Evaluation the Antibacterial Activity of the Brine, Nisin Solution, and Ozonated Water Against E. coli O157:H7 in the Experimentally Local Produced Soft Cheese

This study aimed to evaluate the antibacterial efficacy of brine solution in compared to nisin and ozonated water treatments against E. coli O157:H7 in experimentally local produced soft cheese (ELPSC). A total of 25 raw cow milk samples were collected from farmers and local markets in Baghdad city and utilized to manufacturing 25 of ELPSC samples following the traditional procedure in rural Iraq without salting or heat treatment. Bacteriological analysis for potential E. coli O157:H7 contamination was performed and confirmed by cultural, biochemical, and serological tests. Antibacterial activity of brine solution (7% w/v), nisin solution (100 IU/mL), and ozonated water (0.6 ppm) was performed for positive samples after 2 h contact time at ambient temperature. Positive isolates of E. coli O157:H7 were detected and confirmed as 3 out of 25 cheese samples (12%), with initial average bacterial count of 6.146 log10 CFU/g. The initial bacterial count was significantly (P<0.05) reduced to 3.778, 4.380 and 4.544 log10 CFU/g when subjected to brine salting, nisin solution, and ozonated water, respectively. Cheese samples exposed to brine had a significantly (P<0.05) higher reduction in the bacterial growth than those exposed to nisin or ozonated water treatments for each alone. More effective reduction in E. coli O157:H7 bacterial growth was not observed when brining exposure time elongated at ambient temperature to 6 h. These findings may be useful in raising the public knowledge about the use of brine solution as a domestic antibacterial approach for minimizing the possible risk of E. coli O157:H7 contamination in the local produced soft cheese.


INTRODUCTION
o enhance food quality and extend shelf life, different preservation techniques are designed to control the growth of undesirable pathogenic microorganisms. These techniques include heating, salting, freezing, drying, and fermentation process (1). Cheeses are ready-to-eat food without any heat treatment before consumption (2). Many enteric pathogens can multiply in dairy products made from unpasteurized raw milk, which are highly marketable and the most common route for transmission of these pathogens from farmer dairy animals to humans (3).
Numerous studies have reported that both pathogenic and non-pathogenic strains of E. coli bacteria can contaminate the cheese products (2). E. coli is found in the gastrointestinal tract of man and animals, but some species are known as pathogenic such as E. coli O157:H7. This pathogenic microorganism can cause many serious clinical symptoms such as bloody diarrhea, fever, and hemolytic uremic syndrome which could lead to death in both children and elderly (4). Hurdle technology, when two or more preservation methods are used simultaneously, can

A B S T R A C T
This study aimed to evaluate the antibacterial efficacy of brine solution in compared to nisin and ozonated water treatments against E. coli O157:H7 in experimentally local produced soft cheese (ELPSC). A total of 25 raw cow milk samples were collected from farmers and local markets in Baghdad city and utilized to manufacturing 25 of ELPSC samples following the traditional procedure in rural Iraq without salting or heat treatment. Bacteriological analysis for potential E. coli O157:H7 contamination was performed and confirmed by cultural, biochemical, and serological tests. Antibacterial activity of brine solution (7% w/v), nisin solution (100 IU/mL), and ozonated water (0.6 ppm) was performed for positive samples after 2 h contact time at ambient temperature. Positive isolates of E. coli O157:H7 were detected and confirmed as 3 out of 25 cheese samples (12%), with initial average bacterial count of 6.146 log10 CFU/g. The initial bacterial count was significantly (P<0.05) reduced to 3.778, 4.380 and 4.544 log10 CFU/g when subjected to brine salting, nisin solution, and ozonated water, respectively. Cheese samples exposed to brine had a significantly (P<0.05) higher reduction in the bacterial growth than those exposed to nisin or ozonated water treatments for each alone. More effective reduction in E. coli O157:H7 bacterial growth was not observed when brining exposure time elongated at ambient temperature to 6 h. These findings may be useful in raising the public knowledge about the use of brine solution as a domestic antibacterial approach for minimizing the possible risk of E. coli O157:H7 contamination in the local produced soft cheese.

