with BOE in comparison to the coatings without BOE. Composite coatings of Gel/MS/BOE showed lower FFA in the fillets followed by Gel/Ch/BOE and MS/Ch/BOE. The lowest TVB‐N belonged to MS/Ch/BOE followed by Gel/Ch/BOE and Gel/MS/BOE which were 17.05, 17.39, and 19.40 mg/100 g at the end of the storage. Among the samples, pure BOE, Gel/MS/BOE, Gel/Ch/BOE, and MS/Ch/BOE showed the lowest peroxide value and the coatings containing chitosan had a slower rate of hydroperoxide generation. Drip loss showed a descending trend in all coated samples except for an enhancement in control and BOE‐coated fillets, 6.42% and 6.39%, respectively, on day 12 of storage. Samples coated with Gel/MS and Gel/MS/BOE had the lowest drip loss during the storage period (5.96% and 5.98%, respectively). It should be noted that coatings containing chitosan had higher antimicrobial and antioxidant effects. The effect of the coatings as antimicrobial barriers and preservative agents were as follows: Gel/Ch/BOE > MS/Ch/BOE > Gel/MS/BOE. It can be concluded that the applied composite coatings in this work have a high potential to maintain and improve the quality of raw chicken fillets during storage in the refrigerator.


| INTRODUC TI ON
In recent years, along with the advancement of technology in the food packaging industry, the use of edible biodegradable coatings and films has been developed to maintain the quality of the products.
These edible coatings can be prepared from a variety of biopolymer compounds, including proteins, polysaccharides, fibers, and lipids which are found abundantly in nature (Ng & Kurisawa, 2021). These biopolymers possess the potential of creating an obstacle against the penetration of water molecules, oxygen, the release of compounds responsible for aroma and flavor, slow down the oxidation process, reduce moisture loss and weight loss, and prevent the occurrence of surface microbial contamination. Also, these types of coatings have a positive role in maintaining the sensory characteristics and texture quality as well as increasing the shelf life of the product. Foodstuffs such as fish, shrimp, fruit, and vegetables are packed using these biopolymer coatings in pure or combined (composite) form and sometimes incorporated with preservative compounds such as essential oils and herbal extracts (Smaoui et al., 2022). These new approaches are used in combination with other food preservation methods such as cooling (in the refrigerator) and freezing (in the freezer) to store products (Khwaldia et al., 2010).
Chitosan is a biopolymer and linear polysaccharide consisting of glucosamine molecules obtained from the deacetylation of chitin. Due to its inherent antimicrobial properties and high potential to form a film, it is used extensively in food packaging (Nowzari et al., 2013). Gelatin is a protein derivative that is formed from the partial hydrolysis of collagen. It consists of glycine, proline, and 4-hydroxyproline units. Gelatin powder is transparent, colorless, and almost tasteless. Depending on the process used, two types of gelatin are obtained, namely, type A (of acid hydrolysis) and type B (of alkaline hydrolysis). Gelatin is one of the most common food coatings and is used in the preservation of various meat products due to its high ability to form a film, high water-binding capacity, availability, and low price (Gedarawatte et al., 2021). It has been reported that composite coatings containing chitosan and gelatin used in food preservation had a very good mechanical performance. This property has been attributed to the formation of polyelectrolyte complexes through electrostatic interactions between protonated amine groups of chitosan and negatively charged side substituents in gelatin or collagen (e.g., carboxylate groups; Pereda et al., 2011).
Starch is a polysaccharide macromolecule consisting of glucose units. Starch-based coatings are biodegradable, low-cost, and environmentally friendly (Suhag et al., 2020). However, due to being hydrophilic, these coatings are weak barriers to the penetration of water molecules. To solve this problem, starch is modified by thermal, chemical, or enzymatic methods. Modified starch has a higher capacity to retain water and resistance to shrinkage. In several studies, the use of modified starch with other polymers such as gelatin and essential oils or plant extracts with antimicrobial effects has been reported as a suitable technique for food preservation (Kumar et al., 2021;Suhag et al., 2020).
In research studying the effect of chitosan-gelatin coatings on the rate of oxidative acceleration in rainbow trout fillets during storage in the refrigerator (4 ± 1°C), the results showed that chitosangelatin coatings maintained the quality characteristics of fish fillets, controlled the bacterial population and also increased the shelf life of the samples (Nowzari et al., 2013). In another study, a composite edible coating containing chitosan and starch was used to minimize weight loss and delay microbial spoilage in blackberries. The data showed that the composite edible coating of chitosan and starch (1:1) was effective in minimizing weight loss and mold decay. The researchers announced that the use of a combined edible coating of chitosan and starch is a suitable method to prevent moldy rot and increase the shelf life of black berries in cold storage conditions (Ojeda et al., 2021). Also, it is reported that a mixture of gelatin (10%) and chitosan (1%) sprayed on beef as a coating exerted antioxidant and antimicrobial effects and increased the shelf life of the product (Gedarawatte et al., 2021).
The preservative activity of edible coatings can be increased by adding natural antimicrobial and antioxidant compounds. Byproducts of the citrus processing industry are naturally rich sources of phenolic compounds. The peel, which makes up about half of the fruit's mass, contains the highest amount of flavonoids in citrus.
Extracts obtained from the peel of citrus fruits such as orange, bitter orange, lemon, lime, and mandarin have a high potential as antioxidants in preventing oxidative reactions (Zou et al., 2016). Raw food products such as chicken fillet due to its high content of unsaturated fatty acids (omega 3), high protein and free amino acids content, and considerable moisture are prone to adverse microbial, biochemical, and tissue changes during the distribution, sale, and storage which limits the shelf life of the product (Ahmadi et al., 2020). Bitter orange is one of the most cultivated citrus in north of Iran and its peel is considered agricultural waste but is proved to contain phenolic compounds (Zou et al., 2016). As phenolic compounds possess antioxidant and antimicrobial potential and there is no study about incorporating bitter orange peel extract in biopolymers (to be used as natural preservative), we aimed to evaluate the antioxidant and antimicrobial effect of biopolymer coating (containing gelatin, chitosan, and modified starch) incorporated with phenolic compounds of bitter orange peel on the chicken fillets during the storage period in the refrigerator.

