Search Results (43)

Artisanal raw milk cheeses harbour complex microbial communities that drive cheese making and shape sensory quality. Previous work on Idiazabal cheese identified rennet as a major microbial source, although all reservoirs contributed to varying degrees. However, their impact in terms of enzyme-encoding genes related to technological quality of cheese remained unexplored. Building on that, this study draws on metagenome-assembled genomes (MAGs) from cheeses and dairy environments to comprehensively identify enzyme-encoding genes involved in key biochemical processes. In cheese MAGs (Lacticaseibacillus paracasei), protease-encoding genes were dominated by ATP-dependent metalloproteases (M41), carbohydrate-active enzyme-encoding genes (CAZymes) by glycoside hydrolases (GH) and glycosyltransferases (GT), while esterase, lipase, and related-enzyme-encoding genes were restricted to sparse ‘GDXG’, type-B and esterase D families. Dairy environments emerged as major reservoirs of enzyme-encoding genes, with notable differences among sample types (p ≤ 0.001). The richest sources of protease-encoding genes were grass (610 genes), linked primarily to Pantoea agglomerans, and rennet (318), mainly related to Basfia sp. and Moraxella sp., dominated by metalloproteases (M23, M38) and serine proteases (S15). The largest reservoirs of CAZyme-encoding genes were food contact surfaces (1550), associated mainly with Salinisphaera sp. and Dietzia sp., and rennet (1505), related to, e.g., Bacteroides pyogenes, Alloprevotella sp., and Lentilactobacillus buchneri. Food contact surfaces were also the richest source of esterase, lipase and related-enzyme-encoding genes (1209), mainly linked to Dietzia sp., Corynebacterium sp., and Brevibacterium aurantiacum. Similarly, aroma-related enzyme-encoding genes (e.g., oppA, pepA, GH13, esterase D) were consistently detected in environmental matrices. These results provide novel insights into dairy microbiomes as functional reservoirs of aroma precursors, revealing their relevance for artisanal PDO cheese production and future biotechnological applications. Full article

Background/Objectives: Staphylococcus aureus (S. aureus) intramammary infection remains a major global dairy problem due to its contagious nature, its ability to persist and colonize teat/skin and mucosal niches, and the often-limited bacteriological cure achieved with antimicrobial therapy. Beyond udder health, it is relevant to public health because it can enter raw milk chains and serve as a reservoir for antimicrobial resistance determinants that may circulate between dairy animals and humans. Methods: We assessed S. aureus’ ecology in raw ewe milk from 75 sheep farms in Epirus (Greece) by sampling clinically healthy controls (group A) and clinical mastitis cases pre-treatment (group B), followed by resampling at the first post-withdrawal milking after penicillin/streptomycin treatment (group C1—therapeutic protocol 1), oxytetracycline treatment (group C2—therapeutic protocol 2), or enrofloxacin treatment (group C3—therapeutic protocol 3). Results: S. aureus detection was high and comparable across groups (A 23.0%, B 22.0–30.0%, C 20.0–22.0%), and paired analyses showed no significant pre–post shifts in detection/burden within therapeutic protocols (all p > 0.05). Nevertheless, persistence remained evident. The chromosomal gene mecA was detected in S. aureus strains in all groups, ranging from 13.6% in controls to 54.5% post-withdrawal in group C1, and was also present in the pre-treatment group. In paired sampling animals, mecA was mostly stable, with rare emergence or loss. Across antibiotic classes, within-animal resistance transitions were generally uncommon and non-significant (p > 0.05); β-lactam resistance was fully stable (p = 1.00). Descriptively, resistance to protein synthesis inhibitors tended to decline after therapy in protocol 1 and protocol 3, while protocol 3 showed post-treatment gains in fluoroquinolone resistance. By contrast, virulence-associated phenotype traits shifted after therapy: enterotoxigenicity increased post-withdrawal (especially in the C3 group), Staphylococcal Enterotoxin A (SEA) and Staphylococcal Enterotoxin B (SEB) appeared only post-therapy, Staphylococcal Enterotoxin D (SED) increased significantly in paired isolates (p = 0.002), and strong biofilm adherence increased (in C3, p = 1.5 × 10⁻⁵). Conclusions: The detection of S. aureus after therapy suggests that one possibility is that antimicrobial exposure may select for, or otherwise reshape, the residual intramammary population, rather than reliably eliminating it—an outcome that remains clinically relevant for udder health. Moreover, the persistence of mecA/methicillin-resistant Staphylococcus aureus (MRSA)-compatible profiles indicates that milk released to the food chain after withdrawal compliance may still harbor S. aureus with enhanced preservation capacity and significant food safety relevance. Full article

