Search

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.

Domestication is an excellent model for studying adaptation processes, constituting recent adaptation and diversification, as well as degeneration of unused functions. Geotrichum candidum is a fungus used for cheese-making and is also found in other environments such as soil and plants. By analyzing whole genome data from 98 strains, we found that all strains isolated from cheese formed a monophyletic clade. Within the cheese clade, we identified three differentiated populations and we detected footprints of recombination and hybridization. The genetic diversity in the cheese clade was the lowest but remained high compared to other domesticated fungi, indicating milder bottlenecks. Commercial starter strains were scattered across the cheese clade, not constituting a single clonal lineage. The cheese clade was phenotypically differentiated from other populations, with a slower growth on all media even cheese, a prominent production of attractive cheese flavors and a lower proteolytic activity. Furthermore, cheese populations displayed contrasted phenotypes, with one cheese population producing a typical blue cheese flavor and another one displaying denser and fluffier colonies, excluding more efficiently cheese spoiler fungi. Cheese populations lost two beta lactamase-like genes, involved in xenobiotic clearance, and displayed transposable element expansion, likely due to relaxed selection. Our findings suggest the existence of genuine domestication in G. candidum, which led to diversification into three varieties with contrasted phenotypes. Some of the traits acquired by cheese strains indicate convergence with other, distantly related fungi used for cheese maturation.

The dairy industry produces large amounts of wastewater, including white and cleaning wastewater originating principally from rinsing and cleaning-in-place procedures. Their valorization into process water and non-fat milk solids, in the case of white wastewater, or the renewal of cleaning solutions could be achieved using pressure-driven membrane processes. However, it is crucial to determine the intrinsic characteristics of wastewaters, such as proximate composition and bacterial composition, to optimize their potential for valorization. Consequently, white and cleaning wastewaters were sampled from industrial-scale pasteurizers located in two different Canadian dairy processing plants. Bacterial profiles of dairy wastewaters were compared to those of tap waters, pasteurized skim milk and unused cleaning solutions. The results showed that the physicochemical characteristics as well as non-fat milk solids contents differed drastically between the two dairy plants due to different processing conditions. A molecular approach combining quantitative real-time polymerase chain reaction (qPCR) and metabarcoding was used to characterize the bacteria present in these solutions. The cleaning solutions did not contain sufficient genomic DNA for sequencing. In white wastewater, the bacterial contamination differed depending on the dairy plant (6.91 and 7.21 log10 16S gene copies/mL). Psychrotrophic Psychrobacter genus (50%) dominated white wastewater from plant A, whereas thermophilic Anoxybacillus genus (56%) was predominant in plant B wastewater. The use of cold or warm temperatures during the pasteurizer rinsing step in each dairy plant might explain this difference. The detailed characterization of dairy wastewaters described in this study is important for the dairy sector to clearly identify the challenges in implementing strategies for wastewater valorization.

Domestication is an excellent model for studying adaptation processes, involving recent adaptation and diversification, convergence following adaptation to similar conditions, as well as degeneration of unused functions.Geotrichum candidumis a fungus used for cheese making and is also found in other environments such as soil and plants. By analyzing whole-genome data from 98 strains, we found that all strains isolated from cheese formed a monophyletic clade. Within the cheese clade, we identified three genetically differentiated populations and we detected footprints of recombination and admixture. The genetic diversity in the cheese clade was similar as that in the wild clade, suggesting the lack of strong bottlenecks. Commercial starter strains were scattered across the cheese clade, thus not constituting a single clonal lineage. The cheese populations were phenotypically differentiated from other populations, with a slower growth on all media, even cheese, a prominent production of typical cheese volatiles and a lower proteolytic activity. One of the cheese clusters encompassed all soft goat cheese strains, suggesting an effect of cheese-making practices on differentiation. Another of the cheese populations seemed to represent a more advanced stage of domestication, with stronger phenotypic differentiation from the wild clade, harboring much lower genetic diversity, and phenotypes more typical of cheese fungi, with denser and fluffier colonies and a greater ability of excluding cheese spoiler fungi. Cheese populations lacked two beta lactamase-like genes present in the wild clade, involved in xenobiotic clearance, and displayed higher contents of transposable elements, likely due to relaxed selection. Our findings suggest the existence of genuine domestication inG. candidum, which led to diversification into different varieties with contrasted phenotypes. Some of the traits acquired by cheese strains indicate convergence with other, distantly related fungi used for cheese maturation.

