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PubMed2005

Isolation and characterization of Shiga toxin-producing Escherichia coli strains from raw milk cheeses in France.

AIMS: To evaluate Shiga toxin-producing Eschericha coli (STEC) prevalence in 1039 French raw milk cheeses including soft, hard, unripened and blue mould cheeses, and to characterize the STEC strains isolated (virulence genes and serotypes). METHODS AND RESULTS: STEC strains were recovered from cheese samples by colony hybridization. These strains were then serotyped and genetically characterized. These strains (32 STEC) were then recovered from 18 of 136 stx-positive samples: 19 strains had stx2 variant genes stx(2vh-a) (n = 2), stx(2NV206) (n = 2), stx(2EDL933) (n = 4) and stx2d (n = 11). Thirty strains had the stx1 gene and one strain, the eae gene. Combinations of stx2 and stx1 genes were present in 17 (81%) of the STEC strains. Nineteen strains belonged to the O6 serogroup and the other strains belonged to the O174, O175, O176, O109, O76, O162 and O22 serogroups in decreasing frequency. CONCLUSIONS: No conclusion can be drawn at the moment concerning the potential risk to consumers because the O6:H1 serotype has already been found associated with the haemolytic uremic syndrome and almost no isolate had the eae gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The large number of STEC strains recovered from the cheese samples evaluated in this study emphasizes the health risks associated with raw milk cheeses. This further emphasizes the immediate need to identify and implement effective pre- and postharvest control methods that decrease STEC carriage by dairy cattle and to eliminate contamination of their cheeses during processing.

Animals↗
PubMed2006

Biodiversity of bacterial ecosystems in traditional Egyptian Domiati cheese.

Bacterial biodiversity occurring in traditional Egyptian soft Domiati cheese was studied by PCR-temporal temperature gel electrophoresis (TTGE) and PCR-denaturing gradient gel electrophoresis (DGGE). Bands were identified using a reference species database (J.-C. Ogier et al., Appl. Environ. Microbiol. 70:5628-5643, 2004); de novo bands having nonidentified migration patterns were identified by DNA sequencing. Results reveal a novel bacterial profile and extensive bacterial biodiversity in Domiati cheeses, as reflected by the numerous bands present in TTGE and DGGE patterns. The dominant lactic acid bacteria (LAB) identified were as follows: Leuconostoc mesenteroides, Lactococcus garvieae, Aerococcus viridans, Lactobacillus versmoldensis, Pediococcus inopinatus, and Lactococcus lactis. Frequent non-LAB species included numerous coagulase-negative staphylococci, Vibrio spp., Kocuria rhizophila, Kocuria kristinae, Kocuria halotolerans, Arthrobacter spp./Brachybacterium tyrofermentans. This is the first time that the majority of these species has been identified in Domiati cheese. Nearly all the dominant and frequent bacterial species are salt tolerant, and several correspond to known marine bacteria. As Domiati cheese contains 5.4 to 9.5% NaCl, we suggest that these bacteria are likely to have an important role in the ripening process. This first systematic study of the microbial composition of Domiati cheeses reveals great biodiversity and evokes a role for marine bacteria in determining cheese type.

Biodiversity↗
PubMed1976

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↗
PubMed1978

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↗
PubMed2000

Effect of three factors in cheese production (pH, salt, and heat) on Mycobacterium avium subsp. paratuberculosis viability.

Low pH and salt are two factors contributing to the inactivation of bacterial pathogens during a 60-day curing period for cheese. The kinetics of inactivation for Mycobacterium avium subsp. paratuberculosis strains ATCC 19698 and Dominic were measured at 20 degrees C under different pH and NaCl conditions commonly used in processing cheese. The corresponding D values (decimal reduction times; the time required to kill 1 log(10) concentration of bacteria) were measured. Also measured were the D values for heat-treated and nonheated M. avium subsp. paratuberculosis in 50 mM acetate buffer (pH 5.0, 2% [wt/vol] NaCl) and a soft white Hispanic-style cheese (pH 6.0, 2% [wt/vol] NaCl). Samples were removed at various intervals until no viable cells were detected using the radiometric culture method (BACTEC) for enumeration of M. avium subsp. paratuberculosis. NaCl had little or no effect on the inactivation of M. avium subsp. paratuberculosis, and increasing NaCl concentrations were not associated with decreasing D values (faster killing) in the acetate buffer. Lower pHs, however, were significantly correlated with decreasing D values of M. avium subsp. paratuberculosis in the acetate buffer. The D values for heat-treated M. avium subsp. paratuberculosis ATCC 19698 in the cheese were higher than those predicted by studies done in acetate buffer. The heat-treated M. avium subsp. paratuberculosis strains had lower D values than the nonheated cells (faster killing) both in the acetate buffer (pH 5, 2% [wt/vol] NaCl) and in the soft white cheese. The D value for heat-treated M. avium subsp. paratuberculosis ATCC 19698 in the cheese (36.5 days) suggests that heat treatment of raw milk coupled with a 60-day curing period will inactivate about 10(3) cells of M. avium subsp. paratuberculosis per ml.

