Search Results (13)

Cheese ripening involves microbial changes, with starter lactic acid bacteria (SLAB) initiating fermentation and nonstarter lactic acid bacteria (NSLAB) driving flavor and texture development. However, heat-resistant spores of Clostridium and Bacillus can survive pasteurization and cause spoilage during ripening. This study evaluated NSLAB dynamics in the presence of spores during cheese ripening. Cheddar cheese samples at pilot-scale level (110 L) with four treatments, namely control, with spores of B. licheniformis (T1), with spores of Cl. tyrobutyricum (T2), and both spores (T3) at 2.0 Log₁₀ CFU/mL, were ripened at 7 °C for six months. SLAB declined from 8.0 to 0.2 Log₁₀ CFU/g, while NSLAB increased from 2.0 to 8.5 Log₁₀ CFU/g by month three and maintained their counts up to six months, unaffected by spore presence. Spore counts were ≤1.45 Log₁₀ CFU/g in controls but reached 2.94 ± 0.02 (T2) and 2.48 ± 0.03 (T3), correlating with spoilage signs after five months. MALDI-TOF identified L. rhamnosus (up to 37%) and L. paracasei (up to 25%) as dominant NSLAB across treatments. Physicochemical parameters were not significantly affected by higher spore levels. While NSLAB dominated, they were inadequate to prevent spoilage in spore-inoculated samples exceeding 2.0 logs during cheese ripening. Full article

This review contributes to the knowledge on the complex and adaptive microbial ecosystems within cheese, emphasizing their critical role in determining cheese quality, flavor, and safety. This review synthesizes the current knowledge on the microbial interactions and the dynamics of lactic acid bacteria (LAB), encompassing both starter (SLAB) and non-starter (NSLAB) strains, which are pivotal to the curd fermentation and ripening processes. The adaptability of these microbial consortia to environmental and technological stressors is explored, highlighting their contributions to acidification, proteolysis, and the development of distinctive organoleptic characteristics. Historical and technological perspectives on cheesemaking are also discussed, detailing the impact of milk treatment, starter culture selection, and post-renneting procedures on microbial activity and biochemical transformations. This review underscores the importance of microbial diversity and cooperative interactions in fostering ecosystem resilience and metabolic functionality, and it addresses the challenges in mimicking the technological performance of natural starters using selected cultures. By understanding the ecological roles and interactions of cheese microbiota, this review aims to guide improvements in cheese production practices. Additionally, these insights could spark the development of innovative strategies for microbial community management. Full article

Enterococci are a group of microorganisms that have a controversial position from some scientific points of view. The species of the greatest clinical importance are E. faecalis and E. faecium, which are common agents of nosocomial infections. However, enterococci also have important applications in the dairy industry, as they are used as non-starter lactic acid bacteria (NSLAB) in a variety of cheeses, especially artisanal cheeses. The aim of this study was to determine the presence of representatives from the Enterococcus genus using PCR and MALDI-TOF MS methods on samples of raw milk, processing environment swabs, and cheese from four different artisanal dairy plants in Slovakia. Among the 136 isolates of enterococci, 9 species of genus Enterococci (E. faecalis, E. faecium, E. durans, E. devriesi, E. hirae, E. italicus, E. casseliflavus, E. malodoratus, and E. gallinarum) were identified and were tested for their antimicrobial resistance (AMR) to 8 antibiotics (amoxicillin, penicillin, ampicillin, erythromycin, levofloxacin, vancomycin, rifampicin, and tetracycline); most of them were resistant to rifampicin (35.3%), ampicillin (22.8%), and tetracycline (19.9%). A PCR analysis of vanA (4.41%) and tetM (14.71%) revealed that antimicrobial resistance genes were present in not only phenotypic resistant isolates of enterococci but also susceptible isolates. The investigation of antimicrobial resistance in enterococci during the cheesemaking process can be a source of valuable information for public health in the concept of “One Health”. Full article

