Development of an ad hoc natural whey starter culture for the production of Vastedda della valle del Belìce cheese

Abstract

This work was aimed to characterize the autochthonous lactic acid bacteria (LAB) of PDO Vastedda della valle del Belìce cheeses, produced in several dairy factories, for the development of an ad hoc starter culture preparation for the production of this cheese. To this purpose, winter and spring productions were analysed, in order to isolate LAB adapted to perform the fermentation at low temperatures. Plate counts showed the total microbial counts (TMC) till levels of 9 -1almost 10 CFU g and all cheese samples were dominated by coccus LAB. Not all samples were positive for the presence of enterobacteria, but when they were found their concentrations were at similar levels in both seasons. All colonies with different morphological appearance were isolated and differentiated on the basis of phenotypic characteristics and by randomly amplified polymorphic DNA (RAPD)- PCR analysis. A total of 65 strains were considered and subjected to the genotypic analysis by means of 16S rRNA gene sequencing, which identified 13 LAB species belonging to five genera (Enterococcus, Lactobacillus, Lactococcus, Leuconostoc and Streptococcus). The species most frequently found were Streptococcus macedonicus, Streptococcus thermophilus, Lactococcus lactis and Leuconostoc mesenteroides. The 65 strains were investigated in vitro for their general dairy aptitudes and some strains of the above four species showed technological traits relevant to act as starter strains for this cheese production. Among those strains, 12 LAB (Lactobacillus delbrueckii PON79, PON256 and PON405, Lactococcus lactis PON36, PON46 and PON203, Leuconostoc mesenteroides PON169, PON259 and PON559, Streptococcus thermophilus PON3, PON120 and PON261) were used in different combinations [all strains belonging to each species in triple combinations (Lb, lactobacilli; Lc, lactococci; Ln, leunostocs; St, streptococci), all thermophilic strains (Lb-St, lactobacilli and streptococci) and all mesophilic strains (Lc-Ln, lactococci and leuconostocs)] to produce experimental cheese by means of a dairy pilot plant. The different 7bacterial combinations (final concentration of approximately 10 CFU/mL) were tested in different conditions: 1, after growth in the optimal synthetic media, re- suspended in Ringer’s solution and inoculated in pasteurised ewes’ milk; 2, after growth in whey-based medium (WBM) and inoculated in pasteurised ewes’ milk; 3, after growth in WBM and inoculated in raw ewes’ milk. Plate counts and RAPD analysis were applied to monitor the bacterial evolution during the different trials, while pH was measured to follow their acidifying activities. All lactococci and leuconostocs were able to perform the rapid acidification of the curd in winter conditions; the experimentation was carried in February with 10°C as minimum ambient temperature registered in the room where the curd were left to acidify. A sensory evaluation of the resulting cheeses, obtained after stretching of the acidified curds, indicated the cheeses processed with lactococci in single and multiple combinations as those well appreciated by the judges. These cheeses were then subjected to the analysis of the volatile organic compounds carried out by gas chromatography coupled with mass spectrometry (GC/MS) which clearly showed substantial differences with the control cheese.