Raw Milk Dairy Research Articles

The occurrence of doxycycline residue in raw dairy milk from Malang and Padang Panjang, Indonesia

Antibiotic residue in milk has a negative impact on human health. The objective of this study was to determine the presence of doxycycline residues in fresh dairy milk and to evaluate its dietary intake to Indonesian population through milk consumption. Ninety milk samples were collected from the District of Malang (East Java Province) and sixty-six milk samples from Padang Panjang City (West Sumatera Province). All samples were analyzed for doxycycline residue using high-performance liquid chromatography (HPLC). The estimated daily intake (EDI) was determined by multiplying the mean concentration of antibiotics and the average daily milk consumption dividing it by the weight of population target. The occurrence of doxycycline residue obtained in raw milk samples was 36.67% for samples from Malang and 12.12% for samples from Padang Panjang. The range and average concentration for doxycycline residue in milk samples were 44.8-557.2 ng/g and 203.0 ng/g for samples from Malang and 14.2-64.5 ng/g and 30.1 ng/g for samples from Padang Panjang, respectively. Samples from Malang shows the highest EDI values of doxycycline both for adult, and children population and demonstrated the toxicological risk of consumption doxycycline together with the present other types or groups of antibiotics residue in milk or other types of foods. The results obtained show that, to prevent the negative effects of antibiotic residues on human health, the presence of antibiotic residues in raw dairy milk still needs to be monitored regularly.

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.

Kinetic modeling of inactivation of foodborne bacterial pathogens in serrano artisanal cheese during ripening

Abstract Serrano artisanal cheese is a traditional raw milk dairy product from southern Brazil. For safe consumption of raw milk products, ripening is a critical period for reducing populations of pathogenic and spoilage microorganism and, then, the knowledge on inactivation kinetics of microorganisms is necessary to guarantee food safety and quality. In the present work, inactivation kinetics of foodborne bacterial pathogens required by Brazilian regulations in serrano artisanal cheeses were statistically evaluated during ripening. The Salmonella spp., Listeria monocytogenes, coagulase-positive strain of Staphylococcus aureus and fecal coliforms were analyzed for up to 60 days. Experimental data were fitted using first-order model, Weibull and log-linear + shoulder models. Results showed no presence of Salmonella spp. and L. monogyatogenes in the cheeses through the studied period. The first-order and Weibull models presented good performance to represent coagulase-positive strain of S. aureus and fecal coliforms during ripening, although Weibull distribution showed better outcomes to experimental data. Log-linear+shoulder equation was discarded to describe the reduction of microorganism counts for physical criteria. Kinetics showed an initial resistance of fecal coliforms to be inactivated, unlike to coagulase-positive strain of S. aureus population. Modeling analysis allowed estimating serrano artisanal cheese ripening period to be longer than 33 days for achieving a safe product according to the Brazilian Regulatory Standards.

Raw milk producers with high levels of hygiene and safety.

There is world-wide increasing interest in the consumption of unprocessed, natural food commodities including fresh (unpasteurised) milk and milk products. Consumers are actively seeking out raw milk, partly due to health reasons, but also for taste, freshness, closeness to the producer and to support local agriculture. The need for high levels of hygiene and safety in farms producing raw milk for direct consumption has long been recognised and has led to federal and industry-initiated systems for safe raw milk production. Raw milk producers in North America and Europe have demonstrated that raw milk, intended for direct consumption, can be produced safe and hygienic. The aim of this paper is to describe practices that have been developed for safe raw milk production. The German Vorzugsmilch is a federally regulated programme for legal raw milk production that was established already in the 1930s to provide raw milk with high hygienic standards controlled for zoonotic diseases to consumers. The Raw Milk Institute is a non-profit organisation established in California that has developed a voluntary safe raw milk programme in North America. RAWMI has developed a risk analysis and management system for raw milk dairy farmers to assist farmers in making individually tailored solutions for various production systems. In British Colombia, Canada, small herd share farms have employed good manufacturing practices, a risk management approach and performed monthly samples for pathogens and indicator bacteria to demonstrate safety and consistency. The major components of the raw milk systems applied, and the results of regular milk microbial indicator bacteria are presented. For the German system, the results from standard monthly pathogen tests are compared to zoonotic pathogen tests from other milk sources. The overall results indicate that raw milk can be produced with a high level of hygiene and safety in various systems.

