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[Antigenic analysis of cheese mites with view to a study of cheese-makers' illness (author's transl)].

"Somatic" and "metabolic" antigens prepared from A. farinae and T. casei mites in Auvergne cheese were used for experimental immunisation: the hyper-immune serums obtained permitted 11 to 21 precipitant systems to be distinguished. An enzymatic activity could be identified on certain arcs. Research into antigenic communities showed the existence of 1 to 3 arcs common to the mite antigens, but none with the antigens obtained from the Penicillium cyclopium and Mucor fuscus moulds accompanying the mites. The results obtained thus facilitate an epidemiological investigation among the cheesemakers.

Antigens

Genomic diversity and thermal niches of Aspergillus molds disrupting rind formation of surface-ripened cheeses.

Filamentous fungi play important roles in the development of surface-ripened cheese microbial communities and contribute to the aesthetics and flavors of these products. Much is known about the diversity and ecology of desirable cheese fungi, but our understanding of the natural history of cheese spoilage molds is limited. The goal of this work was to characterize the genomic diversity of Aspergillus species contaminating artisan cheeses and to identify how the abiotic environment of cheese (the substrate itself and temperature) may constrain the growth of Aspergillus. Comparative genomics identified two main species of Aspergillus, A. westerdijkiae and A. ostianus, as the spoilage molds across three different facilities in the Northeastern United States that experienced contamination events. Multiple genomic types of A. westerdijkiae were found across the different cheese production facilities, indicating that these contamination events are not caused by a single clonal strain. All A. westerdijkiae isolates produced ochratoxin A, but concentrations varied greatly across strains. RNA-sequencing of A. westerdijkiae on nutrient-rich lab media (malt extract agar) versus cheese curd agar identified a suite of pathways enriched in expression on cheese, including degradation of amino and fatty acids. Experiments measuring growth over a range of temperatures identified that spoilage Aspergillus species have a higher optimal growth temperature compared to desirable fungal species in cheese rinds and are outcompeted by Penicillium species at temperatures lower than 15°C. Global fungal metabarcoding databases suggest that A. westerdijkiae is not normally found in natural habitats of the Northeastern United States, and it may be introduced to this region.IMPORTANCEOver the past decade, disruptive contamination events of Aspergillus spoilage molds have occurred at cheese production facilities in Massachusetts, Connecticut, and Vermont in the United States, causing aesthetic, flavor, and potential safety issues. Our work highlights independent introductions of different strains of A. westerdijkiae into multiple cheese facilities and suggests that temperature could be used to control the abundance of Aspergillus spoilage molds. Based on our analysis of the global distribution of A. westerdijkiae, it is not invading cheese facilities from local fungal populations and may be a contaminant in materials used for cheese production.

Aspergillus

[Sampling of cheese for aflatoxins (author's transl)].

Due to the greatly differing incidence of molds on cheese and thus the extremely differing amounts of aflatoxin reliable sampling in cheeses is difficult, particularly if only one semi-hard of hard cheese is available. Experiments were undertaken now to sample--hopefully without losses and suitable for commercial application--in order to be able to ascertain the possible aflatoxin content of a cheese. Based on storage results with artificially contaminated Provolone and Tilsit cheeses as a pretest determination of aflatoxins in scrapings from a 100 cm2 surface are is recommended. At high levels (20-30 microgram/kg) the sampling of several borings at different locations on the cheese is necessary. The aflatoxin content on the surface is not constant, a fact which can be explained by biochemical reactions and by migration of aflatoxins into deeper layers. Washing of cheeses seems not to be of any significant influence on the aflatoxin-content.

Aflatoxins

[On the presence and the content of aflatoxin M in commercial cheese samples (author's transl)].