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.

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.

International audience; The temperature of the feed is known to be an important factor affecting the formation and the growth rates of bacterial communities on dairy filtration membranes. However, decades after the integration of filtration processes in the dairy industry, there is still questioning whether filtration should be performed at cold (48 degrees C) temperature. A biofilm reactor designed to mimic a filtration system was used to provide answers to this question. Bacteria adhering and growing on ultrafiltration membranes in contact with pasteurised skim milk were characterised at 15 degrees C and 50 degrees C through a metabarcoding approach. Our results suggested that the processing time should be limited to 10 h at 50 degrees C to avoid the exponential growth of thermophilic spore-former bacteria, while the use of 15 degrees C combined with daily cleaning procedures appeared the best way to retard the formation of biofilms on membranes.

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.

This study aimed to quantify the effects of biofouling on the performance of an ultrafiltration system during 24-h recirculation of whey solutions at 0 and 0.8% (w/v) protein. Biofouling by Streptococcus thermophilus and the intrinsic microflora of the ultrafiltration system was either allowed or inhibited with 10 m m sodium azide. Permeate fluxes were reduced by biofouling after 24 h, as normalised flux values with biofouling were 29.0% lower than without biofouling for 0%-protein whey, and 6.5% lower for 0.8%-protein whey. Furthermore, the membrane hydraulic resistances increased, allowing the measurement of biofouling-specific irreversible and reversible resistance values (Rbio) which accounted for 30.7–44.4% of the total fouling layer resistances. These results provide evidence that biofouling can negatively affect the performance of a single ultrafiltration operation. Consequently, pre-treatment of dairy fluids, biofilm management and optimised cleaning procedures are needed to limit the negative impacts of membrane biofouling.

This study aimed to quantify the contribution of Streptococcus thermophilus cell deposition to the flux decline observed during ultrafiltration of cheese whey constituents using an unstirred batch cell system. Bacterial cells were suspended in model solutions of sodium chloride, of sodium chloride supplemented with either whey protein isolate, β-lactoglobulin or α-lactalbumin, or of whey permeate. Permeate fluxes were compared with those of sterile solutions. Normalised flux dropped by 20–27 percent points when bacterial cells were filtered with whey proteins and only by 5.0–6.7 percent points in the absence of whey proteins. Whey proteins filled void spaces between bacteria by behaving like an extracellular matrix, reducing the fouling layer's permeability. It was hypothesised that electrostatic interactions between bacteria and protein led to a more cohesive cake. This study provides insight on possible interactions between bacterial cells and whey proteins that might exacerbate fouling during industrial whey ultrafiltration.

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.

Few data are available concerning the composition of biofilms found at the surface of filtration membranes, which, to some extent, explains the long-term failure of numerous strategies developed to control biofouling. This preliminary study intended to design a metagenomic tool targeting the 16S rRNA gene in order to unravel a general portrait of bacterial communities found on spiral-wound membranes used in the dairy industry. A total of seven spiral-wound membrane elements (ultrafiltration, nanofiltration, or reverse osmosis) at the end of their useful lifetimes were collected from different dairy plants. Targeted analysis of the 16S rRNA genes of the metagenome extracted from the membranes revealed their bacterial diversity via high-throughput sequencing technology (Miseq, Illumina). It was found that the nature of the filtered fluid (milk, whey, water) explained 58.6 % of the variance observed between communities found on membranes. Treatments applied on dairy fluids (milk pasteurization, whey bleaching or whey ultrafiltration) induced a selective pressure that affected the diversity of bacterial communities found on membranes and the proportions of spore-former bacteria among them. This work provides the first complete bacterial portrait of the biofilm composition of spiral-wound membranes used in the dairy industry. It suggests that the nature of the filtered fluid and potentially filtration operating parameters may be important elements to consider in order to design new cleaning strategies or preventive measures targeting biofouling.

