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PubMed1994

Propionibacterium species diversity in Leerdammer cheese.

Thirteen 'classical' Propionibacterium strains, isolated from Leerdammer cheese samples, using three different media were characterized phenotypically. The phenotypic data of 74 tests, conducted on 27 propionibacteria, including four type, 10 reference strains and the 13 cheese isolates were analysed by numerical taxonomical techniques, using the simple matching coefficient and single linkage cluster analysis. All the strains were grouped in four major clusters, with a final linkage at the 81% S-level. The clusters were equated with the 'classical' P. acidipropionici, P. freudenreichii, P. jensenii and P. thoenii species. The species were identified by relating them to specific type strains and by comparison of phenotypic characteristics. Differential characteristics of each cluster were determined. Strains of P. acidipropionici, P. freudenreichii and P. jensenii, but no P. thoenii strains were isolated from the Leerdammer cheese samples. No 'cutaneous' propionibacteria were isolated. The largest cluster, representing 46% of the cheese isolates was equated with P. jensenii. Various red/brown pigmented strains, which could be identified as the old 'P. rubrum' species were isolated from the cheese. These strains were, however, phenotypically identified as P. jensenii and also grouped in the P. jensenii cluster.

Cheese↗
PubMed1996

Main microbial flora present as natural starters in Cebreiro raw cow's-milk cheese (northwest Spain).

Thirty samples of Cebreiro, a fresh or short-time-ripened raw cow's-milk cheese produced in Northwest Spain, were analyzed for the presence of aerobic mesophilic bacteria, (AMC) lactic acid bacteria (LAB), enterococci and Micrococcaceae. Mean AMC and LAB counts exceeding 10(9)/g were higher than those reported for other fresh or short-time-ripened cheeses, although Micrococcaceae occurred in lower numbers (< 10(5)/g) than reported for other raw-milk cheeses. Out of a total of 126 LAB representation isolates, 59 were identified as enterococci (38 as Enterococcus faecalis) 30 as lactococci (24 as Lactococcus lactis subsp. lactis), 25 as leuconostocs (14 as Leuconostoc mesenteroides) and 6 were identified as mesophylic lactobacilli. The enterocci in general were more proteolytic and produced more diacetyl/acetoin than the other LAB groups lactic acid bacteria isolated. It seems that a starter for making Cebreiro cheese should contain these microorganisms so as to reproduce the typical characteristics of traditional raw milk Cebreiro cheeses.

Bacteria↗
PubMed2006

Assessment of polychlorinated biphenyls (PCBs) in cheese from Rio Grande do Sul, Brazil.

Polychlorinated biphenyls (PCBs) are among the most toxic environmental pollutants. Food, especially animal based, has been described as the major source for human exposure to PCBs. We investigated PCB levels (congeners 10, 28, 52, 153, 138, and 180) in cheese from 14 cities of Rio Grande do Sul, Brazil. The fat was extracted using sodium sulfate and petroleum ether, and PCBs purification was conducted using florisil and hexane. The identification and quantification were made in a gas chromatograph with microelectron capture detector (GC-microECD). PCB congeners were found (lower bound data as ngg(-1) fat) in the following rank: 52 (15.75)>180 (4.91)>28 (3.43)>153 (3.00)>10 (2.38)>138 (1.37). Summation operatorPCB contamination ranged from 0 to 78.32 ngg(-1) fat. Differences in PCB levels between industrialized (33.32 ngg(-1) fat) and homemade (26.58 ngg(-1) fat) cheese and between two different regions (36.19 vs. 26.95 ngg(-1) fat) were not statistically significant. For university students, the estimated daily intake (EDI) of PCBs from cheese ranged from 0 to 3.13 pgkg(-1) b.w. EDI was significantly higher for industrialized than homemade cheese, and for cheese from Mountain/Porto Alegre region than from Santa Maria/South/Western region. No sample exceeded maximum level of PCBs allowed in dairy products in the European Community.

Brazil↗
PubMed2004

Identification and formation of angiotensin-converting enzyme-inhibitory peptides in Manchego cheese by high-performance liquid chromatography-tandem mass spectrometry.

