Candida krusei is the major contaminant of ultrafiltration and reverse osmosis membranes used for cranberry juice production.

Ultrafiltration (UF) and reverse osmosis (RO) are commonly used for the clarification and concentration of fruit juices. However, one of the main limitations of filtration membranes is biofouling, which reduces membrane efficiency and can contaminate the filtered product and lead to spoilage. In this study, the microbial fouling layers of UF and RO membranes from a Canadian cranberry juice processing plant were characterized. Unlike the microbiota found in cranberry juice, which is dominated by Bacillus sp. and other bacteria, both UF and RO membranes were mainly colonized by several strains of the yeast Candida krusei. A variation in bacterial and yeasts count was observed between tubular UF and spiral-wound RO membranes, and the analysis of the spatial distribution highlighted the homogeneity of the contamination across each membrane. Surprisingly, RO membranes had a higher level of contamination when compared to UF membranes. Furthermore, six strains of C. krusei were further characterized through multilocus sequence typing analysis, five of which exhibited unique allelic profiles and two of which were found to contain a new TRP1 allele.

Analysis of Microbiota Persistence in Quebec's Terroir Cheese Using a Metabarcoding Approach.

Environmental short amplicon sequencing, or metabarcoding, is commonly used to characterize the bacterial and fungal microbiota of cheese. Comparisons between different metabarcoding studies are complicated by the use of different gene markers. Here, we systematically compare different metabarcoding molecular targets using V3-V4 and V6-V8 regions of the bacterial 16S rDNA and fungal ITS1 and ITS2 regions. Taxonomic profiles varied depending on the molecular markers used. Based on data quality and detection capacity of the markers toward microorganisms usually associated with the dairy environment, the ribosomal regions V3-V4 and ITS2 were selected and further used to evaluate variability in the microbial ecosystem of terroir cheeses from the province of Quebec in Canada. Both fungal and bacterial ecosystem profiles were described for 32 different ready-to-eat bloomy-, washed- and natural-rind specialty cheese varieties. Among them, 15 were studied over two different production years. Using the Bray-Curtis dissimilarity index as an indicator of microbial shifts, we found that most variations could be explained by either a voluntary change in starter or ripening culture composition, or by changes in the cheesemaking technology. Overall, our results suggest the persistence of the microbiota between the two years studied-these data aid understanding of cheese microbiota composition and persistence during cheese ripening.

Phenotypic and Genetic Characterization of the Cheese Ripening Yeast Geotrichum candidum.

The yeast Geotrichum candidum (teleomorph Galactomyces candidus) is inoculated onto mold- and smear-ripened cheeses and plays several roles during cheese ripening. Its ability to metabolize proteins, lipids, and organic acids enables its growth on the cheese surface and promotes the development of organoleptic properties. Recent multilocus sequence typing (MLST) and phylogenetic analyses of G. candidum isolates revealed substantial genetic diversity, which may explain its strain-dependant technological capabilities. Here, we aimed to shed light on the phenotypic and genetic diversity among eight G. candidum and three Galactomyces spp. strains of environmental and dairy origin. Phenotypic tests such as carbon assimilation profiles, the ability to grow at 35°C and morphological traits on agar plates allowed us to discriminate G. candidum from Galactomyces spp. The genomes of these isolates were sequenced and assembled; whole genome comparison clustered the G. candidum strains into three subgroups and provided a reliable reference for MLST scheme optimization. Using the whole genome sequence as a reference, we optimized an MLST scheme using six loci that were proposed in two previous MLST schemes. This new MLST scheme allowed us to identify 15 sequence types (STs) out of 41 strains and revealed three major complexes named GeoA, GeoB, and GeoC. The population structure of these 41 strains was evaluated with STRUCTURE and a NeighborNet analysis of the combined six loci, which revealed recombination events between and within the complexes. These results hint that the allele variation conferring the different STs arose from recombination events. Recombination occurred for the six housekeeping genes studied, but most likely occurred throughout the genome. These recombination events may have induced an adaptive divergence between the wild strains and the cheesemaking strains, as observed for other cheese ripening fungi. Further comparative genomic studies are needed to confirm this phenomenon in G. candidum. In conclusion, the draft assembly of 11 G. candidum/Galactomyces spp. genomes allowed us to optimize a genotyping MLST scheme and, combined with the assessment of their ability to grow under different conditions, provides a reliable tool to cluster and eventually improves the selection of G. candidum strains.

Biofouling of ultrafiltration membrane by dairy fluids: Characterization of pioneer colonizer bacteria using a DNA metabarcoding approach.

