Relationships between sensorial characteristics and microbial dynamics in "Registered Designation of Origin" Salers cheese

Raw milk cheeses show a wide diversity of sensorial characteristics, largely determined by the microflora of raw milk. Microbial dynamics in Registered Designation of Origin (R.D.O.) Salers cheese was assessed by DNA and RNA SSCP analysis on nine cheeses. These cheeses showed considerable diversity both in microbial dynamics and sensorial characteristics. Relationships between the sensorial characteristics and the microbial dynamics were studied. A global consideration of bacterial dynamics demonstrated that other bacteria than lactic acid bacteria can play a role in the elaboration of sensorial characteristics. Indeed, high CG% Gram-positive bacteria can be involved. DNA data, as well as RNA data, appeared relevant to attempts to explain sensorial variance. Correlations between sensorial and microbial data were rather complex. Several microbial variables for DNA and RNA analyses, noted at different times of analysis, were correlated to each sensory variable. A global view of cheese microbial community proved to be insufficient in explaining the diversity of the sensorial qualities of R.D.O. Salers cheese.     

Effect of phage on survival of Salmonella enteritidis during manufacture and storage of cheddar cheese made from raw and pasteurized milk.

The ability of Salmonella Enteritidis to survive in the presence of phage, SJ2, during manufacture, ripening, and storage of Cheddar cheese produced from raw and pasteurized milk was investigated. Raw milk and pasteurized milk were inoculated to contain 10(4) CFU/ml of a luminescent strain of Salmonella Enteritidis (lux) and 10(8) PFU/ml SJ2 phage. The milks were processed into Cheddar cheese following standard procedures. Cheese samples were examined for Salmonella Enteritidis (lux), lactic acid bacteria, molds and yeasts, coliforms, and total counts, while moisture, fat, salt, and pH values were also measured. Salmonella Enteritidis (lux) was enumerated in duplicate samples by surface plating on MacConkey novobiocin agar. Bioluminescent colonies of Salmonella Enteritidis were identified in the NightOwl molecular imager. Samples were taken over a period of 99 days. Counts of Salmonella Enteritidis (lux) decreased by 1 to 2 log cycles in raw and pasteurized milk cheeses made from milk containing phage. In cheeses made from milks to which phage was not added, there was an increase in Salmonella counts of about 1 log cycle. Lower counts of Salmonella Enteritidis (lux) were observed after 24 h in pasteurized milk cheese containing phage compared to Salmonella counts in raw milk cheese with phage. Salmonella Enteritidis (lux) survived in raw milk and pasteurized milk cheese without phage, reaching a final concentration of 10(3) CFU/g after 99 days of storage at 8 degrees C. Salmonella did not survive in pasteurized milk cheese after 89 days in the presence of phage. However, Salmonella counts of approximately 50 CFU/g were observed in raw milk cheese containing phage even after 99 days of storage. In conclusion, this study demonstrates that the addition of phage may be a useful adjunct to reduce the ability of Salmonella to survive in Cheddar cheese made from both raw and pasteurized milk.     

Control of Listeria monocytogenes in raw-milk cheeses

The development of Listeria monocytogenes in cheeses made with raw-milk originating from six different farms and according to the Saint-Nectaire cheesemaking technology was studied. Milk was inoculated with two strains of L. monocytogenes at 5 to 10 CFU/25 ml. Microbial and chemical analyses were carried out at appropriate intervals during ripening. L. monocytogenes did not grow in the cores of cheeses prepared with milk originating from three farms. That inhibition could be partially attributed to the pH values and L-lactate content. There was no growth in cheeses with pH below 5.2 and lactate content around 14 mg/g. In all cheeses, L. monocytogenes stopped growing in the cores of cheeses after eight days and some other factors may be involved in the inhibition. No relation was found between L. monocytogenes count and other microbial counts. Growth occurred on cheese surfaces between eight and eighteen days, when the pH significantly increased. The lowest L. monocytogenes growth was found on the surface of cheeses with the lowest pH and without any core growth. Further studies will be performed to clarify the involvement of the microbial community in L. monocytogenes inhibition, in particular during the ripening period.     

