Mentha longifolia Essential Oil and Pulegone in Edible Coatings of Alginate and Chitosan: Effects on Pathogenic Bacteria in Lactic Cheese

Mentha longifolia is a valuable medicinal and aromatic plant that belongs to Lamiaceae family. This study looked at the antibacterial effects of M. longifolia essential oil and pulegone in edible coatings made of chitosan and alginate on the growth of Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli in cheese. For this purpose, first fresh mint plant was collected from the cold region of Jiroft in Kerman province. Plant samples were dried in the shade at ambient temperature, and essential oil was prepared using Clevenger. The essential oil was analyzed by gas chromatography using mass spectrometric (GC/MS) detection. The major composition of M. longifolia oil was pulegone (26.07%), piperitone oxide (19.72%), and piperitone (11.88%). The results showed that adding M. longifolia essential oils and pulegone to edible coatings significantly reduced the growth of bacteria during storage. The bacterial population decreased by increasing the concentration of chitosan, M. longifolia, and pulegone in edible coatings. When the effects of pulegone and M. longifolia essential oils on bacteria were compared, it was found that pulegone had a stronger effect on bacterial population reduction. Coating treatments showed more antibacterial activity on E. coli than other bacteria. In general, the results of this research showed that alginate and chitosan coatings along with M. longifolia essential oil and its active ingredient pulegone had antibacterial effects against S. aureus, L. monocytogenes, and E. coli in cheese.


Introduction
Contaminated milk and dairy products are one of the important sources of infections and food intoxication in humans. One of the causes of contamination of dairy products is their suitable conditions for the growth of many microorganisms. In cheese, depending on the preparation stages and ripening condition, there is a possibility of contamination with various spoilage and disease-causing bacteria, including E. coli, S. aureus, L. monocytogenes, etc. E. coli is part of the natural intestinal flora of all warm-blooded animals. The presence the objective of this study was to determine whether adding M. longifolia essential oil and pulegone to edible coatings made of alginate and chitosan would affect the growth of S. aureus, L. monocytogenes, and E. coli in cheese.

Chemical Compounds of M. longifolia Essential Oil
The M. longifolia essential oil compounds were identified using GC/MS. Table 1 shows the identified chemical compounds and the percentage of each component in the essential oil. According to the obtained results, 40 compounds were identified in M. longifolia essential oil, which comprised 97.8% of the total essential oil. The main compounds of the essential oil are pulegone (26.07%), piperitone oxide (19.72%), piperitone (11.88%), 1,8-cineole (8.21%), cis-piperitone oxide (6.35%), and borneol (5.96%). As Table 1 shows, M. longifolia essential oil is mainly composed of monoterpene compounds (pulegone, piperitone oxide, and piperitone). Descriptive statistics related to chitosan, pulegone, and essential oil in E. coli, S. aureus, and L. monocytogenes are presented in Tables 2-7. Based on the values of standard deviation, minimum, maximum, and mean, The 30th day and The first day had the least and the most variation, respectively. The results showed that the treatments under different concentrations have different characteristics.     According to Table 8 findings, the E. coli population in cheese reduced when storage period, chitosan concentration, and essential oil levels were increased. At the conclusion of the storage time, the lowest number of bacteria related to the treatment contained 10% chitosan and 150 ppm peppermint essential oil. On all days of storage, the control showed the greatest level of bacterial population. Table 8. Effect of chitosan and essential oil on E. coli (cfu/g) in cheese during storage.

Treatments
The Pearson's correlation coefficients are presented in Figure 1. The results showed that The first day with The 20th day (0.83927 *), The 30th day (0.97960 **), and The 20th day with The 30th day (0.73614 *) had the most positive and significant correlation.  The results of Table 9 show that with increasing pulegone concentration, the number of E. coli bacteria in cheese decreased. As the storage period increased, the population of E. coli in cheese samples coated with pulegone and chitosan decreased. On the first and

The Effect of Various Concentrations of Chitosan and Pulegone on E. coli (cfu/g) in Cheese during Storage
The results of Table 9 show that with increasing pulegone concentration, the number of E. coli bacteria in cheese decreased. As the storage period increased, the population of E. coli in cheese samples coated with pulegone and chitosan decreased. On the first and 20th days of storage, increasing the amount of chitosan from 5 to 10% in the coatings did not have a significant effect on the bacterial population. At the conclusion of the storage time, the control and treatment coated with 10% chitosan and 50 ppm pulegone had the greatest and lowest populations of bacteria, respectively. Table 9. Effect of chitosan and pulegone on E. coli (cfu/g) in cheese during storage.