Mycotoxin Detection in Maize, Commercial Feed, and Raw Dairy Milk Samples from Assiut City, Egypt.

This survey was conducted to investigate the contamination by multiple mycotoxins, aflatoxins (AFB1, AFB2, AFG1, and AFG2), ochratoxin A (OTA), and zearalenone (ZEA) in 61 samples of maize and 17 commercial animal feed samples, and of aflatoxin M1 (AFM1) in raw dairy milk samples (n = 20) collected from Assiut City in Upper Egypt. Multi-mycotoxin immunoaffinity columns were used for samples cleanup and mycotoxin purification. An HPLC–FLD system with an on-line post-column photochemical derivatization was used for the detection of the target toxins. AFB1 was detected in both maize (n = 15) and feed (n = 8), with only one maize sample presenting a concentration above the maximum permissible level set by the Egyptian authorities. AFB2 was observed in six maize samples and in one feed sample, with a maximum value of 0.5 μg/kg. ZEA was detected only in feed samples (n = 4), with a maximum value of 3.5 μg/kg, while OTA, AFG1, and AFG2 were under the limits of detection. For milk, all the analyzed samples (100%) were contaminated with AFM1, and 14 samples (70%) presented concentrations above the maximum permissible level in the European Union (EU) (0.05 μg/kg). The concentrations ranged from 0.02 μg/kg to 0.19 μg/kg, except that of one sample, which was under the limit of quantification. The contamination rates in maize and animal feeds are not alarming. In contrast, the consumption of dairy milk samples in Assiut City may pose public health hazards, as AFM1 levels were found to exceed the international permissible limits. Further surveys are highly recommended in order to establish a database for mycotoxin occurrence in Egypt to minimize the possible health risks in animals and humans.

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.

Prevalence and Antibiotic Resistance Pattern of Escherichia coli Isolated from Raw Dairy Milk

E. coli is one of the most important food borne pathogen, which could be transmitted by milk and milk products. To assess the role of dairy milk as the source of drug resistant E. coli, we examined 50 raw dairy milk samples (25-farm milk + 25-market milk) from some selected areas of Bangladesh by cultural, morphological, biochemical and antimicrobial sensitivity tests. In the preliminary observation, the mean total aerobic mesophilic count of market and farm raw milk samples were 8.98 and 8.68 log CFU/ml, while mean coliform count were 4.20 and 3.03 log CFU/ml respectively. Thirty-three E. coli isolates were recovered from collected samples (66% 33 of 50) and this pathogen was more prevalent in market milk (76%, 19 of 25) than farm milk (56%, 14 of 25). In addition, most of the isolated E. coli exhibited resistance against ampicillin and cefotaxime. This result shows that, the raw dairy milk and its products could be a source of human drug resistant E. coli.

Pesticides Residue in Milk and Milk Products: Mini Review

Livestock is an important sub-sector of agriculture that plays a key role in economy of a country by contributing to GDP (Gross Domestic Product) and in total export. Pakistan is the 5 th largest milk producer in the world with a total milk production of about 46.44 billion liters per anum. Almost 68% milk is produced by buffalo and 27% by cow. Pesticides used in agriculture sector may transfer to animal bodies through feed and fodder. A pesticide found in water is another source of residues in milk through drinking water. External control of parasites on animal body, insect control in cattle yard and sheds are direct sources of pesticides exposure for dairy animals. Due to its nutritional and supplementary value milk is being consumed by peoples of different age groups therefore this issue of pesticide residues attain the immediate attention of researcher. Pesticides residues levels in raw dairy milk were discussed in some selected developing and developed countries. It is concluded that human health is associated with exposure to organo phosphorus (OPPs), organo chlorine (OCPs), pyrethroids and carbamate (CB) pesticides via milk or milk products and this issue deserve more attention. Different classes of pesticides OPPs, OCPs, pyrethroids and CBs etc. were reported in raw dairy milk in different countries and also in Pakistan. The results of this review demonstrate the need to establish pesticide residue monitoring programs for milk analysis for human consumption to improve food safety and decrease exposure risks to consumers.