The fate of Listeria monocytogenes during the manufacture of Camembert-type cheese: A comparison between raw milk and milk treated with high hydrostatic pressure

Camembert-type cheese was produced from: raw bovine milk; raw milk inoculated with 2 or 4 log CFU/ml Listeria monocytogenes; raw milk inoculated with L. monocytogenes and subsequently pressure-treated at 500 MPa for 10 min at 20 °C; or uninoculated raw milk pressure-treated under these conditions. Cheeses produced from both pressure-treated milk and untreated milk had the typical composition, appearance and aroma of Camembert. Curd and cheese made from inoculated, untreated milk contained large numbers of L. monocytogenes throughout production. An initial inoculum of 1.95 log CFU/ml in milk increased to 4.52 log CFU/g in the curd and remained at a high level during ripening, with 3.85 log CFU/g in the final cheese. Pressure treatment inactivated L. monocytogenes in the raw milk at both inoculum levels and the pathogen was not detected in any of the final cheeses produced from pressure-treated milk. Therefore high pressure may be useful to inactivate L. monocytogenes in raw milk that is to be used for the production of soft, mould-ripened cheese.

Industrial relevance

This paper demonstrates the potential of high pressure (HP) for treatment of raw milk to be used in the manufacture of soft cheeses. HP treatment significantly reduced the level of Listeria monocytogenes in the raw milk and so allowed the production of safer non-thermally processed camembert-like soft cheese.     

60-day aging requirement does not ensure safety of surface-mold-ripened soft cheeses manufactured from raw or pasteurized milk when Listeria monocytogenes is introduced as a postprocessing contaminant

Because of renewed interest in specialty cheeses, artisan and farmstead producers are manufacturing surface-mold-ripened soft cheeses from raw milk, using the 60-day holding standard (21 CFR 133.182) to achieve safety. This study compared the growth potential of Listeria monocytogenes on cheeses manufactured from raw or pasteurized milk and held for > 60 days at 4 degrees C. Final cheeses were within federal standards of identity for soft ripened cheese, with low moisture targets to facilitate the holding period. Wheels were surface inoculated with a five-strain cocktail of L. monocytogenes at approximately 0.2 CFU/ cm2 (low level) or 2 CFU/cm2 (high level), ripened, wrapped, and held at 4 degrees C. Listeria populations began to increase by day 28 for all treatments after initial population declines. From the low initial inoculation level, populations in raw and pasteurized milk cheese reached maximums of 2.96 +/- 2.79 and 2.33 +/- 2.10 log CFU/g, respectively, after 60 days of holding. Similar growth was observed in cheese inoculated at high levels, where populations reached 4.55 +/- 4.33 and 5.29 +/- 5.11 log CFU/g for raw and pasteurized milk cheeses, respectively. No significant differences (P < 0.05) were observed in pH development, growth rate, or population levels between cheeses made from the different milk types. Independent of the milk type, cheeses held for 60 days supported growth from very low initial levels of L. monocytogenes introduced as a postprocess contaminant. The safety of cheeses of this type must be achieved through control strategies other than aging, and thus revision of current federal regulations is warranted.     

Biocontrol of psychrotrophic enterotoxigenic Bacillus cereus in a nonfat hard cheese by an enterococcal strain-producing enterocin AS-48