| Chemicals and materials
All the reagents, chemicals, and biopolymers were purchased from Scharlau Chemical Co. Bitter orange (Citrus aurantium) was purchased from the local market of Sari (Mazandaran province, north of Iran) and peeled using a kitchen knife. Two hundred grams of the peels was cut into pieces of 2-2.5 cm 2 and kept at −34 ± 1°C to inhibit the oxidation of the bioactive compounds. Four kilograms of fresh chicken fillets (fresh carcass transferred from slaughterhouse to market as chilled and cut into fillets just before purchasing in the market) was obtained from a local supermarket in Sari (Iran) and transferred to the laboratory in an ice box.

| Preparing peel extract
The peel extract was prepared according to the method explained by Marzouk (2013). Briefly, the peel pieces were added to the aqueous ethanol (80% v/v) with the solvent ratio of 1:20 (1 g of peels in 20 mL of ethanol) and homogenized with a blender (MCL-HX-4GM, MeCan Co.). An ultrasound-assisted extraction technique was applied to increase the extraction efficiency. The homogenized peel samples were transferred to a volumetric flask and placed in an ultrasonic bath (S30H model, Alma Co.). The sonication was carried out at a power of 150 W, 40°C for 30 min. The sonicated extracts were filtered using Whatman paper No. 1 under a vacuum. Then, the extracts were mixed with n-hexane and incubated in the ultrasonic bath for 20 min at room temperature to remove the oily components.
The ethanol phase was separated from the oily phase and centrifuged (RF 10000 model, Orum Tajhiz Gostar Co.) at 11,000 g for 10 min at room temperature followed by concentrating with a rotary evaporator (RV10 V-C model, Azin Co.) at 50°C (Marzouk, 2013).

| Total phenolic content
The total phenolic content (TPC) of the peel extract was measured through Folin-Ciocalteu method. The results were reported as mg gallic acid equivalents (GAE) per gram (g) of the peel (Sir Elkhatim et al., 2018).

