# The Influence of Chestnut Flour on the Quality of Gluten-Free Bread
## 3 Results and Discussion
### Microbiological Quality  
Microbiological quality of bread samples with the chestnut flour addition differed significantly depending on the method and the time of storage (Table 5). Immediately after baking, no microorganisms were detected in the bread samples; however, after 7, 14 and 21 days of storage, 3.47-5.87 log cfu/g of mesophilic aerobic microorganisms, as well as 2.11-5.47 log cfu/g of yeast and molds were denoted. Moreover, in the samples containing 20% of chestnut flour, _Bacillus_ spp. was detected, but only after 7, 14 and 21 days of storage. Interestingly, the microbiological quality of the samples in vacuum storage was better than the aerobic conditions, which was seen in the TCV and Y&M counts.  
Many bakery products can be spoiled by different microorganisms, including fungi and bacteria. The fungal contamination is quite common in raw bakery materials; however, it is not considered as the most critical issue, as the life cells of microorganisms can be destroyed by the baking temperature. On the other hand, the postbaking contamination (air, product handling, equipment sanitized) seems to be an important issue in terms of the microbiological safety of bread [55]. For example, in the [PERSON] study [55], the raw materials of wheat flour and cornneal exhibited the fungal counts of approx. 10\({}^{2}\) log cfu/g in 60% of the tested samples. However, in the other studies, the fungal counts have been reported as higher, at approx. 10\({}^{5}\) log cfu/g in similar samples [56; 57]  
Among bacteria contamination, _Bacillus_ spp. is claimed as the main agent responsible for a spoilage process known as ropiess or rope. Rope spoilage occurring due to _Bacillus_ spp. is a major concern in the development of the fungal counts.  
\begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline  & \multicolumn{2}{c}{**Chestnut Flour**} & \multicolumn{2}{c}{**TVC**} & \multicolumn{2}{c}{**Y\&M**} & \multicolumn{2}{c}{**LAB**} & \multicolumn{2}{c}{_Bacillus_ spp.**} \\ \cline{3-10}  & **Addition** & **Air** & **Vacuum** & **Air** & **Vacuum** & **Air** & **Vacuum** & **Air** & **Vacuum** \\ \hline \multirow{4}{*}{0} & 0\% & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & Nd & nd \\  & 5\% & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & Nd & nd \\  & 10\% & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & Nd & nd \\  & 15\% & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & Nd & nd \\  & 20\% & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & \(<\)10 & Nd & nd \\ \hline \multirow{4}{*}{7} & 0\% & 3.53 \(\pm\) 0.21 a & 2.98 \(\pm\) 0.15 b & 2.11 \(\pm\) 0.06 a & 1.60 \(\pm\) 0.09 a & \(<\)10 & \(<\)10 & Nd & nd \\  & 5\% & 3.59 \(\pm\) 0.16 a & 2.47 \(\pm\) 0.23 a & 2.92 \(\pm\) 0.02 b & 2.17 \(\pm\) 0.0  
bacteria may produce spores and therefore survive during the thermal processing of bakery products. Rope takes place when counts of _Bacillus_ spores reach \(10^{3}\) spores/g. Once spores withstand baking, they may further germinate and grow [58]. A good solution for getting rid of _Bacillus_ spp. can be the use of sourdough or natural substances like LAB-based bioingredients, which can lower the pH of dough and therefore decrease the thermal resistance of _Bacillus_ cells and help in the inactivation of the microorganism through heat treatment [59]. In our study, we do not use sourdough in bread production, as the LAB count was \(<10\) log cfu/g in all samples during the whole storage period.  
From a microbiological point of view, the storage conditions (vacuum or air) used as the association to control the microbiological risk and stability of the bread samples had the biggest influence, and therefore we recommend vacuum as the better solution.