Bacillus cereus is a food poisoning bacterium of great concern, especially in milk products. In this study, we describe the efficient control of the psychrotrophic and toxigenic strain B. cereus LWL1 in milk and in a nonfat hard cow's cheese by the enterocin AS-48 producer strain Enterococcus faecalis A-48-32 (Bac+). No viable B. cereus cells were detected after 72 h incubation in milk coinoculated with the AS-48-producing strain and B. cereus. Diarrheic toxin production was also markedly inhibited by the Bac+ strain to eightfold lower levels compared with control cultures of B. cereus. In cheeses manufactured by inoculation with a commercial starter (about 6.8 log CFU/ml) and B. cereus (about 4 log CFU/ml), the latter reached 6.27 log CFU/g after 5 days of maturation, and approximately 8 log CFU/g after 15 days. However, in cheeses made from milk inoculated with the starter along with a mixture of E. faecalis-B. cereus (2/1 ratio), counts of B. cereus decreased by approximately 1.0, 2.0, 4.32, and 5.6 log units with respect to control cheeses after 5, 10, 15, and 30 days of ripening, respectively. Growth of E. faecalis A-48-32 was associated with enterocin AS-48 production and persistence in cheese. Interestingly, growth of starter cultures was not affected by the Bac+ strain, and neither was lactic acid production. These results clearly indicate that E. faecalis A-48-32 produced satisfactory amounts of bacteriocin in cheese and support the potential use of AS-48-producing strains as culture adjuncts to inhibit B. cereus during cheese manufacture and ripening.     

Phage inactivation of Staphylococcus aureus in fresh and hard-type cheeses

Bacteriophages are regarded as natural antibacterial agents in food since they are able to specifically infect and lyse food-borne pathogenic bacteria without disturbing the indigenous microbiota. Two Staphylococcus aureus obligately lytic bacteriophages (vB_SauS-phi-IPLA35 and vB_SauS-phi-SauS-IPLA88), previously isolated from the dairy environment, were evaluated for their potential as biocontrol agents against this pathogenic microorganism in both fresh and hard-type cheeses. Pasteurized milk was contaminated with S. aureus Sa9 (about 10(6)CFU/mL) and a cocktail of the two lytic phages (about 10(6)PFU/mL) was also added. For control purposes, cheeses were manufactured without addition of phages. In both types of cheeses, the presence of phages resulted in a notorious decrease of S. aureus viable counts during curdling. In test fresh cheeses, a reduction of 3.83log CFU/g of S. aureus occurred in 3h compared with control cheese, and viable counts were under the detection limits after 6h. The staphylococcal strain was undetected in both test and control cheeses at the end of the curdling process (24h) and, of note, no re-growth occurred during cold storage. In hard cheeses, the presence of phages resulted in a continuous reduction of staphylococcal counts. In curd, viable counts of S. aureus were reduced by 4.64log CFU/g compared with the control cheeses. At the end of ripening, 1.24log CFU/g of the staphylococcal strain was still detected in test cheeses whereas 6.73log CFU/g was present in control cheeses. Starter strains were not affected by the presence of phages in the cheese making processes and cheeses maintained their expected physico-chemical properties.     

Behavior of Escherichia coli O157:H7 during the manufacture and ripening of feta and telemes cheeses

Pasteurized whole ewe's and cow's milk was used in the manufacture of Feta end Telemes cheeses, respectively, according to standard procedures. In both cases, the milk had been inoculated with Escherichia coli O157:H7 at a concentration of ca. 5.1 log CFU/ml and with thermophilic or mesophilic starter cultures at a concentration of ca. 5.3 to 5.6 log CFU/ml. In the first 10 h of cheesemaking, the pathogen increased by 1.18 and 0.82 log CFU/g in Feta cheese and by 1.56 and 1.35 log CFU/ g in Telemes cheese for the trials with thermophilic and mesophilic starters, respectively. After 24 h of fermentation, a decrease in E. coli O157:H7 was observed for all trials. At that time, the pH was reduced to 4.81 to 5.10 for all trials. Fresh cheeses were salted and held at 16 degrees C for ripening until the pH was reduced to 4.60. Cheeses were then moved into storage at 4 degrees C to complete ripening. During ripening, the E. coli O157:H7 population decreased significantly (P < or = 0.001) and finally was not detectable in Feta cheese after 44 and 36 days and in Telemes cheese after 40 and 30 days for the trials with thermophilic and mesophilic starters, respectively. The estimated times required for one decimal reduction of the population of E. coli O157:H7 after the first day of processing were 9.71 and 9.26 days for Feta cheese and 9.09 and 7.69 days for Telemes cheese for the trials with thermophilic and mesophilic starters, respectively.     

