Selección de bacterias lácticas orientada a la mejora de la calidad del queso IGP castellano

Abstract

Cheese quality depends on raw material, microbiota, processing technology and ripening conditions. The use of lactic cultures is a widespread practice in the production of cheeses, especially in those made from pasteurized milk. The selection of wild lactic acid bacteria, based not only on the study of their acidifying activity, salt tolerance or phage resistance, but also extended to the study of enzymatic activities relevant to the development of flavor and aroma in cheese, as well as other characteristics such as the production of bioactive compounds, could expand the diversity of cultures available for cheese production. This approach is especially interesting in the production of prestigious cheeses, such as PGI Castellano cheese, a Spanish cheese variety characterised by a pressed paste and enzymatic coagulation. In this context, the main objective of this Doctoral Thesis has been to develop lactic acid bacteria cultures for the production of Castellano cheese from strains with good technological characteristics, with a particular focus on the production of flavour compounds, while simultaneously ensuring safety from a food safety perspective. Furthermore, the impact of these cultures on the ripening of Castellano cheese produced with pasteurised milk has been investigated. A total of 66 isolates of lactic acid bacteria from six different species were technologically evaluated. Lactococcus lactis isolates showed the highest acidifying activity and proteolytic activity, while the lactobacilli isolates demonstrated higher endopeptidase and sterolytic activities. Twenty-four isolates were selected and, subsequent to typing, were found to correspond to 20 different strains. The selected strains were subjected to testing for glutamate dehydrogenase activity, which was detected in 13 of them (although activity levels varied widely). Additionally, the ability to produce γ-aminobutyric acid was detected in 14 strains. Furthermore, the antimicrobial activity of the isolates was investigated. This revealed that acid production inhibited the indicator microorganisms, while bacteriocin-like compounds produced by a strain of Lactiplantibacillus plantarum inhibited Enterococcus faecalis. Finally, following an assessment of the safety of the strains, Levilactobacillus brevis strain TAUL1567, which demonstrated resistance to tetracycline and produced tyramine, Lactiplantibacillus paraplantarum strain TAUL1399, which exhibited resistance to ampicillin and two putrescine-producing Lactococcus lactis strains, were excluded from the culture design. From the selected strains, four lactic acid bacteria cultures were designed, comprising both starter (commercial and wild Lactococcus strains) and wild non-starter (Lactiplantibacillus plantarum, Lacticaseibacillus paracasei and Leuconostoc mesenteroides) strains. Five batches of Castellano cheese were produced from pasteurised sheep's milk, comprising a control batch with a commercial starter culture and four experimental batches with the designed cultures. Firstly, the effect of the different cultures on the physicochemical, microbiological and sensory characteristics of Castellano cheese during ripening was investigated. Significant differences were observed in pH, titratable acidity and microbial counts as a consequence of the incorporation of the various non-starter strains. In terms of sensory characteristics, the experimental cheese batch L4, produced with a single wild starter strain (Lactococcus lactis GE44) and two non-starter strains (Lactiplantibacillus plantarum TAUL67 and Leuconostoc mesenteroides TAUL1342), exhibited the best attributes in terms of appearance, aroma, flavour and texture. Secondly, the impact of the designed cultures and ripening on the proteolysis of Castellano cheese was assessed, demonstrating significant differences in nitrogen fractions and free amino acid composition between batches at varying ripening stages, particularly after the initial 30 days. Cheeses containing non-starter strains exhibited the highest free amino acid values at the end of the ripening, particularly in batch L4. Among the principal amino acids, γ- aminobutyric acid was notably prevalent in three of the cheese batches (L1, L2 and L3). Conversely, analysis of the cheese microstructure indicated that proteolysis had an impact on the cheese's microstructure. Furthermore, texture profile analysis demonstrated that the incorporation of the wild strains resulted in an improvement in the hardness, chewiness and gumminess of the cheeses. Finally, the impact of ripening time and different starter cultures on the volatile composition of Castellano cheese was investigated. A non-targeted metabolomic approach was employed to identify 280 volatile metabolites belonging to 11 chemical families, 141 of which had not been previously described in cheese. The volatile profile of Castellano cheese was found to comprise more than 50 % carboxylic acids, 15 % ketones, 12 % alcohols, 5 % aldehydes and 2 % esters. The incorporation of wild lactic acid bacteria strains, particularly lactobacilli strains, resulted in a notable increase in the total content of volatile organic compounds at the end of the ripening period. This was observed to be 40 % and 26 % higher in batches L1 and L2, respectively, in comparison to the control batch. Following a 240-day ripening period, 67 volatile compounds exhibited notable differences between cheese batches, including key compounds such as acetic acid, acetoin, diacetyl, butanediol, and several ethyl esters.