Cheese Microbiology

The composition and development of microbial population during the manufacture and 3 ripening of two batches of a blue-veined cheese was examined by culturing and polymerase 4 chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) (PCR-DGGE). Nine 5 selective and/or differential media were used to track the cultivable populations of total and 6 indicator microbial groups. For PCR-DGGE, the V3 hyper variable region of the bacterial 7 16S rRNA gene and the eukaryotic D1 domain of 28S rDNA were amplified with universal 8 primers, specific for prokaryotes and eukaryotes, respectively. Similarities and differences 9 between the results obtained by the culturing and the molecular method were recorded for 10 some populations. Culturing analysis allows minority microbial groups (coliforms, 11 staphylococci) to be monitored, although in this work PCR-DGGE identified a population of 12 Streptococcus thermophilus that went undetected by culturing. These results show that the 13 characterization of the microbial populations interacting and evolving during the 14 cheesemaking process is improved by combining culturing and molecular methods.

A general graphic model for organizational information systems (IS) is proposed. The model includes generalized decision making and data manipulation functions to regulate generalized organizational processes through flows of information. The general IS model serves as a basic pattern to approach the design of any IS. In particular, alternative IS strudures or designs can be derived from the model. Structures include not only information that will be computerized but also the prescription of the decision making behavior of the information users. The existence of alternatives leads to a problem of evaluation for which a quantitative modeling approach is proposed.

Three yeast strains, 1AD8T, 3AD15 and 3AD23, belonging to a previously unknown yeast species were isolated from two independent batches of the Spanish blue-veined Cabrales cheese, a traditional cheese manufactured without the addition of starter and mould cultures. Physiological characterization revealed that the unknown yeast is not fermentative and does not assimilate lactose; rather it assimilates dl-lactic acid and ethanol, major end products of lactic acid bacteria metabolism in cheese. The novel yeast is anamorphic. Phylogenetic tree reconstruction based on nucleotide sequence comparison of the D1/D2 region of the 26S rRNA gene showed that Pichia terricola and Pichia fermentans are the closest relatives of the unknown species. The name Candida cabralensis sp. nov. is proposed, and the isolate 1AD8T (=CECT 13027T =CBS 11679T) is the type strain of this novel taxon.

The purpose of the present study was to elaborate methods for the detailed examination of proteolysis pathways in cheese (reviewed in Chapter 1) and to demonstrate their usefulness. Many techniques, including solvent fractionation, chromatographic separation and electrophoresis have been used previously and were revisited in this study. Gel electrophoresis can be a powerful technique and was examined in detail. The methods investigated were: 1) a slab gel system using the apparatus of the E-C Apparatus Corporation and a polyacrylamide gel in a Tris-EDTA-borate buffer at alkaline pH and containing urea; 2) a mini-slab gel system using the Bio-Rad mini-Protean II apparatus, a polyacrylamide stacking and resolving gel with a discontinuous (Tris-chloride/Tris-EDTA-borate) buffer system that contained urea; 3) a mini-slab gel system using the Bio-Rad mini-Protean II apparatus, a polyacrylamide stacking and resolving gel and acetic acid-ammonium acetate buffers at acidic pH that contained urea; 4) a mini-slab gel system using the Bio-Rad mini-Protean II apparatus, a polyacrylamide gel with a stacking and resolving gel in Tris-HCl buffers containing sodium dodecyl sulphate (SDS) and a Tris-chloride-glycine electrode buffer. The mini-slab alkaline urea polyacrylamide gel electrophoresis (PAGE) method was considered to be the most suitable for monitoring the loss of intact casein during cheese ripening. However, SDS-PAGE gave good resolution of para-K-casein, ~-lactoglobulin and a-lactalbumin and it could therefore be used for the analysis of cheese in which whey proteins have been incorporated or for monitoring the brealcdown of para-K-casein (Chapter 4) in cheese. Two-dimensional PAGE revealed the presence of more bands than were visible using any single method of electrophoresis. Traces of protein were found to lie beneath the a 11-casein band and this explained why, even after considerable proteolysis, some a 11-casein appeared to remain. Storing cheese samples in such a way that there is a minimum of further change was examined using several different storage methods and temperatures, including storage as: freeze-dried powder at 4°C in the dark, frozen at-9,-16,-35,-75 and-100°C, and dissolved in 6 M urea solution and stored at 4 and-l6°C. The trial ran for 6 months and involved the multiple sampling and detailed analysis of three Cheddar cheeses by reversed phase fast protein liquid chromatography (RP-FPLC) for the water-soluble fraction (WSF) and alkaline urea-PAGE for the protein fraction.