| Preparation of composite coatings containing bitter orange peel extract
Modified starch was prepared by thermal heating (physical process) from a commercial source (Alborz Starch Co.), the gelatin type B (Halal gelatin of bovine bone produced through an alkaline process) was kindly donated by Iran Capsule Co., and chitosan (medium molecular weight, viscosity 300-800 cP) was purchased from Scharlau Chemical Co. Modified starch (MS) solution (30% w/v in distilled water), gelatin (Gel) solution (1% w/v in distilled water), and chitosan (Ch) solution (1% w/v in 1% acetic acid) were prepared. All solutions were separately homogenized using a magnetic stirrer (Hei-PLATE Mix 20 I, Heidolph Instruments Co.) at room temperature (25 ± 1°C) for 1 h. Then, the 50:50 ratio of the solutions was mixed and homogenized at 10,000 g for 10 min applying a homogenizer (model HC-2A, Hoacheng Co.). In the next step, 1% (v/v) of glycerol as the plasticizer and 5% (v/v) bitter orange peel extract (BOE) were added to the above solutions to prepare the coating formula. The prepared coating solutions were mixed and stirred for 1 h applying a magnetic stirrer (Gedarawatte et al., 2021).

| Applying the coatings on the chicken fillets
Chicken fillets were rinsed with cold distilled water for 3 min and the outer surface was dried with a dryer paper. The fillets were cut into 50 ± 5 g pieces in a sterile condition and divided into eight groups: Control, F1 (BOE), F2 (Gel/MS), F3 (Gel/Ch), F4 (MS/Ch), F5 (Gel/MS/BOE), F6 (Gel/ Ch/BOE), and F7 (MS/Ch/BOE). The samples were soaked in their individual coating solutions for 30 s, then taken out and placed on a sterile sieve (for 2 min) to drain. The dipping and drying steps were repeated twice. The control group included chicken fillet pieces dipped in deionized water. The samples were allowed to drain for 5 min in a biological safety cabinet, placed in polyethylene zip-locker bags, and stored in the refrigerator (4 ± 1°C). The chemical, microbial, and sensory evaluations were carried out at 3-day intervals during 12-day storage time.

| pH determination
To measure pH value, 10 g of the fillet samples was homogenized with 25 mL of neutral distilled water, incubated at room temperature for 10 min, and filtered using Whatman paper no. 1. The pH of the filtrate was determined using a pH meter (Wideman et al., 2016).

| Determination of drip loss
Drip loss is water escaping from raw meat during storage. Drip loss was measured by suspending individually 50-g chicken fillet samples in polyethylene bags without any touch with the sides of the bags, for 24 h at 4°C. Then, samples were removed from the bags, gently dried, and weighed. Drip loss was determined as the percentage of weight lost (Rahman et al., 2015).

| Texture analysis
The sample preparation for texture analysis was carried out by taking raw fillet pieces with a circular shape using a cylindrical punch with 3 cm long and 3 cm diameter. The texture of the fillet samples was analyzed for firmness by peak shear force (g) applying a texture analyzer instrument (TA.XTplusC, Stable Microsystems Co.) using a 2 mm rectangular steel probe. Texture softness/firmness was determined by the shear energy (N × mm). Ten fillet samples (n = 10) of each treatment were tested and the readings were carried out in triplicate (Khan et al., 2022).

| Microbial analyses
To prepare decimal dilutions of fillet samples, 10 g of each sample was homogenized with 90 mL of 0.1% peptone water using a stomacher (BagMixer 400 SW, HealthCare Technologies Co.) at 4 g

| Measuring peroxide value (PV)
The peroxide value was determined according to the method explained by Rahman et al. (2015) and was expressed as milliequivalent peroxide per kilogram of the sample (Rahman et al., 2015).

| Statistical analysis
All the experiments were carried out in triplicate. Data analysis was carried out using Two-way analysis of variance (ANOVA) and Duncan's Test in SPSS software version 22.00. Significant differences between the means of each characteristic for the different coatings were determined by Tukey's HSD test. The statistically significant differences between mean values were determined at confidence levels of 99% and 95%.