Treatments
The Pearson's correlation coefficients are presented in Figure 2. The results showed that The first day with The 20th day (0.99118 **), The 30th day (0.96251 **), and The 20th day with The 30th day (0.97579 **) had the most positive and significant correlation. Pearson's correlation coefficients are presented in Figure 2. The results showed that The first day with The 20th day (0.99118 **), The 30th day (0.96251 **), and The 20th day with The 30th day (0.97579 **) had the most positive and significant correlation.  Table 10, it can be concluded that the population of S. aureus in cheese reduced with longer storage times in all treatments. At the conclusion of the storage time,

The Effect of Various Concentrations of Chitosan and M. longifolia Essential Oil on S. aureus (cfu/g) in Cheese during Storage
According to Table 10, it can be concluded that the population of S. aureus in cheese reduced with longer storage times in all treatments. At the conclusion of the storage time, the lowest number of bacteria related to the treatment contained 10% chitosan and 150 ppm essential oil. The treatment coated with 5% chitosan without essential oil had the most bacteria population, but there was no significant difference between this treatment, control and the treatment coated with 10% chitosan without essential oil (p > 0.05). By increasing the amount of essential oil and chitosan in the coatings, the population of S. aureus in the cheese samples decreased. Table 10. Effect of chitosan and essential oil on S. aureus (cfu/g) in cheese during storage.

Treatments
The Pearson's correlation coefficients are presented in Figure 3. The results showed that The first day with The 20th day (0.97726 **), The 30th day (0.89521 **), and The 20th day with The 30th day (0.91058 *) had the most positive and significant correlation.  Pearson's correlation coefficients are presented in Figure 3. The results showed that The first day with The 20th day (0.97726 **), The 30th day (0.89521 **), and The 20th day with The 30th day (0.91058 *) had the most positive and significant correlation. The results of Table 11 show that by increasing the concentration of chitosan and pulegone in coatings, the number of S. aureus bacteria in cheese decreased. At the

The Effect of Various Concentrations of Chitosan and Pulegone on S. aureus (cfu/g) in Cheese during Storage
The results of Table 11 show that by increasing the concentration of chitosan and pulegone in coatings, the number of S. aureus bacteria in cheese decreased. At the conclusion of the storage time, the smallest and the largest of the bacterial count were observed in the treatment coated with 10% chitosan and 50 ppm pulegone and the control, respectively. Increasing the storage period led to a decrease in the bacterial population in all treatments. Table 11. Effect of chitosan and pulegone on S. aureus (cfu/g) in cheese during storage.

Treatments
The Pearson's correlation coefficients are presented in Figure 4. The results showed that The first day with The 20th day (0.99442 **), The 30th day (0.94941 **), and The 20th day with The 30th day (0.97392 **) had the most positive and significant correlation. conclusion of the storage time, the smallest and the largest of the bacterial count were observed in the treatment coated with 10% chitosan and 50 ppm pulegone and the control, respectively. Increasing the storage period led to a decrease in the bacterial population in all treatments. Pearson's correlation coefficients are presented in Figure 4. The results showed that The first day with The 20th day (0.99442 **), The 30th day (0.94941 **), and The 20th day with The 30th day (0.97392 **) had the most positive and significant correlation.

