# The Effect of Carob Extract on Antioxidant, Antimicrobial and Sensory Properties of Bread
## 3 Results and Discussion  
### Microbial Load of Bread Samples  
The number of total aerobic bacteria, sporogenous bacteria, yeasts and moulds and the presence of _Bacillus cereus_ in bread samples are shown in Table 1.  
The highest number of total aerobic bacteria was observed in the control sample (4.84 \(\pm\) 0.53 log CFU/g). As expected, with an increase in the carob extract content in the bread, the number of total aerobic bacteria decreased, confirming the inhibitory activity of the carob extract on bacteria. Similar results were obtained by examining the amount of sporogenous bacteria, which were not detected in any bread sample with the addition of CP. Moreover, in the control sample and the sample with the lowest amount of CP (E0.5%), the presence of _B. cereus_ was observed, while in samples with higher CP amounts _B. cereus_ growth was not observed. In the control sample and sample E0.5%, there was a change in the colour of the nutrient medium from yellow to a characteristic dark blue \begin{table}
\begin{tabular}{l c c c} \hline \hline \multirow{2}{*}{**Sample**} & **Amount of Total Aerobic** & **Amount of** & **Amount of Yeasts and** \\  & **Bacteria (log CFU/g)** & **Sporogenous Bacteria** & **Moulds (log CFU/g)** \\  & & **(log CFU/g)** & **(log CFU/g)** \\ \hline
**Control** & 4.84 \(\pm\) 0.47 a & 3.26 \(\pm\) 1.17 & \(<\)2 & + \\
**E0.5\%** & 4.25 \(\pm\) 0.41 a & \(<\)2 & \(<\)2 & + \\
**E1.5\%** & 3.29 \(\pm\) 0.78 a & \(<\)2 & \(<\)2 & \(-\) \\
**E2.5\%** & 2.54 \(\pm\) 0.52 b,c & \(<\)2 & \(<\)2 & \(-\) \\
**E3.5\%** & 2.49 \(\pm\) 0.87 c & \(<\)2 & \(<\)2 & \(-\) \\ \hline \hline \end{tabular}  
* Results are expressed as mean \(\pm\) SD (n = 3); _a_* values in the same column for crust colour and for crumb colour mean that there are no significant differences between mean values at \(p\) < 0.05 (Duncan’s test). CFU—colony forming unit.  
\end{table} 
Table 1: Results of microbial load of bread samples. (Figure 1a,b). In Figure 1b, the sample with the maximum amount of extract (E3.5%) is shown, demonstrating the absence of _B. cereus_. Spores of _B. cereus_ can be found in raw materials of bakery products [33], and it is known that they are able to survive the baking process [34]. Moreover, [PERSON] et al. [35] showed that in bakery products _B. cereus_ can produce cereulide, the emetic toxin of the mentioned sporogenous bacteria, and therefore, the absence of _B. cereus_ in bread with CP is a clear indicator of the improved microbiologival safety, compared to bread without CP. The antimicrobial effect of carob is associated with the presence of bioactive compounds, mainly phenolic acids, flavonoids and their glycosides, which affect the growth and development of microorganisms. In our previous study, the presence of gallic, caffe, p-coumaric and chlorogenic acid, as well as quercetin, quercetin and rutin, were determined in significant amounts [20]. The antimicrobial activity of some of the polyphenonic compounds mentioned were tested against various microorganisms [36]. It is assumed that the antimicrobial activity of CP is mainly the effect of gallic acid, since it is the dominant polyphenonic compound in carob. [PERSON] et al. [36] showed that gallic acid exhibited a greater antibacterial effect at lower concentrations against tested Gram-positive and Gram-negative bacteria, compared to caffe acid, rutin and quercetin. The antibacterial effectiveness of flavonoids stems from their capability to form complexes with extracellular and soluble proteins, as well as their ability to disrupt bacterial cell walls by interacting with vital enzymes that uphold the integrity of this structure [37]. The obtained results of the microbial load of bread with CP are comparable to results of other studies that examined the effect of the addition of functional materials with similar phenol/flavonoid profiles.