The composition and development of microbial population during the manufacture and 3 ripening of two batches of a blue-veined cheese was examined by culturing and polymerase 4 chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) (PCR-DGGE). Nine 5 selective and/or differential media were used to track the cultivable populations of total and 6 indicator microbial groups. For PCR-DGGE, the V3 hyper variable region of the bacterial 7 16S rRNA gene and the eukaryotic D1 domain of 28S rDNA were amplified with universal 8 primers, specific for prokaryotes and eukaryotes, respectively. Similarities and differences 9 between the results obtained by the culturing and the molecular method were recorded for 10 some populations. Culturing analysis allows minority microbial groups (coliforms, 11 staphylococci) to be monitored, although in this work PCR-DGGE identified a population of 12 Streptococcus thermophilus that went undetected by culturing. These results show that the 13 characterization of the microbial populations interacting and evolving during the 14 cheesemaking process is improved by combining culturing and molecular methods.

We previously reported an outbreak of listeriosis in Austria and Germany due to consumption of 'Quargel' cheese. It comprised 14 cases (including five fatalities) infected by a serotype 1/2a Listeria monocytogenes (clone 1), with onset of illness from June 2009 to January 2010. A second strain of L. monocytogenes serotype 1/2a (clone 2) spread by this product could be linked to further 13 cases in Austria (two fatal), six in Germany (one fatal) and one case in the Czech Republic, with onset of disease from December 2009 to end of February 2010.

This study investigated the antibacterial efficiency of five spice and herb extracts (cinnamon stick, oregano, clove, pomegranate peel, and grape seed) against Listeria monocytogenes, Staphylococcus aureus, and Salmonella enterica in cheese at room temperature (*238C). The lipid oxidation (thiobarbituric acid-reactive substances) of cheese was periodically tested by oxidative analyses. The results showed that all five plant extracts were effective against three foodborne pathogens in cheese. Treatments with these extracts increased the stability of cheese against lipid oxidation. Clove showed the highest antibacterial and antioxidant activity. The reduction of foodborne pathogen numbers and the inhibition of lipid oxidation in cheese indicated that the extracts of these plants (especially clove) have potential as natural food preservatives.

The effect of acid and osmotic shifts on the growth of Listeria monocytogenes was evaluated at 10°C. Two types of shifts were tested: (i) within the range of pH and water activity (aw) levels that allow growth of L. monocytogenes and (ii) after habituation at no-growth conditions back to growth-permitting conditions. A L. monocytogenes cheese isolate, with high survival capacity during cheesemaking, was inoculated (102 CFU/ml) in tryptic soy broth supplemented with 0.6% yeast extract at six pH levels (5.1 to 7.2; adjusted with lactic acid) and 0.5% NaCl (aw 0.995), or four aw levels (0.995 to 0.93, adjusted with 0.5 to 10.5% NaCl) at pH 7.2 and grown to early stationary phase. L. monocytogenes was then shifted (at 102 CFU/ml) to each of the aforementioned growth-permitting pH and aw levels and incubated at 10°C. Shifts from no-growth to growth-permitting conditions were carried out by transferring L. monocytogenes habituated at pH 4.9 or aw 0.90 (12.5% NaCl) for 1, 5, and 10 days to...

Listeria monocytogenes can proliferate at the beginning of cheesemaking as the conditions favor growth. The objective of this study was to establish the growth limits of L. monocytogenes in a cheese matrix, in case of potential contamination of the milk prior to cheese manufacture. A semisoft laboratory scale model cheese system was made at different initial pH and water activity (aw) levels with a mix of two strains of L. monocytogenes. A factorial design of five pH values (5.6 to 6.5), four aw values (0.938 to 0.96), and two L. monocytogenes inoculation levels (1 to 20 CFU/ml and 500 to 1,000 CFU/ml) was carried out. Each combination was evaluated in six independent replicates. In order to determine if there was a dominant strain, isolated colonies from the cheeses were analyzed by pulsed-field gel electrophoresis. The data relating to growth initiation were fitted to a logistic regression model. The aw of milk influenced the probability of growth initiation of L. monocytogenes at...

