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The surface microflora (902 isolates) of Livarot cheeses from three dairies was investigated during ripening. Yeasts were mainly identified by Fourier transform infrared spectroscopy. Geotrichum candidum was the dominating yeast among 10 species. Bacteria were identified using Biotype 100 strips, dereplicated by repetitive extragenic palindromic PC...
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... hundred and fifty-two yeast isolates were assigned to 28 clusters by FTIR spectroscopy (Table 3). The clusters contained strains identified as Candida catenulata, Debaryo- myces hansenii, Geotrichum candidum, Kluyveromyces lactis or Kluyveromyces marxianus, Yarrowia lipolytica, and Can- dida glabrata or Pichia jadinii. ...
Similar publications
For studying the microbiota of four Danish surface-ripened cheeses produced at three farmhouses and one industrial dairy, both a culture-dependent and culture-independent approach were used. After dereplication of the initial set of 433 isolates by (GTG)5-PCR fingerprinting, 217 bacterial and 25 yeast isolates were identified by sequencing of the 1...
Citations
... Three different microbial strains were used as neighbor treatments: Brevibacterium aurantiacum strain JB5 (as previously described in [53]), Penicillium solitum strain #12 (as previously described in [47]), and the yeast D. hansenii strain 135B (as previously described in [50]). All of these strains were isolated from one natural rind cheese made in the same aging facility and commonly co-occur in natural rind cheeses made in different regions of the world [50,54,[98][99][100]. ...
Experimental studies of microbial evolution have largely focused on monocultures of model organisms, but most microbes live in communities where interactions with other species may impact rates and modes of evolution. Using the cheese rind model microbial community, we determined how species interactions shape the evolution of the widespread food- and animal-associated bacterium Staphylococcus xylosus. We evolved S. xylosus for 450 generations alone or in co-culture with one of three microbes: the yeast Debaryomyces hansenii, the bacterium Brevibacterium aurantiacum, and the mold Penicillium solitum. We used the frequency of colony morphology mutants (pigment and colony texture phenotypes) and whole-genome sequencing of isolates to quantify phenotypic and genomic evolution. The yeast D. hansenii strongly promoted diversification of S. xylosus. By the end of the experiment, all populations co-cultured with the yeast were dominated by pigment and colony morphology mutant phenotypes. Populations of S. xylosus grown alone, with B. aurantiacum, or with P. solitum did not evolve novel phenotypic diversity. Whole-genome sequencing of individual mutant isolates across all four treatments identified numerous unique mutations in the operons for the SigB, Agr, and WalRK global regulators, but only in the D. hansenii treatment. Phenotyping and RNA-seq experiments highlighted altered pigment and biofilm production, spreading, stress tolerance, and metabolism of S. xylosus mutants. Fitness experiments revealed antagonistic pleiotropy, where beneficial mutations that evolved in the presence of the yeast had strong negative fitness effects in other biotic environments. This work demonstrates that bacterial-fungal interactions can have long-term evolutionary consequences within multispecies microbiomes by facilitating the evolution of strain diversity.
... The development of these species may be promoted by the conditions of ripening which are very different from the two other technologies (long ripening, frequent washings). These bacteria are mostly frequently found in smear-ripened cheesesLarpin-Laborde et al., 2011). Proteobacteria can be on one hand very interesting for flavor development but their growth needs to be well balanced as they can be involved in spoilage of the final product, with smell and taste defaults(Desmasures et al., 1997) or in the production of biogenic amines(Delbès-Paus et al., 2012). ...
... Three different microbial strains were used as neighbor treatments: Brevibacterium aurantiacum JB5 (as previously described in 53), Penicillium solitum strain #12 (as previously described in 47), and the yeast Debaryomyces hansenii strain 135B (as previously described in 50). All of these strains were isolated from the same natural rind cheese made in the same aging facility and commonly co-occur in natural rind cheeses made in different regions of the world (50,54,(95)(96)(97). ...