Ewe’s milk cheese produced from raw milk holds cultural and economic importance in Southern European countries; however, it poses microbiological challenges. Among spoilage microorganisms, Pseudomonas fluorescens is particularly concerning due to thermostable enzymes that impair the texture, aroma, and stability of cheese, even under refrigeration and salinity. This study evaluated the influence of sodium chloride concentration on Pseudomonas fluorescens given the pivotal role of salt in ensuring cheese stability and safety. Cheeses inoculated with Pseudomonas fluorescens were produced under an experimental design that combined three ripening temperatures with four salt concentrations. Physicochemical composition and microbiological stability were assessed at the end of ripening (20 days). Results showed that the ripening temperature emerged as the most determinant factor, influencing microbial viability and increasing solid retention, proteolysis, and dehydration, leading to harder cheeses. Low temperatures without salt favoured surface colour defects, whereas, although high salt levels contributed to partial control of Pseudomonas spp., they also delayed ripening, resulting in cheeses with a pale, uncharacteristic appearance. Conversely, moderate salinity (2%) combined with higher ripening temperatures promoted uniform maturation, resulting in a stable texture and appearance free of defects. These findings highlight the need to balance salt and ripening conditions to optimise quality and safety in traditional raw ewe’s milk cheeses. Full article

A freeze-dried natural starter culture (NSC) was developed and assessed for its suitability in producing a semi-cooked, 60-day-ripened cheese resembling the protected designation of origin (PDO) Pecorino Sardo. The culture, derived from raw ewe’s milk from a dairy farm involved in the study, without thermal decontamination to preserve indigenous microbiota, was enriched with two strain-level-characterised, biodiverse mixtures of Streptococcus thermophilus (Str-mix) and Lactobacillus delbrueckii (Lb-mix). This study evaluated the technological robustness and adaptability of NSC enriched with biodiverse Str-mix and Lb-mix across three different artisanal dairy-processing environments with varying milk compositions and equipment levels at plants located in different geographic areas. During cheesemaking, technological, physico-chemical, and compositional parameters were monitored, along with microbial characterisation of milks and 1-day cheeses. After 60 days of ripening, cheeses were characterised from the microbiological, physico-chemical, and compositional perspectives. Furthermore, nutritional labelling was established, and consumer acceptance was determined. Results showed that the starter (NSC + Str-mix + Lb-mix) demonstrated strong and reproducible technological performance in all dairies, regardless of the milk’s chemical and microbial composition variability. Sensory quality was preserved in cheeses ripened for up to 180 days. These preliminary findings seem to support the use of freeze-dried, raw-milk-derived natural cultures in artisanal cheesemaking as a way to preserve microbial diversity and to reconnect with traditional practices that enhance both the tangible and intangible assets of modern society. Full article

Serra da Estrela cheese (a raw ewe’s milk) ripened for 45 days was treated at 600 MPa for 6 min (P1) and at 450 MPa for 6 (P2) and 9 min (P3) and kept in refrigerated storage for 15 months. Lactobacillus and Lactococcus viable cell numbers were reduced in 3.2–3.6 and 2.7–3.6 log cycle units, respectively. Lower reductions were verified for total aerobic mesophilic and Enterococcus viable cell numbers in cheeses treated at 450 MPa (2.4–2.5 and 1.2 log reductions, respectively). In HPP cheeses, yeasts and moulds were below the enumeration limit up to 6 months of storage, but at 15 months 3.6–4.2 log cfu/g were quantified in all cheeses, while Enterobacteriaceae were inactivated to below the quantification limit. The increment of pressure treatment caused a greater impact on the microbiota’s viability than the increase in time under pressure. During storage, minor total colour differences were determined for HPP P3 cheese surface relative to control cheeses, Ch_C, at 45 days of ripening. HPP can thus be a good process to apply after cheese manufacture, since it offers a good potential to render raw-milk cheese microbiologically safer, with minimal changes in quality. Full article