A total of 75 peptides included in the fraction with molecular mass below 3000 from an 8-month-old Manchego cheese could be identified using HPLC coupled on line to an ion trap mass spectrometer. Some previously described peptides with antihypertensive and/or angiotensin-converting enzyme (ACE)-inhibitory activity were detected. The formation of five active sequences was followed during cheese ripening in four different batches of Manchego cheese. Two experimental batches of Manchego cheese elaborated with selected bacterial strains with the aim of improve the organoleptic characteristics demonstrated also a good performance in the formation of peptides with ACE-inhibitory activity during cheese ripening.

Amino Acid Sequence↗
PubMed2005

Volatile sulfur compounds in Cheddar cheese determined by headspace solid-phase microextraction and gas chromatograph-pulsed flame photometric detection.

The aim of this study was to develop a methodology for the analysis of volatile sulfur compounds (VSCs) in Cheddar cheese. Solid-phase microextraction (SPME) was employed to extract VSCs from the cheese matrix using a CAR-PDMS fiber. This extraction method was combined with gas chromatography-pulsed flame photometric detection (GC-PFPD) to achieve high sensitivity for sulfur compounds. The impact of extraction parameters, including time, temperature and sample size, was evaluated to determine the best conditions to analyze sulfur compounds in Cheddar cheese. Hydrogen sulfide, methanethiol, and dimethyl sulfide were found to constitute the majority of the overall sulfur profile while dimethyl disulfide and dimethyl trisulfide were present in lesser amounts. Artifact formation of volatile sulfur compounds was found to be minimal. Two commercial cheese samples were analyzed and differences in sulfur content were observed. Overall, SPME-GC-PFPD was found to be a highly sensitive technique for the analysis of sulfur compounds in Cheddar cheese.

Artifacts↗
PubMed2005

High-performance liquid chromatography with post-column derivatisation and fluorescence detection for sensitive determination of aflatoxin M1 in milk and cheese.

A new HPLC method with fluorescence detection using pyridinium hydrobromide perbromide as a post-column derivatising agent has been developed to determine aflatoxin M1 in milk and cheese. The detection limits were 1 ng/kg for milk and 5 ng/kg for cheese. The calibration curve was linear from 0.001 to 0.1 ng injected. The method includes a preliminary C18-SPE clean-up and the average recoveries of Aflatoxin M1 from milk and cheese, spiked at levels of 25-75 ng/kg and 100-300 ng/kg, respectively, were 90 and 76%; the precision (RSDr) ranged from 1.7 to 2.6% for milk and from 3.5 to 6.5% for cheese. The method is rapid, easily automatable and therefore useful for accurate and precise screening of aflatoxin M1 in milk and cheese.

Aflatoxin M1↗
PubMed2005

High-performance liquid chromatography of governing liquid to detect illegal bovine milk's addition in water buffalo Mozzarella: comparison with results from raw milk and cheese matrix.

A method to detect fraudulent addition of bovine milk in water buffalo Mozzarella cheese by gradient high-performance liquid chromatography (RP-HPLC), relying on the measurement of quantity ratios within beta-lactoglobulin protein family, is described. Analyses were performed on raw milk, cheese matrix and cheese governing liquid using a C4 column and UV detection. This work demonstrated that bovine milk addition during cheesemaking can be detected in governing liquid of Mozzarella down to the EU law limit of 1% as well as in raw milk and cheese matrix. A significant lowering of peaks' areas and heights was observed in cheese matrix and governing liquid samples in comparison with the corresponding milk ones, possibly due to proteins' degradation during the cheesemaking process. The results show that, unlike previous works reported, the use of a matrix-specific calibration curve is essential in order to achieve a proper quantitation of beta-lactoglobulin proteins, thus allowing a reliable estimation of bovine milk addition.

Animals↗
PubMed2005

Quantitative polymerase chain reaction used for the rapid detection of Carnobacterium species from French soft cheeses.

An identification method by PCR, specific to the Carnobacterium genus, was optimised by testing it on 28 bacterial strains. Primers from the literature were tested to differentiate Carnobacterium strains present among various bacterial species. The DNA of Carnobacterium species (C. alterfunditum, C. divergens, C. funditum, C. gallinarum, C. inhibens, C. maltaromaticum, C. mobile, C. viridans), specifically amplified by the Cb1-Cb2R primer couple at a hybridization temperature of 69 degrees C, gave only one band of 340 bp. The validation of this technique was carried out on a French soft cheese made with pasteurised milk inoculated with C. maltaromaticum LMA 28. Using classical PCR, detection was not possible for decimal dilutions of the cheese above 1 g L(-1). With Sybr Green I real time PCR, the specificity of the reaction was confirmed by the T(m) value. The standard curve constructed using the main threshold cycle and various concentrations of C. maltaromaticum LMA 28 (ranging from 10(0) to 10(8) cfu mL(-1)) showed good linearity and a sensitivity limit of > or = 10(4) cfu g(-1) of cheese. This technique was applied on commercially available cheeses made from raw cow's milk. The Sybr Green I real time PCR method constitutes an effective and easy-to-perform method to quantify Carnobacterium sp. in cheese.

