Application of Oregano Essential Oil Against Salmonella Enteritidis in Mayonnaise Salad

The aim of this study was applying oregano essential oil (OEO) from the Mediterranean reg ions, rich in Carvacro l and traces of p-Cymene and γ-Terpinene like prevention strategy against Salmonella Enterit idis (SE), controlling its mult iplication in vegetable salad prepared with mayonnaise. An overnight culture at 30°C with 104CFU/mL of SE ATCC 13076 was inoculated in the salad containing mayonnaise and 0.2% (v/v) of OEO. A survival curve of SE in the salad under refrigeration (8°C) and at room temperature (30°C) after 0, 1, 2, 4, 8, 24 hours of incubation was done. A decrease in the multip licat ion rate of SE in the mayonnaise salad with OEO was observed. There was a reduction of > 0.5 log until 4 hours at 30°C and until 24 hours at 8°C in relation to the control. Our data suggest that OEO provides additional p rotection able to increase the safety of vegetable salad with mayonnaise contaminated with SE, but not for those subjected to temperature abuse. From Food Safety point of view this study has identified the use of OEO like natural antimicrobial ab le to reduce the SE growth, an enteropathogen associated with foodborne disease around the world in products derived from eggs such as mayonnaise.


Introduction
Foodborne Diseases (FBD) is caused by eating food that was contaminated by an infectious agent or a toxin produced by it [1]. According to the World Health Organization (WHO), 30% of people in industrialized countries suffer fro m FBD and in 2000 only at least two million people world wide died o f d iarrhea caused or not by Salmonella [2]; [3].
Salmonellosis, a FBD caused by ingestion of Salmonella sp. occupies a prominent position in the field of public health throughout the world for its characteristics of mo rbidity and, in particu lar, the difficu lty to be controlled. Salmonellosis is the third most commonly reported infectious disease in the United States, the first in Brazil (40.1% of bacterias) and the mo st frequ ent ly associat ed with hu man illness in t he Eu ro pean Un io n [4], [5], [6]. M any foods can co ntain Salmonella and the most often related are raw meats, poultry, eggs, milk, dairy products and foods without heat treatment [7]. In the United States, salmonellosis is the cause of about 1.3 million FBD, with appro ximately 15,000 hospitalizations and 500 deaths per year, corresponding to 15.1% of the in cidents fo llo wed by campy lobacterios is (13.8%) and shigellosis (6,4%) [8], [9], [10].
Since the 1980's, these caused outbreaks are associated with the consumption of eggs, egg products and poultry products [11]. Salmonella Enteritid is (SE) is a relevant enteropathogen for the food industry, especially in foods that do not require heat treatment before consumption such as salads prepared with mayonnaise [12]; [6], [10]. Mayonnaise is an egg-based product often involved in outbreaks of food poisoning, since it is consumed on a large scale because it is part of the p reparation of salads, sauces and other dishes [13].
The mult iplication of microorganis ms is determined by the mayonnaise pH, the type and concentration of acidulant, time and storage temperature. The aqueous phase with a pH below 4.1 ensures the microbiological safety, but the risk of contamination by Salmonella sp. only decreases with the use of pasteurized eggs. Other ingredients such as antimicrobial activity with salt, garlic, sugar and mustard mayonnaise also contribute to be considered a more safe product. However, the mayonnaise becomes a problem for public health fro m the mo ment it is mixed with other ingredients for preparing salads because the pH increases and, usually, these salads are packed in unappropriated temperatures [13].
Several studies associated with control of SE in food have been developed in laboratory scale and some already applied at the level of industrial production to ensure food security. SE can be destroyed by heat treatment, but usually the salads with mayonnaise are not submitted to this type of treat ment.
This situation creates the need to use other methods for reduction or elimination of the pathogen within the concept of hurdle technology [14]; [2]; [15], [10].
The mult i-barrier technology can be defined as the application of conservation measures combined to increase the microbiolog ical stability and sensory quality of foods, as well as their economic and nutritional properties. The barriers most commonly used in food preservation are temperature as a factor ext rinsic and water act ivity (A w), acidity (pH), redo x potential, chemical and conservatives competing microorganisms like lactic acid bacteria (LA B) as intrinsic factors. In chilled foods is reco mmended to apply additional constraints to prevent the mu ltiplication of microorganis ms because the temperature of cooling is the biggest obstacle and in some cases, the only one. In case of temperature abuse occurrence in food distribution, that barrier is broken and there may be microbial mult iplication in this food [16][17] [15][18]; [19]. Natural antimicrobials can be used as additional barrier as alternative procedure to control SE in these foods but their application is limited by the strong aroma that can negatively affect the sensory characteristics of the product. Govaris et al [20] analysed the antimicrobial effect of OEO against SE at 0.6 or 0.9% in minced sheep meat during storage at 4° or 10℃ for 12 days. At 4ºC, the population of SE in samples treated with OEO at 0.9% were kept below 1 log cfu/g [20]. Sensory evaluation showed that the addition of OEO at 0.6 or 0.9% in minced sheep meat was organoleptically acceptable [20]. However, the problem of unacceptable sensory characteristics can be reduced if used with other methods of preservation by hurdle technology. Whereas acidic nature of mayonnaise (pH 3.5) imp roves the ability of essential oils to penetrate the bacterial cell membrane and its application may be an additional protection to increase the safety of commercial or ho memade mayonnaise salad, often subjected to temperatures higher than reco mmended [21]. Th is study aims to evaluate the antimicrobial activity of essential oil of oregano on Salmonella Enteritidis in mayonnaise salad refrigerated and subjected to temperature abuse.