Psychrotrophic bacterial populations in Chinese raw dairy milk

Four-hundred and eighty isolates isolated from raw milk samples collected from different farms in China were subjected to random amplified polymorphic DNA analysis and 16S rRNA sequencing for clustering and identification, respectively. All isolates comprised 24 genera and 74 species in total. Pseudomonas was the most prevalent genus, representing 58.8% of all isolates, followed by Acinetobacter (13.3%), Flavobacterium (6.0%) and Sphingobacterium (4.2%). Among the 25 species belonging to Pseudomonas, Pseudomonas fluorescens (15.8%) proved to be the most dominant species, followed by Pseudomonas fragi (7.1%) and Pseudomonas psychrophila (5.4%). Out of these isolates, 12.3% have not been reported in previous studies, highlighting that many psychrotrophic bacteria are rather underexplored in raw milk. In the future, a detailed investigation will be required to characterize these bacterial isolates based on their potential to deteriorate the quality of dairy products.

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.

Detection of β-Lactams and Tetracyclines Antimicrobial Residues in Raw Dairy Milk for Human Consumption in Palestine

Milk and milk products are among the most important daily consumed foodstuffs. The quality and safety of raw milk for human consumption in developed countries is not adequately monitored. The lack of studies about the antimicrobial residues in milk keeps this vital topic under observation. In this brief study, 34 raw dairy milk samples were tested for detection of antimicrobial residues of β-lactams and tetracyclines above Maximum Residual Limits (MRLs). Rapid screening test, IDEXX Snap test kit was used in this study. Eighteen samples were tested for β-lactams residues, of which 22.2 % (4 of 18) appeared to be above MRLs. Sixteen samples tested for tetracycline of which 18.7 % (3 of 16) were above MRLs. This is the first report that highlights the occurrence of antimicrobial residues marketed raw milk for human consumption in Palestine.

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.

Sequence and genetic organization of the 19.3-kb erythromycin- and dalfopristin-resistance plasmid pLME300 from Lactobacillus fermentum ROT1

Lactobacillus fermentum ROT1 was isolated from a raw milk dairy product. It is resistant to novobiocin, tetracycline, erythromycin and dalfopristin. A chromosomal tetracycline-resistance determinant was identified as tetM. A 19,398-bp plasmid (pLME300), present in several erythromycin-resistant strains of Lb. fermentum, was isolated from strain ROT1 and completely sequenced. Based on putative open reading frames, pLME300 contains at least four different functional regions. In region I, ORF1 shows high homologies to replication proteins of different theta-replicating plasmids. In addition, a tandem repeat of a 22-bp sequence appears 4.5 times. In region II, ORF3 may code for a methylase, and ORF4 has homologies to Mrr restriction system proteins of Deinococcus radiodurans and Escherichia coli suggesting a restriction–modification system. Region III harbours antibiotic-resistance genes, coding for a macrolide–lincosamide–streptogramin B (MLS) methylase Erm(LF) and the streptogramin A acetyltransferase Vat(E), which is identical to Vat(E) from Enterococcus faecium. Furthermore, region III shows a 91% nucleotide sequence identity to an erm-vat linkage of E. faecium. Region IV carries ORFs that appear to be involved in plasmid mobilization as characterized by a putative origin of transfer and a mobilization protein. pLME300 is the largest completely sequenced multi-resistance plasmid isolated from any Lactobacillus strain so far.