THE EFFECT OF THE TECHNOLOGICAL PROCESS AND ESSENTIAL OIL OF BASIL AGAINST SALMONELLA ENTERICA SEROTYPE ENTERITIDIS (D) ATCC 13076 IN EGG BASED PASTA

Salmonella cause illness in humans and animals. Most Salmonella serotype are naturally occurring in food. Egg and egg products are an important part of the human diet. Since eggs are used for the production of egg pasta, and due to an insufficient thermal treatment during pasta drying they can be a potential risk for the consumer’s health. Different essential oils of herbs can be used in order to reduce potentially present pathogenic microorganisms. The aim of this paper is to describe the impact of the technological process of production of pasta with eggs under the influence of sweet basil on a decrease of the number of Salmonella enterica serotype Enteritidis (D) ATCC 13076. There is a significant effect of concentration of basil against Salmonella enterica serotype Enteritidis (D) ATCC 13076 (p<0.05). Also there is a significant impact of the process against Salmonella enterica serotype Enteritidis (D) ATCC 13076 (p<0.05). There is not significant differences in the effects of the concentration of basil and technological process against Salmonella enterica serotype Enteritidis (D) ATCC 13076 in the production of pasta (p = 0.737).


MATERIAL AND METHODS
Egg-based pasta technology and the sampling procedure: Egg-based pasta was made by the following recipe: 10 kg wheat grits and 2 kg wheat flour, 24 eggs, 3.2 -3.4 L water and 0.010 kg β-carotene. Ingredients were mixed and 25 g of the dough was inoculated with 0.1 mL of the suspension of the investigated species of bacteria with the initial number of bacteria 10 9 CFU/g. After the inoculation, different amounts of sweet basil essential oil (Fitofarm, Skopje, Republic of Macedinia) were added to the prepared dough, at the final concentrations of 1%, 2.5% and 5%. Pasta was formed by extrusion and then dried in the chamber at the temperature of 46 ºC and relative humidity of 80% for 9h. Afterwards pasta was cooled at the room temperature for 15 min and packed into PE bags. Samples of the pasta with and without the addition of oils were collected during following production stages: dough making, dough extrusion, drying of pasta, pasta cooling, pasta packaging.
Microorganisms: The antimicrobial activity of the oils was investigated with following bacteria Salmonella enterica serotype Enteritidis (D) ATCC 13076 from the MicroBioLogics, Ins. Joins ATCC Proficiency Standard Program, Minesota, USA.
Enumeration of bacteria: The determination of the number of Salmonella enterica serotype Enteritidis (D) ATCC 13076 was performed according to the Methods of carrying out microbiological analysis and super-analysis of food. The quantity of 25g of pasta was mixed with 225 mL of selenite broth (Torlak, Belgrade, Serbia) and incubated for 24 h at 37ºC. Inoculation was carried out by spreading of 0.1 mL of the appropriate dilution on the surface of SS agar (Torlak, Belgrade, Serbia) plates. The enumeration of bacteria was performed after the incubation on 37 ºС during 24 hours.
Statistical Analysis: Data were subjected to analysis using MS Office Excel and the computer programme SPSS 17. Two-factorial experiment showed the significance of differences in means between control and concentration of 1%, 2,5% i 5% of sweet basil and technological proces against Salmonella enterica serotype Enteritidis (D) ATCC 13076 in egg-based pasta.

RESULTS AND DISCUSSION
In the EU, Salmonella Enteritidis and Salmonella Typhimurium are the serovars most frequently associated with human illness (Guard-Petter 2001). Human Salmonella Enteritidis cases are most commonly associated with the consumption of contaminated eggs and poultry meat (EFSA 2013). In order to investigate possible effect of essential oil of sweet basil to the reduction of the growth of Salmonella enterica serotype Enteritidis in egg-based pasta the number of bacteria was determined during different stages of the production process. Pasta was inoculated with Salmonella enterica serotype Enteritidis (D) ATCC 13076 with the initial number of bacteria of 10 9 CFU/g and different concentrations of essential oil were added.
During the production process the number of Salmonella Enteritidis (D) ATCC 13076 in egg-based pasta with sweet basil decreased for 1 log CFU/g (Fig 1). The addition of 1% and 2% of essential oil of sweet basil had no influence to the number of Salmonella enterica serotype Enteritidis (D) ATCC 13076 in pasta. Addition of 5% of essential oil of sweet basil reduced the number of S. Enteridis (D) ATCC 13076 during extrusion for 0.14 log CFU/g. The greatest reduction was observed during cooling, 0.76 log CFU/g, while in the final product it was 0.51 log CFU/g compared to the control (Fig 1). The impact of technological process of production of pasta with eggs against Salmonella enterica serotype Enteritidis (D) ATCC 13076 is shown in Table 1.