Practical Proceedi ngs to Eval uate the Anti microbial Acti vi ty of Oregano Essential Oil
Oregano essential oil (fro m Mediterranean zone) added on mayonnaise with 0.2% (v/v) final concentration [22].
Salmonella Enteritidis ATCC 13076 was maintained under refrigeration in Tryptic Soy Agar (TSA) (Oxo id). The inocula was resuspended in Brain Heart Infusion broth (BHI) (Oxo id) and incubated at 30℃ for 18 hours.
To prepare the mayonnaise were used ingredients in the formulat ion of a standard commercial mayonnaise: soybean oil, sodiu m chloride (NaCl), vinegar 4%, Ethylenediamine tetraacetic acid (EDTA) and emu lsifier, as Table 1.
Four types of emulsifier were tested: egg powder, egg powder, pasteurized egg yolk liquid, which had a quality certificate indicating the absence of Salmonella sp. Mayonnaise was prepared in laminar flo w, with sterilized equipment and solutions. The emulsifier (o r egg yolk) was incorporated aseptically to the liquid phase consisting of vinegar, aqueous solutions of NaCl and EDTA at 4℃. After mixing these ingredients, soybean oil was slowly added to the aqueous phase under stirring in sufficient quantity (~ 70%) to form a stable emulsion. In this procedure was used a domestic " mixer" (Robot Classic -Mallory), prev iously cleaned with alcohol 70%.

Determination of Antimicrobial Acti vity
To carry out the antimicrobial activ ity, 25 mL of culture in saline solution containing SE 10 4 UFC/ mL were inoculated with 225g Selection o f Canned Vegetables (Carrefour SA) and mixed with 75g of mayonnaise containing 0.2% OEO. The procedure was repeated with mayonnaise without OEO, called "control sample". Each o f the mixtures was divided into twelve sterile bags in aliquots of 25g, six of wh ich were stored at 8℃ and six to 30℃. At 0, 1, 2, 4, 8 and 24 hours, one of the bags was subjected to three decimal serial dilutions, wh ich were plated on Xy lose Lysine Deo xycholate agar (XLD) surface, in duplicate. After 24 hours incubation at 30℃, it d id a count of colonies on the p lates of XLD and the results were converted to log CFU/g of mayonnaise.
The pH and Aw were measured in simple mayonnaise and mayonnaise mixed with salad vegetables. All experiments were analy zed three times, expressing the results as averages of three replications. For each batch of mayonnaise prepared, the pH and Aw were monitored.

