TESI Dottorato FAVILLA MARA - Università degli Studi del Molise

Abstract
The aim of this research work was to obtain the improvement of microbiological
quality of traditional Apulian breads through the selection of lactic acid bacteria with
antimicrobial activity and the characterization of bacterial contaminants (Bacillus
spp.) from wheat semolina. Lactic acid bacteria are microorganisms used since
ancient times for preparation of fermented foods such as some types of bread made
with sourdough. The products of metabolism of lactic acid bacteria can extend the
shelf life of bread due to their antimicrobial properties. The study of lactic acid
bacteria in the wheat semolina used in the production of traditional Apulian breads
may lead to the selection of strains with peculiar pro-technological characteristics for
improvement of bakery product shelf life. Moreover, the use of antimicrobial
metabolites as an alternative to sourdough that need a long fermentation time, may
represent a new tool for control microbial contamination of bakery products.
In this study, 30 samples of durum wheat semolina were subjected to
microbiological analysis in order to explore their lactic acid bacteria diversity and to
find strains with antifungal activity. The analysis led to the isolation of 250 isolates
that were differentiated into 17 strains by rep-PCR. A representative strain of each
rep-PCR pattern was used to identify the species. The sequencing of 16S rRNA
gene allowed to identify the following species: Weissella cibaria, W. confusa,
Leuconostoc citreum, L. mesenteroides, Lactobacillus plantarum, L. rossiae and
Lactococcus lactis subsp lactis. In particular, W. cibaria and W. confusa showed the
greatest genotypic variability and were the most widely diffused, since occurring in
the 75% of the semolina samples. The antimicrobial activity of the fermentation
products (FP) obtained by growing the 17 strains representative of the rep-PCR
profiles in a flour-based medium, was evaluated in in vitro experiments against 3
fungal contaminants of bakery products (Aspergillus niger, Penicillium roqueforti and
Endomyces fibuliger) as well as against Bacillus subtilis, the causal agent of “bread
rope”. The analysis led to the selection of 6 LAB strains belonging to the species
Weissella cibaria, Leuconostoc citreum, Lactobacillus rossiae, L. plantarum e
Lactococcus lactis showing antimicrobial properties. Among these strains, the
fermentation product of L. citreum C2-27 showed a strong inhibitory activity towards
the three above fungi, which was comparable to that of calcium propionate (0.3%,
wt/vol), and also inhibited B. subtilis spore germination and delayed the
development of A. niger ITEM5132 directly on bread. Bichemical investigations
carried out on the FPs suggested that antimicrobial activity was mainly related to
lactic acid (26.28 mM) and acetic acid (14.62 mM) production, and to the low pH (pH
= 3.42).
Along with the use of natural preservatives, prevention and reduction of bread
spoilage may be achieved also through the control of microbiological quality of raw
materials. Recently, several companies in Southern Italy have reported problems of
deterioration of bakery products due to the presence of spores of Bacillus spp. in
raw materials. As part of this research, 59 samples of wheat semolina from different
localities in the areas of Bari and Foggia were examined in order to assess the
possible contamination by Bacillus spp. The study showed that approximately the
90% of samples were contaminated by 10 0 -10 3 spores/g. Sixty percent of isolates
were identified as B. subtilis/B. amyloliquefaciens and B. licheniformis, the two
species most commonly associated with ropy bread spoilage.
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