Raw goat milk-derived Lactococcus lactis MK1/3 (CCM 9209) was studied to show its potential for use in the dairy industry. Finding an innovative strain indicates having a new safe, original additive for functional food. The strain has been shown to be safe using a model experiment with Balb/c mice, when no mortality was noted. Its counts were increased continually during 120 days, with the highest value on day 90 (4.38 ± 1.24 colony-forming unit per gram (CFU/g, log 10). In vivo (in the experimental mice), anti-staphylococcal effect was noted with difference 1.82 log cycles. The safety of the strain MK1/3 has been also indicated by the fact that it did not produce damaging enzymes, it has been susceptible to antibiotics, and it has shown low-grade biofilm-forming ability (0.126 ± 0.35). This strain has tolerated bile, and low pH sufficiently. It produced a postbiotic active substance with inhibitory activity against cheese and milk contaminants (Enterococci), reaching antimicrobial activity up to 3200 AU/mL. The count of the strain MK1/3 was higher in yogurts from ewe goat milk (4.66 ± 0.30 CFU/g, log 10), in comparison with its count in yogurts from ewe milk (4.10 ± 0.10 CFU/g, log 10), with no influencing yogurt pH. Its use in 100% starter culture to process fresh cheese based on goat milk was revealed in the standard cheese quality with sufficient amount of lactic acid microbiota. To support the benefit of the strain MK1/3, additional human trials have been reinforced. Full article

Serra da Estrela cheese, with a Protected Denomination of Origin (PDO), is one of the most appreciated traditional raw milk Portuguese cheeses, and it is well known for its unique flavor and texture, which are derived from the use of raw ewe’s milk and its production process. In this work, 45-day-old ripened Serra da Estrela cheeses were processed by high-pressure processing (HPP) at 600 MPa/6 min (P1), 450 MPa/6 min (P2) and 450 MPa/9 min (P3) to study the effect of HPP initially and during 15 months of storage at 4 °C. The proteolysis indexes were, in general, lower in the HPP-treated cheeses than in the control cheeses. The P1 cheeses kept their ripening extension index throughout the 15 months of storage close to that of non-processed cheese at month 0. Progression of the ripening depth and free amino acids indexes was also slowed down by HPP. HPP had no immediate effect on the cheese texture parameters, and minor changes were found up to 3 months of storage; moreover, the P2 cheeses maintained their hardness and consistency levels during the 15-month storage period at values close to those of the control cheeses at month 0. Sensory evaluation by trained panelists showed that the P2 cheeses were softer than the control cheeses; furthermore, for the P3 cheeses, there were no observed treatment effects on the sensory attributes evaluated at the end of storage. Overall, the results uphold the potential of HPP in rendering Serra da Estrela cheese proteolysis levels similar to those of control cheese at 45 days of ripening with minor effects on texture. Full article

Raw goat and ewe’s milk samples were used for the isolation of seven lactic acid bacteria new strains. After testing hemolytic activity and resistance to antibiotics, specific functional properties were evaluated; Lactococcus lactis subsp. lactis FBM_1321 and Lacticaseibacillus paracasei FBM_1327 strains resulted in the highest cholesterol assimilation percentages ranging from 28.78 to 30.56%. In addition, strong adhesion capacity to differentiated Caco-2 cells (1.77–21.04%) was mapped, and the lactobacilli strains exhibited strong antagonistic activity against foodborne pathogens compared to lactococci. The strains were able to grow at low pH and high NaCl concentrations, conditions that prevail in food systems (cell counts ranged from 1.77 to 8.48 log CFU/mL after exposure to pH 3 and from 5.66 to 9.52 log CFU/mL after exposure to NaCl concentrations up to 8%). As a next step, freeze-dried immobilized Lc. paracasei FBM_1327 cells on oat flakes were used for the preparation of functional yoghurt and ayran products. Cell loads of the functional strain remained high and stable in both products (7.69 log CFU/g in yoghurt and 8.56 log CFU/g in ayran after 30 days of storage at 4 °C) throughout their shelf life. No significant changes in the volatile profile were noticed, and the new products were accepted by the panel during the sensory evaluation. Full article

Dairy products from ovine milk are very popular in the Mediterranean countries and are gaining a large portion of the market in EU countries and worldwide. EU legislation permits the dairy processing of raw ovine milk under certain conditions. To study the ecology and prevalence of E. coli in raw ewes’ milk and assess thus the public health risk, samples of milk were taken from 75 different sheep farms in the rural area of Epirus, Greece. The initial sampling was conducted in clinically healthy animals which were noted as controls (group A). From the same farms, samples were taken from animals with clinical mastitis and before treatment (group B). For therapeutic purposes, to some animals, a combination of penicillin and streptomycin was administrated (group C1), or tetracycline (group C2), or enrofloxacin (group C3). Finally, samples of raw milk were taken from the C groups, on the first day after the withdrawal period of the antibiotics used, when the milk is permitted to enter the food chain. In total, 97 isolates of Escherichia coli were recovered from all groups. Analysis revealed an impressive increase in E. coli strains in the milk of group B (39.33%) with respect to group A (5%). Even after treatment, although the prevalence was decreased, it was still found to be higher in the C groups than group A. E. coli O157:H7 strains absent from group A were detected in all other groups in relatively low occurrence rates with respect to other “O” serotypes but non-O157:H7 strains. Virulence factors such as the production of toxins (32.60% for serotoxin 1 and 18.47% for serotoxin 2) and hemolysin (42.39%) as well as biofilm formation capacity (52.17% of the total) and ESDL production (43.47% of the total) were also studied. All strains were also tested for susceptibility against 12 antibiotics by the MIC method and the results showed a high prevalence of resistance and multi-resistance. The presence of various resistant strains to antibiotics and pathogenic “O” serotype strains in the milk when it can enter the food chain again is an alarming conclusion. Full article

