Surface Of Smear-ripened Cheeses Research Articles

The Microbial Diversity on the Surface of Smear-Ripened Cheeses and Its Impact on Cheese Quality and Safety.

Smear-ripened cheeses are characterized by a viscous, red-orange surface smear on their rind. It is the complex surface microbiota on the cheese rind that is responsible for the characteristic appearance of this cheese type, but also for the wide range of flavors and textures of the many varieties of smear-ripened cheeses. The surface smear microbiota also represents an important line of defense against the colonization with undesirable microorganisms through various types of interaction, such as competitive exclusion or production of antimicrobial substances. Predominant members of the surface smear microbiota are salt-tolerant yeast and bacteria of the phyla Actinobacteria, Firmicutes, and Proteobacteria. In the past, classical culture-based approaches already shed light on the composition and succession of microorganisms and their individual contribution to the typicity of this cheese type. However, during the last decade, the introduction and application of novel molecular approaches with high-resolution power provided further in-depth analysis and, thus, a much more detailed view of the composition, structure, and diversity of the cheese smear microbiota. This led to abundant novel knowledge, such as the identification of so far unknown community members. Hence, this review is summarizing the current knowledge of the diversity of the surface smear microbiota and its contribution to the quality and safety of smear-ripened cheese. If the succession or composition of the surface-smear microbiota is disturbed, cheese smear defects might occur, which may promote food safety issues. Hence, the discussion of cheese smear defects in the context of an increased understanding of the intricate surface smear ecosystem in this review may not only help in troubleshooting and quality control but also paves the way for innovations that can lead to safer, more consistent, and higher-quality smear-ripened cheeses.

Genome Sequence of Staphylococcus saprophyticus DPC5671, a Strain Isolated from Cheddar Cheese

ABSTRACTThe draft genome sequence of Staphylococcus saprophyticus DPC5671, isolated from cheddar cheese, was determined. S. saprophyticus is a common Gram-positive bacterium detected on the surface of smear-ripened cheese and other fermented foods.

Tn6198, a novel transposon containing the trimethoprim resistance gene dfrG embedded into a Tn916 element in Listeria monocytogenes

To characterize Tn6198, a novel conjugative transposon from the clinical Listeria monocytogenes strain TTH-2007, which contains the tetracycline and trimethoprim resistance genes tet(M) and dfrG, respectively, and to assess its transferability in vitro and in situ. The complete sequence of Tn6198 was determined using a primer walking strategy. Horizontal gene transfer studies were performed by filter matings, as well as on the surface of smear-ripened cheese and smoked salmon. The presence of Tn916-like circular intermediates was determined by PCR. Antibiotic resistance was determined by the broth microdilution method and microarray hybridization. Sequencing of Tn6198 revealed that a 3.3 kb fragment containing dfrG was integrated between open reading frames 23 and 24 of Tn916. Furthermore, an additional copy of Tn916 was present in L. monocytogenes TTH-2007. Both elements were transferred simultaneously and separately in vitro to recipients L. monocytogenes 10403S and Enterococcus faecalis JH2-2 by conjugation, resulting in either tetracycline- and trimethoprim-resistant or solely tetracycline-resistant transconjugants. On the surface of cheese and salmon, only L. monocytogenes 10403S transconjugants were detected. This study reports the first Tn916-like element associated with a trimethoprim resistance gene, as well as the first fully characterized transposon conferring multidrug resistance in L. monocytogenes. This is of concern, as trimethoprim is administered to listeriosis patients with β-lactam allergy and as Tn6198 has a large potential for dissemination, indicated by both intra-species and inter-genus transfer.

Growth of Aerobic Ripening Bacteria at the Cheese Surface Is Limited by the Availability of Iron

The microflora on the surface of smear-ripened cheeses is composed of various species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. The objective of the present study was to show that iron availability is a limiting factor in the growth of typical aerobic ripening bacteria in cheese. For that purpose, we investigated the effect of iron or siderophore addition in model cheeses that were coinoculated with a yeast and a ripening bacterium. Both iron and the siderophore desferrioxamine B stimulated the growth of ripening bacteria belonging to the genera Arthrobacter, Corynebacterium, and Brevibacterium. The extent of stimulation was strain dependent, and generally, the effect of desferrioxamine B was greater than that of iron. Measurements of the expression of genes related to the metabolism of iron by Arthrobacter arilaitensis Re117 by real-time reverse transcription-PCR showed that these genes were transcribed during growth in cheese. The addition of desferrioxamine B increased the expression of two genes encoding iron-siderophore ABC transport binding proteins. The addition of iron decreased the expression of siderophore biosynthesis genes and of part of the genes encoding iron-siderophore ABC transport components. It was concluded that iron availability is a limiting factor in the growth of typical cheese surface bacteria. The selection of strains with efficient iron acquisition systems may be useful for the development of defined-strain surface cultures. Furthermore, the importance of iron metabolism in the microbial ecology of cheeses should be investigated since it may result in positive or negative microbial interactions.

