The antibacterial effect of whey protein–alginate coating incorporated with the lactoperoxidase system on chicken thigh meat

Abstract Nowadays, the environmental problems due to the use of synthetic films and packages have caused the production of natural edible coatings or films. The aim of this study was to produce an edible whey protein–alginate coating with different concentrations of lactoperoxidase system to control the microbial load and increase the shelf life of chicken thigh meat stored in refrigerated condition (4 ± 1°C). So, after the provision of the alginate–whey protein coating incorporated with the lactoperoxidase system (at concentrations of 2%, 4%, and 6% in alginate–whey protein solution), microbial experiments were conducted for the period of 8 days. Three batches of organisms, including total aerobic mesophilic bacteria, Enterobacteriaceae, and Pseudomonas aeruginosa in samples, were tested by culturing in appropriate conditions. Results indicated that the coating had a substantial inhibitory effect on all lots. Also, the antimicrobial activity of coating increased with increase in lactoperoxidase system concentration in alginate–whey protein coating.

Since the early 90s, researchers found that the whey protein has the ability to form film as a natural polymer composition and can be used as an alternative to synthetic polymers (Murray, 2011). Whey protein-based coatings in addition to improving the nutritional value, have good mechanical properties, produce transparent films or coatings, and have better permeability than the films prepared from carbohydrates and fats (Kaplan, Wardowski, Nagy, & Grierson, 1986;Min, Harris, & Krochta, 2005).
Lactoperoxidase system (LPOS) is a natural antimicrobial system in milk and in human secretions such as saliva and tears (Kussendrager & van Hooijdonk, 2000). The use of LPOS has been suggested as a preservative in foods and pharmaceuticals (Bosch, Van Doorne, & De Vries, 2000). The example is the application of whey protein coating incorporated with LPOS in Pike-Perch fillets (Shokri & Ehsani, 2017).
Chicken thigh meat is one of the most important sources providing the human body protein. Also, poultry meats have high consumption due to their low cost compared to red meats. Therefore, this study was carried out to found the effect of alginate-whey protein coating in controlling the bacterial contaminating of chicken thigh meat stored in the refrigerator (4°C).

| Raw material
Boneless, skinless chicken thigh meats were purchased from the butcher shops. Samples were prepared in an average weight of 10 ± 0.5 g. All samples, including control and treated, were packed in polypropylene bags and kept under refrigerated condition (4 ± 1°C) for 8 days.  , 1999). The ingredients were dissolved in 50 ml of 50 mmol/L phosphate buffer (pH 7.4). The solution was incubated at 23 ± 2°C for 24 hr using a water bath shaker (with shaking at 160 revolutions per minute) to intensify the antimicrobial activity of LPOS (Bosch et al., 2000).

| Whey protein coating preparation
The whey protein coating was prepared as described by Han and Krochta (2007). Whey protein isolate (WPI) powder was dissolved in deionized water at 10% (w/w) concentration. Glycerol (Gly) was added to 10% WPI solution at the ratio of 2%. The solution was heated for 30 min in a 90°C circulatory water bath for denaturation of WPI and forming gel and then was cooled in an ice bath.

| Alginate coating preparation
The alginate coating was prepared as described by Song, Liu, Shen, You, and Luo (2011) with a little modification. In order to avoid the formation of calcium alginate gel before application on samples, two solutions were used to prepare the coating solution.
Solution 1: Thirty grams of alginate with 1000 ml of distilled water was mixed and stirred at a controlled temperature of 80°C until the mixture became clear. Then, 20 ml glycerin was mixed with the prepared sodium alginate solution and stirred thoroughly.
Then, the well-mixed solution was made up to 2000 ml with distilled water.
Solution 2: Two percent (w/v) calcium chloride was also prepared.

| Alginate-whey protein with LPOS coating preparation
After preparing the coatings, LPOS was added at concentration levels of 2, 4, 6, and 8% (v/v) to the second solution and agitated vigorously.

