246 Journalof FoodProtection, Vol. 64, No. 2, 2001, Pages 246– 251 Copyright q,International Association forFood Protection RoquefortineC Occurrence inBlue Cheese CARLO FINOLI, 1 ANGELA VECCHIO, 2* ANTONIETTA GALLI, 2 AND IVAN DRAGONI 3 1Dipartimentodi Ingegneria e TecnologieAgro-Forestali, Universita `degliStudi di Palermo, viale delle Scienze 10,90128 Palermo, Italy; 2Dipartimentodi Scienze eTecnologieAlimentari e Microbiologiche,Universita `degliStudi di Milano, via Celoria 2, 20133 Milano, Italy; and 3Istitutodi Ispezione degli Alimenti di Origine Animale, Universita `degliStudi di Milano, via Celoria 6, 20133 Milano, Italy MS00-186: Received 9June2000/ Accepted 6September2000 ABSTRACT Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/2/246/1671860/0362-028x-64_2_246.pdf by guest on 27 September 2021 Severalstrains of Penicillium areused for the production of mold-ripenedcheeses, and some of themare able to produce mycotoxins.The aims of the research were the determination of roquefortine C andPR toxinin domestic and imported blue cheeses,the identi cation of thepenicillia used as starter,and the investigation of their capacity for producing toxins in culture media.Roquefortine C wasalways found in the cheeses at levels ranging from 0.05 to 1.47 mg/ kg,whereas the PR toxinwas neverfound. The identi cation of thefungal strains present in thedomestic cheeses included Penicilliumglabrum, Penicillium roqueforti, and Penicilliumcyclopium inthe Gorgonzola ‘ ‘dolce’’ and Penicilliumroqueforti inthe Gorgonzola ‘ ‘naturale’’; inone case, the presence of Penicilliumcrustosum wasobserved. The strains isolated from the foreign cheeses belonged to P.roqueforti. Thestrains were able to producebetween 0.18 and 8.44 mg/ literof roquefortinein yeastextract sucrose medium andbetween 0.06 and 3.08 mg/ literand less than 0.05 mg/ literwhen inoculated in milk at 20 8Cfor14 days and 4 8C for 24 days,respectively. Linear relations between production of roquefortinein culture media and cheeses did not emerge. PR toxin rangedfrom less than 0.05 to 60.30 mg/ literin yeast extract sucrose medium and was produced in milk at 20 8C from only onestrain. The low levels and the relatively low toxicity of roquefortine make the consumption of blue cheese safe for the consumer. Mostpenicillia reside in soil or onvegetables, and their aspatulin, penicillic acid, mycophenolic acid, isofumigo- presencein foods usually is connected with an accidental clavinA andB, roquefortine C, and PR toxin (11). The contaminationduring production; in some cases, such as presentresearch has been limited to the last two toxins be- fermentedsalami and ripened cheese, they are addedto causethey are the mycotoxins more easily produced from developspeci c organolepticcharacteristics (7). Some thecultures used as starters for theproduction of Gorgon- cheesesare manufactured with strains of Penicilliumro- zola.Roquefortine has neurotoxic properties (34); PR toxin queforti, i.e.,the blue-veined cheeses such as Gorgonzola, inhibitsRNA andprotein synthesis (18, 19) andis carci- Roquefort,Bleu de Bresse, Cabrales, Danablu, and Edel- nogenicfor rats (22) andmutagenic for Salmonella Typhi- pilzka¨se,and strains of Penicilliumcamemberti are used for murium (33). whitesurface mold cheeses such as Camembert, Brie, and Fungalstrains used as starters in the dairy industry Coulommiers.Once these molds were naturallyfound in were ableto produce roquefortine in vitro (10, 26). The themilk and cheese, whereas today spore suspensions are presenceof roquefortinewas foundalso in cheeses (15, 26, directlysprayed on the surface of cheeses or added to the 28, 32, 35).PRtoxin was isolated rst inthe extracts of milkor curd. The most important biochemical characteris- culturemedium of P.roqueforti by Wei et al. (36) in 1973. ticsare the proteolytic activity, which plays a fundamental Itwas neverfound in blue cheeses (9,23, 26, 27) , because roleduring blue cheese ripening, and the lipolytic activity, itreacts with nitrogenous compounds, converting itself to whichinduces triglyceride hydrolysis and subsequent b- PR-imine,which has a lowertoxicity (1).Siemensand Za- ketoacidand methylketone formation. wistowski (30),ina studyon derivatives of the PR toxin P.camemberti and P.roqueforti were usedwithout incheeses, reported PR-imine at levels of 19 to 42 mg/kg particularproblems in cheesemaking until Gibel et al. (14) in50 ofthe60 samplesanalyzed. The authors demonstrated publisheda studyin whichthey assumed that such cultures thatin vitro, in the presence of bovine serum, the conver- couldrepresent a riskfor consumerhealth because of the sionof thisderivative in themore dangerous original parent formationof toxic metabolites. The authors suggested that was possible.PR toxinwas