Complete genome sequence of Corynebacterium variabile DSM 44702 isolated from the surface of smear-ripened cheeses and insights into cheese ripening and flavor generation

BackgroundCorynebacterium variabile is part of the complex microflora on the surface of smear-ripened cheeses and contributes to the development of flavor and textural properties during cheese ripening. Still little is known about the metabolic processes and microbial interactions during the production of smear-ripened cheeses. Therefore, the gene repertoire contributing to the lifestyle of the cheese isolate C. variabile DSM 44702 was deduced from the complete genome sequence to get a better understanding of this industrial process.ResultsThe chromosome of C. variabile DSM 44702 is composed of 3, 433, 007 bp and contains 3, 071 protein-coding regions. A comparative analysis of this gene repertoire with that of other corynebacteria detected 1, 534 predicted genes to be specific for the cheese isolate. These genes might contribute to distinct metabolic capabilities of C. variabile, as several of them are associated with metabolic functions in cheese habitats by playing roles in the utilization of alternative carbon and sulphur sources, in amino acid metabolism, and fatty acid degradation. Relevant C. variabile genes confer the capability to catabolize gluconate, lactate, propionate, taurine, and gamma-aminobutyric acid and to utilize external caseins. In addition, C. variabile is equipped with several siderophore biosynthesis gene clusters for iron acquisition and an exceptional repertoire of AraC-regulated iron uptake systems. Moreover, C. variabile can produce acetoin, butanediol, and methanethiol, which are important flavor compounds in smear-ripened cheeses.ConclusionsThe genome sequence of C. variabile provides detailed insights into the distinct metabolic features of this bacterium, implying a strong adaption to the iron-depleted cheese surface habitat. By combining in silico data obtained from the genome annotation with previous experimental knowledge, occasional observations on genes that are involved in the complex metabolic capacity of C. variabile were integrated into a global view on the lifestyle of this species.

Assessment of the anti-listerial activity of microfloras from the surface of smear-ripened cheeses

The anti-listerial activity of microfloras from the surface of various smear-ripened cheeses was evaluated using four methods that were then compared. Method A measured the anti-listerial potential of supernatants from short-time liquid cultures, whereas in Method B, a model cheese was co-inoculated with the microflora and Listeria innocua test strains. Method C was based on successive propagations of the microfloras on this model cheese, and Method D on successive propagations of the microfloras together with Listeria test strains. Anti-listerial activity considerably depended on the microflora used. Significant correlations were obtained between Methods A and B and Methods C and D. With Methods C and D, the highest anti-listerial activity was obtained with the microflora from a Livarot-type cheese (FC12). To investigate the cause of the anti-listerial activity of FC12, Fourier Transform InfraRed (FTIR) analyses of microbial populations were performed on the original microflora as well as on the microflora after propagations on the model cheese. The composition of FC12 had changed considerably upon propagation, and in the propagated microflora, the population of yeasts was dominated by Yarrowia lipolytica strains, whereas the population of bacteria was dominated by Vagococcus species.

Agrococcus casei sp. nov., isolated from the surfaces of smear-ripened cheeses

Seven Gram-positive, coryneform bacteria with virtually identical whole-organism protein patterns were isolated from the surface of smear-ripened cheeses. Representatives of these strains were the subject of a polyphasic study designed to establish their taxonomic status. The organisms formed a distinct branch in the Microbacteriaceae 16S rRNA gene tree and were most closely related to members of the genus Agrococcus, sharing sequence similarities of 95.4-98.7 %. The chemotaxonomic profiles of the strains were consistent with their classification in the genus Agrococcus. The combined genotypic and phenotypic data show that the isolates should be classified in the genus Agrococcus as representatives of a novel species. The name Agrococcus casei sp. nov. is proposed for this taxon. Isolate R-17892t2(T) (=DSM 18061(T)=LMG 22410(T)) is the type strain of Agrococcus casei sp. nov.