Between May 10 and August 9 1976, a total of 197 commercial cheese samples were tested on their aflatoxin M1 content; 136 samples (69%) were positive. The highest value was at 0.23 mug/kg, the average value of all positive samples was at 0.09 mug/kg. In more than half of the positive samples (54%), only traces of aflatoxin M1 could be detected, 21% contained up to 0.1 mug/kg and 14% more than 0.1 mug/kg. In soft cheese definitely less aflatoxin was found (57% positive samples) than in other cheese varieties (83--85% positive samples). Supplemental feeding of concentrates during spring season and pasture grazing of dairy cows resulted in more respectively less aflatoxin-positive cheese samples. This was especially true in soft cheese. The other cheeses with longer ripening periods showed the same effect, however, less pronounced with a clear lag phase. Inspite of many positive identity reactions on aflatoxin M1, a routine control of cheeses so far cannot be recommended because of the negative results on mass spectrometry.

Aflatoxins

High-fat and low-fat fermented milk and cheese intake, proteomic signatures, and risk of all-cause and cause-specific mortality.

PURPOSE: This study aimed to examine the associations between the intake of high- and low-fat fermented dairy (cheese and fermented milk), their proteomic profiles, and mortality risk. METHODS: This cohort study included 25,187 participants (mean age 57.7 years, 60.9% females). Fermented dairy intake was assessed by a modified diet history method. In a random subset of this cohort (n&#x2009;=&#x2009;4359), we constructed proteomic signatures for fermented dairy intake using 136 candidate plasma proteins. RESULTS: During 23.5 years of follow-up, 9742 participants died. High-fat cheese (>&#x2009;20% fat) intake was inversely associated with risk of all-cause mortality (HR for an increment of 20&#xa0;g/day, 0.97; 95% CI, 0.96-0.99, P&#x2009;<&#x2009;0.001) and cardiovascular disease mortality (HR, 0.96; 95% CI, 0.93-0.99, P&#x2009;=&#x2009;0.006). Low-fat cheese intake showed an inverse association with all-cause mortality (HR, 0.98; 95% CI, 0.96-1.00, P&#x2009;=&#x2009;0.047). Low-fat fermented milk intake was inversely associated with all-cause mortality (HR for an increment of 250&#xa0;g/day, 0.91; 95% CI, 0.85-0.97, P&#x2009;=&#x2009;0.006), while high-fat fermented milk (>&#x2009;2.5% fat) showed null association. A total of 42, 26, 0, and 39 proteins were identified for the signature of high-fat cheese, low-fat cheese, high-fat fermented milk, and low-fat fermented milk, respectively. Inverse associations with all-cause mortality were observed for all three signatures with identified proteins. The identified proteins were involved in biological pathways related to immune response and inflammation. CONCLUSION: Our study indicated that consuming high-fat cheese, low-fat cheese, and low-fat fermented milk was linked to survival benefits. Plasma proteins improve our understanding of the health effects of fermented dairy.

Humans

Spatial Metabolomics Reveals the Role of Penicillic Acid in Cheese Rind Microbiome Disruption by a Spoilage Fungus.

Microbial interactions in cheese rinds influence community structure, food safety, and product quality. But the chemical mechanisms that mediate microbial interactions in cheeses and other fermented foods are generally not known. Here, we investigate how the spoilage mold Aspergillus westerdijkiae chemically inhibits beneficial cheese-rind bacteria using a combination of omics technologies. In cheese rind community and co-culture experiments, A. westerdijkiae strongly inhibited most cheese rind community members. In co-culture with Staphylococcus equorum, A. westerdijkiae strongly affected bacterial gene expression, including upregulation of a putative bceAB gene cluster that is associated with resistance to antimicrobial compounds in other bacteria. Mass spectrometry imaging (MSI) revealed spatially localized production of secondary metabolites, including penicillic acid and ochratoxin B at the fungal-bacterial interface. Integration of LC-MS/MS and genome annotations confirmed the presence of additional bioactive metabolites, such as notoamides and circumdatins. Fungal metabolic responses varied by bacterial partner, suggesting species-specific chemical strategies. Notably, penicillic acid levels increased 2.5-fold during interaction with Brachybacterium, and experiments with purified penicillic acid showed inhibition of a range of cheese rind bacteria. These findings show that A. westerdijkiae deploys a context-dependent arsenal of mycotoxins and other metabolites, disrupting microbial community assembly in cheese rinds.

