# Antimicrobial and Odour Qualities of Alkylpyrazines Occurring in Chocolate and Cocoa Products
## 3 Antimicrobial Activity of Pyrazines
### Antibacterial Activity
The bactericidal effect appeared at a 1.2% concentration regardless of the period of incubation and at 0.6% starting from the fourth hour of incubation [35]. Pyrazine derivatives were discovered to be the predominant class of chemical constituents in Black pepper root entophytic Bacillus megaterium BP17 volatiles after a dynamic headspace study. Pyrazine and 2,5-dimethylpyrazine were among those with the highest amounts. Ethlypyrazine, methylpyrazine and 2-ethyl-3-methylpyrazine were also found. The antibacterial compounds 2-ethyl-3-methylpyrazine (growth inhibition 72.89% at 335 \(\mathrm{\SIUnitSymbolMicro}\)/mL, 91.65% at 504 \(\mathrm{\SIUnitSymbolMicro}\)/mL and 95.9% at 672 \(\mathrm{\SIUnitSymbolMicro}\)/mL) and 2,5-dimethylpyrazine (growth inhibition 69.75% at 504 \(\mathrm{\SIUnitSymbolMicro}\)/mL and 79.87% at 672 \(\mathrm{\SIUnitSymbolMicro}\)/mL) exhibited impressive activity against the soilborne plant pathogenic bacterium Ralstonia solanacearum, which frequently annihilates Irish potatoes, tomatoes, peppers and eggplants [50]. The idea of applying pyrazines to food is a good concept for the food industry since they have antibacterial and smell- and taste-improving characteristics. For example, 2-isobutyl-3-methylpyrazine was used for the first time as a potential preservative for chicken meat strips by [PERSON] et al. [44]. Studies have shown that coupling maltodextrin with 2-isobutyl-3-methylpyrazine may reduce the microbial contamination of processed meat. Additionally, using this alkylpyrazine may enhance the flavour and aroma of food by introducing roasted undertones. The testing of 2-isobutyl-3-methylpyrazine on _Escherichia coli_ and _Staphylococcus aureus_ showed strong bactericidal properties (_E. coli_ minimum inhibition concentration (MIC)--3 mg/mL, minimum bactericidal concentration (MBC)--3 mg/mL; _S. aureus_ MIC--5 mg/mL, MBC--6 mg/mL), with the same MIC and MBC towards _S. aureus_ as 5-isobutyl-2,3-dimethylpyrazine, which was found by [PERSON] [35] as a strong antibacterial and antifungal agent. An additional comparison of the impact of 2-isobutyl-3-methylpyrazine, 2-isopropyl-5-methylpyrazine, 2-isobutylpyrazine and 2-isopropylpyrazine on _E. coli_ strains (10\({}^{5}\)-10\({}^{7}\) CFU/mL) with 0.3% doses showed 2-isobutyl-3-methylpyrazine to be bactericidal to 10\({}^{6}\) and 10\({}^{7}\) CFU/mL, while other pyrazine derivatives showed to be lethal only on a 10\({}^{5}\) CFU/mL invasion level. The findings demonstrate that incubation time has less of an impact on the application efficiency of pyrazine derivatives than chemical concentration. The study of [PERSON] et al. [51] provides additional support for this theory, showing that the addition of concentrations of 100 and 200 \(\mathrm{\SIUnitSymbolMicro}\)/ of the bacterial volatile product 2,5-dimethylpyrazine, produced by Bacillus pumillus, significantly lowered Phaeomoniella chlamdospora mycelial growth by 55 and 67%, respectively. The study by [PERSON] et al. [52] proposed a completely novel method of visualizing the action of the Pyrazinamide (PA, pyrazine-2-carboxamide) on Mycobacterium tuberculosis depending on the pH. PA is a crucial element of medical protocols for treating tuberculosis for approximately 65 years. It was shown that PA functions as a prodrug by converting into pyrazinoate through an enzymatic process and existing in tubercular cells in equilibrium with pyrazinoic acid, with a correlation between pH and the relative amount of both forms. The graphic image of this process is presented in Figure 3. Over a variety of pH values, the impacts of total pyrazinoic acid (pyrazinoic acid + pyrazinoate) on an M. tuberculosis culture were evaluated. In neutral pH environments, M. tuberculosis showed resistance to PA and required high dosages to have antibacterial activity; however, in acidic pH environments, the concentration of medication required to destroy bacterial colonies was lower. The total amount of growth inhibition caused by pyrazinoic acid rose as the pH of the culture medium was lowered from 7.3 to 6.4.