Quantitative Detection of Corynebacterium casei in Cheese by Real-Time PCR

The flora on the surface of smear-ripened cheeses is composed of numerous species of bacteria and yeasts that contribute to the production of the desired organoleptic properties. Due to the absence of selective media, it is very difficult to quantify cheese surface bacteria, and, consequently, the ecology of the cheese surface microflora has not been extensively investigated. We developed a SYBR green I real-time PCR method to quantify Corynebacterium casei, a major species of smear-ripened cheeses, using primers designed to target the 16S rRNA gene. It was possible to recover C. casei genomic DNA from the cheese matrix with nearly the same yield that C. casei genomic DNA is recovered from cells recovered by centrifugation from liquid cultures. Quantification was linear over a range from 10(5) to 10(10) CFU per g of cheese. The specificity of the assay was demonstrated with DNA from species related to C. casei and from other bacteria and yeasts belonging to the cheese flora. Nine commercial cheeses were analyzed by real-time PCR, and six of them were found to contain more than 10(5) CFU equivalents of C. casei per g. In two of them, the proportion of C. casei in the total bacterial flora was nearly 40%. The presence of C. casei in these samples was further confirmed by single-strand conformation polymorphism analysis and by a combined approach consisting of plate counting and 16S rRNA gene sequencing. We concluded that SYBR green I real-time PCR may be used as a reliable species-specific method for quantification of bacteria from the surface of cheeses.

Evaluation of live-culture-producing lacticin 3147 as a treatment for the control of Listeria monocytogenes on the surface of smear-ripened cheese

A live Lactococcus lactis culture, producing the two-component broad spectrum bacteriocin lacticin 3147, was assessed for ability to inhibit the food pathogen Listeria monocytogenes on the surface of smear-ripened cheese. In initial experiments, the addition of Listeria to a lacticin 3147-containing fermentate produced with L. lactis DPC4275 (a transconjugant strain derived from L. lactis DPC3147) resulted in at least a 4 log reduction of the pathogen in 30 min. Two separate trials were performed in order to assess the most suitable method for application of the potential protective culture to smear-ripened cheese. In the initial trial, the L. lactis was sprayed onto the surface of the cheese either before or after Listeria was deliberately applied. Application of the culture following Listeria challenge, yielded up to a 1000-fold reduction of the pathogen in contrast to the pretreatment where Listeria numbers were unaffected. In a further trial, three applications of the live lacticin 3147-producing culture was used on a cheese surface containing Listeria. Listeria numbers were found to be up to 100-fold lower than in the cheese treated with L. lactis DPC4268 (control). While application of the live lacticin 3147 producer did not give complete elimination of the pathogen the results nonetheless demonstrate the potential of the bioprotectant for improving the safety of smear-ripened cheeses and particularly those that contain low level contamination with Listeria. The application of lacticin 3147 as a live-culture can serve as a bioprotectant for the control of L. monocytogenes on the surface of smear-ripened cheese.

Arthrobacter bergerei sp. nov. and Arthrobacter arilaitensis sp. nov., novel coryneform species isolated from the surfaces of cheeses

Fourteen isolates of two different bacterial species isolated from the surface of smear-ripened cheeses were found to exhibit many characteristics of the genus Arthrobacter. The isolates were aerobic, Gram-positive, catalase-positive, non-spore-forming and non-motile. The cell-wall peptidoglycan contained lysine, alanine and glutamic acid. rrs sequence analysis indicated that the new isolates Re117T and Ca106T are closely related to the Arthrobacter nicotianae group and showed highest sequence similarity (>98 %) to Arthrobacter nicotianae and Arthrobacter protophormiae. However, DNA-DNA hybridization studies indicated that the strains represented two novel genomic species within the genus Arthrobacter and did not belong to A. nicotianae or A. protophormiae (<43 % DNA-DNA relatedness). On the basis of the phylogenetic and phenotypic distinctiveness of the new isolates, these bacteria should be classified as two novel Arthrobacter species, for which the names Arthrobacter bergerei sp. nov. and Arthrobacter arilaitensis sp. nov. are proposed. Type strains have been deposited in culture collections as Arthrobacter bergerei Ca106T (=CIP 108036T=DSM 16367T) and Arthrobacter arilaitensis Re117T (=CIP 108037T=DSM 16368T).