Animals↗
PubMed2005

Growth and survival of Escherichia coli O157:H7 during the manufacture and ripening of raw goat milk lactic cheeses.

The behaviour of Escherichia coli O157:H7 was studied during the manufacture and ripening of raw goat milk lactic cheeses. Cheese was manufactured from raw milk in the laboratory and inoculated with E. coli O157:H7 to a final concentration of 10, 100 and 1000 cfu ml(-1). E. coli O157:H7 was counted by CT-SMAC (Mac Conkey Sorbitol Agar with cefixim and tellurite) and O157:H7 ID throughout the manufacturing and ripening processes. When the milk was inoculated with 10, 100 or 1000 cfu ml(-1), counts decreased to less than 1 log(10) g(-1) in curds just prior to moulding. However, viable E. coli O157:H7 were found in cheeses throughout processing, and even after 42 days of ripening. Results indicate that E. coli O157:H7 survives the lactic cheese manufacturing process. Thus, the presence of low numbers of E. coli O157:H7 in milk destined for the production of raw milk lactic cheeses can constitute a threat to the consumer.

Animals↗
PubMed2005

Comparative characterization of Listeria monocytogenes isolated from Portuguese farmhouse ewe's cheese and from humans.

In order to investigate the possible relationships between Listeria monocytogenes strains isolated from farmhouse ewe's cheese and clinical strains collected, in partially overlapping dates, from the same geographical area in Portugal, a total of 109 isolates from seven ewe's cheese manufactures (n=94) and from humans (n=15) were characterized by serotyping, RAPD, PFGE and allelic analysis of the virulent actA gene. Serotyping indicated the presence of four different serovars: 1/2a, 1/2b, 1/2c and 4b. The 15 clinical isolates were either serovar 4b (86.7%) or serovar 1/2b (13.3%). Among the 94 isolates from cheese and related environments the serovars prevalence was 1/2a (1.1%), 1/2b (17.0%), 1/2c (12.8%) and, unexpectedly, 4b (69.1%). Based on results obtained with PFGE typing of the strains, 25 genotypes were identified, 10 from farmhouses and 15 from human cases. Isolates from serovars 1/2a and 1/2c were assigned to single genotypes, respectively. Within serovars 1/2b and 4b three and 20 genotypes were established, respectively. RAPD typing of the isolates rendered 18 types indicating the lack of accuracy of the primers used in strain differentiation within serovar 4b. The actA gene typing of the strains showed a prevalence of actA gene type I (90.4%) compared with the rest of the strains that were all actA gene type II (9.6%). In spite of the fact that all the farmhouses were completely independent, the distribution of L. monocytogenes genotypes, intra and inter cheese manufactures, was relatively homogeneous, suggesting the existence of resident strains. In contrast, among human isolates there was a great genetic diversity. There was no common genotype between L. monocytogenes implicated in the cases of listeriosis and these cheese-related isolates, suggesting the absence of a causal relationship.

Animals↗
PubMed2006

Viability of commercial probiotic cultures (L. acidophilus, Bifidobacterium sp., L. casei, L. paracasei and L. rhamnosus) in cheddar cheese.

Six batches of cheddar cheese were manufactured containing different combinations of commercially available probiotic cultures from three suppliers. Duplicate cheeses contained the organisms of each supplier, a Bifidobacterium spp. (each supplier), a Lactobacillus acidophilus (2 suppliers), and either Lactobacillus casei, Lactobacillus paracasei, or Lactobacillus rhamnosus. Using selective media, the different strains were assessed for viability during cheddar cheese maturation over 32 weeks. The Bifidobacterium sp. remained at high numbers with the three strains being present in cheese at 4 x 10(7), 1.4 x 10(8), and 5 x 10(8) CFU/g after 32 weeks. Similarly the L. casei (2 x 10(7) CFU/g), L. paracasei (1.6 x 10(7) CFU/g), and L. rhamnosus (9 x 10(8) CFU/g) strains survived well; however, the L. acidophilus strains performed poorly with both decreasing in a similar manner to be present at 3.6 x 10(3) CFU/g and 4.9 x 10(3) CFU/g after 32 weeks. This study indicates that cheddar cheese is a good vehicle for a variety of commercial probiotics but survival of L. acidophilus strains will need to be improved.