Recent results showed that solid food disintegration in the stomach may be affected by food texture which was demonstrated to change during digestion. Cheese is complex as, depending on the variety, its composition and texture can be modulated. Cheddar, light Cheddar, Mozzarella and light Mozzarella cheese particles were digested in vitro . Cheese disintegration and nutrients release were studied throughout the oral, gastric and duodenal digestion steps in presence or absence of enzymes. Cheese disintegration was significantly affected by the enzymatic treatment (with or without enzymes). The addition of enzymes allowed to reach 72% of cheese disintegration at the end of the duodenal digestion while it has attained 30% when no enzymes were added. Cheddar cheese disintegration was the highest among cheeses. This phenomenon was related to its initial higher fat content which resulted in a higher fat release during digestion. The disintegration at the end of each digestion step was also correlated to cheese composition (proteolysis and fat) and to textural parameters (hardness, resilience, adhesiveness and chewiness). Light Cheddar and Mozzarella exhibited similar disintegration and nutrients release at the end of the digestion due to a relatively small fat reduction (6%) which had limited effect on cheese texture. This study provides quantitative evidence regarding the impact of cheese textural changes during digestion on cheese disintegration and macronutrients release which may further affect nutrients anabolic response and some physiological functions.

Solid food disintegration in the stomach has recently been linked to food texture, which changes during digestion. This phenomenon is likely to affect the kinetics of protein digestion and therefore associated postprandial metabolic responses. Depending upon the variety, the cheese protein and lipid content as well as the texture can be modulated, illustrating complexity. Five commercial cheeses, covering a range of textural properties, were selected and characterised. Cheese particles were submitted to an in vitro digestion model to study cheese disintegration and protein/peptide release. Cheese disintegration was affected by cheese texture and composition. At the end of gastric digestion, elastic cheeses (mozzarella) were less disintegrated when compared with ripened and soft cheeses with high fat content (Camembert, aged Cheddar). The protein digestion was different amongst cheeses according to different disintegration rates. Cheese structural and textural properties, attributed to processing parameters, can be used to modulate gastro-intestinal digestion of cheese proteins.

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.

International audience; 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.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 x 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.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.

Summary The objective of this work was to evaluate the quality of sterilised canned pea and corn in electro-activated brine solutions at moderate temperatures. The lowest change in vitamin C was associated with the lowest heat treatment, while the short treatment time resulted in significant changes in texture and colour of vegetables. Best texture profile was obtained with the acid electro-activated brine solution for pea and corn. The neutral electro-activated brine solution resulted in a less firm texture for pea and corn. The green colour and brightness of canned pea were higher when neutral solution was used (a* = −8.4 ± 0.3) than for the acidic one (a* = −3.7 ± 0.6). The yellowness of corn was better with the neutral electro-activated brine solution (b* = 36.32 ± 1.24) than with the acidic one (b* = 28.44 ± 2.39). Thirty-three percent decrease of energy consumption was obtained using the electro-activation technology.

This study examines the possibility of producing optimally electro-activated (EA) solutions using response surface methodology (RSM). RSM yielded models to predict solution properties as functions of the process parameters studied with very high coefficients of determination (R 2 = 0.9393–0.9974), indicating the models provided high correlation between observed and predicted values. The effect of production parameters, such as salt concentration, current and exposure time on the properties of the solutions, was investigated. For oxidation–reduction potential, pH and resistance, salt concentration was the most significant factor followed by exposure time and current. Using a Derringer's function, the best performance for the production of electro-activated solution was obtained at a current intensity of 200 mA in the presence of 0.05 M electrolyte after 38 and 60 min of treatment time, depending on which reactor configuration was used. The current investigation shows an agreement with the addition test of predicted data and confirms that the developed model equation can be used for prediction.