Biofouling of filtration membranes is a major quality and performance issue for the dairy industry. Because biofilms that survive cleaning cycles become resistant over time, prevention strategies limiting the adhesion of bacteria to membranes should be prioritized for sustainable control of biofouling. However, this cannot be achieved because the pioneer bacteria colonizing these membranes are still unknown. Consequently, the objective of this study was to characterize pioneer bacteria on the filtration membrane surface and to measure the effect of filtration operational parameters on their diversity. Thus, milk and cheese whey were filtered for 5 h in concentration mode at 10 and 40°C using a laboratory-scale crossflow filtration system equipped with flat-sheet ultrafiltration membranes. Pioneer colonizer bacteria found on membranes after a chlorinated alkaline cleaning cycle were identified using a metabarcoding approach targeting the 16S ribosomal RNA genes. Our results suggested that prevention strategies targeting biofouling should consider the nature of the filtered fluid and the feed temperature (36.15 and 5.09% of the variances observed on membranes, respectively), as well as the microbial environment of the dairy processing plant. In the future, it is hypothesized that cleaning prevention strategies will be specific to each dairy processor and their operational parameters.

Metatranscriptome analysis of fungal strains Penicillium camemberti and Geotrichum candidum reveal cheese matrix breakdown and potential development of sensory properties of ripened Camembert-type cheese.

BACKGROUND: Camembert-type cheese ripening is driven mainly by fungal microflora including Geotrichum candidum and Penicillium camemberti. These species are major contributors to the texture and flavour of typical bloomy rind cheeses. Biochemical studies showed that G. candidum reduces bitterness, enhances sulphur flavors through amino acid catabolism and has an impact on rind texture, firmness and thickness, while P. camemberti is responsible for the white and bloomy aspect of the rind, and produces enzymes involved in proteolysis and lipolysis activities. However, very little is known about the genetic determinants that code for these activities and their expression profile over time during the ripening process. RESULTS: The metatranscriptome of an industrial Canadian Camembert-type cheese was studied at seven different sampling days over 77 days of ripening. A database called CamemBank01 was generated, containing a total of 1,060,019 sequence tags (reads) assembled in 7916 contigs. Sequence analysis revealed that 57% of the contigs could be affiliated to molds, 16% originated from yeasts, and 27% could not be identified. According to the functional annotation performed, the predominant processes during Camembert ripening include gene expression, energy-, carbohydrate-, organic acid-, lipid- and protein- metabolic processes, cell growth, and response to different stresses. Relative expression data showed that these functions occurred mostly in the first two weeks of the ripening period. CONCLUSIONS: These data provide further advances in our knowledge about the biological activities of the dominant ripening microflora of Camembert cheese and will help select biological markers to improve cheese quality assessment.

The composition of Camembert cheese-ripening cultures modulates both mycelial growth and appearance.

The fungal microbiota of bloomy-rind cheeses, such as Camembert, forms a complex ecosystem that has not been well studied, and its monitoring during the ripening period remains a challenge. One limitation of enumerating yeasts and molds on traditional agar media is that hyphae are multicellular structures, and colonies on a petri dish rarely develop from single cells. In addition, fungi tend to rapidly invade agar surfaces, covering small yeast colonies and resulting in an underestimation of their number. In this study, we developed a real-time quantitative PCR (qPCR) method using TaqMan probes to quantify a mixed fungal community containing the most common dairy yeasts and molds: Penicillium camemberti, Geotrichum candidum, Debaryomyces hansenii, and Kluyveromyces lactis on soft-cheese model curds (SCMC). The qPCR method was optimized and validated on pure cultures and used to evaluate the growth dynamics of a ripening culture containing P. camemberti, G. candidum, and K. lactis on the surface of the SCMC during a 31-day ripening period. The results showed that P. camemberti and G. candidum quickly dominated the ecosystem, while K. lactis remained less abundant. When added to this ecosystem, D. hansenii completely inhibited the growth of K. lactis in addition to reducing the growth of the other fungi. This result was confirmed by the decrease in the mycelium biomass on SCMC. This study compares culture-dependent and qPCR methods to successfully quantify complex fungal microbiota on a model curd simulating Camembert-type cheese.

Genome comparison of Bifidobacterium longum strains NCC2705 and CRC-002 using suppression subtractive hybridization.

Because probiotic effects are strain dependent, genomic explanations of these differences will contribute to understanding their mechanisms of action. The genomic sequence of the Bifidobacterium longum probiotic strain NCC2705 was determined, but little is known about the genetic diversity between strains of this species. Suppression subtractive hybridization (SSH) is a powerful method for generating a set of DNA fragments differing between two closely related bacterial strains. The purpose of this study was to identify genetic differences between genomes of B. longum strains NCC2705 and CRC-002 using PCR-based SSH. Strain CRC-002 produces exopolysaccharides whereas NCC2705 is not known for reliable exopolysaccharide production. Thirty-five and 30 different sequences were obtained from the SSH libraries of strains CRC-002 and NCC2705, respectively. Specific CRC-002 genes found were predicted to be involved in the biosynthesis of exopolysaccharides and metabolism of other carbohydrates, and these genes were not present in the genome of strain NCC2705. The identification of an endo-1,4-beta-xylanase gene in the CRC-002 SSH library is an important difference because xylanase genes have previously been proposed as a defining characteristic of the NCC2705 strain. The results demonstrate that the SSH technique was useful to highlight potential genes involved in complex sugar metabolism that differ between the two probiotic strains.