Behaviour of Listeria monocytogenes during the manufacture and ripening of Manchego and Chihuahua Mexican cheeses

The ability of Listeria monocytogenes to survive the Mexican Manchego and Chihuahua cheese-making processes and its persistence during the ripening stages of both cheeses was examined. Commercial pasteurized and homogenized whole milk was inoculated with Listeria monocytogenes (strain ATCC 19114) to a level between 2 x 10(6) and 9 x 10(6) CFU/ml. The milk was used to make Mexican Manchego and Chihuahua cheeses in a 25-l vat. Mexican Manchego cheese was ripened for 5 days and Chihuahua cheese for 6 weeks at 12 degrees C and 85% RH. Listeria present in the cheese was enumerated by diluting samples in sterile 0.1% peptone water and plating on Oxford agar. Duplicate samples were taken at each step of the manufacturing process. During the first week of ripening samples were taken daily from both cheeses. For Chihuahua cheese, samples were taken weekly after the first week of the ripening stage. During the manufacture of Mexican Manchego cheese, Listeria counts remained relatively constant at 10(6) CFU/ml, while with Chihuahua cheese there was a one log decrease in numbers (10(6) to 10(5) CFU/ml). After pressing both curds overnight, numbers of bacteria decreased in Mexican Manchego cheese to 8.2 x 10(5) but increased in Chihuahua cheese from 1.7 x 10(5) to 1.2 x 10(6) CFU/ml. During the ripening stage, counts of Listeria remained constant in both cheeses. However, since the Chihuahua cheese ripening stage is about 6 weeks, the number of bacteria decreased from 2 x 10(6) to 4 x 10(4) CFU/g. The results show that Listeria monocytogenes is able to survive the manufacture and ripening processes of both Mexican cheeses.     

Monitoring and identification of bacteria associated with safety concerns in the manufacture of São Jorge, a Portuguese traditional cheese from raw cow's milk

The aim of this study was to assess the hygienic quality of raw milk used in the manufacture of S?o Jorge, a Protected Denomination of Origin Portuguese semihard cheese, as well as to ascertain the sanitary conditions prevailing during its processing. Viable counts of Enterobacteriaceae and Micrococcaceae were accordingly obtained, pertaining to 21 independent batches (including samples of raw milk, curd, and cheeses after 1, 3, and 4 months of ripening), from 7 dairy farms. Standard plate counts (log CFU per milliliter or per gram) ranged from 6.1 to 8.6 in raw milk, whereas they ranged from 7.0 to 8.0 in 4-month-old cheeses. Viable counts of Enterobacteriaceae ranged between 5.9 and 7.0 in raw milk and between 0.0 and 1.3 in 4-month-old cheeses. Species identified within this family encompassed Klebsiella oxytoca, Klebsiella cloacae, Klebsiella pneumoniae, Enterobacter sakazakii, and Escherichia coli; Klebsiella ornithinolytica, Klebsiella terrigena, and Serratia odorifera were detected only in raw milk. No Salmonella whatsoever could be detected in any of the samples. Viable counts of Micrococcaceae ranged between 4.7 and 5.9 and between 1.3 and 3.3 in raw milk and 4-month-old cheeses, respectively. Species identified within this family encompassed Staphylococcus sciuri, Staphylococcus epidermidis, Staphylococcus saprophyticus (which was found mainly in ripened cheeses), and Staphylococcus aureus (which was not detected in 4-month-old cheeses). Accompanying physicochemical analyses included determination of moisture, salt, and pH. Statistical analyses revealed a negative correlation between salt content and viable numbers of Enterobacteriaceae in cheese, whereas in the case of Micrococcaceae, a more negative correlation was found between viable numbers and moisture content than between viable numbers and pH. The results of our study indicate, in general, poor milk handling conditions in all farms, given that the indicators total mesophile and Enterobacteriaceae counts were high, between 100- and 1,000-fold those enforced by international standards pertaining to the matrices in question. However, by the time of regular consumption (i.e., after 4 months of ripening), S?o Jorge cheeses exhibit low levels of contamination by Enterobacteriaceae and S. aureus, as well as absence of Salmonella.     

