A study was designed to recover Listeria monocytogenes from pasteurized milk and Minas frescal cheese (MFC) sampled at retail establishments (REs) and to identify the contamination source(s) of these products in the corresponding dairy processing plant. Fifty milk samples (9 brands) and 55 MFC samples (10 brands) were tested from REs located in Juiz de Fora, Minas Gerais, Brazil. All milk samples and 45 samples from 9 of 10 MFC brands tested negative for L. monocytogenes; however, “brand F” of MFC obtained from REs 119 and 159 tested positive. Thus, the farm/plant that produced brand F MFC was sampled; all samples from the milking parlor tested negative for L. monocytogenes, whereas several sites within the processing plant and the MFC samples tested positive. All 344 isolates recovered from retail MFC, plant F MFC, and plant F environmental samples were serotype 1/2a and displayed the same AscI or ApaI fingerprints. Since these results established that the storage coolers served as the contamination source of the MFC, plant F was closed so that corrective renovations could be made. Following renovation, samples from sites that previously tested positive for the pathogen were collected from the processing environment and from MFC on multiple visits; all tested negative for L. monocytogenes. In addition, on subsequent visits to REs 159 and 119, all MFC samples tested negative for the pathogen. Studies are ongoing to quantify the prevalence, levels, and types of L. monocytogenes in MFC and associated processing plants to lessen the likelihood of listeriosis in Brazil.
Here, high-throughput sequencing was employed to reveal the highly diverse bacterial populations present in 62 Irish artisanal cheeses and, in some cases, associated cheese rinds. Using this approach, we revealed the presence of several genera not previously associated with cheese, including Faecalibacterium, Prevotella, and Helcococcus and, for the first time, detected the presence of Arthrobacter and Brachybacterium in goats' milk cheese. Our analysis confirmed many previously observed patterns, such as the dominance of typical cheese bacteria, the fact that the microbiota of raw and pasteurized milk cheeses differ, and that the level of cheese maturation has a significant influence on Lactobacillus populations. It was also noted that cheeses containing adjunct ingredients had lower proportions of Lactococcus species. It is thus apparent that high-throughput sequencing-based investigations can provide valuable insights into the microbial populations of artisanal foods.
Model Cheddar cheeses were prepared from pasteurized milk artificially contaminated with high 104 to 105 CFU/ml) and low (101 to 102 CFU/ml) inocula of three different Mycobacterium paratuberculosis strains. A reference strain, NCTC 8578, and two strains (806PSS and 796PSS) previously isolated from pasteurized milk for retail sale were investigated in this study. The manufactured Cheddar cheeses were similar in pH, salt, moisture, and fat composition to commercial Cheddar. The survival of M. paratuberculosis cells was monitored over a 27-week ripening period by plating homogenized cheese samples onto HEYM agar medium supplemented with the antibiotics vancomycin, amphotericin B, and nalidixic acid without a decontamination step. A concentration effect was observed in M. paratuberculosis numbers between the inoculated milk and the 1-day old cheeses for each strain. For all manufactured cheeses, a slow gradual decrease in M. paratuberculosis CFU in cheese was observed over the ripening period. In all cases where high levels (>3.6 log10) of M. paratuberculosis were present in 1-day cheeses, the organism was culturable after the 27-week ripening period. The D values calculated for strains 806PSS, 796PSS, and NCTC 8578 were 107, 96, and 90 days, respectively. At low levels of contamination, M. paratuberculosis was only culturable from 27-week-old cheese spiked with strain 806PSS. M. paratuberculosis was recovered from the whey fraction in 10 of the 12 manufactured cheeses. Up to 4% of the initial M. paratuberculosis load was recovered in the culture-positive whey fractions at either the high or low initial inoculum.
Listeria monocytogenes is of major concern to the food industry in general and the dairy industry in particular. Little is known about incidence of this pathogenic bacterium in dairy products in Iran.