| Coating of chicken thigh meats
Samples in the weight of 10 ± 0.5 g were immersed in whey protein solution for 60 seconds, and then samples were allowed to drip extra solution for 30 s. After that, samples immersed in solution 1 for 3 s, and then after dripping the extra solution drops, samples subsequently were soaked in solution 2 for 30 s. All samples were preserved in polyethylene bags under refrigerated condition for 8 days.

| Antibacterial activity of coatings
The disk diffusion method with the purpose of finding the antibacterial activity of coatings with different LPOS concentrations was performed. For this purpose, two strains of bacteria, including Pseudomonas fluorescens NCTC 10038 and E. coli NCTC 12241, were used.
LPOS at concentrations of 0.5%, 1%, 2%, 4%, 6%, 8%, and 10% was added to second solution. Bacterial suspensions (Pseudomonas fluorescens and E. coli) were adjusted to 1.5 × 10 8 CFU/ml by 0.5 McFarland solution and then were spread on the surface of Muller Hinton agar using sterile cotton swabs. Afterward, small filter blank disks (6 mm diameters) were impregnated with coating solution and placed on the surface of the medium. The plates were incubated at 37°C for 24-48 hr. The strength of antibacterial activity of coatings was estimated by observing the diameter of the inhibition zone.
Also, the different ratios of alginate to whey protein, including 0%, 25%, 50%, 75%, and 100% alginate solution, were tested to found the best ratio in coating to control bacterial growth.

| Coating formulation
Samples were arranged into five coating formulations as follows:

| Evolution of the microbial spoilage
The effect of whey protein-alginate-LPOS coating on the microbial development in chicken thigh meat samples preserved under refrigerated condition on days 0, 2, 4, 6, and 8 was evaluated.
Microbiological analyses were focused on the following: total aerobic mesophilic bacteria (AMB), Enterobacteriaceae, and Pseudomonas aeruginosa. For this purpose, 10 g of samples were put in a sterile plastic bag with 90 ml of 0.1% peptone water and homogenized in a stomacher for 1 min. Appropriate decimal dilutions were prepared and inoculated over plate count agar (PCA), violet red bile agar (VRBA), and cetrimide agar mediums for culturing AMB, Enterobacteriaceae, and Pseudomonas aeruginosa, respectively. Plates were incubated at 37°C for 24-48 hr.

| Statistical analysis
All tests were performed in duplicate. All data were subjected to the analysis of variance (ANOVA) and Duncan test using SPSS version 16. Significance was accepted at p < .05.

| Disk diffusion test
It was observed that the ≤1% LPOS concentration produced none or small growth inhibition zone, while the concentration of ≥2% showed large inhibition zone. Also, the concentration of 10% produced a viscous and dark yellow solution. Therefore, the levels of 2%, 4%, 6%, and 8% were selected.
Also, it was found that 50-50 proportion of whey protein-alginate solution produced large growth inhibition zone. Therefore, the 50-50 proportion of whey protein-alginate was selected to be used as coating. The results of disk diffusion test are shown in Table 1.

| Enterobacteriaceae changes
Enterobacteriaceae counts were ranged from 4.01 to 7.02 logs CFU/g during 8 days. The results (Figure 1) indicated that bacterial growth reduced with increasing the concentration of LPOS in all samples, so that the number of bacteria in C-8 samples was significantly less (p < .05) than other groups on all days except day 0. Also, after the day 2, C-6 samples demonstrated lower Enterobacteriaceae counts compared to C-0, C-2, and C-4 samples. But, the findings did not show any significant differences (p > .05) between C-0, C-2, and C-4 batches on days 0, 2, and 4. On the other hand, the differences between C-0, C-2, and C-4 groups on days 6 and 8 were low and probably meaningless. Concerning the effect of storage day, the only significant difference (more than 1 log CFU/g) was found among all  That study demonstrated that the higher level of LPOS has more controlling impact on Enterobacteriaceae with the passage of days (Yousefi, Farshidi, & Ehsani, 2018).