For the first time, High-Resolution Magic Angle Nuclear Magnetic Resonance spectroscopy (NMR-HRMAS) was applied to directly identify specific metabolites from a Spanish raw ewe’s milk and enzymatic coagulation pressed-curd cheese (Protected Geographical Indication: Castellano) manufactured by two procedures (traditional/artisanal vs. industrial) and including the ewe’s raw milk. The NMR parameters were optimized to study the complex matrixes of this type of cheese. In addition, conventional overcrowded ¹H-NMR-HRMAS spectra were selectively simplified by a Carr–Purcell–Meiboom–Gill (CPMG) sequence or a stimulated echo pulse sequence by bipolar gradients (DIFF), thus modulating spin–spin relaxation times and diffusion of molecular components, respectively. ¹H-NMR-HRMAS spectroscopy displayed important information about cheese metabolites, which can be associated with different manufacturing processes (industrial vs. traditional) and ripening times (from 2 to 90 days). These results support that this spectroscopy is a useful technique to monitor the ripening process, from raw milk to commercial ripened cheese, using a minimum intact sample, implying the absence of time-consuming sample pretreatments. Full article

Natural starter cultures, characterised by undefined microbiota, can contribute to the technological process, giving peculiar characteristics to artisanal fermented foods. Several species have a long history of safe use and have obtained Qualified Presumption of Safety (QPS) status from the European Food Safety Authority (EFSA), whereas others (non-QPS) could represent a potential risk for consumers’ health and must undergo a safety assessment. In this work, the biodiversity, at species and strain level, by pulsed-field gel electrophoresis (PFGE) and (GTG)₅ rep-PCR, of an undefined natural starter culture, in frozen and lyophilized form, obtained from ewe’s raw milk avoiding thermal treatment or microbial selection, was investigated. The culture was constituted by different biotypes of Enterococcus durans, Enterococcus faecium, Enterococcus faecalis, and Lacticaseibacillus paracasei. Streptococcus oralis and Streptococcus salivarius were also found, over species belonging to the Streptococcus bovis–Streptococcus equinus complex (SBSEC), like Streptococcus gallolyticus subsp. macedonicus, Streptococcus lutetiensis, and Streptococcus equinus. Molecular investigation on virulence and antibiotic resistance genes, as well as minimum inhibitory concentration (MIC) determination, revealed that all the non-QPS strains can be considered safe in the perspective of using this culture for cheesemaking. The obtainment of a natural culture directly from ewe’s raw milk bypassing thermal treatment and selection of pro-technological bacteria can be advantageous in terms of biodiversity preservation, but non-QPS microorganisms can be included in the natural starter and also in cheeses, especially in traditional ones obtained from fermenting raw milk. Following EFSA guidelines, artisanal factories should not be allowed to produce starter cultures by themselves from raw milk, running the risk of including some non-QPS species in their culture, and only selected starters could be used for cheesemaking. A revision of the criteria of QPS guidelines should be necessary. Full article

Goat milk is gradually becoming the preferred milk by consumers worldwide, including Slovakia. It is also demanded as a functional and/or nutraceutical drink as it is rich in bioactive components. However, another new development is the research into the beneficial autochthonous strains used to enrich goat dairy products. Among individual species, representatives in raw goat milk are also involved in lacticaseibacilli. Bacteriocin-like inhibitory substance-producing Lacticaseibacillus paracasei LPa 12/1 was isolated from raw goat milk. This study focuses on its potential as a new additive in dairy products. No mortality was found when checking the safety of the LPa 12/1 strain using Balb/c mice. The strain reached higher counts in ewe–goat milk yoghurt (up to 6.1 cfu/g log 10) than in cow milk yoghurt (almost 5.0 cfu/g log 10). Cow milk yoghurts remained in more stable consistency after LPa 12/1 supplementation compared with ewe–goat milk yoghurts, although specific organoleptic tests were not performed. However, LPa 12/1 has survived sufficiently in both types of yoghurts. This bacteriocin-like inhibitory substance-producing strain LPa 12/1 in its encapsulated form applied in yoghurts seems suitable to supplement dairy products. Full article