A survey was made from 23 September 2006 to 22 June 2007 for Listeria species in ninety samples of traditional and industrial cheeses, in milk and surface where the cheeses were manufactured from unpasteurized raw milk in the province of Isfahan (Iran).
Listeria murrayi, L. grayi and L. ivanovii, were detected in nine traditional cheeses and one raw milk sample. None of the different Listeria species were isolated from the industrial cheeses and their environment.
There are almost good hygienic conditions in domestic cheese manufacturing farmhouses in Isfahan area, but we should try to improve hygienic levels until we have none of the Listeria spp. in our samples.
Listeria; Cheese; Milk; Iran
Raw milk was artificially contaminated with declumped cells of Mycobacterium avium subsp. paratuberculosis at a concentration of 104 to 105 CFU/ml and was used to manufacture model hard (Swiss Emmentaler) and semihard (Swiss Tisliter) cheese. Two different strains of M. avium subsp. paratuberculosis were tested, and for each strain, two model hard and semihard cheeses were produced. The survival of M. avium subsp. paratuberculosis cells was monitored over a ripening period of 120 days by plating out homogenized cheese samples onto 7H10-PANTA agar. In both the hard and the semihard cheeses, counts decreased steadily but slowly during cheese ripening. Nevertheless, viable cells could still be detected in 120-day cheese. D values were calculated at 27.8 days for hard and 45.5 days for semihard cheese. The most important factors responsible for the death of M. avium subsp. paratuberculosis in cheese were the temperatures applied during cheese manufacture and the low pH at the early stages of cheese ripening. Since the ripening period for these raw milk cheeses lasts at least 90 to 120 days, the D values found indicate that 103 to 104 cells of M. avium subsp. paratuberculosis per g will be inactivated.
LiCl-phenylethanol-moxalactam Agar (LPMA), ARS Modified McBride Agar, and Modified Vogel Johnson Agar were compared with previously tested plating media in the enumeration of Listeria monocytogenes from pasteurized whole milk, chocolate ice cream mix, Brie cheese, and raw cabbage. LPMA was most suitable for analyzing Brie cheese and cabbage. Gum base-nalidixic acid-tryptone-soya medium (previously tested) was most suitable for analyzing milk and chocolate ice cream mix.
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.
Shiga toxin-producing Escherichia coli (STEC) is an important cause of food-borne illness. The public health implication of the presence of STEC in dairy products remains unclear. Knowledge of STEC behavior in cheeses would help to evaluate the human health risk. The aim of our study was to observe the growth and survival of experimentally inoculated STEC strains in raw-milk cheeses manufactured and ripened according to five technological schemes: blue-type cheese, uncooked pressed cheese with long ripening and with short ripening steps, cooked cheese, and lactic cheese. Cheeses were contaminated with different STEC serotypes (O157:H7, O26:H11, O103:H2, and O145:H28) at the milk preparation stage. STEC growth and survival were monitored on selective media during the entire manufacturing process. STEC grew (2 to 3 log10 CFU · g−1) in blue-type cheese and the two uncooked pressed cheeses during the first 24 h of cheese making. Then, STEC levels progressively decreased in cheeses that were ripened for more than 6 months. In cooked cheese and in lactic cheese with a long acidic coagulation step (pH < 4.5), STEC did not grow. Their levels decreased after the cooking step in the cooked cheese and after the coagulation step in the lactic cheese, but STEC was still detectable at the end of ripening and storage. A serotype effect was found: in all cheeses studied, serotype O157:H7 grew less strongly and was less persistent than the others serotypes. This study improves knowledge of the behavior of different STEC serotypes in various raw-milk cheeses.