| Pseudomonas aeruginosa changes
Pseudomonas aeruginosa counts were ranged from 3.48 to 4.95 logs CFU/g during 8 days. Therefore, the total growth of Pseudomonas aeruginosa was low. ANOVA analysis ( Figure 2) indicated that, although there was a significant difference (p < .05) between all samples on the second day, but the Pseudomonas aeruginosa number in all groups except C-8 was nearly similar. Also, findings showed that C-8 samples had lower bacterial (p < .05) counts compared to others after day 0.
Pseudomonas aeruginosa is the most frequent gram-negative bacterium associated with nosocomial and life-threatening chronic infections (Rasheed et al., 2016). Oxygen is vital for such organism due to its absolutely aerobic nature. It has been proved that whey protein has antioxidant property, in addition to antimicrobial and anti-inflammatory traits (Kishta, Iskandar, Dauletbaev, Kubow, & Lands, 2013). So, it can prevent the growth of absolute aerobic bacteria depending on its concentration in coatings or films. Regarding the relationship between P. aeruginosa and edible films, it should be mentioned that P. aeruginosa is often used as a model organism for the study of biofilms (Rasheed et al., 2016).
According to our survey, any study about the antimicrobial effect of the whey protein-alginate coating with LPOS on the Pseudomonas has not been carried out. However, in a study, it was shown that glucose oxidase (5 U/ml) decreased the number of P. aeruginosa cells from 3.03 × 10 5 CFU/ml to 1.13 × 10 4 CFU/ml, and the bactericidal activity of glucose oxidase increased by decreasing the pH to 5 or 6 or by combining the glucose oxidase with the lactoperoxidase in biofilms (Johansen, Falholt, & Gram, 1997).
F I G U R E 1 Enterobacteriaceae changes of chicken thigh meat as affected by whey protein-alginate coating incorporated with the LPOS during refrigerated storage (4 ± 1°C). Values are the mean ± SD F I G U R E 2 Pseudomonas aeruginosa changes of chicken thigh meat as affected by whey protein-alginate coating incorporated with the LPOS during refrigerated storage (4 ± 1°C). Values are the mean ± SD In our study, whey protein-alginate coating incorporated with LPOS can be introduced as a semisuccessful protection agent against P. aeruginosa. According to Figure 2, increasing the LPOs concentrations from C-0 to C-6 samples slightly decreased the P. aeruginosa counts. But, C-8 sample had the significantly (p < .05) lower P. aeruginosa in comparison with others. In other hand, it was semisuccessful agent.

| Total aerobic mesophilic bacteria (AMB) changes
AMB counts were ranged from 4.61 to 7.89 logs CFU/g during 8 days.
According to the results (Figure 3), differences between all batches were small until day 4. In addition, results did not show any significant difference (p > .05) between C-0 and C-2 samples on all days.
After day 4, C-4 and C-6 groups demonstrated significant lower AMB counts (p < .05) compared with C-0 and C-2 samples, although the significant difference between them (C-4 and C-6) was not seen.
Also, the findings showed that C-8 samples had meaningfully lower number of AMB than other groups after day 4.
Regarding the viability of AMB under the protection of antimicrobial coatings, Shokri, Ehsani, and Jasour (2015)  with 6% LPOS has significant lower AMB counts compared with control samples without LPOS on days 8, 12, and 16. Also, they did not see any differences between samples until the fourth day of storage in refrigerator (Yousefi, Farshidi, et al., 2018).
In other survey, Shokri et al. investigated the efficacy of whey protein coating incorporated with LPOS on bacterial growth in Pike-Perch fillets during refrigeration. Contrary to our results, Shokri et al.
indicated that the 2.5% LPOS in whey protein coating has meaningful impact on total viable bacteria. This result is probably because of differences in the type of sample and coating used in two studies (Shokri & Ehsani, 2017).

| CON CLUS IONS
The whey protein-alginate coating incorporated with the lactoperoxidase system in different levels could significantly control the bacterial growth of tested bacteria, particularly Enterobacteriaceae and total aerobic mesophilic bacteria. Also, the study resulted in the increase in antibacterial effect with increasing the LPOS level, so that the most effective coating was related to whey protein-alginate coating with 8% LPOS.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.
Some quality characteristics of Kasar cheese manufactured from milk preserved by activation of lactoperoxidase/thiocyanate/hydrogen peroxide (LP) system.