Experimental studies of microbial evolution have largely focused on monocultures of model organisms, but most microbes live in communities where interactions with other species may impact rates and modes of evolution. Using the cheese rind model microbial community, we determined how species interactions shape the evolution of the widespread food- and animal-associated bacterium Staphylococcus xylosus . We evolved S. xylosus for 450 generations alone or in co-culture with one of three microbes: the yeast Debaryomyces hansenii , the bacterium Brevibacterium aurantiacum , and the mold Penicillium solitum . We used the frequency of colony morphology mutants (pigment and colony texture phenotypes) and whole-genome sequencing of isolates to quantify phenotypic and genomic evolution after 15 weeks of the evolution. The yeast D. hansenii strongly promoted diversification of S. xylosus ; by the end of the experiment, all populations co-cultured with the yeast were dominated by pigment and colony morphology mutant phenotypes. Populations of S. xylosus grown alone, with Brevibacterium , or with Penicillium did not evolve novel phenotypic diversity. Whole-genome sequencing of individual mutant isolates across all four treatments revealed numerous unique mutations in the operons for the SigB, Agr, and WalKR global regulators, but only in the D. hansenii treatment. Phenotyping and RNA-seq experiments demonstrated that these mutations altered pigment and biofilm production, spreading, stress tolerance, and metabolism of S. xylosus . Fitness experiments revealed trade-offs of these mutations across biotic environments caused by antagonistic pleiotropy, where beneficial mutations that evolved in the presence of the yeast Debaryomyces had strong negative fitness effects in other biotic environments.
IMPORTANCE
Substantial phenotypic and genomic variation exists within microbial species, but the ecological factors that shape this strain diversity are poorly characterized. We demonstrate that the biotic context of a widespread Staphylococcus species can impact the evolution of strain diversity. This work demonstrates the potential for microbes in food production environments to rapidly evolve to novel substrates and biotic environments. Our findings may also help understand how other Staphylococcus species may evolve in multispecies microbiomes.
... samples worldwide (Borelli et al., 2006;Lopandic et al., 2006;Larpin-Laborde et al., 2011;Mei et al., 2014;Chombo-Morales et al., 2016;Dugat-Bony et al., 2016). The use of qPCR in this study provided the first quantitative assessment of indigenous yeast presence in both the rind and the core of cheeses with different rind varieties (bloomy-, washed-, and natural-rind cheeses), and from milk undergoing different heat treatments (raw, thermized, and pasteurized). ...
... jadinii was rarely reported in cheese. It had been isolated from, but not quantified in, the rind of a Livarot cheese (a soft, red washed-rind cheese made from raw milk), during an early stage of ripening before salting (Larpin-Laborde et al., 2011). It was also reported in the core of a Portuguese Serpa cheese (Gonçalves Dos Santos et al., 2017). ...
Indigenous microorganisms are important components of the complex ecosystem of many dairy foods including cheeses, and they are potential contributors to the development of a specific cheese's sensory properties. Among these indigenous microorganisms are the yeasts Cyberlindnera jadinii, Pichia kudriavzevii, and Kazachstania servazzii, which were previously detected using traditional microbiological methods in both raw milk and some artisanal specialty cheeses produced in the province of Québec, Canada. However, their levels across different cheese varieties are unknown. A highly specific and sensitive real-time quantitative PCR assay was developed to quantitate these yeast species in a variety of specialty cheeses (bloomy-rind, washed-rind, and natural-rind cheeses from raw, thermized, and pasteurized milks). The specificity of the quantitative PCR assay was validated, and it showed no cross-amplification with 11 other fungal microorganisms usually found in bloomy-rind and washed-rind cheeses. Cyberlindnera jadinii and P. kudriavzevii were found in the majority of the cheeses analyzed (25 of 29 and 24 of 29 cheeses, respectively) in concentrations up to 10⁴ to 10⁸ gene copies/g in the cheese cores, which are considered oxygen-poor environments, and 10¹ to 10⁴ gene copies/cm² in the rind. However, their high abundance was not observed in the same samples. Whereas C. jadinii was present and dominant in all core and rind samples, P. kudriavzevii was mostly present in cheese cores. In contrast, K. servazzii was present in the rinds of only 2 cheeses, in concentrations ranging from 10¹ to 10³ gene copies/cm², and in 1 cheese core at 10⁵ gene copies/g. Thus, in the ecosystems of specialty cheeses, indigenous yeasts are highly frequent but variable, with certain species selectively present in specific varieties. These results shed light on some indigenous yeasts that establish during the ripening of specialty cheeses.
