# The Effect of Carob Extract on Antioxidant, Antimicrobial and Sensory Properties of Bread
## 3 Results and Discussion
### Challenge Test  
To obtain a more comprehensive understanding of the antimicrobial potential of carob extract as a natural preservative, a microbiological inoculation test was conducted. This test monitored changes in the product during its shelf life after the intentional/targeted inoculation of the product with the desired microorganism. The application of this microbiological test is significant in the development of new products with the addition of raw materials that have potential antimicrobial effects [40].  
Figure 1: The presence of B. cereus in the control sample (**a**) and the absence of _B. cereus_ in the sample with 3.5% powdered carob extract (**b**).  
Mould spoilage is the most common form of microbiological spoilage in food products, especially bread, and leads to large economic losses as well as potential health hazards to consumers due to the production of mycotoxins by moulds. For the bread inoculation test, moulds that are the most common contaminants of bread were selected--_Aspergillus flavus_ and _Penicillium verrucosum_[41]. Figure 2 shows the development of Penicillium verrucosum mould on bread slices over time and Figure 3 shows the development of _Aspergillus flavus_. Figure 2 shows that moulds appeared on all samples on the third day, with an increase in colony size observed in the following days. The highest mould growth was observed in the control sample, followed by the sample with the lowest amount of CP (0.5%). From day four until the end of the monitoring period, on samples with a higher concentration of extract mould, growth was significantly lower compared to control and the E0.5% sample, indicating the inhibitory effect of CP on the growth of _Penicillium verrucosum_.  
Figure 3: Development of _Aspergillus flavus_ over time.  
Figure 2: Development of _Penicillium verrucosum_ over time.  
A flavus_ was visually detected on day 2, and its growth on bread samples with CP was comparable to the control until day 3. From day 4, the mycelia diameter on samples with CP, regardless of the applied amount, was significantly different from the control sample. At the end of monitoring period, mould growth was highest in sample E1.5% and lowest in sample E2.5% (Figure 3). The obtained results indicated a lower inhibitory effect of CP addition in bread on _A. flavus_ development compared to _P. verrucosum_.  
[PERSON] et al. [42] and [PERSON] et al. [43] concluded that plant extracts have strong antifungal activity and that the inhibitory effect of plant extracts on the examined pathogenic fungi is proportional to the applied concentrations. Unexpectedly, the maximum concentration of CP did not result in the highest inhibition of _A. flavus_ in our study; rather, it even led to the stimulation of fungal growth. It is assumed that this is due to the microorganisms' response to environmental stress, which leads to their more intensive reproduction [43]. These results suggest that in the case of _A. flavus_, additional research should be conducted, including the application of various concentrations of the CP.  
The inhibitory effect on mould growth in bread with CP could be attributed to the antifungal effect of ellagitannins [44], which are also present in carob pods and were reported in the literature [45]. The antifungal activity in bread fortified with rambutan peel extract contributed to the extension of bread shelf life, due to the inhibitory compound (corilagin), which protected bread from spoilage [44].