Cheese↗
PubMed2003

Bacterial community dynamics during production of registered designation of origin Salers cheese as evaluated by 16S rRNA gene single-strand conformation polymorphism analysis.

Microbial dynamics during processing and ripening of traditional cheeses such as registered designation of origin Salers cheese, an artisanal cheese produced in France, play an important role in the elaboration of sensory qualities. The aim of the present study was to obtain a picture of the dynamics of the microbial ecosystem of RDO Salers cheese by using culture-independent methods. This included DNA extraction, PCR, and single-strand conformation polymorphism (SSCP) analysis. Bacterial and high-GC% gram-positive bacterial primers were used to amplify V2 or V3 regions of the 16S rRNA gene. SSCP patterns revealed changes during the manufacturing of the cheese. Patterns of the ecosystems of cheeses that were provided by three farmers were also quite different. Cloning and sequencing of the 16S rRNA gene revealed sequences related to lactic acid bacteria (Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Lactobacillus plantarum, and Lactobacillus pentosus), which were predominant during manufacturing and ripening. Bacteria belonging to the high-GC% gram-positive group (essentially corynebacteria) were found by using specific primers. The present molecular approach can effectively describe the ecosystem of artisanal dairy products.

Cheese↗
PubMed2004

Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in cheddar cheese.

Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese. Previous work has shown that alpha-keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds. However, dairy lactobacilli can convert unstable alpha-keto acids to more-stable alpha-hydroxy acids via the action of alpha-keto acid dehydrogenases such as d-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the Lactobacillus casei d-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L. casei ATCC 334. Enzyme assays confirmed that alpha-keto acid dehydrogenase activity was significantly higher in pTRKH2:dhic transformants than in wild-type cells. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter plus L. casei ATCC 334, and starter plus L. casei ATCC 334 transformed with pTRKH2:dhic. After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis. The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese. Results further indicated that enhanced expression of d-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of alpha-keto acids, but sensory work indicated that this effect retarded cheese flavor development.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2002

Milk calcium taken with cheese increases bone mineral density and bone strength in growing rats.

We investigated the calcium bioavailability of milk calcium, taken with or without cheese. Twenty-four 6-week-old male rats for a meal-feeding experiment were trained to consume an AIN-76 diet within 2 h (2 times per day) for 2 weeks. The rats were then divided into three experimental groups, each fed 2 types of experimental diets: Control group, Cheese group, and Ca-Cheese group. The rats were each alternately given 2 types of experimental diets at 2-h meal-feeding for 31 days. The breaking force and energy of the femur in the Ca-Cheese group were significantly higher than in the control group. The bone mineral density (BMD) of the lumbar spine and the femur in the Ca-Cheese group was also significantly higher than in the other two groups. These results indicate that milk calcium taken with cheese increases bone strength and BMD efficiently, results that may be useful for the prevention of osteoporosis.

Animals↗
PubMedDate not supplied

Microencapsulated enzyme systems for the acceleration of cheese ripening.

Enzymes and substrates encapsulated in either milkfat-coated microcapsules or liposomes have been investigated for potential use as agents to accelerate cheese ripening. Milkfat-coated microcapsules have been used to efficiently encapsulate cell-free extracts, viable cells, purified enzymes, and spores. Encapsulation efficiency was dependent on the conditions used during capsule production. Addition of these microcapsules to cheese has resulted in increased levels of flavour compounds such as diacetyl, acetoin, methanethiol, and methyl ketones, compared to levels in control cheeses. Limitations due to cofactors have been overcome by co-encapsulating enzymes which recycle needed cofactors. Liposomes have been used to carry cell-free extracts and enzymes into cheese. Trials with different types of liposomes revealed that enzymes could be entrapped more efficiently and liposomes retained in the cheese curd better when multilammelar vesicles rather than small unilamellar vesicles or reverse phase evaporation vesicles were used. The stability of liposome preparations was found to be adversely affected by increased pH, temperature, and sodium chloride concentrations, as well as by negative surface charge. Encapsulation efficiency was found to increase by using a dehydration-rehydration procedure for liposome preparation. Temperature sensitive liposomes were investigates as a means of obtaining controlled release of the enzymes into the cheese.

