Antibacterial activity of nisin , oregano essential oil , EDTA , and their combination against Salmonella Enteritidis for application in mayonnaise

Salmonella Enteritidis (SE) is one of the most important serovars associated with Salmonella gastroenteritis outbreaks in Brazil. the use of natural antimicrobials can be an alternative method of SE control. the antimicrobial effect of two oregano essential oils (oEo1 and oEo2) at 0.1%, 0.2%, 0.5%, 1.0%, or 2.0%; nisin (Nisaplin®) at 0, 6.25, 12.5, or 25 ppm; ethylenediaminetetraacetic acid (EDtA) at 0.0037%, 0.0056%, 0.0075%, 0.0110%, or 0.0150%; and their combination against SE in vitro was studied to be applied in mayonnaise and russian salad made with the same mayonnaise during storage at 8o or 30oC for 24 hours. OEO was very efficient against SE at all tested concentrations, while nisin and EDtA showed no effect against SE. ten volatile components were identified in the two OEOs using gas chromatography coupled to mass spectrometry with electron impact ionization, with carvacrol being the major component of both samples. oEo2, containing p-cymene (15.95%) and y-terpinene (6.90%), besides carvacrol (61.66%), resulted in larger inhibition zone than the other oEo (oEo1 don’t contains p-cymene or y-terpinene). Nisin in combination with oEo1 or oEo2 had an antagonistic effect at all concentrations. the presence of nisin caused a reduction in essential oil antimicrobial activity (p≤0.05). Sensory evaluation showed that consumers prefer 0.2% oEo in mayonnaise instead of 0.5% and 1.0% concentrations. thus, oEo only, at a concentration of 0.2%, was applied in mayonnaise against SE. the russian salad prepared with mayonnaise plus oEo at 0.2% (wt/wt) caused a reduction of SE when compared with the salad prepared with mayonnaise without oEo. these results indicate that the use of oEo as a biopreservative (natural antimicrobial) can enhance food safety, serving as an additional barrier in helping the good Manufacturing Practices and the Hazard Analysis Critical Control Point program, fundamental to food safety.


INTRODUCTION
Recognition of new food-borne pathogens has changed understanding of the epidemiology of food-borne diseases in the past 2 decades. Globally, bacterial pathogens that have gained prominence over the past 20 years include Salmonella Enteritidis (SE), Campylobacter spp., Escherichia coli O157:H7, and related verocytoxigenic spp. 1,2 . Since 1991, SE has been one of the most important serovars involved in Salmonella gastroenteritis outbreaks in Brazil 3 . While SE is mainly associated with eggs and poultry products, a wide range of other food items have been implicated as vehicles of SE infection in humans, including meat 4,1 .
There is a shortage of new methods to reduce or eliminate foodborne pathogens. The use of antimicrobials combined with existing methods, such as hurdle technology and controlling the numbers and growth of SE, thus remains an important objective for the food industry 5,6,7,8 .
The use of natural antimicrobial compounds such as bacteriocins and essential oils of edible plants has attracted interest from the food industry because, for centuries, indigenous plants have been used in herbal medicine to cure many diseases, including enteritis. Oregano essential oil (OEO), from the herb Origanum vulgare, has been shown to possess antimicrobial activities against pathogens including SE 9,10,11,12,13,14 .
Among bacteriocins, nisin, a lantibiotic class of bacteriocin produced by strains of Lactococcus lactis, has found practical application as a food preservative in a number of products.
However, the practical application of nisin is limited because of its low stability, reduced activity at high pH, and poor efficacy in certain food matrices. Nisin also has limited effect against Gram-negative bacteria 15,16,17 .
Bacteriocin in combination with essential oil can achieve good microbiological safety in food while decreasing the doses of each compound in the product, due to the ability of nisin and the essential oil to act on the cytoplasmic membrane, with additive or synergistic effect, allowing the application of both compounds at lower levels without diminishing their inhibitory effects. Therefore, combined use could be a strategy to overcome these restrictions and further extend the range of applications of nisin in food processing 12,15,16 .
Ethylenediaminetetraacetic acid (EDTA) is a chelator that can be used as food additive and can potentiate the effect of weak acid preservatives against Gram-negative bacteria (Ntzimani, Giatrakou, and Savvaidis, 2010). Treatment of Gram-negative bacteria with EDTA makes them sensitive to agents to which they are normally resistant. EDTA also increases the sensitivity of Gram-negative bacteria to nisin 18,19 .
The aim of this work was to study the antimicrobial activities of OEO, nisin, EDTA, and their combination against SE in vitro, to select the best antimicrobial agent for use in mayonnaise, as well as to evaluate the acceptability of mayonnaise and Russian salad with mayonnaise containing selected antimicrobial agents.

