Raw Milk Dairy Products Research Articles

Enterococci from Raw-Milk Cheeses: Current Knowledge on Safety, Technological, and Probiotic Concerns.

The present study is focused on the safety, technological characteristics, and probiotic evaluation of Enterococcus species from different artisanal raw milk dairy products, mainly cheeses with ripening. Apart from proteolytic and lipolytic activities, most enterococci show the ability to metabolize citrate and convert it to various aromatic compounds. Long-ripened cheeses therefore have a specific flavor that makes them different from cheeses produced from thermally treated milk with commercial starter cultures. In addition, enterococci are producers of bacteriocins effective against spoilage and pathogenic bacteria, so they can be used as food preservatives. However, the use of enterococci in the dairy industry should be approached with caution. Although originating from food, enterococci strains may carry various virulence factors and antibiotic-resistance genes and can have many adverse effects on human health. Still, despite their controversial status, the use of enterococci in the food industry is not strictly regulated since the existence of these so-called desirable and undesirable traits in enterococci is a strain-dependent characteristic. To be specific, the results of many studies showed that there are some enterococci strains that are safe for use as starter cultures or as probiotics since they do not carry virulence factors and antibiotic-resistance genes. These strains even exhibit strong health-promoting effects such as stimulation of the immune response, anti-inflammatory activity, hypocholesterolemic action, and usefulness in prevention/treatment of some diseases.

NGS-Based Metagenomic Study of Four Traditional Bulgarian Green Cheeses from Tcherni Vit

This is the first scientific study of the unique Bulgarian Cherni Vit green cheese based on 16S and ITS2 NGS metagenomics, accomplished on the Illumina HiSeq 2 × 250 bp paired end reads in order to obtain an overview of the microbial composition. The results revealed a rich microbiota, composed of on average of 120 eubacterial and 16 fungal species. Firmicutes were represented by the families Strepotococcaceae, Lactobacillaceae and Staphylococcaceae, while the Actinobacteria were represented mainly by the genera Brevibacterium and Corynebacterium. Low counts of Proteobacteria were also detected, mainly halophiles of the genera Cobetia and Psychrobacter. Almost all (99.99%) of the detected fungi belonged to Ascomycota, represented almost equally by the families Eurotiomycetes (exclusively by Penicillium roqueforti), Saccharomycetes (mainly by the genera Debaryomyces, Pichia, Candida and Kluyveromyces) and Sordariomycetes (mostly by Scopulariopsis flava). The performed diversity analyses revealed that despite some differences, which can be attributed to environmental factors, and the time of the ripening, all of the four batches displayed very rich but similar microbiota. We found a potentially beneficial microbiota with the potential to inhibit the growth of pathogenic bacterial species, as well as to neutralize possibly produced mycotoxins, as described in numerous research on raw milk dairy products.

Genomic Investigation of Virulence Potential in Shiga Toxin Escherichia coli (STEC) Strains From a Semi-Hard Raw Milk Cheese.

Shiga-toxin-producing Escherichia coli (STEC) represents a significant cause of foodborne disease. In the last years, an increasing number of STEC infections associated with the consumption of raw and pasteurized milk cheese have been reported, contributing to raise the public awareness. The aim of this study is to evaluate the main genomic features of STEC strains isolated from a semi-hard raw milk cheese, focusing on their pathogenic potential. The analysis of 75 cheese samples collected during the period between April 2019 and January 2020 led to the isolation of seven strains from four stx-positive enrichment. The genome investigation evidenced the persistence of two serotypes, O174:H2 and O116:H48. All strains carried at least one stx gene and were negative for eae gene. The virulence gene pattern was homogeneous among the serogroup/ST and included adherence factors (lpfA, iha, ompT, papC, saa, sab, hra, and hes), enterohemolysin (ehxA), serum resistance (iss, tra), cytotoxin-encoding genes like epeA and espP, and the Locus of Adhesion and Autoaggregation Pathogenicity Islands (LAA PAIs) typically found in Locus of Enterocyte Effacement (LEE)-negative STEC. Genome plasticity indicators, namely, prophagic sequences carrying stx genes and plasmid replicons, were detected, leading to the possibility to share virulence determinants with other strains. Overall, our work adds new knowledge on STEC monitoring in raw milk dairy products, underlining the fundamental role of whole genome sequencing (WGS) for typing these unknown isolates. Since, up to now, some details about STEC pathogenesis mechanism is lacking, the continuous monitoring in order to protect human health and increase knowledge about STEC genetic features becomes essential.

CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS ISOLATES FROM TRADITIONAL DAIRY PRODUCTS OF SMALL-SCALE ALPINE FARMS

This study investigated the prevalence of Staphylococcus aureus in raw milk dairy products handcrafted in traditional alpine small-scale farms, and characterised the enterotoxigenicity and resistance to methicillin. Among the analysed samples, 69% exceeded the international microbiological recommendations. The highest counts were observed for cheese or fatty products (~106 cfu/g). Conversely, lower contamination levels concerned raw milk and whey cheese (~102 cfu/g). A total of 163 S. aureus isolates were collected, and the prevalence of MRSA was low (1.7%) but not negligible. The finding of enterotoxins genes in 67% of the isolates is of concern for the public health.

Molecular characterization of O157:H7, O26:H11 and O103:H2 Shiga toxin-producing Escherichia coli isolated from dairy products

Pathogenic Shiga toxin-producing E. coli (STEC) are recognized worldwide as environment and foodborne pathogens which can be transmitted by ingestion of ready-to-eat food such as raw milk-derived products. STEC show a prevalence rate in dairy products of 0.9%, yet comparably few outbreaks have been related to dairy products consumption. In this study, we used rt-qPCR to identify the virulence potential of O157, O26 and O103 STEC strains isolated from raw-milk dairy products by analyzing virulence-related gene frequencies and associations with O-island (OI) 44, OI-48, OI-50, OI-57, OI-71 and OI-122. Results showed that 100% of STEC strains investigated harbored genes associated with EHEC-related virulence profile patterns (eae and stx, with either espK, espV, ureD and/or Z2098). We also found similarities in virulence-related gene content between O157:H7 and O103:H2 dairy and non-dairy STEC strains, especially isolates from human cases. The O26:H11-serotype STEC strains investigated harbor the arcA-allele 2 gene associated with specific genetic markers. These profiles are associated with high-virulence seropathotype-A STEC. However, the low frequency of stx2 gene associated with absence of other virulence genes in dairy isolates of O26:H11 remains a promising avenue of investigation to estimate their real pathogenicity. All O26:H11 attaching-effacing E. coli (AEEC) strains carried CRISPRO26:H11SP_O26_E but not genetic markers espK, espV, ureD and/or Z2098 associated with the emerging potentially high-virulence “new French clone”. These strains are potentially as “EHEC-like” strains because they may acquire (or have lost) stx gene. In this study, O157:H7, O103:H2 and O26:H11 STEC strains isolated from dairy products were assigned as potential pathogens. However, research now needs to investigate the impact of dairy product environment and dairy processing on the expression of their pathogenicity.

Survival kinetics of Mycobacterium bovis during manufacture and ripening of raw milk Cheddar and Caerphilly cheese produced on a laboratory-scale

Persistence of Mycobacterium bovis was investigated in UK raw milk cheeses. Replicating traditional cheese production methods under stringent CL3 containment conditions, Cheddar and Caerphilly cheeses were produced with Myco.bovis inoculated raw milk. High-inoculum investigations used three Myco.bovis genotypes; later low-inoculum investigations used only Myco.bovis AF2122/97. High-inoculum Cheddar (n=9) and Caerphilly (n=9) were matured for a minimum of 12 and 4months respectively; maturation of low-inoculum Cheddar (n=3) and Caerphilly (n=3) was up to 11weeks. Survival of Myco.bovis was monitored by enumeration at different points throughout cheese manufacture and ripening. D values were calculated as follows: 57 and 59days in high-inoculum Cheddar and Caerphilly, respectively, and 41 and 24days in low-inoculum Cheddar and Caerphilly respectively. Mycobacterium bovis is concentrated in cheese curd and a proportion lost with the whey. Reduction in viability during manufacturing is limited, while significant Myco.bovis inactivation occurs during maturation. Inactivation was improved, during Caerphilly ripening, when acid development was enhanced by increasing the proportion of starter culture. Mycobacterium bovis inactivation data obtained could be used to inform assessment of the risk posed to consumers by raw milk dairy products.