Bifidobacterium↗
PubMed2005

Application of SSCP-PCR fingerprinting to profile the yeast community in raw milk Salers cheeses.

Bacteria and yeasts are important sensory factors of raw-milk cheeses as they contribute to the sensory richness and diversity of these products. The diversity and succession of yeast populations in three traditional Registered Designation of Origin (R.D.O.) Salers cheeses have been determined by using phenotypic diagnoses and Single-Strand Conformation Polymorphism (SSCP) analysis. Isolates were identified by phenotypic tests and the sequencing of the D1-D2 domains of the 26S rRNA gene. Ninety-two percent of the isolates were identified as the same species in both tests. Yeast-specific primers were designed to amplify the V4 region of the 18S rRNA gene for SSCP analysis. The yeast species most frequently encountered in the three cheeses were Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Candida zeylanoides and Debaryomyces hansenii. Detection of less common species, including Candida parapsilosis, Candida silvae, Candida intermedia, Candida rugosa, Saccharomyces unisporus, and Pichia guilliermondii was more efficient with the conventional method. SSCP analysis was accurate and could be used to rapidly assess the proportions and dynamics of the various species during cheese ripening. Each cheese was clearly distinguished by its own microbial community dynamics.

Animals↗
PubMed2003

Solid-phase microextraction of volatile compounds in "Terrincho" ewe cheese. Comparison of different fibers.

Solid-phase microextraction coupled to gas chromatography-mass spectrometry (GC-MS) was applied to study the volatile compounds in "Terrincho" ewe cheese. Six types of fibers were tested and the main extraction parameters were studied. Carboxen-polydimethylsiloxane fiber 75 microm (CAR-PDMS) achieved the most complete profile of ewe cheese volatile compounds. The optimised conditions used for characterization of "Terrincho" ewe cheese were: sample vial equilibration at 20 degrees C for 20 min, followed by CAR-PDMS fiber exposure to the headspace above the sample for 30 min and finally thermal desorption of the adsorbed substances, in the injector port for GC-MS analysis. This technique was a useful tool for the differentiation of 11 "Terrincho" ewe cheeses, all taken from the same cheesemaking season with 30 days of ripening but from three different farmhouses, according to their volatile fraction. Results obtained were statistically treated by categorical principal component analysis. Subsequently, 49.15% of the variation in data was due to the first dimension (k = 12.7) and the second dimension (k = 8.88) accounted for 34.2% of the total information. Volatile profiles among samples indicated cheese group separation according to farmhouse of production.

Animals↗
PubMed2003

Detection and quantification of bovine, ovine and caprine milk percentages in protected denomination of origin cheeses by reversed-phase high-performance liquid chromatography of beta-lactoglobulins.

A method for detecting and quantifying bovine, ovine and caprine milk mixtures in milk and cheeses by means of reversed-phase high-performance liquid chromatography (RP-HPLC) of beta-lactoglobulins is described. Gradient elution was carried out with a flow rate of 0.5 ml/min and a temperature of 45 degrees C, using a mixture of two solvents: solvent A (0.1% TFA in water) and solvent B (0.09% TFA in 80% aqueous acetonitrile, v/v). The effluent was monitored at 215 nm. Under the conditions used different chromatographic patterns were obtained for bovine, ovine and caprine whey proteins. Each milk type presented different retention times for beta-lactoglobulin peaks. Binary mixtures of bovine and ovine or bovine and caprine raw milks containing 1, 2, 5, 10, 20, 30, 50, 75 and 95% (v/v) of bovine milk, as well as ovine and caprine milk mixtures containing 1, 2, 5, 10, 20, 30, 50, 75 and 95% (v/v) of ovine milk were used for cheese making. Cheeses were prepared and ripened, according to traditional methods. Milk mixtures, fresh and ripened cheeses were analyzed. A linear relationship was established between log 10 of beta-lactoglobulin peaks ratio (calculated as peak area values ratio) and log 10 of the relative percentage of bovine or ovine milk. The ratio between beta-lactoglobulin peaks was not affected by the degree of ripening. Thus, enabling the quantification of milk type percentage, with a detection limit of 2%. This technique allowed quantification of milk species within the concentration range of 5-95%. The method was successfully applied for authenticity evaluation and quantitative determination of ovine and caprine milk percentages of commercial protected denomination of origin (PDO) cheeses.