Soft-ripened cheeses belong to the type of food most often contaminated with Listeria monocytogenes, and they have been implicated in several outbreaks of listeriosis. Bacteriophages represent an attractive way to combat foodborne pathogens without affecting other properties of the food. We used the broad host range, virulent Listeria phage A511 for control of L. monocytogenes during the production and ripening phases of both types of soft-ripened cheeses, white mold (Camembert-type) cheese, as well as washed-rind cheese with a red-smear surface (Limburger-type). The surfaces of young, unripened cheese were inoculated with 101–103 cfu/cm2
L. monocytogenes strains Scott A (serovar 4b) or CNL 103/2005 (serovar 1/2a). Phage was applied at defined time points thereafter, in single or repeated treatments, at 3 × 108 or 1 × 109 pfu/cm2. With Scott A (103 cfu/cm2) and a single dose of A511 (3 × 108 pfu/cm2) on camembert-type cheese, viable counts dropped 2.5 logs at the end of the 21 day ripening period. Repeated phage application did not further inhibit the bacteria, whereas a single higher dose (1 × 109 pfu/cm2) was found to be more effective. On red-smear cheese ripened for 22 days, Listeria counts were down by more than 3 logs. Repeated application of A511 further delayed re-growth of Listeria, but did not affect bacterial counts after 22 days. With lower initial Listeria contamination (101–102 cfu/cm2), viable counts dropped below the limit of detection, corresponding to more than 6 logs reduction compared to the control. Our data clearly demonstrate the potential of bacteriophage for biocontrol of L. monocytogenes in soft cheese.
Listeria monocytogenes; bacteriophage; food safety; soft-ripened cheese
Cheddar cheese was prepared with Lactococcus lactis subsp. lactis MM217, a starter culture which contains pMC117 coding for pediocin PA-1. About 75 liters of pasteurized milk (containing ca. 3.6% fat) was inoculated with strain MM217 (ca. 106 CFU per ml) and a mixture of three Listeria monocytogenes strains (ca. 103 CFU per ml). The viability of the pathogen and the activity of pediocin in the cheese were monitored at appropriate intervals throughout the manufacturing process and during ripening at 8°C for 6 months. In control cheese made with the isogenic, non-pediocin-producing starter culture L. lactis subsp. lactis MM210, the counts of the pathogen increased to about 107 CFU per g after 2 weeks of ripening and then gradually decreased to about 103 CFU per g after 6 months. In the experimental cheese made with strain MM217, the counts of L. monocytogenes decreased to 102 CFU per g within 1 week of ripening and then decreased to about 10 CFU per g within 3 months. The average titer of pediocin in the experimental cheese decreased from approximately 64,000 arbitrary units (AU) per g after 1 day to 2,000 AU per g after 6 months. No pediocin activity (<200 AU per g) was detected in the control cheese. Also, the presence of pMC117 in strain MM217 did not alter the cheese-making quality of the starter culture, as the rates of acid production, the pH values, and the levels of moisture, NaCl, and fat of the control cheese and the experimental cheese were similar. Our data revealed that pediocin-producing starter cultures have significant potential for protecting natural cheese against L. monocytogenes.
Method of homogenization (Waring blender versus stomacher), type of diluent (tryptose broth [TB] versus aqueous 2% trisodium citrate), and temperature of diluent (20 versus 40 degrees C) were compared for recovery of Listeria monocytogenes from freshly made and ripened Colby cheese. By using direct plating on McBride listeria agar, significantly higher numbers of L. monocytogenes were recovered when cheese samples were (i) homogenized for 2 min with the blender rather than the stomacher (P less than 0.01), (ii) diluted in trisodium citrate rather than TB (P less than 0.01), and (iii) diluted in diluents at 40 rather than 20 degrees C (P less than 0.05). Based on these results, a new diluent/enrichment medium was developed by adding 2% trisodium citrate to TB (TBC). Despite superior results with the blender, biosafety concerns led to use of the stomacher for homogenization of cheese samples; hence, the stomaching time was increased to 3 min. Results obtained by direct plating indicated that recovery of L. monocytogenes from Colby cheese and from curd samples taken during manufacture of brick cheese increased when samples were diluted 1:10 in TBC at 45 degrees C and stomached for 3 min, as compared with similarly treated samples diluted in TB at 25 degrees C. A similar comparison of both diluents for recovery of L. monocytogenes from cold-pack cheese food yielded bacterial counts which were not significantly different. Recovery of L. monocytogenes from cold-enriched (at 4 degrees C for up to 8 weeks) samples of Colby cheese and cold-pack cheese food was generally similar for samples homogenized in TBC or TB.