The Plasmid Complement of the Cheese Isolate Lactococcus garvieae IPLA 31405 Revealed Adaptation to the Dairy Environment.

Lactococcus garvieae is a lactic acid bacterium found in raw-milk dairy products as well as a range of aquatic and terrestrial environments. The plasmids in L. garvieae have received little attention compared to those of dairy Lactococcus lactis, in which the genes carried by these extrachromosomal elements are considered of adaptive value. The present work reports the sequencing and analysis of the plasmid complement of L. garvieae IPLA 31405, a strain isolated from a traditional, Spanish, starter-free cheese made from raw-milk. It consists of pLG9 and pLG42, of 9,124 and 42,240 nucleotides, respectively. Based on sequence and structural homology in the putative origin of replication (ori) region, pLG9 and pLG42 are predicted to replicate via a theta mechanism. Real-time, quantitative PCR showed the number of copies per chromosome equivalent of pLG9 and pLG42 to be around two and five, respectively. Sequence analysis identified eight complete open reading frames (orfs) in pLG9 and 36 in pLG42; these were organized into functional modules or cassettes containing different numbers of genes. These modules were flanked by complete or interrupted insertion sequence (IS)-like elements. Among the modules of pLG42 was a gene cluster encoding specific components of a phosphoenolpyruvate-phosphotransferase (PEP-PTS) system, including a phospho-β-galacosidase. The cluster showed a complete nucleotide identity respect to that in plasmids of L. lactis. Loss of pLG42 showed this to be involved in lactose assimilation. In the same plasmid, an operon encoding a type I restriction/modification (R/M) system was also identified. The specificity of this R/M system might be broadened by different R/M specificity subunits detected in pLG9 and in the bacterial chromosome. However, challenges of L. garvieae IPLA 31405 against L. lactis phages proved that the R/M system was not involved in phage resistance. Together, these results support the hypothesis that, as in L. lactis, pLG42 contribute towards the adaptation of L. garvieae to the dairy environment.

Survival of Escherichia coli O26:H11 exceeds that of Escherichia coli O157:H7 as assessed by simulated human digestion of contaminated raw milk cheeses

Shiga toxin producing Escherichia coli (STEC) are an important cause of human foodborne outbreaks. The consumption of raw milk dairy products may be an important route of STEC infection. For successful foodborne transmission, STEC strains must survive stress conditions met during gastrointestinal transit in humans. The aim of this study was to evaluate the survival of two STEC strains of serotypes O157:H7 and O26:H11 during simulated human digestion in the TNO gastro-Intestinal tract Model (TIM) of contaminated uncooked pressed cheeses. The survival of cheese microflora during in vitro gastrointestinal transit was also determined for the first time. The level of STEC increased from 2log10CFU/ml to 4log10CFU/g during the first 24h of cheese making and remained stable at around 4log10CFU/g during cheese ripening and conservation. During transit through the artificial stomach and duodenum, levels of STEC decreased: 0.2% of E. coli O157:H7 and 1.8% of E. coli O26:H11 were recovered at 150min in the gastric compartment, compared with 14.3% for the transit marker. Bacterial resumption was observed in the jejunum and ileum: 35.8% of E. coli O157:H7 and 663.2% of E. coli O26:H11 were recovered at 360min in the ileal compartment, compared with 12.6% for the transit marker. The fate of STEC was strain-dependent, the survival of E. coli O26:H11 being 13 times greater than that of E. coli O157:H7 at the end of digestion in the cumulative ileal deliveries. These data provide a better understanding of STEC behavior during gastrointestinal transit in humans after ingestion of contaminated cheese.

Phenotypic and genotypic characteristics of Lactococcus lactis strains isolated from different ecosystems

Lactococcus lactis is the lactic acid bacteria most used in the manufacture of dairy products because of its fast lactose fermentation and flavor production. In this study, L. lactis strains isolated from different sources and previously characterized and evaluated for their capability to produce flavor were compared with respect to their phenotypic and genotypic characteristics. The variability of phenotypes and genotypes in the L. lactis strains were influenced by the source of isolation. In general, strains that shared the same genotype presented different phenotypes, and only some genotypes were clearly related to the source of isolation. On the other hand, phenotypic characteristics such as coagulant activity and utilization of lactose, glucose, and arginine were closely related to the ecosystem of origin. Coagulant activity and lactose utilization, phenotypic characteristics that are of utmost importance for efficient flavor production by starters in the manufacture of dairy products, were better for strains isolated from raw milk dairy products than for those isolated from vegetables.

