# Plant Extracts for the Control of _Listeria monocytogenes_ in Meat Products
## 2 Materials and Methods  
### Plant Extracts, Strains and Culture Conditions  
The plant extract employed is a natural concentrated product containing active ingredients obtained by hydro-distillation of fresh and dried garlic (_Allium sativum_ L.) and onion (_Allium cpa_ L.), and considered safe (GRAS) by the Food and Drug Administration (FDA). The garlic and the onion plant extracts were used, as they are often employed, to flavor sausages. To make the extracts, 30 g of garlic and onion, respectively, were placed in infusion in a flask containing 300 mL of sterile distilled water for one hour. After this time, the mixture was stirred and allowed to stand at room temperature for 24 h. Four extraction cycles using dichloromethane (30 mL) and one extraction with diethyl ether (30 mL) were performed on each. All the fractions were collected into a sterile flask and dried over anhydrous sodium sulphate. After 24 h, the extracts were concentrated under reduced pressure in a rotary evaporator and were stored at \(-20\)\({}^{\circ}\)C for further analysis. The compounds of extract from garlic and onion were analyzed and identified by GC-FID and GC-MS.  
A cocktail of five _Listeria monocytogenes_ strains from different sources was used in the study based on their different sensitivity to the extracts tested as shown by the minimum inhibitory concentration (MIC) values. The mixture of _Listeria_ included _L. monocytogenes_ CSA10, _L. monocytogenes_ 10P and _L. monocytogenes_ 123 (belonging to the experimental station for the food preserving industry--S.S.I.C.A., Parma, Italy SSICA collection), previously isolated from sausages and cooked ham, and _L. monocytogenes_ Scott A (from National Laboratories of Foods and Drugs) and _L. monocytogenes_ ATCC 7644 (from American Type Culture Collection).  
Overnight cultures of _Listeria_ strains were grown at 37 \({}^{\circ}\)C in 5 mL of Trypic Soy Broth (TSB, Oxoid, Milan, Italy), and collected and centrifuged (Eppendorf Centrifuge 5417r, Eppendorf s.r.l., Milan, Italy) at 4000 rpm (66.70 Hz) at 4 \({}^{\circ}\)C for 15 min. The pellet of each strain was suspended in 9 mL of sterile peptone saline solution (Oxoid, Milan, Italy), enumerated as colony-forming units per milliliter (cfu/mL) on Trypic Soy Agar (TSA, Oxoid, Milan, Italy) and stored at a refrigerated temperature (4 \({}^{\circ}\)C) for 4 h before contaminating the samples.  
The inoculum for the sausage contamination was carried out by adding the cocktail of _Listeria_ to the standard dough then stirred by hand to obtain a uniform distribution of about \(10^{5}\) cfu/g of the pathogen. In this case, a high contamination charge (\(10^{5}\) cfu/g) is required considering that the product, consisting of minced meat, presents a large exposed surface.  
To validate the concentration and the uniform distribution of the artificial contamination, the inoculated pathogens were counted (cfu/g) by taking 25 g samples in six different positions of the standard dough immediately after preparation (time 0). Approximately 4 h after bagging and before drying and ripening treatments, other three samples per line were analyzed removing three 25 g portions from the core, the top and the bottom of sausages. The portions of each sausage were diluted in a 1:10 ratio with sterile peptone saline medium and homogenized for 2 min in a stomacher (Lab Blender, Seward, London, UK). _L. monocytogenes_ viable counts were determined by spreading on Palcam Agar Base (Oxoid, Milan, Italy) plates and incubated aerobically at 37 \({}^{\circ}\)C for 48 h. The samplings were then repeated at established times (1, 3, 8, 17, 21 and 28 days) in the same way. For the cooked ham, the contamination was performed by spreading the _L. monocytogenes_ mixture on the surface of square slices of the product and dispensed with a sterile glass spatula, to obtain a uniform inoculum of about \(10^{3}\) or \(10^{2}\) cfu/g.