Behavior of Escherichia coli O157:H7 during the manufacture and aging of Gouda and stirred-curd Cheddar cheeses manufactured from raw milk

This study was conducted to examine the fate of Escherichia coli O157:H7 during the manufacture and aging of Gouda and stirred-curd Cheddar cheeses made from raw milk. Cheeses were manufactured from unpasteurized milk experimentally contaminated with one of three strains of E. coli O157:H7 at an approximate population level of 20 CFU/ml. Samples of milk, whey, curd, and cheese were collected for enumeration of bacteria throughout the manufacturing and aging process. Overall, bacterial counts in both cheese types increased almost 10-fold from initial inoculation levels in milk to approximately 145 CFU/g found in cheeses on day 1. From this point, counts dropped significantly over 60 days to mean levels of 25 and 5 CFU/g in Cheddar and Gouda, respectively. Levels of E. coli O157:H7 fell and stayed below 5 CFU/g after an average of 94 and 108 days in Gouda and Cheddar, respectively, yet remained detectable after selective enrichment for more than 270 days in both cheese types. Changes in pathogen levels observed throughout manufacture and aging did not significantly differ by cheese type. In agreement with results of previous studies, our results suggest that the 60-day aging requirement alone is insufficient to completely eliminate levels of viable E. coli O157:H7 in Gouda or stirred-curd Cheddar cheese manufactured from raw milk contaminated with low levels of this pathogen.     

Inactivation of spores of Bacillus cereus in cheese by high hydrostatic pressure with the addition of nisin or lysozyme

The objective of this work was to study high hydrostatic pressure (HHP) inactivation of spores of Bacillus cereus ATCC 9139 inoculated in model cheeses made of raw milk, together with the effects of the addition of nisin or lysozyme. The concentration of spores in model cheeses was approximately 6-log10 cfu/g of cheese. Cheeses were vacuum packed and stored at 8 degrees C. All samples except controls were submitted to a germination cycle of 60 MPa at 30 degrees C for 210 min, to a vegetative cells destruction cycle of 300 or 400 MPa at 30 degrees C for 15 min, or to both treatments. Bacillus cereus counts were measured 24 h and 15 d after HHP treatment. The combination of both cycles improved the efficiency of the whole treatment. When the second pressure-cycle was of 400 MPa, the highest inactivation (2.4 +/- 0.1 log10 cfu/g) was obtained with the presence of nisin (1.56 mg/L of milk), whereas lysozyme (22.4 mg/L of milk) did not increase sensitivity of the spores to HHP. For nisin (0.05 and 1.56 mg/L of milk), no significant differences were found between counts at 24 h and 15 d after treatment. Considering that mesophilic spore counts usually range from 2.6 to 3.0 log10 cfu/ml in raw milk, HHP at mild temperatures with the addition of nisin may be useful for improving safety and preservation of soft curd cheeses made from raw milk.     

