Gouda cheese spoilage prevention: Biodegradable coating induced by Bunium persicum essential oil and lactoperoxidase system

Abstract This study aimed to prepare an inhibitory edible coating for Gouda cheese based on whey protein containing lactoperoxidase system (LPOS) and Bunium persicum essential oil (EO) in order to control postpasteurization contamination. Using a full factorial design, the effects of LPOS and EO on microbiological characteristics and chemical indices of manufactured Gouda cheeses were evaluated during 90 days of storage time. Listeria, lactic acid bacteria, Enterobacter, Escherichia, and Pseudomonas species were considered as potential pathogenic and spoilage indicators of produced Gouda cheese samples. Chemical properties of cheeses were assessed using the free fatty acid, peroxide value, and thiobarbituric acid experiments. The results showed that bacteria counts remained constant in cheese samples coated with EO and also EO–LPOS. However, the survival of gram‐positive lactic acid bacteria and Enterobacter spp. was more pronounced in LPOS‐based coating. The most effective treatments on oxidation stability parameters in cheese samples were EO‐ and EO–LPOS coatings. By the addition of B. persicum EO and LPOS, further inhibition of the lipid oxidation of the cheese samples was achieved. Lipolysis, as a result of lipid degradation, was more pronounced in the control, whey‐coated, and whey–LPOS‐coated samples in comparison with whey–EO‐ and whey–EO–LPOS‐coated samples during the final days of storage time. These results indicate that antibacterial, lipid oxidation, and oxygen barrier properties of the coatings were developed by the addition of B. persicum EO and LPOS.

Nowadays the utilization of coating in cheese packaging is one of the common approaches to preserve the quality of cheese during shelf life. Furthermore, nature-ripened Gouda cheeses are coated regularly during ripening and storage time (Wemmenhove et al., 2016). Different natural components including polysaccharides, proteins, and lipids (alone or in combination) can be utilized as edible coating materials. The efficiency of an edible coating in preserving food quality is mainly related to its moisture and gas barrier properties (Aloui & Khwaldia, 2016). Whey proteins, a biodegradable coatings and films material, are a major by-product of the cheese industry. Heat-denatured whey proteins produce transparent and flexible films with excellent water vapor, gas and oil barrier properties (Ramos, Fernandes, Silva, Pintado, & Malcata, 2012). Incorporating antimicrobial compounds into biopolymer edible coatings could improve the quality and shelf life of food products (Aloui & Khwaldia, 2016). Rather than incorporating the antimicrobial agents directly into food, blending them in film or coating solutions induces a functional effect on the food surface. Due to the consumers' concerns about health, there is particular interest in food industry to use natural food preservatives such as antimicrobial enzymes and bacteriocins (Shokri, Ehsani, & Jasour, 2015).
The main objective of this study was to investigate the effects of edible coatings containing LPOS and B. persicum EO as antimicrobial agents on the quality indices and microbial characteristics of Gouda cheese during storage.

| Gouda cheese preparation
In this research, the required treatments were based on five coating formulations which were assigned randomly during the study: 1. Control: 0% EO-LPOS (C)
Cheese slices were dipped in the well-stirred coating solution for 60 s. The ratio of cheese to the solution was 1:2.
After taking away the immersed cheese samples from the solution, they were drained well, packed in polyethylene bags, and kept at 4±1°C for 90 days.

| Extraction of B. persicum EO
Initially, dried seeds (100 g) were ground into powder in a grinder, and then by using a Clevenger-type apparatus, they were exposed to steam distillation for 2.5 hr. In the next step, the obtained EO was well drained from water and dried over anhydrous sodium sulfate until the last traces of moisture were evaporated. At last, the substance was stored in dark glass bottles at 4°C for more experiments (Ehsani, Hashemi, Naghibi, Mohammadi, & Khalili Sadaghiani, 2016). The characteristics of essential oil were determined by GC/ MS (30 m × 250 μm × 0.25 μm).

| Bacterial inoculation
Lyophilized cultures of E. coli (ATCC 43894) and L. monocytogenes (ATCC 19118) were obtained from the culture collection of the Department of Microbiology, Faculty of Veterinary Medicine, Urmia, Iran, and inoculated on the surface of cheese samples before coating with the dilution of 10 6 and 10 3 (CFU/ml), respectively.

| Preparation of LPOS
The weight ratios of LPOS components for lactoperoxidase, glucose oxidase, glucose, potassium thiocyanate, and hydrogen peroxide were 1.00, 0.35, 108.70, 1.09, and 2.17, respectively. Following that, the components were dissolved separately in 50 ml phosphate buffer (pH 6.2), and then, the concentrations of the components were altered on the basis of 15.5 mg LPO (Cissé, Montet, Tapia, Loiseau, & Ducamp-Collin, 2012). To increase the antimicrobial activity of LPOS, the solution was incubated at 23°C for 24 hr under vibration at 160 rpm (Shokri et al, 2015).

| Preparation of whey protein solution
In order to prepare whey protein solution (WPS), 10 g of whey protein was mixed with 100 ml of distilled water and stirred at a controlled temperature of 90°C until a clear mixture was obtained. Just the same amount of whey protein, glycerol was added to the solution. The amount of LPOS in the whey protein solution was 5% (v/v) (Shokri et al, 2015). In the whey protein solution, the 0.5% v/v concentration of B. persicum EO was applied as coating-forming solution.