Aspergillus westerdijkiae

Survival of foot-and-mouth disease virus in cheese.

Persistence of foot-and-mouth disease virus during the manufacture of Cheddar, Mozzarella, Camembert cheese prepared from milk of cows experimentally infected with the virus was studied. Cheese samples were made on a laboratory scale with commercial lactic acid starter cultures and the microbial protease MARZYME as a coagulant. Milk was heated at different temperatures for different intervals before it was made into cheese. Food-and-mouth disease virus survived the acidic conditions of Cheddar and Camembert cheese processing but not that of Mozzarella. Foot-and-mouth disease virus survived processing but not curing for 30 days in Cheddar cheese preparaed from heated milk. However, the virus survived curing for 60 days but not for 120 days in cheese (pH 5) prepared from unheated milk. Foot-and-mouth disease virus survived in Camembert cheese (pH 5) for 21 days at 2 C but not for 35 days.

Animals

[Determination of aflatoxins in cheeses].

To investigate cheeses for the presence of aflatoxins we chose the very sensitive method of Tuinstra and Bronsgeest (1975) used for the determination of aflatoxin M1 in milk. The method was slightly modified and the presence of aflatoxins was determined in 54 samples of different cheeses. Aflatoxin M1 was found out in 24% of the investigated samples. Most of positive samples were found among the soft cheeses (53.8 3/4), then in processed cheeses (13.6%) and in hard cheeses (12.5%). Aflatoxin M1 was not found in the group of mouldy cheeses and Olomouc cake cheeses, which were investigated in a smaller range. Positive findings did not exceed concentrations of 10 ng per kg, i.e. they did not even reach the value of permissible concentration as proposed in the Czech Socialist Republic for foods (5 microgram per kg).

Aflatoxins

[Staphylococci in cheese made from raw milk. (author's transl)].

The presence of markedly varying numbers of coagulase-positive staphylococci per gram (less than 10(2) -- greater than 10(6)) of cheese made from raw milk is reported. In twenty cases, the strains isolated from the cheese were examined for their ability to produce toxins. Eight strains were found to produce toxin A, no toxin being isolated from cultures of the twelve other strains. In two cases, the studies were done because of food poisoning which had probably been caused by the cheese. The number of staphylococci was more than 10(6) in one case (cheese about four weeks old) and approximately 1.5 x 10(4) in another (matured cheese). Subsequent studies showed that rather large amount of enterotoxin A were present in the second cheese. The first cheese was not examined for the presence of toxin.

Cheese

Acid phosphatases activity in cheese and starters.

The acid phosphatase activity levels in a number of Greek cheeses and in Cheddar cheeses were found to be unaffected by storage for up to 18 months and 12 months respectively. In Cheddar cheese, starter organisms made an insignificant contribution to this activity. Studies of acid phosphatase prepared from Streptococcus cremoris-lactis NCDO 762 starter cultures showed that the enzyme was of high molecular weight and largely particle-bound. The pH of optimum activity was 5-2 and the enzyme was inhibited by F-minus,Al-3+, a number of heavy metals, oxidizing agents and sulphydryl-modifying reagents. Kinetic measurements at pH 5-2 gave a Km value for p-nitrophenyl phosphate of 1-2 mM. Orthophosphate, pyrophosphate and isoelectrically precipitated casein behaved as competitive inhibitors to the hydrolysis of p-nitrophenyl phosphate with Ki values of 1-2 mM, 1-0 mM, 1-0 MM and 1-1 mM respectively. In spite of this binding to the enzyme, casein provided a very poor substrate for the starter acid phosphatase. The properties of acid phosphatase present in Cheddar cheese made with Str. cremoris NCDO 924 starter were consistent with the enzyme being exclusively of milk origin and small differences between this and the acid phosphatase previously isolated from bovine milk were attributable to the binding of peptides produced during the cheese maturation to the enzyme molecules. It was concluded that in cheese, phosphatase action was due largely to the enzyme of milk origin, with that provided by the starter being of minor importance.

