# Microbiological Safety and Health Properties of Markleted Fermented Root Vegetables
## 2 Materials and Methods
### Determination of Microorganisms  
Sample solution (100 mL) from ready-made fermented products was taken randomly, every time after mixing. Samples were collected aseptically. Then, a series of dilutions were prepared in geometric progression (from 10\({}^{-1}\) to 10\({}^{-6}\)), according to the procedure described in PN-90 A-75052 [39]. Buffered peptone water (Graso Biotech, Starogard Gdanski, Poland) was used as the diluent. The prepared dilutions were inoculated by the pour plate method using nutrient agar (Himedia, Mumbai, India) to determine the number of mesophilic bacteria (37 \({}^{\circ}\)C, 48 h, according to PN-EN 204 ISO4833:2004 [40]); the number of psychrophilic bacteria (4.5 \({}^{\circ}\)C, 5-7 days, according to PN-ISO 17410:2004 [41]); the number of thermophilic bacteria (55 \({}^{\circ}\)C, 48 h, according to PN-A-75052-06:1990 [42]); and the number of acidifying bacteria of the lactic type, according to PN-90 A-75052/07 208 [43], (37 \({}^{\circ}\)C, 72 h, elevated CO\({}_{2}\) concentration; MRS agar-De Man-Rogosa-Sharpe agar; Biomaxima, Lublin Poland). The surface-spread method was used to determine the number of yeasts and molds, according to PN-90 A-75052/08; PN ISO 21527-1:2009; PN ISO 21527-2:2009 [44; 45; 46] (20-25 \({}^{\circ}\)C, 4-5 days; Sabouraud agar with chloramphenicol and gentamicin (Graso Biotech, Starogard Gdanski, Poland); the presence of bacteria of the genus _Enterococcus_, according to PN-EN 15788:2009 217 [47] (37 \({}^{\circ}\)C, 48 h; Slanetz Barteley agar (Graso Biotech, Starogard Gdanski, Poland)); and the number of _Bacillus cereus_ bacteria, according to PN-EN ISO 7932:2005 [48] (30 \({}^{\circ}\)C,  
Figure 4: Optimization of CUPRAC assay. 48 h; MYP agar-Mannitol egg Yolk Polymynxin agar (Biomaxima, Lublin, Poland)). In order to determine the presence and number of bacteria of the genus _Leuconostoc_, inoculation was performed using the pour plate method with the sucrose medium (25 \({}^{\circ}\)C, for 5 days; homemade sucrose medium for _Leuconostoc_ detection: meat extract 0.3%, peptone 1%, NaCl 0.5%, sucrose 10%, and agar 2% were dissolved in 100 mL of distilled water, pH 7.0, sterile at 121 \({}^{\circ}\)C for 15 min). In turn, the presence and number of mesophilic and thermophilic spore-forming anaerobic bacteria were detected according to PN-ISO 15213:2005 [49] (a 10 mL of initial sample was heated at 70 \({}^{\circ}\)C for 20 min). After cooling, the sample was diluted in a geometrical progression, and next, each dilution was inoculated in a 9:1 ratio with iron sulphite agar (Graso Biotech, Starogard Gdarski, Poland). When the medium had set, approximately 1 mL of water agar was layered on the samples, and thus, prepared samples were incubated for 48 h at 37 \({}^{\circ}\)C (for mesophilic bacteria) and 55 \({}^{\circ}\)C (for thermophilic bacteria). The most probable number method was used to detect the presence and number of coliforms according to PN-EN ISO 4831:1998 [50], using MUG (4-methylumbelliferyl-\(\beta\)-D-glucuron) Lauryl Sulpate Broth (Himedia, Mumbai, India). The initial sample and the next two tenfold dilutions were inoculated on the medium in a 9:1 ratio in triplicate and incubated at 37 \({}^{\circ}\)C for 48 h. Positive samples were checked for _E. coli_ according to PN-EN ISO 7251:2005 [51], using EC broth (_Escherichia coli_ broth; Biomaxima, Lublin, Poland) and peptone water with tryptophan (Biomaxima, Lublin, Poland). The samples mentioned above were identified biochemically with the API20E bioMerieux Polska Sp. z o.o. test. The presence of acetic fermentation bacteria was determined according to PN-90 A-75052/15 [52] on a home-made medium (peptone 2% and yeast extract 1% dissolved in 100 mL of distilled water, pH 6.8-7.0, sterilized at 121 \({}^{\circ}\)C for 20 min, and 0.8% ethyl alcohol was added to the cooled medium). Determination of the acetic fermentation bacteria was performed using the pour plate method, and the media were incubated at 30 \({}^{\circ}\)C for 5 days to determine the presence of _Salmonella_ spp.