Listeria monocytogenes is a widely distributed foodborne pathogen that causes listeriosis in risk groups, such as young, old, pregnant women, neonates and the immunocompromised, with a mortality rate of 20-30% despite correct antimicrobial treatment (Swaminathan and Gerner-Smidt 2007). It has been responsible for several foodborne outbreaks of listeriosis reported in Europe, USA and Japan.

The dynamics of the physicochemical characteristics of foods help to determine the fate of pathogens throughout processing. The aim of this study was to assess the behaviour of Listeria monocytogenes during cheesesmaking and ripening and to model the growth observed under the dynamic conditions of the cheese. A laboratory scale cheese was made in 4 independent replicates from pasteurised or raw cow's milk, artificially contaminated with L. monocytogenes. No growth of L. monocytogenes occurred during raw milk cheese-making, whereas growth did occur in pasteurised milk. During ripening, growth occurred in raw milk cheese, but inactivation occurred in pasteurised milk cheese. The behaviour observed for L. monocytogenes was modelled using a logistic primary model coupled with a secondary cardinal model, taking into account the effect of physicochemical conditions (temperature, pH, water activity and lactate). A novel statistical approach was proposed to assess the optimal growth rate of a microorganism from experiments performed in dynamic conditions. This complex model had an acceptable quality of fit on the experimental data. The estimated optimum growth rates can be used to predict the fate of L. monocytogenes during cheese manufacture in raw or pasteurized milk in different physicochemical conditions. The data obtained contributes to a better understanding of the potential risk that L. monocytogenes presents to cheese producers (growth on the product, if it is contaminated) and consumers (the presence of high numbers) and constitutes a very useful set of data for the completion of chain-based modelling studies.

Enterolithiasis, or presence of stone concretions in the gastrointestinal tract, is an important but relatively uncommon clinical condition. It is most commonly found in animals especially equines. In humans clinical presentation of this condition is varied and sometimes masquerading other intra abdominal pathologies. Clinically insignificant incidentally noted enteroliths may shift attention from primary non-enterolith related pathology of the patient that may be responsible for a current clinical presentation. Here we are reporting four cases of incidentally detected enterolithiasis at our hospital with review of literature.

The bactericidal activity on Listeria spp. of nine disinfectants used in the food industry was studied by previously published methods. The disinfectants were diluted to the test concentation in sterile standard hard water. Various types of chemical agents were evaluated, including phenolic compounds, alcohols, quaternary ammonium compounds, surface-active agents, aldehydes and disochlorine tablets. The following strains isolated from cheese were studied : Listeria innocua, L. welshimeri, L. monocytogenes 1/2a, 1/2b, 1/2c and 4b. The results show that the listerias are not particularly resistant to disinfectants but the efficacy of some agents is affected by organic matter.

Ninety-five isolated strains of Lactic acid bacteria (LAB) were identified from Lighvan cheese. The LAB evolution showed the dominance of lactococci and lactobacilli in the first stage and substitution of these genera by enterococci at the end of ripening. The most predominant strains were Enterococcus faecium (22.44%), Lactococcus lactis ssp. lactis (20.4%), Lactobacillus plantarum (18.36%) and E. faecalis (14.28%), respectively. Eleven and 51 different carbohydrate fermentation profiles were observed according to API 20 STREP and API 50 CH, respectively. API 20 STREP dendogram showed identical fermentation profiles of some E. faecalis and E. faecium strains, indicating that these strains might be well adapted to the whole cheese manufacture.

The composition and development of microbial population during the manufacture and 3 ripening of two batches of a blue-veined cheese was examined by culturing and polymerase 4 chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) (PCR-DGGE). Nine 5 selective and/or differential media were used to track the cultivable populations of total and 6 indicator microbial groups. For PCR-DGGE, the V3 hyper variable region of the bacterial 7 16S rRNA gene and the eukaryotic D1 domain of 28S rDNA were amplified with universal 8 primers, specific for prokaryotes and eukaryotes, respectively. Similarities and differences 9 between the results obtained by the culturing and the molecular method were recorded for 10 some populations. Culturing analysis allows minority microbial groups (coliforms, 11 staphylococci) to be monitored, although in this work PCR-DGGE identified a population of 12 Streptococcus thermophilus that went undetected by culturing. These results show that the 13 characterization of the microbial populations interacting and evolving during the 14 cheesemaking process is improved by combining culturing and molecular methods.