Bacteria↗
PubMed2000

Whiteness change during heating and cooling of Mozzarella cheese.

Whiteness (L-value) changes in low-fat and low-moisture, part-skim Mozzarella cheeses during heating (7 to 60 degrees C) and cooling (60 to 7 degrees C) were evaluated. In low-fat Mozzarella, a large increase in whiteness was observed during heating, and a decrease in whiteness was observed during cooling. In low-moisture, part-skim Mozzarella, the whiteness changes during heating and cooling were smaller. Serum phase was removed from low-fat and low-moisture, part-skim Mozzarella cheeses. White protein gels were formed when the isolated serum phase from either low-fat or low-moisture, part-skim Mozzarella was heated. The white gel that formed was composed predominantly of casein and casein proteolysis products. The gel might have been produced by heat-induced, hydrophobic protein-protein interactions, and it tended to dissociate when cooled. Formation of a gel during heating increased light scattering, which increased the L-value. The gel dissociated during cooling and no longer scattered light, which decreased the L-value. We hypothesized that a gel, which was reversible, formed in the serum phase of cheese during heating and might have been responsible for the observed changes in the L-value of low-fat Mozzarella cheese during heating and cooling. The additional fat in low-moisture, part-skim Mozzarella compared with low-fat Mozzarella masked some of the color changes in the serum phase of low-moisture, part-skim Mozzarella. A model was developed to describe the contributions of the casein matrix plus serum phase of Mozzarella cheese and the contribution of fat to the changes in whiteness of Mozzarella cheese during heating and cooling.

Cheese↗
PubMed2000

Influence of salting procedure on the composition of Muenster-type cheese.

Muenster-type cheeses were salted with a traditional saturated brine solution or by direct addition of salt to the curd. Cheeses were evaluated at 0, 30, 60, 90, 120, and 180 d of age for numbers and type of microflora, casein hydrolysis, and amounts of free fatty acids. No significant differences were found in the populations of starter, lactobacilli, or yeast for the brine- and direct-salted cheeses. The amounts of free fatty acids liberated were similar for both cheeses. The hydrolysis of alpha s1-casein was complete at 90 d of age, whereas only 40% of the beta-casein was hydrolyzed at 180 d of age. The inner layer of the brine-salted cheeses had the highest number of starter microorganisms, followed by the middle and outer layers, respectively. The salt concentrations were similar in the three layers after 4 mo of age. Results of this study showed that comparable Muenster-type cheese could be produced with either of the salting procedures. With direct salt addition to curd, a 59% reduction was observed in salt emissions from the Muenster manufacturing process.

Animals↗
PubMed2000

Viability of probiotic (Bifidobacterium, Lactobacillus acidophilus and Lactobacillus casei) and nonprobiotic microflora in Argentinian Fresco cheese.

We evaluated the suitability of Argentinian Fresco cheese as a food carrier of probiotic cultures. We used cultures of Bifidobacterium bifidum (two strains), Bifidobacterium longum (two strains), Bifidobacterium sp. (one strain), Lactobacillus acidophilus (two strains), and Lactobacillus casei (two strains) in different combinations, as probiotic adjuncts. Probiotic, lactic starter (Lactococcus lactis and Streptococcus thermophilus), and contaminant (coliforms, yeasts, and molds) organisms were counted at 0, 30, and 60 d of refrigerated storage. Furthermore, the acid resistance of probiotic and starter bacteria was determined from hydrochloric solutions (pH 2 and 3) of Fresco cheese. The results showed that nine different combinations of bifidobacteria and L. acidophilus had a satisfactory viability (count decreases in 60 d <1 log order) in the cheese. Both combinations of bifidobacteria and L. casei cultures assayed also showed a satisfactory survival (counts decreased <1 log order for bifidobacteria but no decrease was detected for L. casei). On the other hand, the three combinations of bifidobacteria, L. acidophilus, and L. casei tested adapted well to the Fresco cheese environment. When a cheese homogenate at pH 3 was used to partially simulate the acidic conditions in the stomach, the probiotic cultures had an excellent ability to remain viable up to 3 h. At pH 2, the cell viability was more affected; B. bifidum was the most resistant organism. This study showed that the Argentinian Fresco cheese could be used as an adequate carrier of probiotic bacteria.