... The biodiversity of the bacteria isolated during this research, comprising six main species, is typical of this kind of cheese. Indeed, the bacterial community of the surface of washed-rind cheeses generally comprises several distinct groups such as non-starter lactic acid bacteria (e.g., Leuconostoc spp.), staphylococci (e.g., S. xylosus, S. equorum), coryneform bacteria (e.g., Glutamicibacter arilaitensis, Brevibacterium aurantiacum, Corynebacterium variabile, Microbacterium gubbeenense) and Gram-negative bacteria (e.g., Alcaligenes faecalis, Halomonas spp., Psychrobacter spp., Hafnia alvei, Proteus spp., Vibrio spp., Pseudoalteromonas spp.) [24,[58][59][60]. ...
Smear-ripened cheeses host complex microbial communities that play a crucial role in the ripening process. Although bacteriophages have been frequently isolated from dairy products, their diversity and ecological role in such this type of cheese remain underexplored. In order to fill this gap, the main objective of this study was to isolate and characterize bacteriophages from the rind of a smear-ripened cheese. Thus, viral particles extracted from the cheese rind were tested through a spot assay against a collection of bacteria isolated from the same cheese and identified by sequencing the full-length small subunit ribosomal RNA gene. In total, five virulent bacteriophages infecting Brevibacterium aurantiacum, Glutamicibacter arilaitensis, Leuconostoc falkenbergense and Psychrobacter aquimaris species were obtained. All exhibit a narrow host range, being only able to infect a few cheese-rind isolates within the same species. The complete genome of each phage was sequenced using both Nanopore and Illumina technologies, assembled and annotated. A sequence comparison with known phages revealed that four of them may represent at least new genera. The distribution of the five virulent phages into the dairy-plant environment was also investigated by PCR, and three potential reservoirs were identified. This work provides new knowledge on the cheese rind viral community and an overview of the distribution of phages within a cheese factory.
... From the microbiological point of view, cheese and dairy products, both fermented in a natural way and with the addition of starters and/or adjunct cultures, contain a complex mixture of microbial communities, including those relevant for carrying out the processes, or responsible of cheese deterioration, opportunistic and pathogenic organisms, which develop and change during production and maturation [7,8]. From the systematic point of view, cheese bacteria as lactic acid bacteria, enterococci and staphylococci belong to the phylum Firmicutes; bifidobacteria, propionibacteria and corynebacteria to the phylum Actinobacteria; enterobacteria to the phylum Proteobacteria [9,10]. Archaea have rarely been found in the cheese microbiota, with few members belonging to the genera Thermocladium and Sulfurisphaera of the phylum Crenarchaeota, and to the genus Methanohalobium of the phylum Euryarchaeota [11,12]. ...
Cheese and dairy products require a rigorous observation of the procedures, strictly linked to the different indigenous components, such as the initial raw materials, the process chain, the ripening temperature, the water activity (aw), the pH and the contamination of the environment and operators. Microorganisms are key agents in the transformation of milk and in the subsequent phases which confer typicity and stability to cheese and dairy products. Contamination by pathogenic microorganisms may occur, compromising the safety of the final products. Meanwhile, beneficial microorganisms present in cheese and dairy products can produce antimicrobial compounds, thus avoiding spoilage of the products, ensuring their safety for human consumption. This mini review reports a description of the microorganisms involved in the fermentation of milk and in the subsequent processes concerning cheeses and derivatives, highlighting the aspects that microorganisms play in terms of quality, typicality and safety. New aspects emerged from this study, suggesting possible insights and future research. These include cultural approaches on the one hand, which allow for the isolation and characterization of new microbial strains that confer peculiarities in terms of quality and typicality to cheeses and dairy products, and the isolation of lactic acid bacteria that produce bacteriocins as important tools for combating microbial pathogens. On the other hand, investigations on cheese and dairy products using metagenomic approaches with DNA extraction followed by amplification and sequencing of microbial genes, allow the description and monitoring of the entire microbiota involved in the transformation processes of cheese and dairy products. Therefore, the combination of cultural dairy microbiology and metagenomic approaches can lead to improving the characteristics of cheeses and dairy products, while maintaining respect for traditions.