| pH
The results of pH measurement in chicken fillets treated with biopolymer coatings are presented in Table 1. There were significant differences between the samples coated with pure peel extract (F1) and the samples coated with extract-incorporated biopolymers and the control (p < .01). F3 (Gel/Ch) and F7 (MS/Ch/BOE) showed pH values similar to the control on the 3rd day of the storage (p > .01).
As seen, in all treatments, there was a significant decrease in pH value toward the end of the storage time (p < .01). The decrease in pH of treatments, especially those containing BOE, is due to the presence of organic compounds such as organic acids and the conversion of glycogen of the muscle to lactic acid in cease of oxygen supply. Also, during storage in the refrigerator, a slight decrease in pH may occur due to the formation of carbonic acid from the CO 2 resulting from the metabolism of spoilage-causing microorganisms (Chmiel et al., 2018). The lowest final pH value was found in F1 (BOE) and F2 (Gel/MS) and F5 (Gel/MS/BOE) and the highest pH was observed in the control, F3 (Gel/Ch), and F4 (MS/Ch) (p < .01). It seems that among the treatments, those including chitosan had higher pH values at the end of the storage time (p < .01). We assumed that the decrease in pH in chicken fillets could be due to the growth of LAB during storage which is also claimed by other studies (Clarke et al., 2017;Gedarawatte et al., 2021). This is similar to the findings  particularly in combination with gelatin, showed lower pH which may be due to the trivial degradation of starch and is used as a substrate for lactic acid generation (Tkaczewska et al., 2022).

| Drip loss
According to Table 2, the drip loss of the fillet samples on the first day was approximately 6.12%-6.16% (Table 2). Drip loss showed a descending trend in all coated samples except for an enhancement in control and F1 (BOE-coated fillets), 6.42% and 6.39%, respectively,  (Wei et al., 2019). In this work, the chicken fillets coated with starch, gelatin, and chitosan reflected significantly lower drip loss compared to BOE-coated samples and control that indicates higher muscle integrity and water holding of the texture .
On the other hand, microbial activity leads to the hydrolysis of collagens and myofibrillar proteins on poultry meat surface and the release of intercellular fluids during storage (Roslan et al., 2019). The lower drip loss shows lower microbial activity in biopolymer-coated fillets.

| Texture
The texture and structure of meat are considered important freshness properties that depend on several parameters such as firmness, springiness, chewiness, and also the internal cross-linking of the connective tissue and the detachment of muscle fibers (Cheng et al., 2014). The mean texture firmness of the fillets on day 0 was 4.02 ± 0.10 N which gradually decreased in all the fillet samples during storage (p < .05; Table 3

| Total phenolic content
The industries of food and agricultural products generate considerable amounts of byproducts rich in phenolic compounds that are valuable natural sources of antioxidants and antimicrobials such as polyphenols.
In sweat and bitter oranges, the peel accounts for almost 30% of the fruit mass which contains the highest concentration of flavonoids in comparison to the other parts of the fruit. In this study, we quantified the peel TPC in ethanolic extract of bitter orange peel as 145.28 mg GAE/g. It TA B L E 2 Drip loss (%w/w) of the chicken fillets treated with bitter orange extract and biocomposite coatings during storage at 4 ± 1°C. is reported that polymethoxylated flavonoids and flavonoid C-and Oglycosides are the most abundant flavonoids (Haggag et al., 1999;Xi et al., 2017). According to Sawalha et al. (2009), naringin and neohesperidin are the major polyphenols in bitter orange peels (Sawalha et al., 2009).

| Total viable count
The population of total viable bacterial (TVC) in chicken fillets is presented in

| Psychrotrophic bacteria
The dominant group of microorganisms that causes spoilage in fresh meat, poultry, and fish products during cold storage is psychrotrophic (Saenz-García et al., 2020). As presented in Table 5 Nowzari et al. (2013), the psychrotrophic count in the trout fillets coated with chitosan and gelatin was about 6.5 log CFU/g on day 16 of cold storage with a significant difference compared to control and samples coated with pure gelatin or pure chitosan (Nowzari et al., 2013). It is reported that chitosan is an effective antimicrobial agent (Pereda et al., 2011) which

| Pseudomonas spp.
It is obvious from the results of Table 6  showed significantly higher antibacterial activity compared to the control and F2-F4 (p < .05). The antimicrobial activity of the bitter orange extract is attributed to phenolic compounds, tannins, saponins, and flavonoids that are biologically active. It has been reported that tannin in citrus peel extracts forms irreversible bonds with proline-rich proteins leading to the inhibition of protein synthesis in the cells (Shimada, 2006). Our data indicated that the coatings consisted of chitosan had higher effect against Pseudomonas growth. The mechanism of antibacterial action of chitosan may be due to the disruption of the lipopolysaccharide layer of the cellular outer membrane in gram-negative bacteria; on the other hand, chitosan can act as a barrier against oxygen and moisture transfer (Pereda et al., 2011).