Acid Phosphatase

Characteristics of bacteria isolated by the anaerobic roll-tube method from cheeses and ground beef.

In this study the methods of Hungate were used to quantitate the anaerobic bacteria present in commercially available ground beef, cheddar cheese, and German hand cheese. Of 235 anaerobic roll-tube isolates from ground beef and German hand cheese, all were facultative anaerobes. Of 213 anaerobic roll-tube isolates from cheddar cheese, 91% were facultative anaerobes and 9% were obligate anaerobes. Using results of biochemical tests, 14 or the 17 obligately anaerobic isolates from cheddar cheese were Propionibacterium acnes, two were strains of Propionibacterium that could not be speciated, and one was tentatively identified as a strain of Streptococcus evolutus. Obligate anaerobes were estimated to be present in the cheddar cheese at a level of about 10(6)/g. The possible significance of these levels of P. acnes in nonsterile foods is discussed.

Anaerobiosis

Association of Yersinia enterocolitica with the manufacture of cheese and occurrence in pasteurized milk.

Raw milk in southern Ontario frequently contains Yersinia enterocolitica. The potential for transmission of this organism by cheese manufactured from unpasteurized milk was evaluated by examination of milk and cheese curd samples from cheese manufacturing plants and finished cheddar and Italian cheeses. The incidence of Y. enterocolitica was lower in cheese curd samples (9.2%) than in raw milk (18.2%). Most of the curd samples showed a positive phosphatase test, indicating production from raw milk. One curd sample yielded Y. enterocolitica after 4 weeks of storage at 4 degrees C but was negative after 8 weeks. All samples of cheddar and Italian cheeses, most of which showed a positive phosphatase test, were negative for Y. enterocolitica. One out of 265 samples (0.4%) of pasteurized fluid dairy products contained Y. enterocolitica.

Animals

Pimaricin and mycostatin for retarding cottage cheese spoilage.

Two antifungal agents, pimaricin and mycostatin, added to Cottage cheese through the wash water at concentrations of 20, 50, or 100 mug/ml of wash water or added through the cheese dressing at 1, 2, or 5 mug/g retarded the growth of Aspergillus niger and Saccharomyces cerevisiae and improved the shelf-life of the cheese. In general, cheese with highest concentration of antifungal agent and stored at lowest temperature had best keeping quality. Pimaricin was slightly more effective than mycostatin in inhibiting fungi; inhibition was greater if the antifungal agents were added to the cheese dressing and the cheese was stored at low temperature; and A. niger was more sensitive to the inhibitors than S. Cerevisiae.

Aspergillus niger

Instability of PR toxin in blue cheese.

PR toxin was unstable in solvent extracts of blue cheese and in strongly acidic solutions. However, it was appreciably stable over a 2.5 hr period in moderately acidic methanol-water extracts (pH 2--3) of blue cheese. Using this extraction mixture, it was determined that PR toxin was not stable in blue cheese itself. PR toxin reacted with model neutral and basic amino acids and the formation of PR imine from PR toxin in the presence of blue cheese was demonstrated. While PR imine added to blue cheese could be recovered on analysis after 5 min, over a 2--5 day period it too was unstable in the cheese.

Cheese

[Microbiological studies of brynza cheese].