A new detection system, the magnetic immuno-polymerase chain reaction (PCR) assay (MIPA) has been developed to detect Listeria monocytogenes in food. This method separates Listeria cells from PCR-inhibitory factors present in enrichment broths containing food samples by using magnetic beads coated with specific monoclonal antibodies (MAbs). The separated bacteria were lysed, and the supernatant containing the bacterial DNA was subjected to the PCR. Detection of L. monocytogenes in three naturally contaminated cheese samples with two different MAbs and PCR primers specific for the gene encoding the delayed-hypersensitivity factor showed that with MAb 55 all three samples were positive whereas with MAb A two samples were positive. A further improvement of the method was obtained by using a PCR step based on the listeriolysin O gene. A MIPA employing MAb 55 and the listeriolysin O gene primer set detected L. monocytogenes after 24 h of culture in Listeria Enrichment Broth samples from Port Salut artificially contaminated with 40 CFU/25 g. We could detect 1 CFU of L. monocytogenes per g of cheese after a second enrichment for 24 h in Fraser broth. The analysis time including both enrichments is approximately 55 h.
A 500-base-pair DNA fragment of a presumptive beta-hemolysin gene of Listeria monocytogenes has been used to identify this organism by a modified colony hybridization technique. We have cloned this DNA fragment into M13 bacteriophage vectors and sequenced it by a dideoxynucleotide sequencing technique. From this sequencing information, several oligodeoxyribonucleotides were synthesized and used as synthetic probes to identify L. monocytogenes. The probes were specific for L. monocytogenes and did not react with any other Listeria strains in a colony hybridization assay. In particular, one of these probes (AD07) was used to detect L. monocytogenes in artificially contaminated raw-milk and soft-cheese samples.
Background and Objectives
In this study, the antibacterial effect of essential oil of tarragon (Artemisia dracunculus) on Staphylococcus aureus and Escherichia coli was evaluated in culture media and Iranian white cheese.
Materials and Methods
The tarragon essential oil (EO) obtained by the steam distillation method and its antibacterial activity was evaluated in 96-well microtiter plates containing brain heart infusion broth. The enumeration of S. aureus and E. coli in cheese samples were carried out on the following media: Baired parker agar for S.aureus, incubated at 37°C for 24 h; and MacConkey sorbitol agar for E. coli, incubated at 37°C for 24 h. Iranian white cheese was produced from fresh and whole pasteurized cow milk (2.5%). Bacteria (103 cfu/mL) were inoculated to different batches. Cheese was treated with different concentrations of EO (15 and 1500 µg/mL) and separated into four parts in an equal manner. The sensory evaluation was done by a panel of four judges.
According to the results obtained, minimum inhibitory concentrations (MIC) for E. coli and S. aureus were 2500 and 1250 µg/mL, respectively. Also, minimum bactericidal concentration (MBC) for the mentioned microorganisms were 5000 and 2500 µg/mL, respectively. All the EO concentrations for each bacteria result in reducing bacterial count of cheese samples compared to control (P<0.05). Also, with increasing concentration of EO in cheese samples, the bacterial count was reduced further (P<0.05).
Based on our findings, tarragon essential oil has antibacterial effect on two important pathogen bacteria (S. aureus and E. coli) and can be applied as a preservative in foods such as cheese.
