ABSTRACT
A total of 114 lactic acid bacteria were isolated at one and 21 days of ripening from a traditional raw cow's milk cheese without the addition of starter culture, produced by three artisanal cheese-makers in Azores Island (Pico, Portugal). Identification to species and strain level was accomplished by16S rRNA gene and PFGE analysis. Carbohydrate utilization profiles were obtained with the relevant API kits. Isolates were evaluated according to safety and technological criteria. The most frequently observed genus identified by 16S rRNA sequencing analysis was Enterococcus, whereas API system mostly identified Lactobacillus. The highest percentages of antibiotic resistance were to nalidixic acid (95%), and aminoglycosides (64-87%). All isolates were sensitive to several beta-lactam antibiotics and negative for histamine and DNase production. Gelatinase activity was detected in 49.1% of isolates, 43% were able to degrade casein and 93% were α-hemolytic. Most enterococci presented virulence genes, such as gelE, asaI, ace. Diacetyl production was found to be species dependent and one strain (Leu. citreum) produced exopolysaccharides. Selected strains were further studied for technological application and were found to be slow acid producers in milk and experimental cheeses, a desirable trait for adjunct cultures. Two strains were selected on the basis of technological and safety application as adjunct cultures in cheese production and presented the best cheese aroma and flavor in consumer preference tests. This is the first effort to characterize Pico cheese LAB isolates for potential application as adjunct cultures; the results suggest the potential of two strains to improve the quality of this traditional raw milk product.
Subject(s)
Cheese/microbiology , Food Safety , Genetic Variation , Lactobacillaceae/genetics , Raw Foods/microbiology , Animals , Anti-Bacterial Agents/pharmacology , Cattle , Drug Resistance, Multiple, Bacterial , Enterococcus/drug effects , Enterococcus/genetics , Enterococcus/isolation & purification , Food Microbiology , Food Quality , Lactobacillaceae/classification , Lactobacillaceae/isolation & purification , Lactobacillus/drug effects , Lactobacillus/genetics , Lactobacillus/isolation & purification , Portugal , RNA, Ribosomal, 16S , Taste , Vancomycin/pharmacology , Virulence/geneticsABSTRACT
AIM: Evaluate technologically relevant properties from bacteriocin-producing strains to use as starter/adjunct cultures in cheese making. METHODS AND RESULTS: Eight isolates obtained from Pico cheese produced in Azores (Portugal) were found to produce bacteriocins against Listeria monocytogenes and three isolates against Clostridium perfringens. They were identified as Lactococcus lactis and Enterococcus faecalis and submitted to technological tests: growth at different conditions of temperature and salt, acid production, proteolysis, lipolysis, coexistence, enzymatic profile and autolytic capacity. Safety evaluation was performed by evaluating haemolytic, gelatinase and DNase activity, resistance to antibiotics and the presence of virulence genes. Some isolates presented good technological features such as high autolytic activity, acid and diacetyl production. Lactococcus lactis was negative for all virulence genes tested and inhibit the growth of all Lactic acid bacteria (LAB) isolates. Enterococci were positive for the presence of some virulence genes, but none of the isolates were classified as resistant to important antibiotics. CONCLUSIONS: The bacteriocin-producing Lc. lactis present good potential for application in food as adjunct culture in cheese production. The study also reveals good technological features for some Enterococcus isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriocin-producing strains presented important technological properties to be exploited as new adjunct culture for the dairy industry, influencing flavour development and improve safety.