The properties of electro-activated (EA) aqueous solutions as well as the dynamics of their changes were considered in the current study using aqueous solutions of NaCl and NaHCO 3 . The concentrations of the salt solutions were 0.5, 0.25, 0.125 and 0.05 M. The tests were performed at the DC current densities of 25, 37.5, and 50 A/m 2 . The electro-activation reactor consisted of three individual cells assembled together and separated by anion-exchange (AEM) and cation-exchange (CEM) membranes. During the experiments, four configurations of the membrane placements and solutions concentrations were studied. The obtained results showed the dynamics of the electro-activation process that allows obtaining electro-activated solutions with targeted properties such as pH and oxydo-reduction potential (ORP). It was possible to obtain electro-activated solutions at the anodic side (acid anolyte) with pH of 3.0, 3.5, and 4.0 and ORP of +1100 ± 15 mV when NaCl solution was used as electrolyte. Furthermore, several types of electro-activated solutions with high redox potential (ORP = +921 ± 12 mV) and neutral pH (6.48 ± 0.05) were obtained on the anode side when sodium carbonate was used. At the same time, two types of solutions, one with acid pH (2.14 ± 0.14) and the other one with alkaline pH (10.46 ± 0.03) with ORP = +689 ± 10 and 110 ± 21 mV, respectively, were obtained in the central compartment which considered as electro-activated solutions obtained by means of noncontact electro-activation.

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.

Electro-activation (EA) of a maple sap/syrup beverage was studied. The purpose of the study was to assess the product parameters, such as pH, ORP, transmittance and degree Brix, while minimizing energy consumption. The experiments were conducted using three different configurations of the reactor that differed by the position of the anion (AEM) and cation (CEM) exchange membrane relative to the electrodes as well as the nature of the electrolyte (NaCl vs. Na2CO3) in the central cell of the reactor. The results showed that the type of configuration, the electric current and the temperature influenced the parameters of the electro-activated beverage. At 23 °C, the beverage was acidified to pH 3.89 with a Redox potential (ORP) of 417.33 mV. At 55 °C and 150 mA, the minimum pH of 3.78 and an ORP of 329.67 mV were obtained. The electric resistance of the electro-activation reactor (EAR) decreased during electro-activation, indicating a gain of energy efficiency corresponding to an electric resistance of 266.76 Ω. Moreover, the reactor configuration and electric current affected the presence or absence of a fouling of the ion exchange membrane at the anodic side. The beverages' transmittance increased slightly during EA without any effect on the degree Brix.

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.

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.

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.

Identifying and isolating yeasts and moulds within a fungal community is challenging. The main goal of the present study was to assess a new approach for the detection and identification of fungi involved in Camembert cheese rind formation to replace the use of traditional microbiological techniques. Two molecular methods, terminal-restriction fragment length polymorphism (T-RFLP) and automated ribosomal intergenic spacer analysis (ARISA), were adapted and compared for their potential to determine fungal composition directly from cheese samples. The two techniques in combination with principal component analysis showed differences in the fungal composition of cheeses when comparing surface with centre, different batches, manufacturing processes and ripening times. Moreover, cheese stabilisation induced changes in the flora at the cheese centre, and difference in size (150 g versus 1 kg) modified surface flora. Nine fungal genera were identified in cheese samples: Cladosporium, Debaryomyces, Geotrichum, Kluyveromyces, Mucor, Penicillium, Pichia, Saccharomyces and Yarrowia.

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.

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.

Phage p2, a member of the lactococcal 936 phage species, infects Lactococcus lactis strains by binding initially to specific carbohydrate receptors using its receptor-binding protein (RBP). The structures of p2 RBP, a homotrimeric protein composed of three domains, and of its complex with a neutralizing llama VH domain (VHH5) have been determined (S. Spinelli, A. Desmyter, C. T. Verrips, H. J. de Haard, S. Moineau, and C. Cambillau, Nat. Struct. Mol. Biol. 13:85-89, 2006). Here, we show that VHH5 was able to neutralize 12 of 50 lactococcal phages belonging to the 936 species. Moreover, escape phage mutants no longer neutralized by VHH5 were isolated from 11 of these phages. All of the mutations (but one) cluster in the RBP/VHH5 interaction surface that delineates the receptor-binding area. A glycerol molecule, observed in the 1.7-Å resolution structure of RBP, was found to bind tightly ( K d = 0.26 μM) in a crevice located in this area. Other saccharides bind RBP with comparable high affinity. These data prove the saccharidic nature of the bacterial receptor recognized by phage p2 and identify the position of its binding site in the RBP head domain.

