Longitudinal Study of Lactococcus Phages in a Canadian Cheese Factory.

The presence of virulent phages is closely monitored during cheese manufacturing, as these bacterial viruses can significantly slow down the milk fermentation process and lead to low-quality cheeses. From 2001 to 2020, whey samples from cheddar cheese production in a Canadian factory were monitored for the presence of virulent phages capable of infecting proprietary strains of Lactococcus cremoris and Lactococcus lactis used in starter cultures. Phages were successfully isolated from 932 whey samples using standard plaque assays and several industrial Lactococcus strains as hosts. A multiplex PCR assay assigned 97% of these phage isolates to the Skunavirus genus, 2% to the P335 group, and 1% to the Ceduovirus genus. DNA restriction profiles and a multilocus sequence typing (MLST) scheme distinguished at least 241 unique lactococcal phages from these isolates. While most phages were isolated only once, 93 of them (out of 241, 39%) were isolated multiple times. Phage GL7 was isolated 132 times from 2006 to 2020, demonstrating that phages can persist in a cheese factory for long periods of time. Phylogenetic analysis of MLST sequences showed that phages could be clustered based on their bacterial hosts rather than their year of isolation. Host range analysis showed that Skunavirus phages exhibited a very narrow host range, whereas some Ceduovirus and P335 phages had a broader host range. Overall, the host range information was useful in improving the starter culture rotation by identifying phage-unrelated strains and helped mitigating the risk of fermentation failure due to virulent phages. IMPORTANCE Although lactococcal phages have been observed in cheese production settings for almost a century, few longitudinal studies have been performed. This 20-year study describes the close monitoring of dairy lactococcal phages in a cheddar cheese factory. Routine monitoring was conducted by factory staff, and when whey samples were found to inhibit industrial starter cultures under laboratory conditions, they were sent to an academic research laboratory for phage isolation and characterization. This led to a collection of at least 241 unique lactococcal phages, which were characterized through PCR typing and MLST profiling. Phages of the Skunavirus genus were by far the most dominant. Most phages lysed a small subset of the Lactococcus strains. These findings guided the industrial partner in adapting the starter culture schedule by using phage-unrelated strains in starter cultures and removing some strains from the starter rotation. This phage control strategy could be adapted for other large-scale bacterial fermentation processes.

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.

Comparative Study of Different Sampling Methods of Biofilm Formed on Stainless-Steel Surfaces in a CDC Biofilm Reactor.

The formation of biofilms in dairy processing plants can reduce equipment efficiency, contribute to surface deterioration, and contaminate dairy products by releasing the microorganisms they contain, which may cause spoilage or disease. However, a more representative identification of microbial communities and physico-chemical characterization requires to detach and recover adequately the entire biofilm from the surface. The aim of this study is to develop an efficient technique for in-plant biofilm sampling by growing a strain of Pseudomonas azotoformans PFl1A on stainless-steel surface in a dynamic CDC biofilm reactor system using tryptic soy broth (TSB) and milk as growth media. Different techniques, namely, swabbing, scraping, sonic brushing, synthetic sponge, and sonicating synthetic sponge were used and the results were compared to a standard ASTM International method using ultrasonication. Their efficiencies were evaluated by cells enumeration and scanning electron microscopy. The maximum total viable counts of 8.65 ± 0.06, 8.75 ± 0.08, and 8.71 ± 0.09 log CFU/cm2 were obtained in TSB medium using scraping, synthetic sponge, and sonicating synthetic sponge, respectively, which showed no statistically significant differences with the standard method, ultrasonication (8.74 ± 0.02 log CFU/cm2). However, a significantly (p < 0.05) lower cell recovery of 8.57 ± 0.10 and 8.60 ± 0.00 log CFU/cm2 compared to ultrasonication were achieved for swabbing and sonic brushing, respectively. Furthermore, scanning electron microscopy showed an effective removal of biofilms by sonic brushing, synthetic sponge, and sonicating synthetic sponge; However, only the latter two methods guaranteed a superior release of bacterial biofilm into suspension. Nevertheless, a combination of sonication and synthetic sponge ensured dislodging of sessile cells from surface crevices. The results suggest that a sonicating synthetic sponge could be a promising method for biofilm recovery in processing plants, which can be practically used in the dairy industries as an alternative to ultrasonication.

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.

Identification of texture parameters influencing commercial cheese matrix disintegration and lipid digestion using an in vitro static digestion model.