High-pressure processing of Turkish white cheese for microbial inactivation

High-pressure processing (HPP) of Turkish white cheese and reduction of Listeria monocytogenes, total Enterobacteriaceae, total aerobic mesophilic bacteria, total molds and yeasts, total Lactococcus spp., and total Lactobacillus spp. were investigated. Cheese samples were produced from raw milk and pasteurized milk and were inoculated with L. monocytogenes after brining. Both inoculated (ca. 10(7) to 10(8) CFU/g) and noninoculated samples were subjected to HPP in a high-pressure food processor at 50 to 600 MPa for 5 and 10 min at 25 degrees C. Reductions in L. monocytogenes, total aerobic mesophilic bacteria, Lactococcus spp., and Lactobacillus spp. in both pasteurized- and raw-milk cheese samples and reductions in total molds and yeasts and total Enterobacteriaceae counts in raw-milk cheese samples increased with increased pressure (P < or = 0.05). The maximum reduction of the L. monocytogenes count, ca. 4.9 log CFU/g, was obtained at 600 MPa. Because of the highly inhibitory effect of pasteurization, the total molds and yeasts and total Enterobacteriaceae counts for the cheese samples produced from pasteurized milk were below the detection limit both before and after HPP. There was no significant difference in inactivation of L. monocytogenes, total aerobic mesophilic bacteria, Lactococcus spp., and Lactobacillus spp. under the same treatment conditions for the raw milk and pasteurized milk cheeses and for 5- and 10-min treatment times (P > 0.05). No significant change was detected in pH or water activity of the samples before and after HPP. Our findings suggest that HPP can be used effectively to reduce the microbial load in Turkish white cheese.     

Inhibition of a methicillin-resistant Staphylococcus aureus strain in Afuega'l Pitu cheese by the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729

Methicillin-resistant Staphylococcus aureus strains are a potential threat for food safety because foodborne illness caused by methicillin-resistant Staphylococcus aureus has been reported even though these strains were only associated with nosocomial infections until recently. This article focuses on the inhibitory effect of the nisin Z-producing strain Lactococcus lactis subsp. lactis IPLA 729 on the growth of Staphylococcus aureus CECT 4013, a methicillin-resistant strain. S. aureus was inhibited by the presence of the nisin producer IPLA 729 in buffered Trypticase soy broth, milk, and Afuega'l Pitu cheese, an acid-coagulated cheese manufactured in Asturias, Northern Spain. A reduction of 3.66 log units was observed in Trypticase soy broth at the end of the incubation period. In milk, viable counts of S. aureus were undetectable or were reduced by 2.16 log units in 24 h depending on the initial inoculum (1.8 x 10(4) and 7.2 x 10(6) CFU/ml). The staphylococcal strain was also undetected in test cheeses in which the nisin Z producer was present whereas 2 log units were detected in control cheeses at the end of ripening.     

Simplification of a complex microbial antilisterial consortium to evaluate the contribution of its flora in uncooked pressed cheese

A complex microbial consortium derived from raw milk and composed of populations classified in 4 groups (lactic acid bacteria (A), Gram positive catalase positive bacteria (B), Gram negative bacteria (C) and yeasts (D)) can contribute to the inhibition of Listeria monocytogenes in the core of an uncooked pressed cheese. To identify what groups may be involved in the inhibition, the consortium was simplified by successively omitting one group at a time. Pasteurized milk was inoculated with these more or less complex consortia and their effects on L. monocytogenes count, pH, acids and volatile compounds in the core of uncooked pressed cheese were evaluated. The growth of L. monocytogenes was the highest in cheeses prepared with pasteurized milk and only St. thermophilus. Inhibition in other cheeses was expressed by comparison with growth in these ones. All the consortia containing both lactic acid bacteria (group A) and Gram positive catalase positive bacteria (group B)--ABCD, ABD, ABC, AB--were more inhibitory than those containing lactic acid bacteria on its own (A) or associated only with yeasts (AD) or/and Gram negative (ADC). Consortia without lactic acid bacteria were weakly inhibitory or had no effect. Gram positive catalase positive bacteria alone were not inhibitory although most of the species became established in the cheeses. The Lactobacillus population (Lb. casei, Lb. plantarum, Lb. curvatus and Lb. farciminis) was predominant in cheeses (9 log CFU/g) with a higher count than Leuconostoc (7 log CFU/g) and Enterococcus (7 log CFU/g). Lactobacillus counts were negatively correlated with those of L. monocytogenes (r=-0.84 at 18 days) and with the level of D-lactic acid. There was no correlation between L. monocytogenes and Leuconostoc or Enterococcus counts. Complex consortium ABCD and AB not only had a stronger inhibitory power in cheeses than consortium AD, they were also associated with the highest levels of L-lactic and acetic acids. All cheeses inoculated with lactic acid bacteria differed from those without by higher levels of ethyl formiate, pentane and alcohols (2-butanol, 2-pentanol), and lower levels of ketones (2-hexanone, 2,3-butanedione) and aldehydes (2-methyl-butanal). Levels of 2-methyl-butanal, 2-butanol and 2-pentanol were higher in ABCD and AB cheeses than in AD cheeses. Beside their contribution to the inhibition, their effect on cheese flavour must be evaluated.     