The Effect of Various Concentrations of Chitosan and M. longifolia Essential Oil on L. monocytogenes (cfu/g) in Cheese during Storage
The results of Table 12 show that the number of L. monocytogenes in cheese decreased by increasing the storage period in all treatments. At the end of the storage time, the highest amount of bacterial count was observed in the control, which had no significant difference with the treatment coated with 10% chitosan without essential oil. By increasing the essential oil concentration in the coatings, the number of L. monocytogenes decreased. Increasing the concentration of chitosan also led to a decrease in the bacterial population. Pearson's correlation coefficients are presented in Figure 5. The results showed that The first day with The 20th day (0.98381 **), The 30th day (0.87148 **), and The 20th day with The 30th day (0.85096 *) had the most positive and significant correlation.  Table 13's results show that at the conclusion of the storage period, the treatment coated with 10% chitosan and 50 ppm pulegone had the lowest number of bacteria, which had no significant difference with the treatments coated with 10% chitosan and 25 ppm  Table 13's results show that at the conclusion of the storage period, the treatment coated with 10% chitosan and 50 ppm pulegone had the lowest number of bacteria, which had no significant difference with the treatments coated with 10% chitosan and 25 ppm pulegone and the treatment coated with 10% chitosan and 10 ppm pulegone (p> 0.05). Over the course of all storage times, the control treatment had the highest bacterial count. On the first and 20th day, at 5% and 10% chitosan concentrations, increasing the amount of pulegone in the coatings did not have a significant effect on the bacterial population. On the 30th day, in the concentration of 10% chitosan, increasing the pulegone concentration from 10 to 50 ppm did not have a significant effect on the number of L. monocytogenes. Table 13. Effect of chitosan and pulegone on L. monocytogenes (cfu/g) in cheese during storage.

Treatments
The Pearson's correlation coefficients are presented in Figure 6. The results showed that The first day with The 20th day (0.96618 **), The 30th day (0.84423 *), and The 20th day with The 30th day (0.93431 **) had the most positive and significant correlation. Pearson's correlation coefficients are presented in Figure 6. The results showed that The first day with The 20th day (0.96618 **), The 30th day (0.84423 *), and The 20th day with The 30th day (0.93431 **) had the most positive and significant correlation.

Material and Methods
Materials used in the study were S. aureus ATCC 29213, and L. monocytogenes

Material and Methods
Materials used in the study were S. aureus ATCC 29213, and L. monocytogenes

Collection of Plants and Production of Essential Oils
After the scientific confirmation of the species by plant science experts at University of Jiroft Herbarium, M. longifolia was taken in May 2021 from a cold area of Jiroft City. The aerial part of this plant was dried in the shade and ambient temperature. After drying, the plant was ground. About 100 g of dried sample was placed in 400 mL distilled water and submitted to hydrodistillation for 3 h using a Clevenger-type apparatus [24]. The yield of essential oil extraction from M. longifolia was 1%(v/w), and it was colorless. Gas chromatography (Shimadzu single quadrupole GCMS-QP2010 SE, Kyoto, Japan) equipped with a mass spectrometer (GC-MS) was used to determine the chemical composition of M. longifolia essential oil. Compounds were separated on HP-5 MS capillary column (30 m × 0.25 mm, film thickness 0.25 µm; Little Falls). A sample of 1.0 µL was injected in the split mode with split ratio 1:100. Helium was used as a carrier gas at a flow rate of 1.0 mL/min. The injection temperature was 230 • C. Compounds were further identified and authenticated using their complete mass fragmentation data compared to the NIST02.L and WILEY7n.L mass spectral libraries and published mass spectra and, wherever possible, by coinjection with authentic standards [24].

Preparation of Cheese
Lactic cheese samples were prepared at Pegah Dairy Company of Jiroft. The steps were as follows: First, raw milk was standardized (the amount of fat (3%) and dry matter (15%) was adjusted). Then the milk temperature rose to 96 • C. A mixture of sour yogurt and vinegar was added to the hot stirring milk until the milk was completely coagulated. The clot was cut, drained, and poured into plastic molds, and within 5 h, the molds were returned to complete dehydration. Cheese pieces were placed in salt solution (16%) at 5 • C, and after 72 h, the relevant tests (pH, moisture content, salt content) were performed on them [16]. The pH, moisture, and salt content of the cheese samples were 5.5, 65, and 4%, respectively [25].

Treatment of Cheese Samples
The cheese samples were cut into cubes (length, width, and height of 3 cm) and coated by immersion method, during which the samples were immersed in the coating mixtures for 1 min until all surfaces of the cheese samples were completely covered with the coating material. Coating mixtures were sodium alginate solution (25%) with concentrations of 0, 5, and 10% chitosan and different concentrations of M. longifolia essential oil (0, 100, and 150 ppm) as well as different concentrations of pulegone (10, 25, and 50 ppm). The samples were then placed in an incubator (Fan Azma Gostar, Tehran, Iran) under controlled temperature and humidity (about 12 • C and relative humidity of 85%) for approximately 8 h until the coatings were dry [26].
In the pre-test, we used higher concentrations of M. longifolia essential oil and pulegone in the coatings, and a sensory evaluation test was also performed. The maximum concentration of M. longifolia essential oil and pulegone was determined according to the results obtained from the sensory evaluation. By increasing the concentration more than 150 ppm for M. longifolia essential oil and 50 ppm for pulegone, the flavor and taste scores decreased. In this way, the studied concentrations were determined for essential oil and pulegone.

