Cotija Cheese Research Articles

Microbiological Evaluation of Two Mexican Artisanal Cheeses: Analysis of Foodborne Pathogenic Bacteria in Cotija Cheese and Bola de Ocosingo Cheese by qPCR.

Cotija and Bola de Ocosingo are artisanal ripened cheeses produced in Mexico. Both are made with raw bovine milk from free-grazing cows and with no starter cultures. Unlike culture-based techniques, molecular methods for pathogen detection in food allow a shorter turnaround time, higher detection specificity, and represent a lower microbiological risk for the analyst. In the present investigation, we analyzed 111 cheese samples (95 Cotija and 16 Bola de Ocosingo) by qPCR (TaqMan®) after an enrichment-culture step specific to each foodborne bacterium. The results showed that 100% of the samples were free of DNA from Listeria monocytogenes, Brucella spp., Escherichia coli enterotoxigenic (ETEC), and O157:H7; 9% amplified Salmonella spp. DNA; and 11.7%, Staphylococcus aureus DNA. However, the threshold cycle (Ct) values of the amplified targets ranged between 23 and 30, indicating DNA from non-viable microorganisms. Plate counts supported this assumption. In conclusion, 100% of the cheeses analyzed were safe to consume, and the enrichment step before DNA extraction proved essential to discern between viable and non-viable microorganisms. Hygienic milking, milk handling, cheese manufacturing, and ripening are crucial to achieve an adequate microbiological quality of cheeses made with raw milk.

Study of an Enterococcus faecium strain isolated from an artisanal Mexican cheese, whole-genome sequencing, comparative genomics, and bacteriocin expression

Enterococci are ubiquitous microorganisms in almost all environments, from the soil we step on to the food we eat. They are frequently found in naturally fermented foods, contributing to ripening through protein, lipid, and sugar metabolism. On the other hand, these organisms are also leading the current antibiotic resistance crisis. In this study, we performed whole-genome sequencing and comparative genomics of an Enterococcus faecium strain isolated from an artisanal Mexican Cotija cheese, namely QD-2. We found clear genomic differences between commensal and pathogenic strains, particularly in their carbohydrate metabolic pathways, resistance to vancomycin and other antibiotics, bacteriocin production, and bacteriophage and CRISPR content. Furthermore, a bacteriocin transcription analysis performed by RT-qPCR revealed that, at the end of the log phase, besides enterocins A and X, two putative bacteriocins not reported previously are also transcribed as a bicistronic operon in E. faecium QD-2, and are expressed 1.5 times higher than enterocin A when cultured in MRS broth.

Functional Social Organization to Obtain the Region of Origin Cotija Cheese Collective Brand

Objective: To identify the functional elements which impact the processes of patrimonial activation to obtain the Region of Origin Cotija Cheese Collective Brand, through collective social action as the basis of territorial development in the Sierra of the states of Jalisco and Michoacán. Design/Methodology/Approach: A qualitative study was conducted, based on the tradition of rural sociology, where the categories of analysis of the Localized Agrifood Systems approach were used. For this purpose, semi-structured interviews with different social actors were applied where there was an inquiry about the technical, organizational, economic and political aspects that have an impact in obtaining seals of quality, destined to the protection and differentiation of emblematic foods. Results: After an attempt to obtain the Denomination of Origin, given the particularities of the Localized Agrifood System involved in the production of Cotija cheese, the decision was to obtain a Collective Brand whose prerequisites adjusted better to the characteristics of their productive systems. Obtaining the seal was an important achievement in terms of the horizon that this signal generated for producers and for the territory. However, this is only the beginning in a process of constant valuation that must be redefined throughout time. Study Limitations/Implications: It is an exploratory study that only addresses the internal dimension of the productive system of Cotija cheese; a deeper analysis ought to broaden the study of the social networks implied in the processes of valuation, including actors outside the territory. Findings/Conclusions: Mexico’s agrifood heritage is a strategic resource that must be protected for cultural, economic, food security and health reasons. This should be assumed as an obligation of the State in co-responsibility with academia, producers and consumers. Obtaining a collective brand is only one step within an unfinished valuation process that must be maintained through collective action and market stimulus.