... The biodiversity of the bacteria isolated during this research, comprising six main species, is typical of this kind of cheese. Indeed, the bacterial community of the surface of washed-rind cheeses generally comprises several distinct groups such as non-starter lactic acid bacteria (e.g., Leuconostoc spp.), staphylococci (e.g., S. xylosus, S. equorum), coryneform bacteria (e.g., Glutamicibacter arilaitensis, Brevibacterium aurantiacum, Corynebacterium variabile, Microbacterium gubbeenense) and Gram-negative bacteria (e.g., Alcaligenes faecalis, Halomonas spp., Psychrobacter spp., Hafnia alvei, Proteus spp., Vibrio spp., Pseudoalteromonas spp.) [24,[58][59][60]. ...
Smear-ripened cheeses host complex microbial communities that play a crucial role in the ripening process. Although bacteriophages have been frequently isolated from dairy products, their diversity and ecological role in such type of cheese remain underexplored. In order to fill this gap, the main objective of this study was to isolate and characterize bacteriophages from the rind of a smear-ripened cheese. Thus, viral particles extracted from cheese rind were tested against a collection of bacterial isolates through a spot assay. In total, five virulent bacteriophages infecting Brevibacterium aurantiacum, Glutamicibacter arilaitensis, Leuconostoc falkenbergense and Psychrobacter aquimaris species were obtained. All exhibit a narrow host range, being only able to infect a few cheese-rind isolates within the same species. The complete genome of each phage was sequenced using both Nanopore and Illumina technologies, assembled and annotated. Sequence comparison with known phages revealed that four of them may represent at least new genera. The distribution of the five virulent phages into the dairy-plant environment was also investigated by PCR and three potential reservoirs were identified. This work provides new knowledge on the cheese rind viral community and an overview of the distribution of phages within a cheese factory.
... Less is known, however, about the activity of other components of the cheese microbiota, even of species that reach high cell densities during ripening; e.g., Macrococcus caseolyticus, Brevibacterium aurantiacum, Corynebacterium casei, or Arthrobacter spp. in smearripened cheese varieties (Mounier et al., 2005). Alone or in combination with LAB (or even yeasts and molds), strains of these species, which have been found to be more adapted to cheese than commercial secondary cultures (Larpin-Laborde et al., 2011), are currently being employed as ripening starters to reproduce the typical flavor profiles of traditional cheeses. Well-characterized Tetragenococcus spp. ...
High throughput sequencing has recently revealed the presence of Tetragenococcus-related DNA sequences in dairy environments such as brine and cheeses. In the present work, a selective medium was developed to isolate Tetragenococcus spp. from two ripened, traditional, Spanish, blue-veined cheese varieties made from raw milk. The strains recovered belonged to either Tetragenococcus koreensis or Tetragenococcus halophilus species. Twenty of these isolates (15 of T. koreensis and 5 of T. halophilus) were then subjected to a battery of phenotypic and genetic tests, and six strains (4 T. koreensis and 2 T. halophilus) to genome sequencing. Wide genetic and phenotypic diversity was noted. All strains grew poorly in milk, producing small quantities of lactic and acetic acids. Most strains used lactose as a carbon source and ferment milk citrate. In agreement, genome analysis detected in the genome of the six strains analyzed gene clusters harboring several lactose/galactose-related genes and genes encoding citrate metabolic enzymes (permease, citrate lyase, and oxaloacetate decarboxylase). Most of the tested strains were resistant to erythromycin and clindamycin, and a few to other antimicrobial agents, but neither known mutations nor acquired genes conferring resistance to antibiotics were identified in their genomes. Neither were genes coding for pathogenicity or virulence factors detected. Decarboxylase-encoding genes involved in biogenic amine production were not identified, in keeping with the strains' negative biogenic amine-producer phenotype. Genome comparison revealed vast arrays of genes (similar in number to those described in other lactic acid bacteria) coding for components of proteolytic and lipolytic systems. Tetragenococcus strains showing desirable traits plus the absence of detrimental features might be exploitable in the form of secondary, adjunct or ripening cultures to ensure the typical bouquet of traditional blue-veined cheeses is obtained, or to diversify the final flavor in other varieties.