KHUDHIR ZS
be used to limit the growth and survival of pathogenic microbes in food (5).
Nisin is a polycyclic antibacterial peptide of 34 amino acids, including the uncommon residues such as lanthionine, methyllanthionine, produced by some strains of Lactococcus lactis subsp. lactis (6). According to the US Food and Drug Administration (US FDA), nisin is generally recognized as safe (GRAS) for use as a preservative in processed cheeses (Federal Register: 53 FR 11247, April 6, 1988). Currently, this bacteriocin is licensed in many countries as a natural food biopreservative mainly for dairy products (6). Antibacterial activity of nisin against Grampositive bacteria is superior to that of Gram-negative bacteria; nisin is difficult to penetrate through the outer membrane of Gram-negative bacteria, this may result to low activity of nisin against these microorganism (7).
Ozone is a powerful oxidizing substance composed form oxygen gas which is quickly gives up nascent oxygen molecule, used as antimicrobial agent to kill bacteria, fungi and inactivated the viruses (8). Ozone regarded as a promising preservative method with no chemical-residues and powerful antibacterial agent specially in the food industry, water treatment, and medical uses, as the most Gram-positive and -negative bacteria, spores of bacteria, fungi, spores of fungi, viruses, and protozoans are vulnerable to ozone (10).
Brining method is immersing the cheese completely inside the sodium chloride solution. Brined soft cheeses are commonly consumed in rural and urban locations throughout the Middle East, including Iraq. Local soft cheese in Iraq is manufactured by raw bovine milk that is coagulated by a commercially available rennet and usually not exposed to heat treatments. Because local soft cheese is produced following traditional methods, there is a greater risk of infection with pathogenic food-borne bacteria. The main objective of this study was to evaluate the effect of brining process under ambient storage temperature to recommend the most suitable salting temperature/time for improving the quality of the locally produced soft cheese compared to nisin solution and ozonated water as other preservative methods to hinder the survival of pathogenic E. coli in this kind of local product that made in the rural areas in Iraq.

Soft Cheese Manufacturing
A total of twenty-five fresh raw cow milk samples (three liters each) were obtained from farmer houses and local markets in Baghdad city and immediately transferred in cooling using an icebox to the Milk Hygiene Laboratory, Department of Veterinary Public Health, College of Veterinary Medicine, University of Baghdad, Iraq. Experimentally local produced soft cheese (ELPSC), 25 samples, was manufactured according to the traditional procedure used in Iraqi's rural areas. Without salting or heat treatment, a commercial rennet powder was blended with sterile water and added to the milk. The milk was allowed to coagulate for 40 to 60 minutes before being squeezed through sterile cotton cloths to remove the whey from the cheese.

Antibacterial Activity
The antibacterial activity of different solutions was evaluated after immersing the cheese samples in the nisin solution that prepared from commercial standard stock solution (100 mL, Sigma), 2.5% (w/w) of nisin was prepared sterilized by Millipore filter paper (No. 0.02 μm) then transferred into 1000 mL of sterile distilled water (12).
The final concentration of antibacterial solutions of nisin, brine solution, and ozonated water were 100 IU/mL (7% w/v) 7 g of sodium chloride in the sterile distal water and 0.6 ppm respectively with contact time for 2 h at ambient temperature (~30 °C). All the solutions used in this study were prepared daily as demand.
Viability of E. coli O157:H7 was evaluate in the experimentally local produced soft cheese as one hundred g of each cheese samples was placed into three closed plastic containers that were filled with (7% w/v) brine solution, nisin solution at 100 IU/mL, and dipping the cheese pieces in the third container during the bubbling by the ozonated water (aqueous solutions) at 0.6 ppm by insert the stone of ozone generator inside the plastic container containing 1000 mL of sterile distilled water, all the containers were maintained at ambient conditions before and after each of the above treatments, the plastic containers fleshed by ozone as disinfected method.