Microbiological Characteristics and Behavior of Staphylococcus aureus, Salmonella spp., Listeria monocytogenes and Staphylococcal Toxin during Making and Maturing Cotija Cheese

Cotija cheese is an artisanal matured Mexican cheese from unpasteurized milk. This work determined the microbiological characteristics and behavior of Staphylococcus aureus, Salmonella spp., Listeria monocytogenes and staphylococcal toxin during cheese elaboration and ripening. Sixteen 20-kg cheeses were used, eight inoculated with 6 log CFU/mL of each pathogen, and eight uninoculated, and samples were taken at each stage of the process. In the uninoculated samples, the survival of S. aureus and L. monocytogenes decreased after 30 days of ripening. The average counts of S. aureus in milk, curd, and serum were 7 log MPN /mL, and 8.7 log MPN /g in cheese, decreasing from day 15. Salmonella spp. counts (initial inoculum: 7.2 log MPN /mL) decreased after 24 h, and L. monocytogenes counts (8.7 log MPN/g at 24 h) decreased from day 15 in the cheese. Salmonella spp. and L. monocytogenes were not detected in any sample after 60 days of ripening, unlike S. aureus, which was detected at the end of the study. Lactic acid bacteria counts were 9 log CFU/mL in milk and whey and 7.2 log CFU/g in cheese. Pathogens behavior during the ripening process reduces the health risks by consuming products made with unpasteurized milk when subjected to ripening.

Statistical Methodology for the Decrease of Variability in the Cheese Sector, Case Study: Cotija Cheese Production in Tonalá, Chiapas

Statistical Methodology for the Decrease of Variability in the Cheese Sector, Case Study: Cotija Cheese Production in Tonalá, Chiapas

Genomic and functional characterisation of two Enterococcus strains isolated from Cotija cheese and their potential role in ripening.

Enterococcus spp. are present in the native microbiota of many traditional fermented foods. Their ability to produce antibacterial compounds, mainly against Listeria monocytogenes, has raised interest recently. However, there is scarce information about their proteolytic and lipolytic potential, and their biotechnological application is currently limited because enterococcal strains have been related to nosocomial infections. In this work, next-generation sequencing and optimised bioinformatic pipelines were used to annotate the genomes of two Enterococcus strains-one E. faecium and one E. faecalis-isolated from the Mexican artisanal ripened Cotija cheese. A battery of genes involved in their proteolytic system was annotated. Genes coding for lipases, esterases and other enzymes whose final products contribute to cheese aroma and flavour were identified as well. As for the production of antibacterial compounds, several peptidoglycan hydrolase- and bacteriocin-coding genes were identified in both genomes experimentally and by bioinformatic analyses. E. faecalis showed resistance to aminoglycosides and E. faecium to aminoglycosides and macrolides, as predicted by the genome functional annotation. No pathogenicity islands were found in any of the strains, although traits such as the ability of biofilm formation and cell aggregation were observed. Finally, a comparative genomic analysis was able to discriminate between the food strains isolated and nosocomial strains. In summary, pathogenic strains are resistant to a wide range of antibiotics and contain virulence factors that cause host damage; in contrast, food strains display less antibiotic resistance, include genes that encode class II bacteriocins and express virulence factors associated with host colonisation rather than invasion.

ACEI and antioxidant peptides release during ripening of Mexican Cotija hard cheese

<p>Cotija cheese is an artisanal Mexican cheese produced with raw cow´s milk. Our objective was to measure the antioxidant and angiotensin converting enzyme (ACE) inhibitory activities of the peptides released during its ripening. For that, Cotija cheeses were ripened 6 months in a chamber at 25 ºC without humidity control. Weekly samples were taken to determine acid soluble nitrogen (ASN), non-protein nitrogen (NPN) and ethanol soluble nitrogen (EtOH-SN) indexes, by Kjeldahl method. Antioxidant and ACE inhibitory activities were measured by spectrophotometry and HPLC methods, respectively. Peptides in each nitrogen fraction were determined by HPLC. Our results showed that during ripening of Cotija cheeses peptides with antioxidant and ACE inhibitory activities were released and increased through ripening time reaching a maximum of 79.8 % of 2,2- diphenyl-1-picrylhydrazyl (DPPH) discoloration and 100 % of ACE inhibition at the end of ripening. Both activities were highly correlated with the types of peptides present in each fraction.</p>

Metagenomic analysis of a Mexican ripened cheese reveals a unique complex microbiota