... In this study, Enterobacterales were mainly represented by isolates of the genera Proteus and Morganella, whereas Citrobacter, Enterobacter, Serratia, Providencia and Hafnia were isolated to a much lesser extent. Besides Enterobacterales, Gram-negative bacteria regularly detected on the surface of surface-ripened cheeses belonged mainly to Proteobacteria families such as Alcaligenaceae, Caulobacteraceae, Halomonadaceae, Moraxellaceae, Oceanospirillaceae, Pseudoalteromonadaceae, Pseudomonadaceae, Vibrionaceae and Xanthomonadaceae [12,13,15,20,24,[42][43][44]. On Livarot, a ...
... French surface-ripened soft cheese, Gram-negative bacteria accounted for 32% of the overall bacteria isolated from the cheese surface [15]. A recent study on the temporal differences in the microbial composition of Époisses cheese rinds during ripening and storage also revealed a dominance of Gram-negative species. ...
... Proteus spp., in particular Proteus vulgaris strains, were frequently isolated from the surface of surface-ripened cheeses, and their potential role in cheese ripening has already been intensively discussed [12][13][14][15]36,40,50]. Proteus vulgaris was able to successfully colonize and dominate the surface of pilot-scale cheese while significantly contributing to its organoleptic properties through the production of aldehydes [14]. ...
The smear of surface-ripened cheese harbors complex microbiota mainly composed of typical Gram-positive aerobic bacteria and yeast. Gram-negative bacteria are usually classified as unwanted contaminants. In order to investigate the abundance and impact of Gram-negative bacteria naturally occurring in the smear of surface-ripened cheese, we performed a culture-based analysis of smear samples from 15 semi-hard surface-ripened cheese varieties. The quantity, diversity and species distribution of Proteobacteria in the surface smear of the analyzed cheese varieties were unexpectedly high, and comprised a total of 22 different species. Proteus and Morganella predominated most of the analyzed cheese varieties, while Enterobacter, Citrobacter, Hafnia and Serratia were also found frequently. Further physiological characterization of Proteus isolates revealed strong proteolytic activity, and the analysis of volatiles in the smear cheese surface headspace suggested that Enterobacterales produce volatile organic flavor compounds that contribute to the organoleptic properties of surface-ripened cheese. Autochthonous members of Enterobacterales were found in 12 of the 15 smear samples from surface-ripened cheeses, suggesting that they are part of the typical house microbiota that shape the organoleptic properties of the cheese rather than represent unwanted contaminants. However, further investigation on safety issues of the individual species should be performed in order to manage the health risk for consumers.
... Fungal diversity increased substantially following increased exposure in the milking barn environment and decreased after overnight ripening to one dominated by a few genera, including Kluyveromyces, Exophiala, and Candida. Kluyveromyces lactis and Kluyveromyces marxianus are commonly identified in dairy samples (23), and Kluyveromyces lactis is often added as a ripening culture in cheese production to metabolize residual lactose in the cheese curd (39). Candida is also prevalent in cheeses, with some strains demonstrating strong proteolytic and lipolytic activity (40,41). ...
Throughout the 20th century, especially in the United States, sanitation practices, pasteurization of milk, and the use of commercial defined-strain starter cultures have enhanced the safety and consistency of cheese. However, these practices can reduce cheese microbial diversity.