Contro l 1%
KNOWLEDGE -International Journal Vol. 20.5 Bansko, December, 2017 The impact of different concentration of sweet basil in pasta with eggs on a decrease of the number of Salmonella enterica serotype Enteritidis (D) ATCC 13076 is shown in Table 2.  Salmonellae are readily destroyed by heat pasteurization of foods at high a w . As the a w is reduction with addition of solutes or by removal of water, heat resistance increases markedly. In foods such chocolate, several seconds on 105 ºC may be required to reduce Salmonella counts by 1 log CFU/g. There is a high probability of infections at doses of >10 5 cells but in foods containing high levels of fat and/or protein such as chocolate, salami and cheddar cheese infection can result from ingesting as few as <10-100 cells (Teunis et al. 2010). In the EU in 2004-2009 was 8 outbreaks with pasta (25% with Salmonella spp.), (EFSA 2012). To minimize the potential risk of

KNOWLEDGE -International Journal
Vol. 20.5 Bansko, December, 2017 salmonellosis due to the consumption of egg and egg products, good manufacturing and handling practices in production of pasta with eggs should always be observed.
After drying the pasta at 46 ° C, humidity of 80% for a period of 9 hours and the inhibition of the various concentrations of the essential oil of basil on SS agar was colony, which indicates that the conditions for the drying of pasta against Salmonella Enteritidis (D) ATCC 13076 were not rigorous and that this strain of pathogenic bacteria is not destroyed. After cooling and packaging of pasta in plastic bags presence of Salmonella was also recorded (Fig 1).
Comparison within the group (different process: mixing, extrusion, drying, cooling, packaging, and storage and distribution) in the two-factorial experiment showed that there was statistically significant at p <0.05 between the mixing and all other processes (p = 0.000), between extrusion and other processes (p = 0.000), and all other drying processes (p = 0.000), cooling with mixing, extrusion and drying (p = 0.000), packaging with mixing, extrusion and drying (p = 0.000) and storage and distribution with mixing, extrusion and drying (p = 0.000) ( Table  1).
Essential oils of spices and herbs have been used as food additives, as flavoring agent and as natural food preservatives since ancient times. A number of spices have antimicrobial activity against different types of microorganisms (Škrinjar andNemet 2009, Tajkarami et al. 2010). Comparison of the effect of different concentrations of the sweet basil against Salmonella enterica serotype Enteritidis (D) ATCC 13076 in egg-based pasta showed that the mean difference was significant at the 0,05 level between the control, basil concentration of 1%, 2.5% and 5%. Between the control and the concentration of basil at 2.5%, the mean difference was significant (p<0.05), for the control and basil concentration of 5% (p<0.05). For a concentration between 1% and 5% significance level of the difference was p<0.05 (Table 2). Essential oils of cinnamon, oregano and mustard are efficient in the reduction of the number Salmonella in beef (Turgis, et al. 2008), while a concentration of 2 μL/mL cinnamon, geranium, lemongrass and palmarosa oils decreased the number of Salmonella Enteritidis in fruit juice (Raybaudi-Massilia et al. 2006). Essential oils of Thymus vulgaris, Mentha piperita, Rosmarinus officinalis, showed strong antimicrobial activity (both bacteriostatic and bacteriocidal effect) against Salmonella Enteritidis and Escherichia coli in concentrations ranged from 0,125 to 2% (v/v) (Niculae et al. 2009).
There is not significant differences in the effects of the concentration of basil and technological process on Salmonella Enteritidis ATCC 13076 (Table 3) in the production of pasta (p = 0.737). There is a significant effect of concentration of basil on Salmonella Enteritidis ATCC 13076 (p = 0.002). Also there is a significant impact of the process on Salmonella Enteritidis ATCC 13076 (p = 0.000).

CONCLUSION
This study has been described the individual stages of the tecnological process of production pasta with eggs with basil (mixing, extrusion, drying of pasta, cooling, packaging, storage and distribution) against Salmonella enterica serotype Enteritidis (D) ATCC 13076. There is not significant differences in the effects of the concentration of basil and technological process on Salmonella Enteritidis ATCC 13076 (Table 3) in the production of pasta (p = 0.737). There is a significant effect of concentration of basil on Salmonella Enteritidis ATCC 13076 (p = 0.002). Also there is a significant impact of the process on Salmonella Enteritidis ATCC 13076 (p = 0.000).