Influence of bacterial dynamics upon the final characteristics of model Portuguese traditional cheeses Cláudia I. Pereira, João A. Graça, Natacha S. Ogando, Ana M.P. Gomes * , F. Xavier Malcata CBQF/Escola Superior de Biotecnologia – Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, P-4200-072 Porto, Portugal Keywords: Lactic acid bacteria (LAB) Proteolysis Organic acids Microstructure abstract The microbiological profile in raw milk cheeses is typically characterized by a multitude of microbial groups, with interactions among them throughout ripening that are not fully understood to date. Inci- dence of undesired microorganisms in raw cheesemaking milk, as is the case of either spoilage or even pathogenic ones, is a common trait in Portuguese traditional cheeses. Hence, they will likely contribute to the physicochemical changes occurring therein and, consequently, to the characteristics of the final product. In order to gain insight into their role, model cheese systems, manufactured as far as possible according to artisanal practices (except that the initial microbial load and biodiversity were controlled), were experimentally tested. Single contaminants, or a consortium thereof, were inoculated at two levels in sterilized raw ewe's milk, and duly combined with inocula containing one or two lactic acid bacteria normally found in those traditional cheeses. The physicochemical composition, organic acid profile, and evolution of both protein breakdown and rheology were monitored throughout a 60 d-ripening period. Modifications brought about within the cheese matrix as a result of microbial metabolism, especially those arising from the interaction between lactic acid bacteria and unwanted microorganisms, included the enhanced release of peptides and free amino acids, which in turn led to higher viscoelastic moduli. The final model cheeses could be well discriminated, based on the impact of the various inocula considered upon the levels of organic acids. Conversely, proteolysis and viscoelastic properties appeared to be essentially independent of the initial microflora. One of the major problems associated with raw milk cheeses is the presence of undesired microorganisms throughout the manu- facture and ripening processes. Use of raw milk, and thus absence of any standardizing thermal process, coupled with different milking and handling protocols, and thus variation in hygienic conditions prevailing in the farmhouses, lead to extensive and unpredictable variability. Portuguese cheeses, bearing Protected Denomination of Origin (PDO) status, are examples of such prod- ucts. They are manufactured traditionally from whole raw milk and depend critically on their indigenous microflora for development of the final characteristics. The contribution of such microflora, coupled with that of milk composition, which depends on the local animals' diet and physiological condition, eventually account for the unique flavour and texture of those traditional cheeses. Several microorganisms – including bacteria, yeasts and moulds, are present in cheese throughout ripening. Hence, they contribute to maturation, either directly via their metabolic activity, or indi- rectly via release of enzymes into the cheese matrix, after autolysis. Although most of the microflora of raw milk comprises lactic acid bacteria (LAB) – e.g. Lactococcus and Lactobacillus spp., passive inclusion of such undesired microorganisms as coliforms, Staphy- lococcus spp., Pseudomonads or even Listeria spp. raises potential public health hazards. Hence, they are of major concern towards microbiological safety of the final product (Almeida et al., 2007). Although the aforementioned cheeses are consumed after a minimum ripening time of ca. 45 d at temperatures of ca. 10 °C (Macedo et al., 1993) – which are conditions lethal to most con- taminantic microorganisms, their action while viable will eventu- ally affect the final cheese features. Addition of a tailor-made starter culture may bring about favourable contributions to control growth of unwanted microorganisms. In particular, the action of LAB via initial fermentation of lactose and breakdown of proteins, or via more complex catabolic reactions later during ripening, is a well- known contributor to the organoleptic features perceived in the final cheese (Menéndez et al., 2000). Furthermore, yeasts have for long been recognized to play an important role in Serra da Estrela cheese (Macedo et al., 1993; Freitas and Malcata, 2000). On the other hand, much of the existing knowledge is derived from studies * Corresponding author. Tel.: +351 22 558 00 84; fax: +351 22 509 03 51. E-mail address: amgomes@esb.ucp.pt (A.M.P. Gomes). Introduction