Cheese characteristics, such as composition or textural properties, can impact the matrix degradation rate which could modulate the bioaccessibility of fatty acids during digestion. The aim of this study was to identify texture parameters influencing cheese degradation in a gastrointestinal environment. A static in vitro digestion model has been used on nine commercial cheeses: young and aged cheddar, regular and light cream cheese, parmesan, feta, camembert, mozzarella, and sliced processed cheese. At the end of gastric digestion, camembert and mozzarella presented the lowest matrix disintegration whereas aged cheddar, regular and light cream cheeses showed the highest. For all cheeses, the fatty acid release was fast during the first 30 min of duodenal digestion and slowed down afterwards. A partial least square regression revealed that springiness, cohesiveness, and hardness were negatively correlated to the rate of cheese disintegration during gastric digestion. In addition, textural parameters were not correlated with free fatty acid release. By modulating cheese texture, it could be possible to influence matrix disintegration during gastrointestinal digestion which could have an impact on lipids release.

Identification of Peptides Implicated in Antibacterial Activity of Snow Crab Hepatopancreas Hydrolysates by a Bioassay-Guided Fractionation Approach Combined with Mass Spectrometry.

Snow crab (Chionoecetes opilio) by-products are a rich source of biomolecules, such as lipids, proteins, and chitin, which have not been extensively investigated. This study aims to identify antibacterial peptides to enhance the value of C. opilio by-products. After hydrolysis of different component parts using Protamex®, and concentration by solid-phase extraction, the resulting fractions were tested for antibacterial activity against Escherichia coli, Listeria innocua, and Vibrio parahaemolyticus. Hepatopancreas was the only tissue to display antibacterial activity detected using this protocol. Four fractions obtained with and without enzymatic hydrolysis of hepatopancreas followed by SPE C18 fractionation and elution with 50 and 80% acetonitrile demonstrated bacteriostatic activity against L. innocua HPB13, from concentrations of 0.30 to 43.05 mg/mL of peptides/proteins. Eleven peptides sharing at least 80% amino acid homology with four antimicrobial peptides were identified by mass spectrometry. Two peptides had homology to crustin-like and yellowfin tuna GAPDH antimicrobial peptides belonging to the marine organisms Penaeus monodon and Thunnus albacares, respectively. Other peptide sequence homologies were also identified: Odorranain-C7 from the frog Odorrana grahami and a predicted antibacterial peptide in the Asian ladybeetle Harmonia axyridis. These active peptides may represent a novel group of bioactive peptides deserving further investigation as food preservatives.

Differential impact of the cheese matrix on the postprandial lipid response: a randomized, crossover, controlled trial.

Background: In a simulated gastrointestinal environment, the cheese matrix modulates dairy fat digestion. However, to our knowledge, the impact of the cheese matrix on postprandial lipemia in humans has not yet been evaluated.Objective: In healthy subjects, we compared the impact of dairy fat provided from firm cheese, soft cream cheese, and butter on the postprandial response at 4 h and on the incremental area under the curve (iAUC) of plasma triglycerides.Design: Forty-three healthy subjects were recruited to this randomized, crossover, controlled trial. In random order at intervals of 14 d and after a 12-h fast, subjects ingested 33 g fat from a firm cheese (young cheddar), a soft cream cheese (cream cheese), or butter (control) incorporated into standardized meals that were matched for macronutrient content. Plasma concentrations of triglycerides were measured immediately before the meal and 2, 4, 6, and 8 h after the meal.Results: Cheddar cheese, cream cheese, and butter induced similar increases in triglyceride concentrations at 4 h (change from baseline: +59%, +59%, and +62%, respectively; P = 0.9). No difference in the triglyceride iAUC0-8 h (P-meal = 0.9) was observed between the 3 meals. However, at 2 h, the triglyceride response caused by the cream cheese (change from baseline: +44%) was significantly greater than that induced by butter (change from baseline: +24%; P = 0.002) and cheddar cheese (change from baseline: +16%; P = 0.0004). At 6 h, the triglyceride response induced by cream cheese was significantly attenuated compared with that induced by cheddar cheese (change from baseline: +14% compared with +42%; P = 0.0004).Conclusion: This study demonstrates that the cheese matrix modulates the impact of dairy fat on postprandial lipemia in healthy subjects. This trial was registered at clinicaltrials.gov as NCT02623790.

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.

Study of the antibacterial activity of electro-activated solutions of salts of weak organic acids on Salmonella enterica, Staphylococcus aureus and Listeria monocytogenes.