TA B L E 5
Psychrotrophic bacteria count (log CFU/g) of the chicken fillets treated with bitter orange extract and biocomposite coatings during storage at 4 ± 1°C.

| Enterobacteriaceae count
The population of Enterobacteriaceae in the samples during cold storage is presented in Table 7. It is known that alkaloid compounds and flavonoids of the ethanolic extract of citrus peel possess an extensive range of biological activities such as cytostatic, antimicrobial, and antioxidant in addition to toxicity against foreign cells and organisms. Also, terpenoids in ethanolic extracts are involved in cell membrane degradation through the disruption of lipophilic compounds (Kademi & Garba, 2017). We concluded that BOE and biocomposite/BOE expressed significant inhibitory effects on the growth and proliferation of Enterobacteriaceae. MS/Gel/BOE. Moreno et al. (2018) reported that composite coating of starch and gelatin on chicken fillets actively reduced the population of LAB which was significantly lower than the control; our results are in accordance with their findings (Moreno et al., 2018).

| Lactic acid bacteria (LAB) count
Also, Jonaidi Jafari et al. (2018) reported the lowest LAB count in the chicken fillets treated with chitosan coatings incorporated with propolis extract compared to pure chitosan, pure propolis, and control (Jonaidi Jafari et al., 2018).

| Chemical evaluations
3.6.1 | Free fatty acids content Free fatty acids (FFAs) are derived from lipid hydrolysis and cleavage of triacylglycerols due to lipase activity in high temperatures and the presence of moisture. FFAs act as pro-oxidants and speed up the rate of hydroperoxide degradation; therefore, high FFA content in the fats causes further oxidation and results in the development of offflavor and unpleasant taste (Chew & Nyam, 2020). FFA may involve in the reactions with myofibrillar proteins which leads to protein aggregation (Özyurt et al., 2009). According to the data presented in Table 9, the initial FFA value of the fillet samples was around 0.2% of oleic acid and significant differences were found among the sam-   (Mohan et al., 2012). In a study by Nowzari et al. (2013)  with Ziziphora clinopodioides and Thymus daenensis essential oils compared to uncoated samples (Rajabian et al., 2019). It can be concluded that composite coatings incorporated with BOE are more effective than pure BOE in controlling TVB-N of the chicken fillets.

| Peroxide value (PV)
The PV is the most common chemical test for evaluating the oxidative deterioration of fats and oils. Although hydroperoxides (as the primary products of oxidation) degrade to a mixture of volatile and nonvolatile molecules and react to the endoperoxides, the PV measurement is a useful method of monitoring oxidative deterioration changes (Gilbraith et al., 2021). The degradation of the hydroperoxides leads to the formation of a variety of secondary products such as furans, ketones, carbonyl compounds, hydrocarbons, and other compounds that are responsible for off-flavor and rancidity in food (Kong & Singh, 2016).
Hydroperoxide formation rate in the samples coated with BOE (F1) and BOE/biopolymer (F5, F6, and F7) was trivial during the first 3-6 days of the storage period (  during storage and significant differences were observed between samples with Gel/Ch and the control or pure Gel and pure Ch. Our results were in similarity to theirs that all coated fillets had lower PV than the control and shows that Gel/Ch coating could efficiently decrease lipid oxidation in fillets (Nowzari et al., 2013). It is demonstrated that chitosan, starch, and gelatin may be considered as the natural barriers against oxygen permeation and chitosan is presumed as a potential natural antioxidant for stabilizing lipid-containing foods (Jonaidi Jafari et al., 2018;Perez Sira & Dufour, 2017).

| CON CLUS IONS
The pure extract of bitter orange peel had a considerable amount of phenolic compounds and expressed high antioxidant and antimicrobial activity in the chicken fillets. Combining BOE with biopolymer composites exerted higher preservative effect on the samples, increased BOE's activity, and also improved texture and drip loss.

ACK N O WLE D G E M ENTS
The authors are grateful to Zoleykha Shirvani of Amol University of Special Modern Technologies for her technical support.

FU N D I N G I N FO R M ATI O N
This work has been supported by a research grant from the Amol University of Special Modern Technologies, Amol, Iran.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that they do not have any conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.

E TH I C S S TATEM ENT
Not applicable.

TA B L E 11
Peroxide value (meq/kg) of the chicken fillets treated with bitter orange extract and biocomposite coatings during storage at 4 ± 1°C.