Culture conditions
Salmonella enterica subsp. enterica serovar Enteritidis American Type Culture Collection 13076 samples were stored in tryptone soya agar at 4ºC, resuspended in brain heart infusion (BHI) broth and incubated without agitation at 30ºC overnight.

Chemical analysis of OEO
The composition of both OEOs was determined by gas chromatography coupled to mass spectrometry with electron impact ionization (GC-MS-IE).
To identify the constituents of OEO1 and OEO2, dilutions of samples with hexane and 1 μL of each sample were injected automatically (ALS injector HP-1100; Hewlett Packard, Palo Alto, CA, USA) in split mode (100:1). Separation and analysis were performed with HP 5890 gas chromatograph II coupled to mass spectrometer (HP 5973 MSD) and EI at 70 eV, monitored by the ChemStation software. Helium was used as carrier gas with constant flow of 1 cm 3 /min. An HP-5 fused silica capillary column (30 m x 0.32 mm x 0.25 mm) was used, at temperatures of 250ºC for the injector and 250ºC for the interface between the chromatograph and the detector. The temperature cycle was initial column temperature of 40ºC for 2 min, increased at the rate of 5ºC/min up to 90ºC and then at 10ºC/ min to 250ºC, which was maintained for 5 minutes, for a total run of 35 minutes.

Determination of antimicrobial activity in vitro
The agar well test was used to determine the antimicrobial activities of the investigated compound 20 . Nisin, OEO, EDTA, or their combination were deposited into wells (6 mm) cut into the agar

Antimicrobial activity in Russian salad
Ingredients of a standard commercial formulation were used to prepare the mayonnaise: soybean oil, sodium chloride (NaCl), 4% vinegar, EDTA, and emulsifier, as shown in Table 1.
Egg yolk powder was used as emulsifier, with a quality cer-

Chemical analysis of mayonnaise and Russian salad
The pH and Aw (water activity) were measured in mayonnaise and Russian salad. All experiments were conducted three times, and the results were expressed as the averages of three replications. The pH and Aw were monitored for each batch of mayonnaise prepared.

Sensory evaluation
To conduct sensory evaluation, the project was submitted to and Corporation -Embrapa (Rio de Janeiro, Brazil).
All conditions of the test environment, hedonic test conditions, and acceptance scoring were the same as for the mayonnaise acceptability tests. In this evaluation, the testers were also asked to express their intent to purchase and provided a space for comments about the product. All data were calculated as percentages.

Statistical analysis
The determination of antimicrobial activity was performed three times in triplicate (nine measurements). The experimental results were subjected to analysis of variance and the Duncan test

Composition of OEO and antimicrobial effect
Using GC-MS-IE, ten volatile components were identified.   Nisin and EDTA alone, as well as EDTA in combination with nisin, showed no effect against SE. EDTA in combination with OEO1 showed no difference in relation to OEO1 only (p > 0.05) (Table 3).
Nisin combined with EDTA had no effect against SE. In turn, nisin in combination with OEO caused a reduction of the antimicrobial activity of the essential oil (p ≤ 0.05). An antagonistic effect was observed against SE. The growth inhibition zones obtained by the agar well test are presented in Tables 5 and 6

and Figures 3 and 4.
Considering these results, we decided to apply only OEO2 in mayonnaise and not the antimicrobials combined.