Tarragon; essential oil; antibacterial effect; Staphylococcus aureus; Escherichia coli; cheese
The quality of distinctive artisanal cheeses is closely associated with the territory of production and its traditions. Pedoclimatic characteristics, genetic autochthonous variations, and anthropic components create an environment so specific that it would be extremely difficult to reproduce elsewhere. Pecorino cheese is included in this sector of the market and is widely diffused in Italy (∼62.000t of production in 2010). Pecorino is a common name given to indicate Italian cheeses made exclusively from pure ewes’ milk characterized by a high content of fat matter and it is mainly produced in the middle and south of Italy by traditional procedures from raw or pasteurized milk. The microbiota plays a major role in the development of the organoleptic characteristics of the cheese but it can also be responsible for the accumulation of undesirable substances, such as biogenic amines (BA). Bacterial amino acid decarboxylase activity and BA content have to be investigated within the complex microbial community of raw milk cheese for different cheese technologies. The results emphasize the necessity of controlling the indigenous bacterial population responsible for high production of BA and the use of competitive adjunct cultures could be suggested. Several factors can contribute to the qualitative and quantitative profiles of BA’s in Pecorino cheese such as environmental hygienic conditions, pH, salt concentration, water activity, fat content, pasteurization of milk, decarboxylase microorganisms, starter cultures, temperature and time of ripening, storage, part of the cheese (core, edge), and the presence of cofactor (pyridoxal phosphate, availability of aminases and deaminases). In fact physico-chemical parameters seem to favor biogenic amine-positive microbiota; both of these environmental factors can easily be modulated, in order to control growth of undesirable microorganisms. Generally, the total content of BA’s in Pecorino cheeses can range from about 100–2400 mg/kg, with a prevalence of toxicologically important BA’s, tyramine and histamine. The presence of BA is becoming increasingly important to consumers and cheese-maker alike, due to the potential threats of toxicity to humans and consequent trade implications.
Italian ewe cheese; microbial groups; biogenic amines
The aim of this study was to develop a predictive model simulating growth over time of the pathogenic bacterium Listeria monocytogenes in a soft blue-white cheese. The physicochemical properties in a matrix such as cheese are essential controlling factors influencing the growth of L. monocytogenes. We developed a predictive tertiary model of the bacterial growth of L. monocytogenes as a function of temperature, pH, NaCl, and lactic acid. We measured the variations over time of the physicochemical properties in the cheese. Our predictive model was developed based on broth data produced in previous studies. New growth data sets were produced to independently calibrate and validate the developed model. A characteristic of this tertiary model is that it handles dynamic growth conditions described in time series of temperature, pH, NaCl, and lactic acid. Supplying the model with realistic production and retail conditions showed that the number of L. monocytogenes cells increases 3 to 3.5 log within the shelf life of the cheese.
Butyric acid fermentation, the late-blowing defect in cheese, caused by the outgrowth of clostridial spores present in raw milk, can create considerable loss of product, especially in the production of semihard cheeses like Gouda cheese, but also in grana and Gruyère cheeses. To demonstrate the causative relationship between Clostridium tyrobutyricum and late blowing in cheese, many cheesemaking experiments were performed to provoke this defect by using spores from several strains of the major dairy-related clostridia. A method of PCR amplification of a part of the 16S rRNA gene in combination with hybridization with species-specific DNA probes was developed to allow the specific detection of clostridial sequences in DNAs extracted from cheeses. The sensitivity was increased by using nested PCR. Late blowing was provoked in experimental cheeses with 28 of the 32 C. tyrobutyricum strains tested, whereas experimental cheeses made with spores from C. beijerinckii, C. butyricum, and C. sporogenes showed no signs of butyric acid fermentation. In all experimental and commercial cheeses with obvious signs of late blowing, DNA from C. tyrobutyricum was detected; in some cheeses, signals for C. beijerinckii were also found. It was concluded that only C. tyrobutyricum strains are able to cause butyric acid fermentation in cheese.