Ozone Equipment
Ozone was generated using Aqua-6 Ozone Generator a commercially available from A2Z, Ozone Inc., USA, with generation limit between 0-0.6 and 0.6-3 ppm. The ozone concentration that used in this current study was at 0.6 ppm with contact time for 2 h at ambient temperature, all the containers that used in the current study were disinfected by flushed with the ozonated water and dried well before each treatment.

Microbiological Assay
After immersion of cheeses samples at ambient temperature in the different antibacterial solutions, each of cheese pieces were sampled for bacteriological analysis, the bacteria that selected in the current study was E. coli O157:H7, bacterial contamination were determined by using the spreading plate assay with triplicates (11). The cheese samples were cut by a sterile knife to smaller pieces and weighed (100 g) then cheese pieces (10 g) were homogenized in the sterile polyethylene lab stomacher bags for 5 min with 90 mL mixture of warmed (40-45 °C) sodium citrate (2%), and sterile peptone water (0.1%) by using the Laboratory Stomacher to provide the dilution of 10 -1 , tenfold serial dilutions were made (10 -2 to 10 -7 ) precisely and 0.1 mL of 10 5 and 10 6 dilutions was spread on the surface of the selective culture media (Chromo-agar™ E. coli O157:H7, Paris). The isolates were subjected to the biochemical and serological tests such as Gram stain, potassium cyanide (KCN) test, indole, catalase, oxidase, and motility. Additionally, the E. coli O157:H7 latex test (Remel) was performed serologically for both O157 and flagellar antigen ( Table 1). All analysis were conducted in triplicate.

Statistical Analysis
Data were statistically analyzed using the SPSS software version 22.00 (IBM SPSS Inc., Chicago, IL, USA). Data were subjected to one-way ANOVA and means deemed significant were separated by Fisher's least significant differences (LSD) at P≤0.05. Data were illustrated as the means of three replicates with standard error (Mean±SE). The bacterial count results were converted to log10 CFU/g and analyzed.