Sensory evaluation
The results indicated that 77% of tasters liked mayonnaise's aroma without the addition of OEO1, while 50% liked it with 0.5% OEO1 and 40% found it pleasant with 1% OEO1 (Figures 5 -7).
The acceptance of the mayonnaise aroma decreased when the concentration of OEO1 was increased from 0.5% to 1%. However, it is important to note that the acceptability of the product was just regular without OEO1 (< 80%).
The results indicated that 97% liked mayonnaise's flavor without addition of OEO1, while only 24% liked the flavor with 0.5% OEO1 and 3% liked it with 1% OEO1 (Figures 8 -10). The acceptability of the flavor decreased considerably as the content of OEO1 increased.
These results indicate the impossibility of using substantial concentrations of OEO1 in mayonnaise to improve the antimicrobial effect. Therefore, the microbiological analyses to determine the survival of SE in Russian salad were carried out with 0.2% OEO2, which showed no difference in relation to 0.5% OEO2 (p > 0.05).      (6 mm). **each value is the average of nine measurements. ***for the same line, values followed by the same uppercase letter did not differ significantly from each other (p < 0,05); for the same column, values followed by the same lower case letter were not statistically different from each other (p < 0.05).
The aroma and flavor acceptability of Russian salad with mayonnaise containing 0.2% OEO2 showed optimal acceptability: in this test, 95% liked the aroma ( Figure 11) and 93% liked the flavor (Figure 12).
The tasters were asked to comment about the product. Regarding the aroma, only six consumers made comments: they found no change between this or any other mayonnaise and identified a "slight oregano aroma." Twenty-nine consumers commented about the flavor. Some of them felt that the vegetables affected the mayonnaise flavor. In contrast, others said that the mayonnaise was "spicy, but did not overpower the flavor of the food." Some also stated that the mayonnaise flavor was very pronounced, suggesting decreasing the amount of OEO2. Other consumers identified a disagreeable aftertaste in the sample containing OEO2. A single consumer defined the initial taste as acidic but said the final flavor was pleasant. Some consumers found the flavor to be different from what they were accustomed to, but pleasant, while others defined a "slight taste" that improved the vegetables' flavor. Some other consumers suggested culinary preparations such as green salads, sandwiches, .3 c *The diameter of inhibition zone includes well (6mm). **for the same column, values followed by the same lowercase letter were not statistically different from each other (p < 0.05). *The diameter of inhibition zone includes well (6mm). **for the same column, values followed by the same lowercase letter were not statistically different from each other (p < 0,05).  22 A,a *inhibition zone includes well (6 mm). **each value is the average of nine measurements. ***for the same line, values followed by the same uppercase letter did not differ significantly from each other (p < 0,05); for the same column, values followed by the same lowercase letter were not statistically different from each other (p < 0.05). and snacks, which made us believe that using mayonnaise with OEO2 is possible.
Regarding buying intentions, only 74% of consumers said they would purchase the mayonnaise with 0.2% OEO, while 10% said they might buy it and 16% stated they would not ( Figure 13).

Chemical analysis of mayonnaise and Russian salad
Chemical analysis of mayonnaise and Russian salad Chemical analyses of mayonnaise without OEO and Russian salad with or without 0.2% OEO showed an increase in pH and Aw (Table 7) when vegetables were mixed in the mayonnaise to make Russian salad, demonstrated that the safety of mayonnaise decreases in culinary preparations, as indicated by the presence of microbial multiplication.

Antimicrobial activity in Russian salad
Antimicrobial activity was measured in Russian salad inocu- The reducti-on in the count of SE was less than 0.5 log CFU/g for a period of 24 hours at 8ºC and for a period of 4 hours at 30ºC compared to the control (without OEO2) (Figures 14   and 15).
The antimicrobial efficacy of essential oils can be attributed to the chemical composition and the proportions in which the components are present and their interactions 4,6,25,26,27 . Synergism between carvacrol and its biological precursor p-cymene was observed in Bacillus cereus vegetative cells because p-cymene probably enables carvacrol to be more easily transported into the cell 28 .
Nisin or EDTA only had no effect against SE. It is already known that nisin has no effect against Gram-negative bacteria, including SE 1,29 .
The agar well test is a qualitative assay and can introduce errors in testing EDTA only, whereas the inhibition of bacteria by EDTA is presumed to be due to chelation of divalent cations found in the cell wall 18 . However, Salmonella Infantis is sensitized either in the presence of EDTA only or when combined with plant extracts 9 .
Nisin combined with EDTA at all tested concentrations presented no effect against SE. SE inhibition zones were not observed, showing that both antimicrobial agents studied, alone or in   nisin and OEO might prevent the OEO from reaching its target of activity or could interfere with its structure or configuration, leading to a reduction in its specific activity 15 .
On the other hand, the combination of nisin and thymol (OEO constituent) was very effective against vegetative cells of B.
cereus, showing a synergistic effect 30 . In another study, when OEO was used in combination with nisin, a synergistic effect was observed against Listeria monocytogenes, i.e., the oil enhanced the activity of the bacteriocin 31 .
In Russian salad, the presence of 0.2% OEO resulted in a reduction in the SE count, thus forming an effective barrier that inhibits the growth of the pathogen. Increased safety by increasing the concentration of OEO in these products is not always possible because they can become sensorially unacceptable. It follows, therefore, that to reduce the risk of infection by SE in recipes with mayonnaise, other ways must be used besides high temperature and addition of OEO as a natural antimicrobial.
In conclusion, these results indicate that OEO antimicrobial activity against SE is enhanced when combined with nisin or EDTA. Nisin and OEO both have potential applications in improving food safety, but their activity together depends on the target pathogen.