Cotija cheese is a Mexican handcrafted product made from raw cow milk whose ripening process occurs spontaneously and, presumably, it is influenced by environmental conditions. Its sensory characteristics and safety are probably the result of the balance between microbial populations and their metabolic capacity. In this work, we studied the dominance and richness of the bacteria in the Cotija cheese microbiome, as well as their metabolic potential by high-throughput sequencing. By the analysis of 16S ribosomal sequences, it was found that this metagenome is composed mainly of three dominant genera: Lactobacillus, Leuconostoc and Weissella, and more than 500 of non-dominant genera grouped in 31 phyla of both bacteria and archaea. The analysis of single-copy marker genes reported a similar result for dominant genera, although with greater resolution that reached the species level. Pathogenic bacteria such as Salmonella, Listeria monocytogenes, Brucella or Mycobacterium were not found. The Cotija cheese microbiome has the metabolic capacity for the synthesis of a wide range of flavor compounds, mainly involved with the metabolism of branched chain amino acids and free fatty acids. Genes associated with bacteriocin production and immunity were also found. Arguably, this is one of the most diverse metagenomes among the microbial communities related to fermented products.

Partial Characterization of Bacteriocin Produced by Halotolerant Pediococcus acidilactici Strain QC38 Isolated from Traditional Cotija Cheese.

During a screening of lactic acid bacteria producing bacteriocin from Cotija cheese, the strain QC38 was isolated. Based on the 16S rRNA gene nucleotide sequencing (516 pb accession no KJ210322) and phylogenetic analysis, the isolate was identified as Pediococcus acidilactici. Neutralized cell-free supernatant was tested for antimicrobial activity against 17 Gram-negative and Gram-positive pathogens. Growth inhibition was achieved against Listeria monocytogenes (supplier or indication or source), Staphylococcus aureus, Vibrio vulnificus, Vibrio cholerae O1 Ogawa, Vibrio cholerae NO 01 and Salmonella enterica subsp. Enterica serovar Typhimurium. Bacteriocin-like substance, after heating at 121°C for 15 min it remained stable and its antimicrobial activity was observed at pH ranging from 1.0 to 10.0 but inactivated by α-chymotrypsin and proteinase K. Strain QC38 was able to grow in 1-9% NaCl concentration. The plate overlay assay showed an approximate size of bacteriocin-like substance between 3.4 and 6.5 kDa. P. acidilactici QC38 harboured a plasmid that contains a gene for a pediocin (PA-1).

Effects of controlling ripening conditions on the dynamics of the native microbial population of Mexican artisanal Cotija cheese assessed by PCR-DGGE

Diversity and dynamics of autochthonous microbiota of Cotija, a traditional Mexican raw-milk cheese that resembles Parmesan, were compared in cheeses during 90 days of ripening under traditional and controlled conditions (temperature and relative humidity). Molecular assessments by PCR-DGGE 26S and 16S rRNA encoding regions were performed on various sections of the cheese at different ripening times. A complex microbial profile was found differentially distributed in the cheese. Several spices of lactic-acid-bacteria (LAB) from the genera Leuconostoc, Lactobacillus, Weissella, Aerococcus, Enterococcus, Carnobacterium and the yeasts Candida etchellsii, Pichia kudriavzevii and Moniliella suaveolens were found in the cheese matrix, with Leuconostoc mesenteroides being dominant. The cheese surface was populated by some of these microorganisms before the characteristic crust was formed, but Kluyveromyces lactis, Kodamaea ohmeri together with non-lactic-acid-bacteria (nLAB) Psychrobacter celer and Corynebacter variabile became distinctive in this part of the cheese. The microbial spatial/temporal variations showed similar patterns in samples from both ripening conditions, which suggested a robust ecosystem that is likely responsible for the safety, sensorial richness, and potential bio-functionality of Cotija. This knowledge could open the possibility for standardizing the ripening operation to avoid defects and improve the marketing of the product.

English

Milk and dairy products such as Cotija cheese are susceptible to contamination by Staphylococcus aureus. Some isolates of these bacteria carry the genes for enterotoxins and also the toxic shock syndrome toxin (TSST-1), additionally, many of these strains are potentially resistant to antibiotics. In the present study, a total of 50 samples of Cotija cheese sold at the Central Market "Baltazar R Leyva Mancilla” in the State of Guerrero were collected to determine the amount of bacteria growing in the cheese (expressed as CFU/g of cheese), the biotype, the antibiotype and the genotype. The results show that the amount of bacteria varies from 12x103 to 3x106CFU/g exceeding the permitted limits for ripened cheeses. Three biotypes were identified, 100% were susceptible to oxacylin, vancomycin, ciprofloxacin, trimethoprim-methoxazole, amikacin and clindamycin but tetracycline resistant. Only one of the four identified genotypes was positive to the TSST-1 gene. This is the first report of S. aureus isolated from Cotija cheese in the state of Guerrero, Mexico. Finally, our data evidenced cotija cheese as a vehicle carrying a large number of pathogenic S. aureus strains, suggesting that the public policy on food safety must be firmly reviewed. Key words: Staphylococcus aureus, biotype, antibiotype, genotype, tsst-1, cheese.&nbsp