RESULTS AND DISCUSSION
The percentage of E. coli O157:H7 that isolated from the soft cheese samples was 12%; out of 25 examined samples, 3 were positive to cultural, biochemical, and serological tests ( Table 1). Results of antibacterial activity of different solutions against E. coli O157:H7 in the ELPSC that immersed inside the solutions at ambient temperatures at zero time (control) and after 2 h of exposure time are shown in Table 2. The initial average values of E. coli O157:H7count in the soft cheese samples was 6.146 log10 CFU/g. Bacterial population in the cheese samples decreased during the immersion process in the brine, nisin solution and ozonated water to 3.778, 4.380, and 4.544 log10 CFU/g, respectively. There was no significant (P>0.05) difference decrease in the bacterial population when compared the antibacterial activity of nisin solution and ozonated water.
Immersed the cheese pieces in the sodium chloride solution at concentration of (7 %w/v) for 2 h had significant (P<0.05) decrease in the bacterial viability compared to the nisin and ozonated water for each treatment alone as shown in (Table 2).
The average values of the viability of E. coli O157:H7 in the cheese samples that subjected to activity of brine solution (7 % w/v) after 2 h and 6 h of contact time at ambient temperature were 3.778 and 3.376 log10 CFU/g, respectively (Table 3).  There are many factors that influence the survival and multiplication of pathogenic microorganisms in cheese, such as acidity degree, type of preservatives, methods of preservation, water activity, temperature of storage competing flora, and salt levels (13). E. coli and Salmonella bacteria, for example, belong to the Enterobacteriaceae family, do not tolerate high salt concentrations (14). Raw milk is the crucial factor that contributing to cheeseassociated outbreaks, and local homemade cheese is a popular product usually made from raw cows' milk. This kind of unaged cheese is produced without salt addition and marketed in Iraq's rural and urban markets. Due to many factors interfering during the local cheese making process, such as traditional milking procedures, abuse temperatures during storage, and transportation to local cheese markets using equipment polluted from farm conditions, this local cheese is signified as a health risk for the people and farmers' families. All these variables could be deemed post-contamination stages, particularly in the case of this homemade product that is traditionally produced on farms and consumed without any heat treatments. The prevalence of E. coli O157:H7 in the ELPSC were 3 out of 25 samples (12%). This isolation percentage was high when compared with another study conducted in the Scotland on the twenty-eight samples of Artisanal farmhouse cheeses, the E. coli O157:H7 was not detected in this kind of cheese (15). Another study illustrated that the isolation percentage of E. coli was 62% from herby cheese samples subjected for sale in both of Van and Hakkari provinces, while E. coli O157:H7 was not detected from the same samples (16). Dairy products were implicated in seven outbreaks, four outbreaks from consuming raw milk while other three outbreaks were implicated as cheese, raw butter, and ice cream by post-contamination of these dairy products (16). The prevalence of E. coli O157:H7 in the soft cheese samples may attributed to the initial bacterial load of raw milk samples that collected from the rural area. Another study (17) illustrated that from 1998 to 2011 KHUDHIR ZS cheeses made from unpasteurized milk caused 11% of outbreaks of Shiga toxin-producing E. coli.
In the current study, the data revealed that the count of E. coli O157:H7 in the ELPSC was decreased from 6.146 to 3.778 and 3.376 log10 CFU/g, respectively, after 2 and 6 h of storage in the brine solution (Table 3). Due to increasing demand for natural and unpreserved foods many of people in all over the world consuming the unpasteurized products (18). Sodium chloride as brine solution can be used as familiar rural preservative for extends the shelf-life of local produce soft cheese, in the current study the cheese pieces were maintained in the sodium chloride solution as domestic antibacterial methods, E. coli O157:H7 isolates were sensitive to brining process (7 % w/v) for 2 h as short term contact time but without complete inhibition at this concentration, there was no increase in the inhibition level when the cheese dipping in the brine solution for longer exposure time (6 h ) at the same concentration. Previous studies revealed that increases in the cheese content of salt occurs faster in the first hours of salting process and slower after 24 h of the maintenance in the salt solution, the absorption of salt decreases with the increases of salting time, that may attributed to the decreases in the brine and the cheese moisture due to the differences in the salt content (19). Khudhir and Hammad (20) reported that the E.coli O157:H7 was insensitive to the antibacterial action of nisin at concentration of 50 IU/mL while E. coli O157:H7 became more susceptible to its action after subjecting to activation of lactoperoxidase system these result gives an indication that antibacterial potency of nisin can increase due to the synergistic activity of nisin with another natural inhibitor. Another study recorded that high nisin concentration (470 mM) alone can eliminate the viability E. coli O157:H7 (21).
Aqueous solution of ozone is used as disinfectant in the food applications specially for extend the shelf life many kinds of food (22). The activity of ozonated water as disinfection agent has been reported for food according to the type and texture of food, ozone concentrations and the contact time (23). The advantages of ozone treatment are usually proposed for various kinds of food, such as meat products as well as fresh vegetables and fruits. The half-life of ozone in distilled water at 20 °C is reported as 20 to 165 min (24). Because ozone is an unstable gas at room temperature, it quickly degrades. It is highly unstable when dissolved in the water and the solubility of ozone in the water is 13 times more than of oxygen at temperature 0-30 °C. Many studies illustrated that bactericidal activity of ozone was lost with the present of organic matter in water with increase in another substances includes dissolved organic carbon, amines, lipids, proteins, and carbohydrates (25).
The contamination with E. coli O157:H7 in the experimental soft cheese can be related to the contamination of local raw milk that used for made such this local product. Brining process (7% w/v for 2 h) was the most effective as demotic antibacterial solution for reducing E. coli O157:H7 counts in the soft cheese, with no significant increase in the antibacterial activity after longer exposure for 6 h at ambient temperature, followed by the nisin solution and ozonated water at 100 IU/mL, and 0.6 ppm, respectively. These results will be helpful to increase the people awareness to use brine solution at concentration 7% as an effective antibacterial washing method to reduce potential E. coli O157:H7 contamination facilitated by production local produced soft cheese.