This study aimed to investigate enterococci recovered from eight Portuguese cheeses made with raw ewe’s milk, regarding antibiotic resistance, virulence genes, minimum inhibitory concentration (MIC) of benzalkonium chloride (BAC), biofilm formation capacity, and biofilm eradication (MBEC) by BAC. Antimicrobial resistance against seven antibiotics of five groups was evaluated using the disk diffusion method. The presence of the genes that encode resistance to the antibiotics penicillin (blaZ), erythromycin (ermA, ermB, and ermC), vancomycin (vanA and vanB), aminoglycoside (aac(6′)-Ie-aph(2″)-Ia), and β-lactam (pbp5) and the genes that encode virulence factors, frsB, cylA, gelE, esp, and agg, were investigated via multiplex PCR. The susceptibility of planktonic cells to BAC was evaluated by the MIC and MBC values of the isolates, using the broth microdilution method. To assess the biofilm-forming ability and resistance of biofilms to BAC, biofilms were produced on stainless steel coupons, followed by exposure to BAC. The results showed a high resistance to the antibiotics vancomycin (87.5%), erythromycin (75%), tetracycline (50%), and penicillin (37.5%). Multidrug resistance was observed in 68.8% of the isolates. Genes encoding the virulence factors FrsB (frsB) and gelatinase E (gelE) were detected in all isolates. The esp and cylA genes were found in 56.3% and 37.5% of the isolates, respectively. All isolates exhibited a biofilm-forming ability, regardless of incubation time and temperature tested. However, after 72 h at 37 °C, E. faecium and E. faecalis biofilms showed significant differences (p ≤ 0.05). Although most isolates (62.5%) were susceptible to BAC (MIC ≤ 10 mg/L), biofilms of the same isolates were, generally, resistant to the higher concentration of BAC (80 mg/mL) tested. This study using Enterococcus isolates from a ready-to-eat food, such as cheese, reveals the high percentages of vancomycin resistance and multidrug resistance, associated with the presence of virulence genes, in isolates also capable of producing biofilms resistant to BAC, an important active ingredient of many disinfectants. These results emphasize the need for effective control measures to ensure the safety and quality of dairy products. Full article

Serra da Estrela is the oldest and most recognizable traditional protected designation of origin (PDO) cheese from Portugal. It has been extensively studied over the years, but the latest microbial characterization is 20 years old. Hence, this work aimed to perform an updated characterization of Serra da Estrela PDO cheeses and raw materials. Our analysis showed that lactic acid bacteria content on Serra da Estrela cheeses exceeded 8.8 log CFUsg⁻¹, in all analyzed samples, with lactococci, lactobacilli and Leuconostoc spp. predominating over enterococci strains. Moreover, lactococci and lactobacilli abundance increased across the production season, while enterococci dropped considerably in late manufactures. Lastly, Leuconostoc spp. content remained unchanged in all analyzed periods. A correspondence analysis showed that L. paracasei, L. lactis, E. durans, E. faecium and L. mesenteroides are transversal in Serra da Estrela cheese manufacturing and were closely associated with milk, curd and cheese matrices. Additionally, L. casei, L. plantarum and L. curvatus were specifically associated with cheese matrices, possibly active during ripening and contributing for the development of these cheeses’ organoleptic characteristics. Full article

High-pressure processing (HPP) can be used as a nonthermal pasteurization technique to overcome microbial safety issues of the raw ewes’ milk Serra da Estrela cheese without negatively influencing its quality, in particular, the lipid composition partly responsible for Serra da Estrela cheese’s sensorial and textural attributes. The aim of this work was to assess HPP’s effect (600 MPa/6 min and 450 MPa/6 and 9 min) on the qualitative and quantitative lipid profiles of Serra da Estrela cheese during 15 months of refrigerated storage. Total triglycerides content (65–66 g TG/100 g) was similarly determined for HPP-treated (450 MPa/6 min) and control cheeses. Similar total contents of saturated, monounsaturated, and polyunsaturated fatty acids were reported for all cheeses during storage. A high total conjugated linoleic acid content (1.29–1.65 g FA/100 g fat) was quantified in all cheeses during storage; all cheeses revealed similar atherogenic and thrombogenic indices (~2.3 and ~2.6, respectively). HPP can be used to process Serra da Estrela cheese at conditions that assure microbial safety without influencing cheese lipid profiles. Full article