Nonstarter lactic acid bacteria (NSLAB) are major contributors to the unique characteristics (e.g., aroma, flavor, texture) of dairy and nondairy fermented products. Lc. paracasei SRX10 is an NSLAB strain originally isolated from a traditional Greek cheese and previously shown to exhibit favorable biotechnological characteristics. More specifically, the strain showed tolerance to simulated gastrointestinal conditions, exopolysaccharide (EPS) biosynthetic capacity, and lack of hemolytic activity and was used in the production of yoghurt and feta cheese with distinct organoleptic characteristics. The aim of the present study was to investigate these traits at the genome level through whole-genome sequencing (WGS), annotation, and comparative genomics. Functional annotation of the genome revealed that Lc. paracasei SRX10 can utilize different carbon sources, leading to the generation of flavor compounds, including lactic acid, acetate, ethanol, and acetoin. Similarly, full clusters for fatty acid biosynthesis, protein and peptide degradation, as well as genes related to survival under extreme temperatures, osmotic shock, and oxidative stress were annotated. Importantly, no transferable antibiotic resistance genes or virulence factors were identified. Finally, strain-specific primers based on genome-wide polymorphisms were designed for the efficient and rapid identification of Lc. paracasei SRX10 via multiplex PCR in fermented products. Full article

This mini review deals with some controversial non-starter lactic acid bacteria (NSLAB) species known to be both human and animal pathogens but also health-promoting and probiotic. The focus is on Lactococcus garvieae, two Streptococcus species (S. uberis and S. parauberis), four Weissella species (W. hellenica, W. confusa, W. paramesenteroides, and W. cibaria), and Mammalicoccus sciuri, which worldwide, are often found within the microbiotas of different kinds of cheese, mainly traditional artisanal cheeses made from raw milk and/or relying on environmental bacteria for their ripening. Based on literature data, the virulence and health-promoting effects of these bacteria are examined, and some of the mechanisms of these actions are reviewed. Additionally, their possible roles in cheese ripening are also discussed. The analysis of the literature data available so far showed that, in general, the pathogenic and the beneficial strains, despite belonging to the same species, show somewhat different genetic constitutions. Yet, when the safety of a given strain is assessed, genomic analysis on its own is not enough, and a polyphasic approach including additional physiological and functional tests is needed. Full article

The microbial ecology fundamentals of raw milk and long-ripened cheeses consist of a complex interaction between starter lactic acid bacteria (SLAB) and non-starter LAB (NSLAB). Although NSLAB aromatic properties are paramount, other phenotypic traits need to be considered for their use as adjunct cultures, such as the capability to endure technological parameters encountered during cheesemaking. The present study focused on the isolation and characterization of NSLAB from spontaneously fermented raw cow’s milk coming from 20 dairies that produce Grana Padano PDO cheese. From 122 isolates, the screening process selected the 10 most diverse strains belonging to Lacticaseibacillus spp. to be phenotypically characterized. The strains were tested for their growth performance in milk in combination with the application of technological stresses, for their ability to produce volatile compounds after their growth in milk, and for their ability to use different nutrient sources and resist chemicals. The complex characterization qualified the strains 5959_Lbparacasei and 5296_Lbparacasei as the best candidates to be used as adjunct strains in the production of raw milk and long-ripened cheeses, provided that antibiotic resistance is measured before their employment. Other strains with interesting aromatic capabilities but lower heat resistance were 5293_Lbparacasei, 5649_Lbparacasei and 5780_Lbparacasei, which could be candidates as adjunct strains for uncooked cheese production. Full article

This literature review aimed to collect investigations on the in vivo evidence for bacteria associated with fermented dairy foods to behave as probiotics with beneficial effects in the prevention and treatment of various diseases. All main bacterial groups commonly present in high numbers in fermented milks or cheeses were taken into account, namely starter lactic acid bacteria (SLAB) Lactobacillus delbrueckii subsp. bulgaricus and lactis, L. helveticus, Lactococcus lactis, Streptococcus thermophilus, non-starter LAB (NSLAB) Lacticaseibacillus spp., Lactiplantibacillus plantarum, dairy propionibacteria, and other less frequently encountered species. Only studies regarding strains of proven dairy origin were considered. Studies in animal models and clinical studies showed that dairy bacteria ameliorate symptoms of inflammatory bowel disease (IBD), mucositis, metabolic syndrome, aging and oxidative stress, cancer, bone diseases, atopic dermatitis, allergies, infections and damage caused by pollutants, mild stress, and depression. Immunomodulation and changes in the intestinal microbiota were the mechanisms most often involved in the observed effects. The results of the studies considered indicated that milk and dairy products are a rich source of beneficial bacteria that should be further exploited to the advantage of human and animal health. Full article