As the cheese market faces strong international competition, the optimization of production processes becomes more important for the economic success of dairy companies. In dairy productions, whey from former cheese batches is frequently re-used to increase the yield, to improve the texture and to increase the nutrient value of the final product. Recycling of whey cream and particulated whey proteins is also routinely performed. Most bacteriophages, however, survive pasteurization and may re-enter the cheese manufacturing process. There is a risk that phages multiply to high numbers during the production. Contamination of whey samples with bacteriophages may cause problems in cheese factories because whey separation often leads to aerosol-borne phages and thus contamination of the factory environment. Furthermore, whey cream or whey proteins used for recycling into cheese matrices may contain thermo-resistant phages. Drained cheese whey can be contaminated with phages as high as 109 phages mL-1. When whey batches are concentrated, phage titers can increase significantly by a factor of 10 hindering a complete elimination of phages. To eliminate the risk of fermentation failure during recycling of whey, whey treatments assuring an efficient reduction of phages are indispensable. This review focuses on inactivation of phages in whey by thermal treatment, ultraviolet (UV) light irradiation, and membrane filtration. Inactivation by heat is the most common procedure. However, application of heat for inactivation of thermo-resistant phages in whey is restricted due to negative effects on the functional properties of native whey proteins. Therefore an alternative strategy applying combined treatments should be favored – rather than heating the dairy product at extreme temperature/time combinations. By using membrane filtration or UV treatment in combination with thermal treatment, phage numbers in whey can be reduced sufficiently to prevent subsequent phage accumulations.
bacteriophages; dairy technology; whey recycling; inactivation; resistance
An outbreak of Salmonella dublin infection occurred in England and Wales in October to December 1989. Forty-two people were affected, mainly adults, and most lived in south-east England. Microbiological and epidemiological investigations implicated an imported Irish soft unpasteurized cows' milk cheese as the vehicle of infection. A case-control study showed a statistically significant association between infection and consumption of the suspect cheese (p = 0.001). Salmonella dublin was subsequently isolated from cheeses obtained from the manufacturer's premises. Initial control measures included the withdrawal of the cheese from retail sale and a Food Hazard Warning to Environmental Health Departments, as well as a press release, from the Department of Health. Subsequently, a decision was taken by the manufacturer to pasteurize milk used in the production of cheese for the UK market and importation of the cheese resumed in June 1990.
The bacteriological quality of 198 ripened soft or semi-soft goat cheeses obtained from dairy farms and the retail trade was investigated. The cheeses were examined for total counts of aerobic bacteria, coliform bacteria (37 and 44 degrees C respectively), enterococci, coagulase positive staphylococci, Bacillus cereus and Clostridium perfringens. Cheeses obtained from dairy-farms were also determined for pH value. In terms of all tests performed, cheeses made of heat-treated milk with starter culture had the best prospects for fulfilling the criteria for 'fit for consumption'. Cheeses made of raw milk without starter culture made up the most unsatisfactory group from a food-hygiene point of view. Bacteriological guidelines for on-farm manufactured goat cheese are suggested.
The bacteriocin produced by Pediococcus acidilactici PAC 1.0, previously designated PA-1 bacteriocin, was found to be inhibitory and bactericidal for Listeria monocytogenes. A dried powder prepared from PAC 1.0 culture supernatant fortified with 10% milk powder was found to contain bacteriocin activity. An MIC against L. monocytogenes and lytic effects in broth cultures were determined. Inhibition by PA-1 powder occurred over the pH range 5.5 to 7.0 and at both 4 and 32 degrees C. In addition, inhibition of L. monocytogenes was demonstrated in several food systems including dressed cottage cheese, half-and-half cream, and cheese sauce.