Evaluation of biogenic amines and microbial counts throughout the ripening of goat cheeses from pasteurized and raw milk

The effect of the hygienic quality of milk on changes in microbial counts and biogenic amine content was evaluated during ripening of goat cheeses manufactured from pasteurized and raw milks at 1, 14, 30, 60 and 90 d. The original milk, rennet, curd and whey were also included in the study. The pH, salt content and extent of proteolysis in the cheese were also evaluated. Spermidine and spermine were the main amines in raw milk, while they were minor amines in cheeses. Other amines increased markedly during ripening, tyramine being the main amine in cheese made from raw milk and cadaverine and putrescine in those produced from pasteurized milk. Enterobacteriaceae counts decreased during ripening whereas those of lactic acid bacteria increased, especially lactobacilli and enterococci. Cheese made from raw milk showed higher microbial counts during ripening than those made from pasteurized milk, especially for Enterobacteriaceae and enterococci, counts being 2 or 3 log units higher. Raw milk cheese showed remarkably higher biogenic amines compared with pasteurized milk cheeses. Therefore, pasteurization of milk causes a decrease in final biogenic amine content of cheese as a result of the reduction of its microbial counts.     

Characteristics of microbial biofilm on wooden vats ('gerles') in PDO Salers cheese

The purpose of this study was to characterize microbial biofilms from 'gerles' (wooden vats for making PDO Salers cheese) and identify their role in milk inoculation and in preventing pathogen development. Gerles from ten farms producing PDO Salers cheese were subjected to microbial analysis during at least 4 periods spread over two years. They were distinguished by their levels of Lactobacillus (between 4.50 and 6.01 log CFU/cm(2)), Gram negative bacteria (between 1.45 and 4.56 log CFU/cm(2)), yeasts (between 2.91 and 5.57 log CFU/cm(2)), and moulds (between 1.72 and 4.52 log CFU/cm(2)). They were then classed into 4 groups according their microbial characteristics. These 4 groups were characterized by different milk inoculations (with either sour whey or starter culture, daily or not), and different washing procedures (with water or whey from cheese making). The farm gerles were not contaminated by Salmonella, Listeria monocytogenes or Staphylococcus aureus. Only one slight, punctual contamination was found on one gerle among the ten studied. Even when the milk was deliberately contaminated with L. monocytogenes and S. aureus in the 40 L experimental gerles, these pathogens were found neither on the gerle surfaces nor in the cheeses. Using 40 L experimental gerles it was shown that the microbial biofilms on the gerle surfaces formed in less than one week and then remained stable. They were mainly composed of a great diversity of lactic acid bacteria (Leuconostoc pseudomesenteroides, Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus hilgardii,…), Gram positive catalase positive bacteria (Curtobacterium flaccumfaciens, Curtobacterium oceanosedimentum Citrococcus spp., Brachybacterium rhamnosum, Kocuria rhizophila, Arthrobacter spp.…) and yeast (Kluyveromyces lactis, Kluyveromyces marxianus). In less than 1 min, even in a 500 L farm gerle, the gerle's microbial biofilm can inoculate pasteurized milk with micro-organisms at levels superior to those in raw milk.     