The Streptococcus thermophilus virulent pac -type phage 2972 was isolated from a yogurt made in France in 1999. It is a representative of several phages that have emerged with the industrial use of the exopolysaccharide-producing S. thermophilus strain RD534. The genome of phage 2972 has 34,704 bp with an overall G+C content of 40.15%, making it the shortest S. thermophilus phage genome analyzed so far. Forty-four open reading frames (ORFs) encoding putative proteins of 40 or more amino acids were identified, and bioinformatic analyses led to the assignment of putative functions to 23 ORFs. Comparative genomic analysis of phage 2972 with the six other sequenced S. thermophilus phage genomes confirmed that the replication module is conserved and that cos - and pac -type phages have distinct structural and packaging genes. Two group I introns were identified in the genome of 2972. They interrupted the genes coding for the putative endolysin and the terminase large subunit. Phage mRNA splicing was demonstrated for both introns, and the secondary structures were predicted. Eight structural proteins were also identified by N-terminal sequencing and/or matrix-assisted laser desorption ionization—time-of-flight mass spectrometry. Detailed analysis of the putative minor tail proteins ORF19 and ORF21 as well as the putative receptor-binding protein ORF20 showed the following interesting features: (i) ORF19 is a hybrid protein, because it displays significant identity with both pac - and cos -type phages; (ii) ORF20 is unique; and (iii) a protein similar to ORF21 of 2972 was also found in the structure of the cos -type phage DT1, indicating that this structural protein is present in both S. thermophilus phage groups. The implications of these findings for phage classification are discussed.

The bacteriophage lysis cassette, which comprises a lysin and a holin gene, was analyzed in 18 Lactococcus lactis phages. A muramidase motif was found in the lysins of c2-like phages, while an amidase motif was observed in the lysins of 936-like phages. Both amidase and muramidase types were detected among the P335 phages. The P335 lysins were separated into three groups based on amino acid sequence identity. A class I holin was recognized in 936-like and c2-like phages, whereas P335-like phages possess class II holins. The P335 holins were further divided into four groups based on sequence identity. Only the holins of 936-like phages contained putative dual-start motifs. The unusual lysis cassette of the highly virulent P335-like phage ul36 contains a unique holin (orf74B) upstream of a lysin which is present in several other P335-like phages. Using the lambdadelta Sthf system, we demonstrated that gpORF74B induces cell lysis at the same time as lambdadelta Sthf::S105, the effector of lambda lysis. Transcriptional analysis of ul36 lysis cassette showed that first transcripts are detected 35 min after infection of L. lactis cells. The lysis clock of phage ul36 appears to be controlled by the late expression of the holin and lysin genes.

Three genetically distinct groups of Lactococcus lactis phages are encountered in dairy plants worldwide, namely, the 936, c2, and P335 species. The multiplex PCR method was adapted to detect, in a single reaction, the presence of these species in whey samples or in phage lysates. Three sets of primers, one for each species, were designed based on conserved regions of their genomes. The c2-specific primers were constructed using the major capsid protein gene ( mcp ) as the target. The mcp sequences for three phages (eb1, Q38, and Q44) were determined and compared with the two available in the databases, those for phages c2 and bIL67. An 86.4% identity was found over the five mcp genes. The gene of the only major structural protein ( msp ) was selected as a target for the detection of 936-related phages. The msp sequences for three phages (p2, Q7, and Q11) were also established and matched with the available data on phages sk1, bIL170, and F4-1. The comparison of the six msp genes revealed an 82.2% identity. A high genomic diversity was observed among structural proteins of the P335-like phages suggesting that the classification of lactococcal phages within this species should be revised. Nevertheless, we have identified a common genomic region in 10 P335-like phages isolated from six countries. This region corresponded to orfF17-orf18 of phage r1t and orf20-orf21 of Tuc2009 and was sequenced for three additional P335 phages (Q30, P270, and ul40). An identity of 93.4% within a 739-bp region of the five phages was found. The detection limit of the multiplex PCR method in whey was 10 4 to 10 7 PFU/ml and was 10 3 to 10 5 PFU/ml with an additional phage concentration step. The method can also be used to detect phage DNA in whey powders and may also detect prophage or defective phage in the bacterial genome.