Inoculation of the Desired Bacteria into Cheese Samples
To inoculate the bacteria (S. aureus, L. monocytogenes, and E. coli O157) into the cheese texture, bacterial suspensions containing 10 5 CFU/g were injected into 8 points of cheese samples with a sterile syringe. After that, the samples were placed in polypropylene containers and kept at 5 • C [27]. Sampling and culture were performed once every 10 days for 1 month.

Bacterial Count in Cheese Samples
In order to count the bacteria, 1 g of the cheese sample was thoroughly homogenized in 9 mL of physiological saline. Then, 0.1 mL of this solution was cultured on specific media for each bacterium.

Statistical Analysis
Analysis of variance (ANOVA) was used with the Statistix ver. 10 software to see whether there were any significant differences between the results. Differences at p ≤ 0.05 were considered significant. All experiments were performed in triplicate.

Discussion
Cheese is a ready-to-eat food product that is not subjected to any other treatment to ensure its safety before consumption. Contamination of cheese with foodborne pathogens may occur in several stages (before production, during production, and during storage period). Therefore, information on the main sources of pathogens and the mechanisms by which they infect the dairy chain is needed if contamination of any cheese is to be prevented [31]. The use of different additives in cheese can partially inhibit its bacterial population. Essential oils, extracts, and powders of herbs are compounds that can be added to cheese to reduce microbial contamination and increase sensory properties. In this study, the effect of an edible coating containing alginate, chitosan (0, 5, and 10%), M. longifolia essential oil (0, 100, and 150 ppm), and pulegone (0, 10, 25, and 50 ppm) on the growth of E. coli, S. aureus, and L. monocytogenes in cheese was examined during three storage times. In general, by increasing the storage period (30 days) in all treatments, the number of bacteria in the cheese decreased. This decrease was more pronounced in treatments with increasing concentrations of chitosan, essential oil, and pulegone. The effect of the studied treatments on reducing the growth of E. coli was greater than the other two bacteria. In general, the pulegone active ingredient was more effective in reducing the growth of bacteria than the M. longifolia essential oil.
Studies have demonstrated the antibacterial properties of chitosan, M. longifolia essential oil, and the pulegone active component. Evaluation of the antimicrobial effects of M. longifolia essential oil against E. coli, S. aureus, and Candida albicans showed that the pulegone and 1,8-cineole compounds are important in this regard [32]. In our previous study on the same essential oil, the compounds of piperitenone oxide (26.07%), pulegone (19.72%), piperitenone (11.88%), and 1,8-cineole (8.21%) were the major compounds present in the essential oil that can play a very important role in its antimicrobial activity [33]. M. longifolia essential oil has been found to have strong antibacterial properties against a variety of bacteria, including Staphylococcus, Pseudomonas, Bacillus, and E. coli, as well as some fungal strains including Aspergillus, Fusarium, and Penicillium [34].
It has been reported that one of the key characteristics of essential oils and active substances is the hydrophobic property, which led to the change and destruction of the cell membrane structure and their greater permeability. This is concerning the action of these substances and their compounds in the death of pathogenic bacteria. The result is that the majority of the ions and other essential components of the cell leak out, which ultimately causes the bacterium's death [35]. It will lead to defects in the synthesis of many cell-wall polysaccharide compounds, and inhibit cell growth and morphogenesis [36]. The antimicrobial performance of essential oils in vitro depends on various factors such as antimicrobial components, type of microorganism, culture medium, amount of inoculum, pH, temperature, and food composition [37,38]. It was related [39] that, in a study on the antibacterial effect of M. longifolia essential oil against several foodborne pathogens, pulegone, 1,8-cineole, and menthofuran were the most prevalent constituents of essential oils. Their results also showed that the most sensitive bacterium to M. longifolia essential oil was E. coli, which is consistent with our results. In another study, it was found that M. longifolia from the mint family had antibacterial properties on Staphylococcus and Listeria species. The results of this study similarly confirm the results of our study [40]. Studies have shown that the effect of different essential oils is concentration-dependent so that in low concentrations, phenolic compounds act on the enzymatic activity, particularly those involved in energy generation, but in high concentrations these can cause protein denaturation [41]. In one study, the antibacterial effects of pulegone and 1,8-cineole against S. aureus and Salmonella typhimurium were investigated, and the results showed that 1,8cineole has a stronger antibacterial effect on Gram-positive bacteria than Gram-negative bacteria, while pulegone has a higher antibacterial effect on Gram-negative bacteria [42], which is consistent with the results of the present study.
Comparing the results of M. longifolia essential oil and pulegone as the effective ingredient and the main composition of M. longifolia essential oil, it was found that this substance had more antibacterial properties than the complete essential oil in much lower concentrations. Pulegone is a monoterpene ketone found in the leaves and flowers of several members of the mint family [16]. Terpenes are capable of penetrating the bacterial cell wall, leading to the denaturation of proteins and disintegration of the cell membrane, leading to cytoplasmic leakage, cell lysis, and eventually cell death [43]. Based on the published reports, pulegone can effectively destroy S. aureus, S. typhimurium, and E. coli [44].
The antibacterial effects of trans-cinnamaldehyde, 1,8-cineole, and pulegone against Streptococcus equi subsp. equi were investigated [45]. According to the results, transcinnamaldehyde, 1,8-cineole, and pulegone possess antibacterial capabilities and may serve as a convenient and reasonably priced alternative to synthetic antibiotics. According to the results of the current study, chitosan in edible coatings inhibited bacterial growth as compared to the control, and antibacterial activity improved by increasing chitosan content.
In general, the antimicrobial activity of essential oils is expressed by several mechanisms:

1.
Dissolving in the cytoplasmic membrane and interfering with the protein structure of the enzyme and destroying the microorganism; 2.
Interruption in activities related to succinate and reactions related to NADH; 3.
Disturbing electron transfer in the respiratory chain; 4.
Creating a break in oxidative phosphorylation.
The lipophilic property of essential oils can explain its increased membrane permeability or destruction due to the activity of enzymes in the cell membrane such as protein kinase [46].
Chitosan's polycationic composition is thought to be the source of its antibacterial properties. The protonated amino group in chitosan interacts electrostatically with the negative residues on cellular surfaces to achieve antibacterial activity [47]. With increasing levels of deacetylation, chitosan contains more protonated amino groups, which affects its antibacterial efficacy [48]. The growth of aerobic bacteria can be inhibited by coverings made of chitosan. Chitosan coatings keep oxygen away from pathogenic bacteria because they block air penetration [49,50]. The antibacterial properties of chitosan film enhanced with oregano and thyme essential oils have been investigated [51]. Their results showed that even at the lowest concentrations, the chitosan film with essential oils may suppress bacterial and fungal development. In comparison to the doses required to stop the de-velopment of the beneficial bacteria of Lactobacillus rahmnosu and Enterococcus faecium, all antimicrobial agents' MIC and MBC against E. coli and S. aureus were extremely low.
In some studies [52][53][54], the antibacterial effectiveness of chitosan as an edible coating against L. monocytogenes on the surface of ready-to-eat roast beef was examined. Results showed that L. monocytogenes on the surface of roast beef may be controlled by chitosan coatings. In [55], the effects of an edible chitosan coating on the quality and shelf life of sliced mango fruit were investigated. Mango slices were exposed to aqueous solutions containing 0%, 0.5%, 1%, and 2% chitosan. The results showed that coating with chitosan efficiently stopped the growth of microorganisms.

Conclusions
In this research, the impact of edible coatings of alginate and chitosan along with M. longifolia essential oil and the active ingredient of pulegone on the growth of some pathogenic bacteria in lactic cheese was investigated. The results generally demonstrated that the population of investigated microorganisms decreased by increasing the concentration of chitosan, M. longifolia, and pulegone in edible coatings. When the effects of pulegone and M. longifolia essential oils on bacteria were compared, it was found that pulegone had a stronger impact on bacterial population reduction. Coating treatments showed more antibacterial activity on E. coli than other bacteria. From this study, it can be concluded that chitosan coating along with M. longifolia essential oil and its active ingredient pulegone had antibacterial effects against S. aureus, L. monocytogenes, and E. coli in cheese. As a result, it can be utilized as a strong and natural food preservative.