This work assessed the antibacterial activity of electro-activated solutions of salts of weak organic acids (potassium acetate, potassium citrate and calcium lactate) on Salmonella enterica, Staphylococcus aureus and Listeria monocytogenes. This activity was compared in terms of minimal inhibitory (bactericidal) concentration to the effect of commercial acetic, citric and lactic acid at equivalent titratable acidity. Staining live/dead BacLight method was used to consider physiological state of bacteria following the evaluation of pathogenic strains during exposure to the tested solutions. The results demonstrated strong inhibitory activity of all electro-activated solutions. After 10 min of exposure to electro-activated potassium acetate, a reduction of ≥6 log CFU/ml of all bacteria was observed. The electro-activated potassium citrate demonstrated the lowest minimal inhibitory concentration. Nevertheless, its inactivation power was significantly higher than that of conjugated citric acid. Although electro-activated calcium lactate was found less effective in comparison with its conjugated acid form, after 10 min of contact with the tested pathogens, it induced a population reduction of 2.23, 2.97 and 5.57 log CFU/ml of S. aureus, L. monocytogenes and S. enterica, respectively.

Electro-activation of potassium acetate, potassium citrate and calcium lactate: impact on solution acidity, Redox potential, vibrational properties of Raman spectra and antibacterial activity on E. coli O157:H7 at ambient temperature.

AIMS: To study the electro-activation of potassium acetate, potassium citrate and calcium lactate aqueous solutions and to evaluate their antimicrobial effect against E. coli O157:H7 at ambient temperature. METHODS AND RESULTS: Potassium acetate, potassium citrate and calcium lactate aqueous solutions were electrically excited in the anodic compartment of a four sectional electro-activation reactor. Different properties of the electro-activated solutions were measured such as: solutions acidity (pH and titratable), Redox potential and vibrational properties by Raman spectroscopy. Moreover, the antimicrobial activity of these solutions was evaluated against E. coli O157:H7. The results showed a pH decrease from 7.07 ± 0.08, 7.53 ± 0.12 and 6.18 ± 0.1 down to 2.82 ± 0.1, 2.13 ± 0.09 and 2.26 ± 0.15, after 180 min of electro-activation of potassium acetate, potassium citrate and calcium lactate solution, respectively. These solutions were characterized by high oxidative ORP of +1076 ± 12, +958 ± 11 and +820 ± 14 mV, respectively. Raman scattering analysis of anolytes showed stretching vibrations of the hydrogen bonds with the major changes within the region of 3410-3430 cm-1. These solutions were used against E. coli O157:H7 and the results from antimicrobial assays showed high antibacterial effect with a population reduction of ≥6 log CFU/ml within 5 min of treatment. CONCLUSIONS: This study demonstrated the effectiveness of the electro-activation to confer to aqueous solutions of organic salts of highly reactive properties that differ them from their conjugated commercial acids. The electro-activated solutions demonstrated significant antimicrobial activity against E. coli O157:H7. SIGNIFICANCE AND IMPACT OF STUDY: This study opens new possibilities to use electro-activated solutions of salts of weak organic acids as food preservatives to develop safe, nutritive and low heat processed foods.

Effect of electro-activated brine solution on the migration of metallic ions from the cans to the product in sterilized canned sweet corn.

Tinplate cans were used to study if electro-activated brine solution (EAS) is more corrosive than conventional one by ICP analysis. The results showed different effects of EAS on cans, alone or filled with product. Acidic EAS (pH 2-3) and Redox +900 to +1200 mV highly reacted with the cans. The concentrations of Zn, Fe, and Cu in the solution were 0.028, 28.81, and 0.022 ppm, respectively. No Sn migration was observed in this case. When neutral or acidic chlorine-free EAS was used, no significant difference was observed in comparison with the corrosivity of standard NaCl brine. Alkaline EAS with pH>10 and negative E (≤-966 mV) did not affect Zn, Fe, and Cu migration. However, it affected tin migration. Nevertheless, it is important to mention that even if some corrosion was observed, it was in the limit of the permitted level of concentration when the cans were filled with a product.

Efficacy of two Staphylococcus aureus phage cocktails in cheese production.