Queijo Serra da Estrela is a PDO Portuguese cheese produced through coagulation of raw ewe’s milk using cardoon (Cynara cardunculus L.) flowers. The present research was aimed at depicting an up-to-date and comprehensive overview of the microbiota of Queijo Serra da Estrela cheese. To this end, viable counting and metataxonomic analysis were carried out on cheeses sampled from four Portuguese artisan producers. Physico-chemical and morpho-textural analyses were also performed, together with the analysis of volatile organic compounds (VOCs). Finally, non-starter lactic acid bacteria (NSLAB) isolated from the cheeses were characterized for their enzymatic activities using a semi-quantitative method. According to the metataxonomic analysis, Lactococcus lactis and Lactococcus piscium were the species occurring at the highest relative abundance. The isolates collected from the cheeses were assigned to Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus lactis, Levilactobacillus brevis, Latilactobacillus graminis, Leuconostoc mesenteroides, and the Lacticaseibacillus casei group. The enzymatic characterization of these cultures highlighted esterase, aminopeptidase, acid phosphatase, beta-galactosidase, alpha-glucosidase, and beta-glucosidase among the major enzymatic activities. Fungal populations were dominated by Debaryomyces hansenii and Kurtzmaniella zeylanoides; however, species rarely found in cheese (e.g., Candida boidinii, Vishniacozyma victoriae, and Starmerella) were also detected. The volatile compounds characterizing the analyzed cheeses were carboxylic acids and esters, followed by carbonyl compounds and alcohols. Full article

Background: The identification of bacterial species in fermented PDO (protected designation of origin) cheese is important since they contribute significantly to the final organoleptic properties, the ripening process, the shelf life, the safety and the overall quality of cheese. Methods: Ten commercial PDO feta cheeses from two geographic regions of Greece, Epirus and Thessaly, were analyzed by 16S metagenomic analysis. Results: The biodiversity of all the tested feta cheese samples consisted of five phyla, 17 families, 38 genera and 59 bacterial species. The dominant phylum identified was Firmicutes (49% of the species), followed by Proteobacteria (39% of the species), Bacteroidetes (7% of the species), Actinobacteria (4% of the species) and Tenericutes (1% of the species). Streptococcaceae and Lactobacillaceae were the most abundant families, in which starter cultures of lactic acid bacteria (LAB) belonged, but also 21 nonstarter lactic acid bacteria (NSLAB) were identified. Both geographical areas showed a distinctive microbiota fingerprint, which was ultimately overlapped by the application of starter cultures. In the rare biosphere of the feta cheese, Zobellella taiwanensis and Vibrio diazotrophicus, two Gram-negative bacteria which were not previously reported in dairy samples, were identified. Conclusions: The application of high-throughput DNA sequencing may provide a detailed microbial profile of commercial feta cheese produced with pasteurized milk. Full article

Grana Padano (GP) is the most appreciated and marketed cheese with Protected Designation of Origin in the world. The use of raw milk, the addition of undefined cultures (defined as ‘sieroinnesto naturale’), the peculiar manufacturing proces, and the long ripening make the cheese microbiota play a decisive role in defining the quality and the organoleptic properties of the product. The knowledge on the microbial diversity associated with GP has been the subject, in recent years, of several studies aimed at understanding its composition and characteristics in order, on the one hand, to improve its technological performances and, on the other hand, to indirectly enhance the nutritional quality of the product. This review aims to briefly illustrate the main available knowledge on the composition and properties of the GP microbiota, inferred from dozens of studies carried out by both classical microbiology techniques and metagenomic analysis. The paper will essentially, but not exclusively, be focused on the lactic acid bacteria (LAB) derived from starter (SLAB) and the non-starter bacteria, both lactic (NSLAB) and non-lactic, of milk origin. Full article

