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The use of several autochthonous strains of lactic acid bacteria, including Lactobacillus paracasei subsp. paracasei as adjunct of the starter in the manufacture of Manchego cheese, was evaluated in an attempt to improve the aroma of the industrial Manchego cheese. Volatile composition and odour characteristics were evaluated and compared to those in Manchego cheese manufactured with a commercial starter (CS) culture and with raw milk cheese manufactured without starter. Manchego cheeses manufactured with two autochthonous strains of Lactococcus lactis subsp. lactis displayed a similar volatile profile and odour characteristics to the cheese made with the CS. The use of the strain Lactobacillus paracasei subsp. paracasei CECT 7882 as adjunct of the Lactococcus strains produced cheeses with higher amounts of some free fatty acids and alcohols, acetoin, lactones, phenylacetaldehyde, 2-phenylethanol and linalool, and higher scores of the odour intensity, odour quality, and ewe’s milk odour than the CS cheeses. It resulted in an intensification and improvement of industrial Manchego cheese aroma.

Thirty Lactobacillus isolates from artisanal Manchego cheeses, 10 belonging to Lactobacillus plantarum and 20 belonging to Lactobacillus paracasei subsp. paracasei, were examined for potential use as adjunct cultures. Isolates were screened for acidifying and amino-biogenic capacity, for proteolytic, lipolytic, autolytic and aminopeptidase activities, and for sensory characteristics in UHT coagulated milk. Most of the lactobacilli displayed high aminopeptidase and autolytic activities and low acidifying activity. Only one isolate was able to produce tyramine. When growing on milk 53.3% of the Lb. paracasei subsp. paracasei isolates were able to produce pleasant flavours. Strains PBL210 (Lb. plantarum) and PBL213 and PBL226 (Lb. paracasei subsp. paracasei) presented the best technological characteristics, and also were still viable and with unaltered properties after freeze-drying and storage for 12 months. For these reasons, these strains could be good candidates for inclusion as adjuncts in a starter culture to manufacture industrial Manchego cheese.

Aims: Genotypic and technological characterization of wild lactococci isolated from artisanal Manchego cheese during the ripening process for selection of suitable starter cultures. Methods and Results: A total of 114 isolates of lactococci were typed using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Sixteen distinct RAPD-PCR patterns, at a similarity level of 73%, were obtained. On the basis of species-specific PCR reaction, the isolates were assigned to the species Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris with L. lactis subsp. lactis being predominant at both dairies. Twenty-six isolates were technologically characterized to select those with the best properties. Most of them showed good technological properties although some could produce tyramine. Conclusions: The presence of coincident genotypes at both dairies has been demonstrated, which would suggest that they are well adapted to the Manchego cheese environment. Interesting differences were found in the technological characterization and the potential role of autochthonous lactococci strains as starter culture has been displayed. Significance and Impact of the Study: The great economic importance of Manchego cheese encouraged a deeper knowledge of its microbiota, to select strains with the best properties to use as starter cultures in industrial Manchego cheeses, preserving the autochthonous characteristics.

Enterococci represent a considerable proportion of the microbiota in Manchego cheeses. In this study, a total of 132 enterococci isolated from good quality Manchego cheeses from two dairies at different ripening times were genotypically characterized and identified using molecular techniques. Representative isolates from the clusters obtained after genotyping were assayed for some enzymatic activities considered to have a potential role in cheese ripening, and for 2,3-butanedione and acetoin production, evaluation of odor intensity and appearance in milk and safety evaluation. Enterococcus faecalis was the predominant specie, accounting for 81.8% of the total isolates, while Enterococcus faecium, Enterococcus hirae and Enterococcus avium were present in low proportions. The number of genotypes involved at each ripening time varied both between dairies and with the ripening times; genotype E. faecalis Q1 being present in almost all the samples from both dairies. Eight isolates showed a higher proteolytic activity and 3 isolates produced high quantities of acetoin-diacetyl, for which reason they are interesting from a technological standpoint. A low antibiotic resistance was found and almost all the strains were susceptible to clinically important antibiotics. On the contrary, only four isolates (E. faecalis C4W1 and N0W5, and E. faecium N32W1 and C16W2) did not harbor some of the virulence genes assayed.

Twenty-seven Leuconostoc (Ln.) isolates from Manchego cheese were characterized by phenotypic and genotypic methods, and their technological abilities studied in order to test their potential use as dairy starter components. While phenotypic diversity was evaluated by studying the biochemical characteristics of technological interest (i.e. acidifying and aminopeptidase activities), genotypic diversity was evidenced by using Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). Additional technological abilities such as lipolytic, proteolytic and autolytic activities, salt and pH tolerance and production of dextran, flavour compounds and biogenic amines, were investigated. The marked differences among strains reflected the existing biodiversity in naturally fermented products. After statistically evaluating their performance, strains C0W2, belonging to Ln. lactis, and C16W5 and N2W5, belonging to Ln. mesenteroides subsp. dextranicum, revealed the best properties to be used in mixed dairy starter cultures. This study evidences the fact that natural environments can be considered as a proper source of useful strains, for the dairy industry.