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.

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.

The effect of four sugars (glucose, galactose, lactose and fructose) on exopolysaccharide (EPS) production by Bifidobacterium longum subsp. longum CRC 002 was evaluated. More EPS was produced when CRC 002 was grown on lactose in the absence of pH control, with a production of 1080±120 mg EPS l−1 (P−1, respectively. The proposed repeating unit composition of the EPS is 2 galactose to 3 glucose. The effect of sugar and fermentation time on expression of genes involved in sugar nucleotide production (galK, galE1, galE2, galT1, galT2, galU, rmlA, rmlB1 and rmlCD) and the priming glycosyltransferase (wblE) was quantified using real-time reverse transcription PCR. A significantly higher transcription level of wblE (9.29-fold) and the genes involved in the Leloir pathway (galK, 4.10-fold; galT1, 2.78-fold; and galE2, 4.95-fold) during exponential growth was associated with enhanced EPS production on lactose compared to glucose. However, galU expression, linking glucose metabolism with the Leloir pathway, was not correlated with EPS production on different sugars. Genes coding for dTDP-rhamnose biosynthesis were also differentially expressed depending on sugar source and growth phase, although rhamnose was not present in the composition of the EPS. This precursor may be used in cell wall polysaccharide biosynthesis. These results contribute to understanding the changes in gene expression when different sugar substrates are catabolized by B. longum subsp. longum CRC 002.

The complete genomic sequence of the Lactococcus lactis virulent phage ul36 belonging to P335 lactococcal phage species was determined and analyzed. The genomic sequence of this lactococcal phage contained 36,798 bp with an overall G+C content of 35.8 mol %. Fifty-nine open reading frames (ORFs) of more than 40 codons were found. N-terminal sequencing of phage structural proteins as well as bioinformatic analysis led to the attribution of a function to 24 ORFs (41%). A lysogeny module was found within the genome of this virulent phage. The putative integrase gene seems to be the product of a horizontal transfer because it is more closely related to Streptococcus pyogenes phages than it is to L. lactis phages. Comparative genome analysis with six complete genomes of temperate P335-like phages confirmed the heterogeneity among phages of P335 species. A dUTPase gene is the only conserved gene among all P335 phages analyzed as well as the phage BK5-T. A genetic relationship between P335 phages and the phage-type of the BK5-T species was established. Thus, we proposed that phage BK5-T be included within the P335 species and thereby reducing the number of lactococcal phage species to 11.

The cheese microbial ecosystem is complex, and the presence of non-starter adventitious microorganisms in milk may have an influence on the organoleptic characteristics of cheese. The aim of this study was to analyze the composition and diversity of the fungal flora of raw milk destined for cheesemaking from 19 dairy farms in Quebec and to monitor their evolution throughout ripening. Six hundred ten yeast and mold isolates were collected from raw milk and raw milk cheeses over a 9-month period. Based on the sequences of the rDNA ITS1-5.8S-ITS2 region, 67% of the raw milk isolates were yeasts, which were assigned to 37 species across 11 genera, while 33% were molds, which were assigned to 33 species across 25 genera. A semi-quantitative analysis of the yeasts and molds in the raw milk from four farms was performed over a 5-month period. The composition and diversity of the fungal microflora were totally different for each farm, each of which had a unique species profile. To determine whether adventitious yeast strains from the milk could develop in raw milk cheese, a multilocus-sequence-typing (MLST) analysis was performed on 13 Issatchenkia orientalis (syn. Pichia kudriavzevii, anamorph: Candida krusei) isolates. The same MLST genotypes were identified for strains independently isolated from raw milk and raw milk cheese from a farm processing its own milk. This study contributes to the understanding of the natural fungal microflora of raw milk and suggests that non-starter yeasts and molds can transfer from raw milk to raw milk cheese and may influence cheese ripening. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13594-011-0051-4) contains supplementary material, which is available to authorized users.