Staphylococcus aureus is one of the most prevalent pathogenic bacteria contaminating dairy products. In an effort to reduce food safety risks, virulent phages are investigated as antibacterial agents to control foodborne pathogens. The aim of this study was to compare sets of virulent phages, design phage cocktails, and use them in a cocktail to control pathogenic staphylococci in cheese. Six selected phages belonging to the three Caudovirales families (Myoviridae, Siphoviridae, Podoviridae) were strictly lytic, had a broad host range, and did not carry genes coding for virulence traits in their genomes. However, they were sensitive to pasteurization. At MOI levels of 15, 45, and 150, two anti-S. aureus phage cocktails, each containing three phages, one from each of the three phage families, eradicated a 10(6)CFU/g S. aureus population after 14 days of Cheddar cheese curd ripening at 4°C. The use of these phages did not trigger over-production of S. aureus enterotoxin C. The use of phage cocktails and their rotation may prevent the emergence of phage resistant bacterial strains.

Conidiation of Penicillium camemberti in submerged liquid cultures is dependent on the nitrogen source.

OBJECTIVE: To study the ability of a commercial Penicillium camemberti strain, used for Camembert type cheese ripening, to produce conidia during growth in liquid culture (LC), in media containing different sources of nitrogen as, industrially, conidia are produced by growth at the surface of a solid state culture because conidiation in stirred submerged aerobic LC is not known. RESULTS: In complex media containing peptic digest of meat, hyphae ends did not differentiate into phialides and conidia. Contrarily, in a synthetic media containing KNO3 as sole nitrogen source, hyphae ends differentiated into phialides producing 0.5 × 10(7) conidia/ml. Conidia produced in LC were 25 % less hydrophobic than conidia produced in solid culture, and this correlates with a seven-times-lower expression of the gene rodA encoding hydrophobin RodA in the mycelium grown in LC. CONCLUSION: Conidiation of P. camembertii is stimulated in iquid medium containing KNO3 as sole source of nitrogen and therefore opens up opportunities for using liquid medium in commercial productions.

Study of the combined effect of electro-activated solutions and heat treatment on the destruction of spores of Clostridium sporogenes and Geobacillus stearothermophilus in model solution and vegetable puree.

The combined effect of heat treatment and electro-activated solution (EAS) on the heat resistance of spores of Clostridium sporogenes and Geobacillus stearothermophilus was assessed under various heating and exposure time combinations. The acid and neutral EAS showed the highest inhibitory activity, indicating that these solutions may be considered as strong sporicidal disinfectants. These EAS were able to cause a reduction of ≥6 log of spores of C. sporogenes at 60 °C in only 1 min of exposition. For G. stearothermophilus spores, a reduction of 4.5 log was observed at 60 °C in 1 min, while in 5 min, ≥7 log CFU/ml reduction was observed. Inoculated puree of pea and corn were used as a food matrix for the determination of the heat resistance of these spores during the treatments in glass capillaries. The inactivation kinetics of the spores was studied in an oil bath. Combined treatment by EAS and temperature demonstrated a significant decrease in the heat resistance of C. sporogenes. The D100°C in pea puree with NaCl solution was 66.86 min while with acid and neutral EAS it was reduced down to 3.97 and 2.19 min, respectively. The spore of G. stearothermophilus displayed higher heat resistance as confirmed by other similar studies. Its D130°C in pea puree showed a decrease from 1.45 min in NaCl solution down to 1.30 and 0.93 min for acid and neutral EAS, respectively. The differences between the spores of these species are attributable to their different sensitivities with respect to pH, Redox potential and oxygen.

Improving the safety of Staphylococcus aureus polyvalent phages by their production on a Staphylococcus xylosus strain.

Team1 (vB_SauM_Team1) is a polyvalent staphylococcal phage belonging to the Myoviridae family. Phage Team1 was propagated on a Staphylococcus aureus strain and a non-pathogenic Staphylococcus xylosus strain used in industrial meat fermentation. The two Team1 preparations were compared with respect to their microbiological and genomic properties. The burst sizes, latent periods, and host ranges of the two derivatives were identical as were their genome sequences. Phage Team1 has 140,903 bp of double stranded DNA encoding for 217 open reading frames and 4 tRNAs. Comparative genomic analysis revealed similarities to staphylococcal phages ISP (97%) and G1 (97%). The host range of Team1 was compared to the well-known polyvalent staphylococcal phages phi812 and K using a panel of 57 S. aureus strains collected from various sources. These bacterial strains were found to represent 18 sequence types (MLST) and 14 clonal complexes (eBURST). Altogether, the three phages propagated on S. xylosus lysed 52 out of 57 distinct strains of S. aureus. The identification of phage-insensitive strains underlines the importance of designing phage cocktails with broadly varying and overlapping host ranges. Taken altogether, our study suggests that some staphylococcal phages can be propagated on food-grade bacteria for biocontrol and safety purposes.

