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,1
* Nancy-Université, Laboratoire de Science et Génie Alimentaires (LSGA), Ecole Nationale Supérieure d’Agronomie et des Industries Alimentaires-Institut National Polytechnique de Lorraine (ENSAIA-INPL), 2, Avenue de la Forêt de Haye, BP 172, F-54505, Vandoeuvre-lès-Nancy Cedex, France
Institut Universitaire de Technologie (IUT) Nancy-Brabois, Le Montet, F-54600 Villers-lès-Nancy, France
1 Corresponding author: catherine.cailliez{at}ensaia.inpl-nancy.fr
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ABSTRACT |
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Key Words: Carnobacterium • polymerase chain reaction • French soft cheese
In the manufacture of soft cheeses, lactic acid bacteria are involved in all phases of cheese making. Initially present as natural microbiota or added as starter cultures, they actively participate in the development of flavor and texture in a wide range of cheeses (Giraffa, 2004; Marilley and Casey, 2004). Carnobacterium is a lactic acid bacterium that is not used as a starter because of its nonaciduric and psychrotrophic properties. Indeed, it cannot participate actively in the first phase of fermentation, but its growth can be induced during ripening as pH values increase and after a cold-storage period. Studies have shown that the Carnobacterium genus is predominant in French surface-mold-ripened soft Brie cheeses (Millière et al., 1994; Millière and Lefebvre, 1994). This genus has frequently been associated with the spoilage of seafood and meat products (Pilet et al., 1994; Connil et al., 1998; Paludan-Muller et al., 1998; Dalgaard et al., 2003). However, the bacteriocin-production ability of Carnobacterium genus may be considered useful in preventing the growth of food-borne pathogens such as Listeria (Duffes et al., 1999; Brillet et al., 2005; Laursen et al., 2005).
The aim of this work was to determine whether Carnobacterium is a ubiquitous genus present in many traditional soft cheeses and whether a diversity of species exists. Samples were taken from cheeses manufactured from cow’s, goat’s, and ewe’s milk (raw or pasteurized) in different French provinces during 2 periods of the year. A previous study showed that genus-specific primers could be applied for the detection of this genus in cheeses (Cailliez-Grimal et al., 2005). Species-specific primers (Rachman et al., 2004) were then tested and applied to the cheese samples.
Strains were purchased from different collections (Table 1
) and were maintained at –20°C in 15% (vol/ vol) glycerol. They were subcultured in trypticase-soy medium (TS; Biomerieux, Craponne, France) supplemented with 0.6% (wt/vol) yeast extract (YE; Biokar Diagnostics, Beauvais, France) and incubated at their optimal growth temperature (Table 1).
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), were analyzed and their Carnobacterium flora enumerated by plate count and by PCR (see below). Fractions (10 g) of whole cheese (surface and interior) were homogenized in 90 mL of citrate buffer (0.5 M, pH 7.0). The whole suspension was submitted to a serial decimal dilution. Improvements in the detection of Carnobacterium spp. from cheese can be obtained on nonselective media (TS-YE agar or broth) with high pH values (pH 9; Holzapfel, 1992) supplemented with 40 mg/L of nalidixic acid (Sigma, St. Louis, MO) and 5 mg/L of amphotericin (Sigma) and incubated under psychrotrophic conditions (at 7°C for 10 d). For the plate count method, each serial decimal dilution was plated using spiral plating (Whitley Automatic Spiral Plater, WASP 2; AES Laboratoire, Bruz, France). For the enrichment method, 10 g of each cheese was mixed with 90 mL of TS-YE broth medium (described above). The interior pH of the cheese was measured with an electrode pHM210 standard pH meter (Radiometer Analytical, Villerbanne, France).
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Template DNA was obtained by the boiling method (Yost and Nattress, 2000; De Medici et al., 2003). One isolated bacterial colony was picked from the modified TS-YE agar plates with a sterile toothpick and resuspended in 50 µL of sterile ultrapure water (Easypure, RF; Fisher Bioblock, Illkirch, France). From the TS-YE broth culture, a volume of 50 µL was centrifuged in a microcentrifuge at 8,000 x g for 3 min and the cell pellet was resuspended in 100 µL of sterile ultrapure water. These suspensions were boiled for 5 min, and the cellular debris was then pelleted by centrifugation at 8,000 x g for 3 min. Ten microliters of each supernatant was retained for the PCR reaction.
Fractions of whole cheese (10 g) were homogenized as described above. A 1,000-fold-diluted matrix was used for the PCR assay. For the enrichment method, 10 g of each cheese was mixed with 90 mL of enrichment medium, as described above. After 10 d at 7°C, a 1,000-fold-diluted sample was used for PCR.
Deoxyribonucleic acid sequences of the primers (obtained from Eurogentec, Herstal, Belgium) are indicated in Table 3. The amplification was carried out in a thermal cycler iCycler System (Bio-Rad, Marnes-la-Coquette, France). The reaction mixture (50 µL) contained 25 µL of the ready-to-use master mixes (Eurogentec) and 10 µL of the sample (described below), completed to 50 µL with sterile ultrapure water. The PCR program was as follows: 5 min at 95°C for primary DNA denaturation, followed by a series of 30 cycles, each of which comprised 30 s at 95°C (denaturation), 30 s at the corresponding annealing temperature (see Table 3), 30 s at 72°C (extension), and a 7-min final extension step at 72°C. Products were stored at 4°C until analysis. Aliquots (20 µL) of the amplified products were subjected to electrophoresis in 1.5% (wt/vol) agarose gels (Agarose DNA grade; Euromedex, Souffelweyersheim, France) in TAE buffer (40 mM Tris acetate, 1 mM EDTA, pH 8.2). Gels were stained with ethidium bromide (5 µg/mL) and visualized under UV light (312 nm). A 100-bp DNA ladder polymer was used as a molecular mass marker (Eurogentec).