Results
The mayonnaise prepared with the four emu lsifiers described in 2.3 item varied in emulsion stability as well as in pH during storage for 15 days under refrigeration. These data are shown in Table 2. Given these characteristics, the mayonnaise used in the development of the research that has been formulated with Salmonella Enteritidis in M ayonnaise Salad egg yolk powder, pH increased to 4.2, allo wing the mu ltip licat ion of SE. Table 3 and Figure 1 show the survival of SE in vegetable salad with mayonnaise containing 0.2% of OEO during storage under refrigerat ion (8℃). Table 3. Mean scores of Salmonella Enteritidis in vegetable salad with mayonnaise control and vegetable salad with mayonnaise containing 0.2% essential oil of oregano and the brand retained the 8℃ Figure 1. Survival of the SE vegetable salad with mayonnaise control and vegetable salad with mayonnaise containing 0.2% essential oil of oregano and the brand retained on 8℃. Table 4. Mean scores of Salmonella Enteritidis in vegetable salad with mayonnaise control and vegetable salad with mayonnaise containing 0.2% oregano essential oil of the brand and maintained at 30℃     Table 4 and Figure 2 show the survival of SE in vegetable salad with mayonnaise containing 0.2% of OEO during storage at 30℃.
The results indicated that the reduction in the count of SE in the vegetable salad with mayonnaise containing 0.2% OEO was less than 0.5 log CFU / g for a period of 24 hours at 8℃ and for a period of 4 hours at 30℃ co mpared to the control (Figures 1 and 2). It is important to observe that pH of mayonnaise and Aw increased with the addition of salad vegetables ( Table 5), indicating that the microb iological safety of mayonnaise is reduced when it is mixed with other ingredients, allo wing microbial mu lt iplication [23] [11].
Thus, the use of mayonnaise containing OEO for preparing vegetable salad with mayonnaise constitutes an additional hurdle to the mult iplication of SE, although the difference is less than 0.5 log CFU / g between product with and without OEO.

Discussion
There are few similar studies in scientific literature for a comparison of these results with those obtained in this study. Researchers [24] tested the survival of Salmonella Enteritidis inoculated into mayonnaise made with lemon juice (p H 4.4 to 4.5) supplemented with or without chitosan as antimicrobial and stored at 5 or 25℃ for 8 days. As a result, remained the same viability for 8 days at 5℃ in the absence of chitosan and decreased only 0.5 log CFU / g in the presence of chitosan.
Nu merous studies show that the in vitro effectiveness of antimicrobial agents is significantly reduced when tested in food model [24][25] [26][27] [28]. It is known that presence of lip ids, carbohydrates, protein, salt has influence about antimicrobial activ ity. Researchers [29] tested essential oils of clove and cinnamon against SE in cheese with lo w (16%) or high (30%) lip id content of both oils tested were mo re efficient in cheese with low fat. The result found by these researchers may be due to the fact that fat to form a protective barrier around bacteria or the possibility of lip id fraction to absorb the essential oil. Thus, it one can speculated that the mayonnaise is not a suitable product for the application of an essential oil as antimicrobial agent, as is a product with approximately 70% fat hindered the action of the OEO is dispersed in fat globules while SE mult iplies in the aqueous phase. Figure 3 show Su rvival curve of Salmonella Enterit idis in In mixed vegetable salad with mayonnaise handmade, the presence of 0.2% of OEO resulted in a reduction in the count of SE, thus becoming an effect ive barrier to inhib it the growth of the pathogen in the product.
Increased security by increasing the concentration of OEO in these products is not always possible, they may beco me sensory unacceptable. It follows therefore that in reducing the risk of infection by SE in food mayonnaise or mayonnaise-containing foods must be added other barriers besides the temperature and the addition of OEO as a natural antimicrobial.
As conclusion, it is important to observe that the use of biopreservatives (natural antimicrobials) is characterized as an additional barrier to the Good Manufacturing Practices (GMP) and HA CCP program (Hazard Analysis Critical Control Point"), fundamental to Food Safety.