| Bacteriological analysis
Initially, 25 g of cheese samples was brought to a final volume of 250 ml with 0.1% peptone water and then homogenized in a stomacher (Pulsifier ® , UK) for 1 min. Dilution process was done serially in all samples. The method of drop plate by using a proper tool was applied to count the number of all bacteria.

| pH
Through using a pH meter (Eutech ® CyberScan, pH 510, Singapore), the pH value for the homogeneous mixture of distilled water (1:10, w/v) and cheese samples was measured.

| Lipid extraction
During storage days (0, 7, 15, 30, 60, and 90), 10 g of each cheese sample was mixed for 2 min in 20 ml chloroform and 40 ml methanol. Then, 20 ml chloroform and 20 ml methanol were added to the homogenized mixture consecutively. Vacuum filtration using Whatman No. 1 filter paper was applied to separate solid from liquid. After preparing a transparent solution, the solvent was removed by rotary evaporator (Bligh & Dyer, 1959). The prepared lipid samples were sealed in opaque bottles and stored at −80°C until use.

| Thiobarbituric acid
First, 0.2 g of cheese fat was homogenized with 25 ml of butanol solution. The next step was to blend 5 ml of the prepared solution and 5 ml of TBA reagent (200 mg TBA in 100 ml butanol) and heat them in a boiling water bath for 2 hr. Then, they were cooled under running water for 1 min and the absorbance was measured at 539 nm against a blank (consisting of 5 ml of TBA reagent and 5 ml butanol).

| Peroxide value
First, 0.1 g cheese fat was weighed into a 250-ml flask and 25 ml acetic acid/chloroform (ratio of 3:2, v/v) was poured. Then, the mixture was stirred up to complete dissolution of remained lipids. After potassium iodide (1 ml) addition, the solution was maintained in a dark room for 10 min. Distilled water (20 ml) was poured and titrated with sodium thiosulfate, with 1.5% starch as an indicator. The peroxide value was represented as meq peroxide/kg cheese sample (Shin et al., 2012).

| Sensory analysis
To perform the sensory evaluations, 10 trained panelists who were familiar with odor, color, and overall acceptability of cheese were selected. An acceptance test using a 9-point hedonic scale was used to evaluate the overall acceptance, with 1 as "dislike extremely" and 9 as "like extremely." Water was used for mouth rinsing between evaluations of samples (Cui, Wu, Li, & Lin, 2017).

| Statistical analysis
A completely randomized factorial design, three replicates, was proposed with two independent factors: cheese type and storage time (Table 1). Data were subjected to one-way analysis of variance (2) FFA = acid value × 1 2

Cheese type Odor Color
Overall acceptance (from 9)

| TBARS, POV, pH, and FFA
The peroxide value was measured to assess the lipid oxidation of Gouda cheese during storage. The antiperoxidation evaluation was followed by an adaptation of the TBARS assay, which relies on the colorimetric detection of the malondialdehyde formation by polyunsaturated lipid degradation as a result of reactive oxygen species.
Eventually reaction of the malondialdehyde with thiobarbituric acid (TBA) represents a colored compound.
The ANOVA results revealed that cheese type × storage time interaction had a significant effect on TBARS and POV (p < 0.05). The trend shows that both oxidation indicators increased until day 30.
Also, the following decrement of TBARS and POV during storage had no association with cheese types, except in POV of WL cheese which showed a significant correlation between storage time and POV even at 60 days (Figure 2a and b). The reduction in POVs was also reported in an investigation by Lee, Yang, and Song (2016), who prepared The effects of cheese type × storage time interaction on the pH and FFA content were similar and are presented in Figure 2c and d. As demonstrated, during storage, the pH and FFA content were also significantly increased (p < 0.001), which is most apparent in the WL-type cheese. The treatments did not change the

| Sensory evaluation
Sensory characteristics such as odor, texture, and appearance play an important role in the overall acceptability from the consumers'  (Fox et al., 2004). Sensory evaluation scores are presented in Table 1. Our findings about the positive sensory effects of using B. persicum EO in cheese preparation are in line with those reported previously by Ehsani et al. (2016) and Hassanien, Mahgoub, and El-Zahar (2014). They found that B. persicum EO-enriched cheese samples had significantly higher color, odor, flavor, texture, and general acceptability scores than control cheese (Ehsani et al., 2016). Also, Hassanien et al. (2014) found that Domiati soft cheeses supplemented with black cumin oil showed higher sen- sory scores compared to control cheese (Hassanien et al., 2014).
On the other hand, it has been reported that lactoperoxidase system (LPOS), alone and in combination with some plants and their derivatives, can provide beneficial effects for the food industry.

| CON CLUS IONS
Based on our findings in the present study, B. persicum EO was very effective against LAB, Listeria, and Enterobacter growth unlike LPOS-containing coatings. LPOS is recommended to be used in combination with other preservation methods, as it was effective in inhibiting gram-negative bacteria. Decreases in lipid oxidation indices and also free fatty acid content were more pronounced in whey protein coatings involving EO and also EO-LPOS. LPOS as an active gram-negative microorganism inhibitor and EO as a grampositive bacterium inhibitor as well as an oxidation inhibitor are seriously recommended as preservative agents against chemical and microbiological spoilage in cheese. Therefore, B. persicum EO in combination with LPOS can be applied as a natural antimicrobial agent for extending the shelf life of washed and ripened cheeses.

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.

E TH I C A L R E V I E W
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.
The manuscript has not been published previously (partly or in full) or submitted for publication elsewhere.