Studies on the changes occurring in the microflora at the time of 45-day ripening and 4-month storage of cheese produced in pitchers (earthen jugs) were performed. It was established that at the beginning of the ripening period the total number of microorganisms as well as the number of lactic acid producing microorganisms increases, but after the 15th day of ripening until the end of storage their number diminishes. At the time of ripening and storage of cheese curdled in pitchers, coliform bacteria diminish progressively. In case cheese curd is used, these bacteria vanish as soon as ripening comes to an end, while in case lactic acid or butter curd is used they vanish during the 1st month of storage. In spontaneously curdled cheese coliform bacteria vanish during the third month of storage. At the time of cheese ripening in a pitcher the yeast quantity increases, while during storage it varies. At the time of ripening fungae get into the cheese produced in pitchers and their number increases along with the ripening process.

Animals

[The influence of different phosphates on the flow properties of processed cheeses (author's transl)].

Processed cheese was manufactured with different types of phosphates (P1 monophosphate to P4 tetrapolyphosphate) and by various techniques. The limiting viscosity numbers of sodium casein and the casein of processed cheese were determined and the axial ratio calculated. The axial ratio of processed cheese protein was a/b=20,0 and that of sodium casein 10,0 resp. We deduce that the apparent increase of the axial ratio is caused by the depolymerization of casein caused by emulsifying salts. The flow curves of processed cheese were analysed. There is good agreement with the power law of Ostwald tau=k.Dn. Therefore processed cheeses are characterized as pseudoplasts. The influence of melting salts on the flow properties can be described by a differing emulsifying effect (P1 less than or equal P4). Temperature, concentration and salt effects are discussed for the viewpoint of correlation between aggregation-desaggregation processes and dehydration and hydration of casein.

Caseins

[Effect of primaricin on moulds and their aflatoxin formation in cheese].

The prior condition for the application of pimaricin is its heat stability (up to 80 degrees C) and the low penetration (around 2.6 mm), so that it is available for the effect on the cheese surface for a long period. With 13 different aflatoxin forming moulds, the inhibition on the mycel development and thereby on the aflatoxin formation was tested; the different strains were inhibited to various degrees. The effect on cheese was not definite, because the native cheese surface remained free of mould for 8 weeks after pimaricin treatment, whereas cheese slices dipped into pimaricin solution were covered with moulds very soon. The aflatoxin formation itself is only inhibited, if the growth of the moulds is inhibited. Therefore the cheeses have to be treated very early before strong growth of the moulds has started. The aflatoxin formation, however only starts at a certain growth period of the moulds, but at a not completed inhibition, a reduced aflatoxin formation has to be taken into consideration.

Aflatoxins

Detection and growth of enteropathogenic Escherichia coli in soft ripened cheese.

The organism most frequently encountered during the 1971 outbreak of enteropathogenic Escherichia coli (EPEC) in soft ripened cheese was a strain that failed to ferment lactose broth within 48 h. Since existing methods for E. coli are dependent upon fermentation of this sugar, such strains can remain undetected, particularly when present in low numbers. Therefore a cultural testing procedure was developed to insure isolation of both lactose-positive and -negative strains. This method used GN broth, modified by substituting lactose and arabinose for glucose and D-mannitol, as an enrichment medium. MacConkey agar, used as a plating medium, was modified by substituting arabinose for half the lactose. The cultural procedure was used in conjunction with a fluorescent antibody method to screen cheese for the presence of presumptive enteropathogenic E. coli. Suspected isolates were subjected to further biochemical and serological testing and identified as members of specific serogroups. These methods were used for the analysis of over 2,000 wheels of cheese; over 10% of the samples tested were found to contain strains belonging to six different serogroups associated with diarrheal diseases. No attempt was made to confirm pathogenicity by in vivo tests. Enumeration of E. coli in cheese showed that numbers increased during storage. Cheese with less than 10 organisms/g initially increased to over 10-5 at room temperature and over 10-3 at 4 C within 10 days. With higher initial counts, levels up to 10-9 were found at 4 C. These studies showed that the high levels of E. coli encountered in these products cannot be used as a direct indicator of post-processing contamination.

Arabinose