Animals↗
PubMed2003

Development of a fluorescence in situ hybridization method for cheese using a 16S rRNA probe.

A 16S rRNA fluorescence in situ hybridization (FISH) method for cheese was developed to allow detection in situ of microorganisms within the dairy matrix. An embedding procedure using a plastic resin was applied to Stilton cheese, providing intact embedded cheese sections withstanding the hybridization reaction. The use of a fluorescein-labelled 16S rRNA Domain Bacteria probe allowed observation of large colonies of microbial cells homogeneously distributed in the cheese matrix. FISH experiments performed on cheese suspensions provided images of the different microbial morphotypes occurring. The technique has great potential to study the spatial distribution of microbial populations in situ in foods, especially where the matrix is too fragile to allow manipulation of cryosections.

Cheese↗
PubMed2001

Distribution of aflatoxin M1 in cheese obtained from milk artificially contaminated.

Small-scale manufacture of cheese using artificially AFM1 contaminated milk as raw material to study the distribution of such toxin both in whey and in cheese, was carried out. Whole milk with undetectable levels of AFM1 was used. The toxin was added in concentration that varied from 1.7 to 2.0 microg/l of milk. After the home-made production of cheese, the concentration of AFM1 was determined both in whey and in cheese, using the enzymatic immunoassay technique. The greatest proportion, 60%, was detected in whey while 40% AFM1 remained in cheese.

Aflatoxin M1↗
PubMed2001

A survey of the prevalence of Escherichia coli O157 in raw meats, raw cow's milk and raw-milk cheeses in south-east Scotland.

2429 samples of foodstuffs were examined for the presence of verocytotoxigenic Escherichia coli O157 (VTEC O157) by means of immunomagnetic separation (IMS) over a 2-year period commencing April 1997. Specimens comprised 1190 raw meats, 500 raw milks and 739 raw-milk cheeses. The meat and cheese samples were purchased from retail premises in south-east Scotland; raw milk samples were obtained directly from farms. In addition, total E. coli counts were performed on milk and cheese samples, and the pH of cheese specimens measured. The water activity (Aw) was also measured for a representative sample of each cheese type, and for all of the samples with high levels of E. coli. VTEC O157 was isolated from two samples of beef burger, both manufactured on the premises of the same butchers shop. Control studies with artificially inoculated foodstuffs demonstrated a sensitivity of detection of < 5 organisms 25 g(-1). These findings, which contrast with the results of similar studies elsewhere in the UK, suggest that other sources of infection may be important in explaining the high rates of infection with this organism in south-east Scotland.

Animals↗
PubMed2001

Inhibitory effects of eugenol and thymol on Penicillium citrinum strains in culture media and cheese.

In the present work we studied the antifungal effect of eugenol and thymol on the growth and production of citrinin from Penicillium citrinum (NRRL 2274 and NRRL 2269) in culture media and in different Spanish cheeses (Arzúa-Ulloa, Cebreiro and San Simón). The rate of growth was assessed by measuring colony diameters and the production of citrinin was measured using a rapid semi-quantitative fluorometric technique confirmed by RP-HPLC. A stronger inhibitory effect of eugenol than thymol was evident. 200 microg/ml of eugenol in solid culture medium increased the lag time of growth up to 9 days, and decreased the rate of colony growth. In liquid medium, a complete inhibition of fungal growth was observed. By contrast, thymol in the liquid culture medium only affected the growth rate. In Arzúa-Ulloa cheese, 200 microg/ml of eugenol fully inhibited fungal growth, while in Cebreiro cheese no effect was observed for this compound. Regarding the capacity to inhibit mycotoxin production 100 microg/ml eugenol delayed citrinin production until the sixth day, after which a limiting effect persisted. In Arzúa-Ulloa cheese, no citrinin was detected at a concentration of 150 microg/ml of eugenol, but citrinin was detected after 5 days in the case of thymol at the same concentration. In Cebreiro cheese, neither eugenol nor thymol prevented the production of citrinin at the concentrations applied.

Antifungal Agents↗