Cross-contamination of ready-to-eat (RTE) foods with pathogens on contaminated tableware and food preparation utensils is an important factor associated with foodborne illnesses. To prevent this, restaurants and food service establishments are required to achieve a minimum microbial reduction of 5 logs from these surfaces. This study evaluated the sanitization efficacies of ware-washing protocols (manual and mechanical) used in restaurants to clean tableware items. Ceramic plates, drinking glasses and stainless steel forks were used as the food contact surfaces. These were contaminated with cream cheese and reduced-fat milk inoculated with murine norovirus (MNV-1), Escherichia coli K-12 and Listeria innocua. The sanitizing solutions tested were sodium hypochlorite (chlorine), quaternary ammonium (QAC) and tap water (control). During the study, the survivability and response to the experimental conditions of the bacterial species was compared with that of MNV-1. The results showed that current ware-washing protocols used to remove bacteria from tableware items were not sufficient to achieve a 5 log reduction in MNV-1 titer. After washing, a maximum of 3 log reduction in the virus were obtained. It was concluded that MNV-1 appeared to be more resistant to both the washing process and the sanitizers when compared with E. coli K-12 and L. innocua.
Ragusano cheese is a “protected denomination of origin” cheese made in the Hyblean region of Sicily from raw milk using traditional wooden tools, without starter. To explore the Ragusano bacterial ecosystem, molecular fingerprinting was conducted at different times during the ripening and biofilms from the wooden vats called “tinas” were investigated. Raw milks collected at two farm sites, one on the mountain and one at sea level, were processed to produce Ragusano cheese. Raw milk, curd before and after cooking, curd at stretching time (cheese 0 time), and cheese samples (4 and 7 months) were analyzed by PCR-temporal temperature gel electrophoresis (PCR-TTGE) and by classical enumeration microbiology. With the use of universal primers, PCR-TTGE revealed many differences between the raw milk profiles, but also notable common bands identified as Streptococcus thermophilus, Lactobacillus lactis, Lactobacillus delbrueckii, and Enterococcus faecium. After the stretching, TTGE profiles revealed three to five dominant species only through the entire process of ripening. In the biofilms of the two tinas used, one to five species were detected, S. thermophilus being predominant in both. Biofilms from five other tinas were also analyzed by PCR-TTGE, PCR-denaturating gradient gel electrophoresis, specific PCR tests, and sequencing, confirming the predominance of lactic acid bacteria (S. thermophilus, L. lactis, and L. delbrueckii subsp. lactis) and the presence of a few high-GC-content species, like coryneform bacteria. The spontaneous acidification of raw milks before and after contact with the five tinas was followed in two independent experiments. The lag period before acidification can be up to 5 h, depending on the raw milk and the specific tina, highlighting the complexity of this natural inoculation system.
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.
A511::luxAB is a recombinant derivative of a broad-host-range bacteriophage specific for the genus Listeria, transducing bacterial bioluminescence into infected cells. In this study, we have evaluated its use for rapid and easy testing of contaminated foods and environmental samples for the presence of viable Listeria cells, in comparison to the standard plating procedure. With a short preenrichment step of 20 h, the system was capable of detecting very low initial contamination rates in several foods artificially contaminated with Listeria monocytogenes Scott A cells. In ricotta cheese, chocolate pudding, and cabbage, less than one cell per g of food could be clearly identified by comparing the light emission of phage-infected samples to that of controls without lux phage. In foods having a large and complex microbial background flora, such as minced meat and soft cheese, at least 10 cells per g were necessary to produce a positive bioluminescence signal. Of 348 potentially contaminated natural food and environmental samples, 55 were found to be Listeria positive by the lux phage method. The standard plating procedure detected 57 positive samples. Some differences were observed with respect to the individual samples, i.e., the lux phage procedure detected more positive samples among the dairy products and environmental samples, whereas the plating procedure revealed more contaminated meat and poultry samples. Overall, both methods performed similarly, i.e., were equally sensitive. However, the minimum time required for detection of Listeria with the luciferase phage assay was 24 h, which is much shorter than the 4 days needed by the standard plating method. Furthermore, a most probable number technique with three parallels, based on the use of A511::luxAB for differentiation of positive and negative tubes, is described. The method enables rapid enumeration of low levels of Listeria cells in several foods tested, against the background of a competing microflora.