foundin culture media up to P.camemberti extractsseemed to be able to induce carci- 200mg/ liter (9). nogeniceffects in mice. P.roqueforti, atpresentreclassi ed Gorgonzolabelongs to the group of the blue cheeses afterpolymerase chain reaction identi cation into three spe- havingin common the speci c characteristicof the for- cies named P.roqueforti,Penicillium carneum, and Peni- mationof bluegreenish veins in their interior ,producedby cilliumpaneum (5) ,isable to produce several toxins, such mold of the P.roqueforti type,with tonality of moreor less intensecolor (12,13, 24, 25) .It isa cheeseof ‘ ‘denomin- *Authorfor correspondence. Tel: 0039022367181; Fax: 0039022361576; azionedi origine’’ (6),originatingfrom Gorgonzola,a town E-mail: [email protected]. nearMilan, and its production is governed by appropriate J.FoodProt., Vol. 64, No. 2 ROQUEFORTINE COCCURRENCE INBLUECHEESE 247 regulationsestablished by theConsortium for theProtection TABLE 1. Mycotoxinrecovery percentages and standard devia- ofGorgonzola Cheese. Only the dairies authorized by the tions consortiumcan market Gorgonzola cheese with the brand Added name ‘‘g,’’whichguarantees its authenticity. Depending on amount Cheese YES medium Milk themanufacturing processes, two varieties of Gorgonzola Mycotoxin (mg/kg) (%) (%) (%) canbe obtained:a traditionaltype, ‘ ‘Gorgonzolanaturale,’ ’ Roquefortine25 97 6 5.2 61 6 5.7 53 6 4.2 witha consistingand homogenous texture of the paste, 200 101 6 4.3 65 6 5.1 62 6 4.8 sometimesslightly chalky, a strongtaste, with molds of a shininggreen color ,verydeveloped and uniformly distrib- PR toxin 500 45 6 4.7 65 6 2.3 48 6 3.7 utedin the paste; and a creamytype, ‘ ‘Gorgonzoladolce,’ ’ 1000 46 6 3.4 69 6 1.8 50 6 2.6 witha soft,smeared texture of the paste, sometimes with smalleyelike holes, a sweettaste, with green-colored molds littledeveloped and uniformly distributed in thepaste. The matography(TLC) andin high-performance liquid chromatogra- 7 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/2/246/1671860/0362-028x-64_2_246.pdf by guest on 27 September 2021 productionof Gorgonzolacheese is ofabout4.2 3 10 kg/ phy(HPLC) eluentfor roquefortine (3) andin methanol for PR yearand 28% is exported (data from theConsortium for toxinfor HPLC analysis.Working standard solutions of roque- theProtection of Gorgonzola Cheese). fortineand PR toxinwere prepared from crystalline toxins (Sigma Theobjectives of this study were toinvestigate the ChemicalCo., St. Louis, Mo.). The purity of the standards was ability of Penicillium speciesto produce mycotoxins in blue determinedin absolute ethanol by UV analysisusing a spectro- cheeses,to isolateand identify the molds, and to determine photometerUV/ VIS Lambda15 Perkin Elmer (Perkin Elmer ifthesemolds produce mycotoxins in variousculture media Corp.,Norwalk, Conn.) and compared with reported spectra (8). incubatedin different conditions. BeforeHPLC analysis,all samples were analyzed by TLC. MATERIALS AND METHODS TLC analysis. HPTLC Kieselgel60 F 254 andFertigplatten Kieselgel60 (Merck) (10 by 10 cm), activated at 110 8C for 2 h, TwentyItalian Gorgonzola cheeses, supplied by the Consor- wereused for TLC ofroquefortine and PR toxin,respectively. tiumfor the Protection of Gorgonzola cheese with technical in- Thedevelopment was monodimensional using, in sequence, n- formation,and 10 foreign blue cheeses, purchased in various Eu- hexane,diethyl ether, and chloroform– methanol– ammonium hy- ropeancountries, have been analyzed. droxide(9:1:0.1) for roquefortine and n-hexane, diethyl ether, and toluene–ethyl acetate– acetic acid (6:4:1) for PR toxin.After de- Isolationand classication of penicillia. Themolds were velopment,plates were air dried. The roquefortine plates were isolatedafter extracting a portionof thegreen zone of the cheese observedunder UV light(UVLS-58 mineral light multiband 254 witha sterileneedle. The mycelium was transferred to a maltagar to366 nm, San Gabriel, Calif.) at 254 nm, and the toxin appeared plateand incubated at 20 8Cfor7 daysaccording to Pitt and Hock- likea darkspot. After spraying plates with 1% CeSO 4 in 6 N ing (21).Thepurity of the strain isolated was tested with a mi- sulfuricacid, roquefortine formed an orange spot (26). For the croscope,and the strain was kept on malt agar slant at 4 8C. The detectionof PR toxin,plates after exposure to UV lightat 254 identication and classi cation of molds were carried out using nmfor about 1 minwere observed at 366 nm; the dark blue thefollowing media: malt extract agar ,25%glycerol nitrate agar , uorescentspot changed to green uorescence (4). andCzapek yeast extract agar according to Pitt and Hocking (21). HPLC analysis. Roquefortineanalysis was carried out ac- Mycotoxinproduction. Thecapability of