Antibacterial activity produced by Enterococcus spp. isolated from an artisanal Mexican dairy product, Cotija cheese

Several Enterococci were isolated from Cotija cheese, which is a Mexican farmhouse-made product prepared from whole raw milk. No thermal step, neither starter cultures, are used in its manufacturing process. From the isolated strains two were outstanding for their extracellular lytic activity against Staphylococcus aureus, and were identified by 16S rDNA sequencing as Enterococcus faecalis and Enterococcus faecium, respectively. They also showed bacteriolytic activity against other pathogenic bacteria, such as Yersinia enterocolitica, Salmonella enterica Typhimurium and Pseudomonas aeruginosa, as demonstrated by agar diffusion tests. By zymography it was shown that they produced proteins with bacteriolytic activity with molecular masses of 95 and 81 kDa, respectively. Protein identification by LC/ESI–MS/MS showed that they correspond to different enzymes with peptidoglycan hydrolase (PGH) activity of N-acetylglucosaminidase. Two of them were considered as putative enzymes and had not been studied before. Concentrated protein preparations from both strains showed lytic activity on S. aureus growth in liquid culture, as well. Considering that the PGHs studied are produced by lactic bacteria isolated from a dairy product and their spectrum of bacterial inhibition, these enzymes have a potential use in food preservation.

Characterization of microbial traits involved with the elaboration of the Cotija cheese

Physicochemical and microbial analyses were conducted for artisanal and semi-industrial manufacturing processes of the Cotija cheese. Differences between manufacturing processes in Cotija cheese affect the microbial composition in the final product. Lactic acid bacteria are important microorganisms in the artisanal Cotija cheese manufacturing. Thirty-one different microorganisms were isolated and identified by ribosomal sequencing analysis during the production of this artisanal cheese. The yeast isolates comprised the following species: Kluyveromyces lactis, Kluyveromyces marxianus, Pichia guillermondii, Rhodotorula mucilaginosa, Galactomyces reessii, and Galactomyces geotrichum. Bacterial isolates were members of the Lactobacillaceae family with the follow species: Pediococcus pentosaceus, Lactobacillus brevis, Lactobacillus plantarum, and Lactobacillus casei. The possible role of these microorganisms in the final flavor and taste, and their utility in the control of undesirable microorganisms in artisanal Cotija cheese is discussed.

Isolation and Partial Characterization of Halotolerant Lactic Acid Bacteria from Two Mexican Cheeses

Isolated strains of halotolerant or halophilic lactic acid bacteria (HALAB) from Cotija and doble crema cheeses were identified and partially characterized by phenotypic and genotypic methods, and their technological abilities were studied in order to test their potential use as dairy starter components. Humidity, a(w), pH, and salt concentration of cheeses were determined. Genotypic diversity was evaluated by randomly amplified polymorphic DNA-polymerase chain reaction. Molecular identification and phylogenetic reconstructions based on 16S rRNA gene sequences were performed. Additional technological abilities such as salt tolerance, acidifying, and proteolytic and lipolytic activities were also investigated. The differences among strains reflected the biodiversity of HALAB in both types of cheeses. Lactobacillus acidipiscis, Tetragenococcus halophilus, Weissella thailandensis, and Lactobacillus pentosus from Cotija cheese, and L. acidipiscis, Enterococcus faecium, Lactobacillus plantarum, Lactobacillus farciminis, and Lactobacillus rhamnosus from doble crema cheese were identified based on 16S rRNA. Quantitative and qualitative assessments showed strains of T. halophilus and L. plantarum to be proteolytic, along with E. faecium, L. farciminis, and L. pentosus to a lesser extent. Lipolytic activity could be demonstrated in strains of E. faecium, L. pentosus, L. plantarum, and T. halophilus. Strains belonging to the species L. pentosus, L. plantarum, and E. faecium were able to acidify the milk media. This study evidences the presence of HALAB that may play a role in the ripening of cheeses.