Bifidobacterium↗
PubMed2001

Tyrosine and phenylalanine catabolism by Lactobacillus cheese flavor adjuncts.

Bacterial metabolism of Tyr and Phe has been associated with the formation of aromatic compounds that impart barny-utensil and floral off-flavors in cheese. In an effort to identify possible mechanisms for the origin of these compounds in Cheddar cheese, we investigated Tyr and Phe catabolism by Lactobacillus casei and Lactobacillus helveticus cheese flavor adjuncts under simulated Cheddar cheese-ripening (pH 5.2, 4% NaCl, 15 degrees C, no sugar) conditions. Enzyme assays of cell-free extracts indicated that L. casei strains catabolize Tyr and Phe by successive, constitutively expressed transamination and dehydrogenation reactions. Similar results were obtained with L. helveticus strains, except that the dehydrogenase enzymes were induced during incubation under cheese-ripening conditions. Micellar electrokinetic capillary chromatography of supernatants from L. casei and L. helveticus strains incubated under simulated cheese-ripening conditions confirmed that Tyr and Phe transamination and dehydrogenation pathways were active in both species and also showed these reactions were reversible. Major products of Tyr catabolism were phydroxy phenyl lactic acid and p-hydroxy phenyl acetic acid, while Phe degradation gave rise to phenyl lactic acid, phenyl acetic acid, and benzoic acid. However, some of these products were likely formed by nonenzymatic processes, since spontaneous chemical degradation of the Tyr intermediate p-hydroxy phenyl pyruvic acid produced p-hydroxy phenyl acetic acid, p-hydroxy phenyl propionic acid, and p-hydroxy benzaldehyde, while chemical degradation of the Phe intermediate phenyl pyruvic acid gave rise to phenyl acetic acid, benzoic acid, phenethanol, phenyl propionic acid, and benzaldehyde.

Cheese↗
PubMed2001

Tryptophan catabolism in Brevibacterium linens as a potential cheese flavor adjunct.

Attempts to develop a desirable reduced fat Cheddar cheese are impeded by a propensity for flavor defects such as meaty-brothy, putrid, fecal, and unclean off-flavors in these products. Recent studies suggest aromatic amino acid catabolism of starter, adjunct, and nonstarter lactic acid bacteria significantly impact off-flavor development. The objective of this study was to delineate pathways for catabolism of tryptophan (Trp) in Brevibacterium linens, a cheese flavor adjunct, and to determine the potential for this organism to contribute to this defect. Growth and production of aromatic compounds from Trp by B. linens BL2 were compared in two incubated conditions (laboratory and a cheese-like environment). A chemically defined medium was used to determine the cellular enzymes and metabolites involved in Trp catabolism. Trp was converted to kynurenine, anthranilic acid, and three unknown compounds in laboratory conditions. The accumulation of other unknown compounds in the culture supernatant in laboratory conditions indicated that B. linens BL2 degraded Trp by various routes. Up to 65% of Trp was converted to anthranilic acid via the anthranilic acid pathway. To assess this potential before cheese making, the cells were incubated in cheese-like conditions (15 degrees C, pH 5.2, no sugar source, 4% NaCl). Trp was not utilized by BL2 incubated in this condition. Enzyme studies using cell-free extracts of cells incubated in laboratory conditions and assayed at optimal and nonoptimal enzyme assay conditions revealed Trp transaminase (EC 2.6.1.27) was active before enzymes of the anthranilic acid pathway were detected. The products of Trp transaminase activity were not, however, found in the culture supernatant, indicating these intermediates were not exported nor accumulated by the cells. Enzymes assayed in nonoptimal conditions had considerably lower enzyme activities than found in laboratory incubation conditions. Based on these results, we hypothesize that these enzymes are not likely to be involved in the formation of compounds associated with off-flavors in Cheddar cheese.

Brevibacterium↗
PubMed2002

Microbial succession of Debaryomyces hansenii strains during the production of Danish surfaced-ripened cheeses.