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Among the 30 cheeses tested, only DNA extracted from 5 cheeses (Brique de Jussac, Petit Munster Val de Weiss, Petit Munster fermier, Camembert Réo, Epoisses) gave an amplicon of 340 bp with Carnobacterium genus-specific Cb1-Cb2R primers. From these cheeses, after 10 d at 7°C, the enumeration of plates showed population levels of Carnobacterium spp. 
105 cfu/g (Table 2
). Colonies on modified TS-YE agar were nonpigmented, convex, circular, and smooth.
After the enrichment period, with the same primers Cb1-Cb2R, DNA extracted from 5 other cheeses [Pérail, Picodon, Petit Munster (Petits amis), Camembert Is-signy, Petit Livarot] also gave one band of 340 bp (Table 2). In this case, the amount of Carnobacterium was 
102 cfu/g.
With the species-specific primers, DNA extracted from these 10 cheeses showed a positive response with the C. maltaromaticum-specific primers Cpis-23S-7 (Figure 1A). With the 5 other species-specific primers, no response was obtained (example with the specific-primer of Carnobacterium divergens; Figure 1B).
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A comparison of their biochemical characteristics is shown in Table 4. Among the 50 isolates from the 10 cheeses (5 colonies isolated from each positive cheese), results from the API 50 CHL showed 6 differences in the fermentation patterns. All isolates were consistent in their ability to produce acid from the following substrates normally fermented by carnobacteria: ß-gentiobiose, saccharose, N-acetyl glucosamine, amygdalin, arbutin, esculin, salicin, maltose, cellobiose, D-mannose, D-fructose, D-glucose, and ribose (Table 4).
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). Isolates from Camembert Réo (C. maltaromaticum reo), Epoisses (C. maltaromaticum epoisses), and Petit Munster (Petits amis; C. maltaromaticum amis) exhibited the same pattern, whereas Petit Munster (Fermier and Val de Weiss; C. maltaromaticum fermier and C. maltaromaticum weiss) and Isigny (C. maltaromaticum isigny) were identical. The other isolates, Pérail (C. maltaromaticum perail), Petit Livarot (C. maltaromaticum livarot), Brique de Jussac (C. maltaromaticum jussac) exhibited different patterns. The ability to produce antagonistic substances against L. monocytogenes CIP 82110 was found with isolates from 3 different cheese samples, 1 from pasteurized ewe’s milk (Pérail) and 2 from raw cow’s milk (Réo and Epoisses). The diameter of inhibition against L. monocytogenes CIP 82110 for the 3 isolates tested was 19 ± 1.0 mm.
The primers Cb1-Cb2R were used successfully to conduct a rapid screening of the genus in a wide variety of soft French cheeses. Thirty cheeses of 19 types that differed in the methods used in manufacture and ripening were analyzed. The presence and the number of Carnobacterium in these cheeses, between 1 and 107 bacteria/g, were compatible with the amounts found previously (Millière et al., 1994; Millière and Lefebvre, 1994). This genus has been detected in cheeses manufactured in various departments in the East (Vosges, Bas-Rhin, Haute-Marne), in the West (Manche, Calvados), and in the South (Aveyron, Drôme, Haute-Loire) of France.
Ten positive samples with Cb1-Cb2R primers were also positive with the species-specific primers of C. maltaromaticum. The other Carnobacterium species (except C. divergens) were isolated from others sources, so it was not surprising that they were absent from the cheese (Collins et al., 1987; Franzmann et al., 1991; Joborn et al., 1999; Holley et al., 2002; Pikuta et al., 2005). On the other hand, the detection of C. divergens, previously found in cheeses (Millière and Lefebvre, 1994; Morea et al., 1999) was expected.
A previous study showed Carnobacterium only in cow’s milk cheese (Millière et al., 1994). In this work, ewe’s and goat’s milk cheeses also contained this genus. The pH values of cheeses tested varied from 5.6 to 8.1, which are in the pH range required for the growth of the genus Carnobacterium. No correlations were found among the ripening of cheese, use of raw or pasteurized milk, and C. maltaromaticum counts. Cheeses containing Carnobacterium did not differ significantly in taste or off-flavors from the same cheeses without Carnobacterium (Camembert, Munster). Eight cheeses were AOC cheeses, a designation that guarantees the quality of the product. The role of the Carnobacterium in these cheeses was therefore unclear.
Isolates from 3 different cheeses (1 from pasteurized ewe’s milk, 2 from raw cow’s milk) exhibited anti-L. monocytogenes activity. In raw milk soft cheese manufacturing, L. monocytogenes could be present at the end of the ripening period, with the rind being the preferential site of colonization. After food processing, this species can potentially grow in refrigerated products stored for extended periods. The anti-Listeria property of isolates from industrial cheeses should then be an advantage. Assuming that no product defect is related to the presence of Carnobacterium, it could be interesting to investigate their positive influence in the preservation of cheese.
Received for publication February 22, 2006. Accepted for publication October 21, 2006.
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