# Plant Organic Acids as Natural Inhibitors of Foodborne Pathogens
## 2 Materials and Methods
### Antibacterial and Antifungal Activity
#### 2.2.2 Minimal Inhibitory Concentrations (MIC)  
To determine the minimal inhibitory concentrations (MIC) of the organic acids, the microdilution method was used with 96-well plates (Nest Scientific Biotechnology, Wuxi, China). The MIC methodology was detailed in our published paper [21] and the procedures were based on previous research [22; 23]. Bacteria were grown in tryptose-soy broth and fungi in Sabouraud broth (Graso Biotech, Owidz, Poland) and serial dilutions of the organic acids were made to reach final concentrations ranging from 100 mg/mL to 0.02 mg/mL in the wells. The inoculum was adjusted to achieve a final concentration of \(10^{5}\) CFU/mL. Plates were incubated at 37 \({}^{\circ}\)C for 24-48 h and MIC values were determined visually, using  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Organic Acid** & **Molecular Formula** & **pH of Prepared Solutions** & **Exemplary Natural Occurrence** \\ \hline Capric acid & C\({}_{6}\)H\({}_{2}\)O\({}_{2}\) & 2.7 & Vegetable oils \\ \hline Chlorogenic acid & C\({}_{16}\)H\({}_{18}\)O\({}_{9}\) & 4.4 & \begin{tabular}{c} Apples, pears, carrots, tomatoes, sweet \\ potatoes, coffee, and tea \\ \end{tabular} \\ \hline Citric acid & C\({}_{6}\)H\({}_{8}\)O\({}_{7}\) & 2.9 & Fruits \\ \hline Formic acid & CH\({}_{2}\)O\({}_{2}\) & 2.3 & Stinging hairs of nettles \\ \hline Fumaric acid & C\({}_{4}\)H\({}_{4}\)O\({}_{4}\) & 4.5 & Mosses and mushrooms \\ \hline Glutamic acid & C\({}_{5}\)H\({}_{9}\)NO\({}_{4}\) & 2.9 & \begin{tabular}{c} Sunflower seeds, flax seeds, peanut, \\ pistachio, almond, broad bean, Brussels \\ sprout, and lentil \\ \end{tabular} \\ \hline Malic acid & C\({}_{4}\)H\({}_{6}\)O\({}_{5}\) & 2.0 & Fruits \\ \hline Nicotinic acid & C\({}_{6}\)H\({}_{5}\)NO\({}_{2}\) & 4.0 & Common in plants \\ \hline Oleic acid & C\({}_{18}\)H\({}_{34}\)O\({}_{2}\) & 2.8 & Olive oil and grape seed oil \\ \hline Oxalic acid & C\({}_{2}\)H\({}_{2}\)O\({}_{4}\) & 1.8 & Fruits \\ \hline Palmitic acid & C\({}_{16}\)H\({}_{32}\)O\({}_{2}\) & 4.5 & Seeds of beans, sunflowers, and cotton \\ \hline Rosmarinic acid & C\({}_{18}\)H\({}_{16}\)O\({}_{8}\) & 5.8 & \begin{tabular}{c} Rosemary, sage, Spanish sage, basil, oregano, \\ thyme, spearmint, and perilla \\ \end{tabular} \\ \hline Salicylic acid & C\({}_{7}\)H\({}_{6}\)O\({}_{3}\) & 3.0 & Common in plants \\ \hline Succinic acid & C\({}_{4}\)H\({}_{6}\)O\({}_{4}\) & 2.4 & Fruits and vegetables \\ \hline Tannic acid & C\({}_{76}\)H\({}_{52}\)O\({}_{46}\) & 2.6 & \
\begin{tabular}{c} Bark of oak, beech, American chestnut, \\ spruce, willow, witch hazel, walnut, \\ blackberry, raspberry leaves, blueberries, \\ shoes, rhizome of cinquefoil, hen’s weed, \\ and snakeweed \\ \end{tabular} \\ \hline Tartaric acid & C\({}_{4}\)H\({}_{6}\)O\({}_{6}\) & 1.9 &
\begin{tabular}{c} Peaches, apples, grapes, cherries, \\ and strawberries \\ \end{tabular} \\ \hline Valeric acid & C\({}_{5}\)H\({}_{10}\)O\({}_{2}\) & 2.8 & Valerian rhizome and angelica root \\ \hline \hline \end{tabular}
\end{table}
Table 1: _Cont._color reactions with 2,3,5-triphenyltetrazolzolium chloride (TTC) (Sigma, Poznan, Poland) to aid in reading. Each test was performed in triplicate.