Surface-ripened cheeses of the Danbo type were analyzed for the presence of yeasts with special emphasis on Debaryomyces hansenii. Samples were taken from pasteurized milk, brine, and inoculation slurries and from cheese surfaces during ripening at a Danish dairy. D. hansenii was found to be the dominant yeast species throughout the ripening period, whereas other yeast species such as Trichosporon spp., Rhodotorula spp., and Candida spp. were found in minor concentrations during early stages of cheese ripening. Mitochondrial DNA RFLP was used to show that several strains of D. hansenii were present from the onset of ripening. Thereafter, a microbial succession among the strains took place during the ripening. After 3 d of ripening, only one strain was found. This particular strain was found to be dominant in 16 additional batches of surface-ripened cheeses. We investigated the cause of the observed microbial succession by determining the variation in strains with regard to their ability to grow on lactate and at different pH and NaCl concentrations. The strains were shown to vary in their ability to grow on lactate. In a full factorial design at three levels with factor levels close to the actual levels on the cheese surface, differences in pH and NaCl tolerances were observed. The dominant strain was found to be better adapted than other strains to the environmental conditions existing in surface-ripened cheeses during production [e.g., lactate as the main carbon source, pH 5.5 to 6.0 and NaCl concentrations of 7 to 10% (wt/vol)].

Adaptation, Physiological↗
PubMed2002

Holstein's milk detection in cheeses inferred from melanocortin receptor 1 (MC1R) gene polymorphism.

For some French Registered Designation of Origin (RDO) cheeses Prim'Holstein's milk is not allowed for cheese making (e.g., Reblochon, Abondance, and Beaufort cheeses). To find molecular markers for Prim'Holstein's milk detection in RDO cheese, four genes affecting coat color in cattle (c-kit, MGH, TYRP1, and MC1R) have been sequenced for three mountain breeds and the Prim'Holstein breed. Only the MC1R gene (E-locus) has shown variation between the four breeds. Among the 25 French and Italian breeds sequenced for the MC1R gene, only the Vosgienne breed has presented the same allele as the black Prim'Holstein breed (E(D)). A quick and easy DNA-based method to detect Holstein's milk in RDO cheese is proposed based on ED allele detection. A DNA extraction from cheese, a preamplification of the gene and a competitive oligonuleotide priming PCR on MC1R mutations were performed. Using an automated sequencer, differences in fluorescence and fragment size reveal the allele type. This simple approach provides good reproducibility and is shown to be relatively sensitive, with a detection limit of about 1% of Holstein's milk in milk curd.

Alleles↗
PubMed2003

The effect of homogenization and milk fat fractions on the functionality of Mozzarella cheese.

Mozzarella cheese was manufactured from milk containing either a low (olein) or a high (stearin) melting point fraction of milk fat or anhydrous milk fat. The fat was dispersed into skim milk by homogenization at 2.6 MPa before being manufactured into cheese. The melting point of the milk fat did not affect the size or shape of the fat globules, nor was there any effect of homogenization on the polymorphic state of the milk fat. There were no changes in milk fat globule size and shape concomitant with the amount of free oil formed. The polymorphic state of the milk fat did affect the amount of free oil formed and the apparent viscosity of the cheese. The lower melting point fraction yielded a larger amount of free oil. The higher melting point fraction yielded a higher viscosity of melted cheese at 60 degrees C. Mozzarella cheese was also manufactured from homogenized milk, nonhomogenized milk, and a 1:1 ratio of the two, without altering the milk fat composition. Increasing the proportion of homogenized milk yielded a lower free oil content and higher viscosity of the cheese.

Animals↗
PubMed2003

Influence of presalting and brine concentration on salt uptake by Ragusano cheese.

The impact of presalting and nonsaturated brine on salt uptake by Ragusano cheese was determined. The study included four treatments: 1) the traditional method using no presalting and saturated brine, 2) presalting and saturated brine, 3) no presalting and 18% brine for 8 d followed by 16 d in saturated brine, and 4) presalting and 18% brine for 8 d followed by 16 d in saturated brine. Cheese blocks were weighed and sampled before brine salting (time 0) and after 1, 4, 8, 16, and 24 d of brining for each treatment. Presalting delivered 60% of the normal level of salt in the center of the block prior to brine salting without decreasing the rate of uptake of salt from either saturated or 18% brine. Use of 18% salt brine for the first 8 d of 24 d of brine salting increased the rate of salt uptake, compared with 24 d in saturated brine. The increased rate of salt uptake with 18% brine compared with saturated brine was related to the impact of salt brine on the moisture content and porosity of the cheese near the surface of the block. Brine with higher salt content causes a rapid loss of moisture from cheese near the surface of the block. Moisture loss causes shrinkage of the cheese structure and decreases porosity, which impedes moisture movement out and salt movement into the block. The use of 18% salt brine for the first 8 d delayed the moisture loss and cheese shrinkage at the exterior of the block and allowed more salt penetration.

Cheese↗