Adjunct cultures are used in cheesemaking to improve the sensory characteristics of the ripened cheeses. In addition, it is known that different adjunct cultures are capable of producing enzymes with the specificity to hydrolyze caseins, leading to the release of various bioactive compounds (bioactive peptides, amino acids, etc.). The objective of this study was to evaluate the effect of adjunct heat-treated starter XT–312 and a cheese culture Lb. helveticus LH-B01 on the proteolytic activity and angiotensin converting enzymes inhibitors (ACE) in cheese models during ripening. Seven different cheese models were evaluated for: proteolytic activity using the spectrophotometric method with ortho-phthaldialdehyde (OPA), soluble nitrogen (SN), trichloroacetic acid-soluble nitrogen (TCA-SN) phosphotungstic acid-soluble nitrogen (PTA-SN), total nitrogen (TN), pH, contents of water, fat, as well as for total bacteria count (TBC), count of Lactococcus genus bacteria, count of Lb. helveticus, and number of non-starter lactic acid bacteria (NSLAB). Presence of adjunct bacterial cultures both in the form of a cheese culture LH-B01 and heat-treated XT–312 starter promoted primary and secondary proteolysis, which resulted in acceleration of the ripening process. ACE inhibitory activity and proteolytic activity was the highest throughout of ripening for cheese model with LH-B01 culture. The cheese models with the adjunct heat-treated starter were characterized by lower TBC, NSLAB and lower count of Lactococcus genus bacteria during ripening, compared to control cheeses. Full article

In many dairy products, Leuconostoc spp. is a natural part of non-starter lactic acid bacteria (NSLAB) accounting for flavor development. However, data on the genomic diversity of Leuconostoc spp. isolates obtained from cheese are still scarce. The focus of this study was the genomic characterization of Leuconostoc spp. obtained from different traditional Montenegrin brine cheeses with the aim to explore their diversity and provide genetic information as a basis for the selection of strains for future cheese production. In 2019, sixteen Leuconostoc spp. isolates were obtained from white brine cheeses from nine different producers located in three municipalities in the northern region of Montenegro. All isolates were identified as Ln. mesenteroides. Classical multilocus sequence tying (MLST) and core genome (cg) MLST revealed a high diversity of the Montenegrin Ln. mesenteroides cheese isolates. All isolates carried genes of the bacteriocin biosynthetic gene clusters, eight out of 16 strains carried the citCDEFG operon, 14 carried butA, and all 16 isolates carried alsS and ilv, genes involved in forming important aromas and flavor compounds. Safety evaluation indicated that isolates carried no pathogenic factors and no virulence factors. In conclusion, Ln. mesenteroides isolates from Montenegrin traditional cheeses displayed a high genetic diversity and were unrelated to strains deposited in GenBank. Full article

Bacteriocinogenic cultures can represent a natural way to increase the safety of cheeses made from raw milk, in which a relevant role in ripening and flavor formation is exerted by the nonstarter lactic acid bacteria (NSLAB). Since the latter can be inhibited by bacteriocin producers, this study evaluated to which extent a nisinogenic culture inoculated at low initial levels can affect the growth rate and peptide degradation activity of the nisin-sensitive cheese isolate Lactobacillus plantarum LZ by comparison with its isogenic variant, L. plantarum LZNI, with increased immunity to nisin. A growth delay of the nisin sensitive strain was observed only when its initial number was 100-fold lower than the nisin producer and nisin was added as an inducer of its own production. In this case, the amount of free α-amino groups was significantly different between cultures of L. plantarum LZ and LZNI only at Day 1. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) highlighted a few differences between the peptide profiles of co-cultures L. plantarum LZ and LZNI. However, results showed that the bacteriocin producer did not dramatically influence the behavior of the sensitive NSLAB and that the evaluation of the effects on microbial contaminants in cheese is worthwhile. Full article