Biochemical profiles, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and multilocus variable number tandem repeat analysis (MLVA) for typing Staphylococcus aureus isolated from dairy products

The study concerns 130 Staphylococcus aureus strains isolated from different raw-milk dairy products (122 isolates) and human samples (eight isolates). Four different typing techniques were applied: biochemical profiles (Biolog GP), restriction fragment length polymorphism of coagulase gene (coaRFLP), random amplified polymorphic DNA (RAPD) and multilocus variable number tandem repeat analysis (MLVA). Moreover multiplex-PCR was used to study the distribution of genes encoding staphylococcal enterotoxins. The results of this study reveal marked genomic and phenotypic variability among the tested S. aureus. The considered techniques were all found useful for strain typing, but, based on discriminatory power as the key parameter of the typing system, MLVA and Biolog GP were found to be the most powerful techniques. The methods showed little concordance in terms of discerning the clusters of related strains.

Evaluation of Aroma Generation of Lactococcus lactis with an Electronic Nose and Sensory Analysis

There is an increased interest in exploring the potential of new Lactococcus lactis strains isolated from different natural ecosystems for the production of aroma compounds. Thus, the objective of this study was to screen the aroma generation of Lactococcus lactis strains isolated from different sources by an electronic nose and sensory evaluation for their potential use in starter cheese cultures. Twenty-three strains of Lactococcus lactis were isolated from dairy sources such as artisanal raw-milk cheeses, nondairy sources, and commercial starter cultures (industrial). All the strains were assessed for their ability to produce aromas by an electronic nose and sensory analysis after their incubation in milk. Some phenotypic characteristics of technological importance such as lactose fermentation, proteolytic activity, and citrate utilization were also evaluated. Lactococcus lactis strains showed clear phenotypic differences related to their isolation source. Strains isolated from raw-milk dairy products or commercial starter cultures presented faster lactose fermentation and proteolytic activity than those presented by strains isolated from nondairy sources. Additionally, strains isolated from dairy and nondairy sources presented better citrate utilization than strains isolated from commercial dairy starters. On the other hand, there was not a clear relationship between the source of isolation and the ability of lactococci strains to produce aroma. Principal components analysis of electronic nose data revealed 4 distinctive groups based on aroma profiles. Additionally, odor intensity scores (yogurt-like and Fresco cheese-like) for these 4 groups revealed the nature of their differences. In general, strains from dairy products presented intense yogurt-like and Fresco cheese-like aromas, with the latter being the most intense for one specific strain. On the other hand, the majority of wild strains from nondairy sources presented a stronger yogurt-like aroma, whereas industrial strains presented low intensity for both aroma descriptors. Additionally, an aroma potentiation effect was observed when mixtures of 2 lactococci strains isolated from different sources acted together.

Comparison of EB and Fraser Enrichment Broths for the Detection of Listeria spp. and Listeria monocytogenes in Raw-Milk Dairy Products and Environmental Samples

This study is a comparison of the isolation frequency of Listeria spp. and Listeria monocytogenes from selected naturally contaminated dairy products, especially soft smear-ripened cheeses from raw milk and samples of feces and rinse samples from the udder taken on the farm, by using an enrichment broth (EB) recommended by the International Dairy Federation and the U.S. Food and Drug Administration (IDF and FDA) or Fraser broth as the selective enrichment. Detection and identification were carried out according to the IDF protocols and a polymerase chain reaction technique. Listeria spp. were detected in 39.8% of the 570 samples while 15.3% were positive for L. monocytogenes. For cheese and curd samples, Fraser enrichment broth gave a statistically significant higher recovery for all Listeria spp. (26 to 21 %) as well as for L. monocytogenes in particular (9 to 1.4%). For raw milk and samples taken from feces and the udder rinse no significant difference was found between EB and Fraser broth. A combination of both enrichments resulted in an increase of recovery over all matrices by 15%.