A food-grade system for production of pediocin PA-1 in nisin-producing and non-nisin-producing Lactococcus lactis strains: application to inhibit Listeria growth in a cheese model system

Food-grade heterologous production of pediocin PA-1 in nisin-producing and non-nisin-producing Lactococcus lactis strains, previously selected because of their technological properties for cheese making, was achieved. Plasmid pGA1, which contains the complete pediocin operon under the control of the strong P32 promoter and is devoid of any antibiotic marker, was introduced into L. lactis ESI 153 and L. lactis ESI 515 (Nis+). Transformation of L. lactis ESI 515 with pGA1 did not affect its ability to produce nisin. The antimicrobial activity of the pediocin-producing transformants on the survival of Listeria innocua SA1 during cheese ripening was also investigated. Cheeses were manufactured from milk inoculated with 1% of the lactic culture and with or without approximately 4 log CFU/ml of the Listeria strain. L. lactis ESI 153, L. lactis ESI 515, and their transformants (L. lactis GA1 and GA2, respectively) were used as starter cultures. At the end of the ripening period, counts of L. innocua in cheeses made with the bacteriocin-producing lactococcal strains were below 50 CFU/g in the L. lactis GA1 cheeses and below 25 CFU/g in the L. lactis GA2 ones, compared with 3.7 million CFU/g for the controls without nisin or pediocin production.     

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.     

Utilization of microfiltration or lactoperoxidase system or both for manufacture of Cheddar cheese from raw milk

The objective of the present study was to determine if application of microfiltration (MF) or raw milk lactoperoxidase system (LP) could reduce the risk of foodborne illness from Escherichia coli in raw milk cheeses, without adversely affecting the overall sensory acceptability of the cheeses. Escherichia coli K12 was added to raw milk to study its survival as a non-pathogenic surrogate organism for pathogenic E. coli. Five replications of 6 treatments of Cheddar cheese were manufactured. The 6 treatments included cheeses made from pasteurized milk (PM), raw milk (RM), raw milk inoculated with E. coli K12 (RME), raw milk inoculated with E. coli K12 + LP activation (RMELP), raw milk inoculated with E. coli K12 + MF (MFE), and raw milk inoculated with E. coli K12 + MF + LP activation (MFELP). The population of E. coli K12 was enumerated in the cheese milks, in whey/curds during cheese manufacture, and in final Cheddar cheeses during ripening. Application of LP, MF, and a combination of MF and LP led to an average percentage reduction of E. coli K12 counts in cheese milk by 72, 88, and 96%, respectively. However, E. coli K12 populations significantly increased during the manufacture of Cheddar cheese for the reasons not related to contamination. The number of E. coli K12, however, decreased by 1.5 to 2 log cycles during 120 d of ripening, irrespective of the treatments. The results suggest that MF with or without LP significantly lowers E. coli count in raw milk. Hence, if reactivation of E. coli during cheese making could be prevented, MF with or without LP would be an effective technique for reducing the counts of E. coli in raw milk cheeses. The cheeses were also analyzed for proteolysis, starter and nonstarter lactic acid bacteria (NSLAB), and sensory characteristics during ripening. The concentration of pH 4.6 soluble nitrogen at 120 d was greater in PM cheese compared with the other treatments. The level of 12% trichloroacetic acid-soluble nitrogen at 120 d was greater in RM, RME, and RMELP cheeses compared with PM, MFE, and MFELP cheeses. This could be related to the fact that cheeses made from raw milk with or without LP (RM, RME, and RMELP) had greater levels of NSLAB compared with PM, MFE, and MFELP cheeses. Cheeses at 60 d, as evaluated by 8 trained panelists, did not differ in bitterness, pastiness, or curdiness attributes. Cheeses at 120 d showed no differences in acid-taste, bitterness, or curdiness attributes. Sensory analysis at 60 d showed that PM and MFELP cheeses had greater overall sensory acceptability than RM and RME cheeses. The overall sensory acceptability of the cheeses at 120 d showed that PM, MFE, and MFELP cheeses were more acceptable than RM and RME cheeses.