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.

Genetic diversity of dairy Geotrichum candidum strains revealed by multilocus sequence typing.

The introduction of multilocus sequence typing (MLST) for strain characterization provided the first sequence-based approach for genotyping many fungi, leading to reproducible, reliable, and exchangeable data. A MLST scheme based on the analysis of six housekeeping genes was developed for genotyping Geotrichum candidum. The scheme was first developed using 18 isolates for which the complete sequences of the alanyl-tRNA synthetase (ALA1), pyruvate kinase (CDC19), acetyl-coA acetyltransferase (ERG10), glutaminyl-tRNA synthase (GLN4), phosphoglucoisomerase (PGI1), and phosphoglucomutase (PGM2) housekeeping genes were determined. Multiple sequence alignments of these genes were used to define a set of loci showing, as closely as possible, the same phylogenetic resolution level as complete gene sequences. This scheme was subsequently validated with 22 additional isolates from dairy and non-dairy sources. Overall, 58 polymorphic sites were indexed among 3,009 nucleotides analyzed. Depending on the loci, four to eight alleles were detected, generating 17 different sequence types, of which ten were represented by a single strain. MLST analysis suggested a predominantly clonal population for the 40 G. candidum isolates. Phylogenetic analysis of the concatenated sequences revealed a distantly related group of four isolates. Interestingly, this group diverged with respect to internal transcribed spacers 1 (ITS1), 5.8S, and ITS2 analysis. The reproducibility of the MLST approach was compared to random amplification of microsatellites by PCR (RAM-PCR), a gel profiling method previously proposed for G. candidum strain typing. Our results found MLST differentiation to be more efficient than RAM-PCR, and MLST also offered a non-ambiguous, unique language, permitting data exchange and evolutionary inference.

Identification and Partial Characterization of Antilisterial Compounds Produced by Dairy Yeasts.

Food-grade yeasts make significant contributions to flavor development in fermented foods. Some yeast species also inhibit undesirable bacteria, yeasts and molds, apparently by producing antimicrobial compounds called mycocins. The aim of this study was to evaluate the ability of wild yeasts, isolated from raw milk and cheese in the Quebec province area, to produce antilisterial compounds. Based on an agar-membrane screening test, 22 of 95 isolates, namely one Candida catenulata, one Candida parapsilosis, five Candida tropicalis, four Debaryomyces hansenii, one Geotrichum candidum, nine Pichia fermentans and one Pichia anomala, exhibited a significant inhibitory effect against Listeria ivanovii HPB28. Four in particular, namely C. tropicalis LMA-693, D. hansenii LMA-916, P. fermentans LMA-256 and P. anomala LMA-827, produced substances extractable from culture supernatant and capable of decreasing 18-h growth of L. ivanovii by, respectively, 97, 92, 84 and 78 %. Heating the extracted material (100 °C for 10 min) decreased these values to 72, 62, 58 and 31 %, respectively, while treatment with trypsin or pronase E decreased them to as little as 27 %. The extracts reduced the numbers of viable Listeria monocytogenes by as much as four log cycles within an hour. Transmission electron microscopy revealed a high proportion of lysis among the cells, apparently due to pore formation. This study clearly shows the potential of these four yeast isolates for use as bio-preservatives in a variety of dairy products.

Antimicrobial and probiotic properties of yeasts: from fundamental to novel applications.

The yeasts constitute a large and heterogeneous group of microorganisms that are currently attracting increased attention from scientists and industry. Numerous and diverse biological activities make them promising candidates for a wide range of applications not limited to the food sector. In addition to their major contribution to flavor development in fermented foods, their antagonistic activities toward undesirable bacteria, and fungi are now widely known. These activities are associated with their competitiveness for nutrients, acidification of their growth medium, their tolerance of high concentrations of ethanol, and release of antimicrobial compounds such as antifungal killer toxins or "mycocins" and antibacterial compounds. While the design of foods containing probiotics (microorganisms that confer health benefits) has focused primarily on Lactobacillus and Bifidobacterium, the yeast Saccharomyces cerevisiae var. boulardii has long been known effective for treating gastroenteritis. In this review, the antimicrobial activities of yeasts are examined. Mechanisms underlying this antagonistic activity as well as recent applications of these biologically active yeasts in both the medical and veterinary sectors are described.