1563

Journal of Food Protection Vol. 71, No. 8, 2008, Pages 1563-1571copyright CO, internationa l Association for Food Protection

60-Day Aging Requirement Does Not Ensure Safety ofSurface'-Mold-Ripened Soft Cheeses Manufactured from Raw orPasteurized Milk When Listeria monocytogefles Is Introduced as

a Postprocessing ContaminantDENNIS J. D'AMICO, MARC J. DRUART, AND CATHERINE W. DONNELLY*

Depar!nent of Nutrition and Food Sciences, University of Vermont, Burlington. Vermont 05405, USA

MS 07-588: Received 5 November 2007/Accepted 13 February 208

ABSTRACT

Because of renewed interest in specialty cheeses, artisan and farmstead producers are manufacturing surface-mold-ripenedsoft cheeses from raw milk, using the 60-day holding standard (21 CFR 133.182) to achieve safety. This study compared thegrowth potential of List eria inonocyto genes on cheeses manufactured from raw or pasteurized milk and held for >60 days at4°C. Final cheeses were within federal standards of identity for soft ripened cheese, with low moisture targets to facilitate theholding period. Wheels were surface inoculated with a five-strain cocktail of L. monocylogenes at approximately 0.2 CFUI

cm2 (low level) or 2 CFU/cm 2 (high level), ripened, wrapped, and held at 4°C. Listeria populations began to increase by day28 for all treatments after initial population declines. From the low initial inoculation level, populations in raw and pasteurizedmilk 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, cheesesheld 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.

According to the Centers for Disease Control and Pre-vention, Listeria inonocyto genes infection had the secondhighest case fatality rate (20%) and the highest hospitaliza-tion rate (92.2%) of all infections caused by foodbornepathogens (29). Numerous outbreaks and sporadic cases oflisteriosis have been linked to the consumption of soft freshcheeses (2, 5, 10, 24, 28) and surface-ripened soft cheeses(16, 37). As a subcategory of soft cheese, surface-ripenedsoft cheeses undergo further ripening through the externalgrowth of yeast, mold, and/or bacteria.

'Surface-mold-rip-

ened soft cheeses include the well known Brie and Cam-embert types. According to a recent risk assessment (45),a moderate health risk is associated with the consumptionof fresh and ripened soft cheeses, and a high health risk isassociated with consumption of unripened soft cheese. In2005, the U.S. Food and Drug Administration (FDA) issueda health advisors' warning consumers not to consume softcheeses made from rs.' milk (42). Although thi warningwas focused 'on illegally produced Mexican-style cheeses,the lack of stañdárdof identity for the various cheese va-rieties can create much confusion soft cheeses comprise awide range of cheese types ranging from Hispanic or Mex-ican-style cheese's ch as unripened queso fresco to sur-face-ripened varieties such as Camembert types, each with

* Author for correspondence. Tel: 802-656-8300: Fax: 802-656-0001E-mail: catherine.donnelly@uvm.cdU.

various associated risks. Specific standards of identity arecritical to food safety because the characteristics of the spe-cific cheese variety dictate the potential for growth and sur-vival of microbial pathogens. Many hard cheeses madefrom raw milk and aged for 60 days or more have an excellent food safety record because of the interaction of fac-tors such as pH, salt content, and water activity that renderthese cheeses microbiologically safe (6). . Current regula-tions (43) permit the manufacture of soft ripened cheesesfrom raw milk provided that these cheeses are aged for 60days at ^1.67°C (35°F) to ensure safety. Raw milk cheesesthat have not been properly aged are illegal in the UnitedStates and cannot be imported. Because of renewed interestin specialty cheeses, domestic artisan and farmstead pro-ducers are manufacturing surface-mold-ripened soft cheesesfróhiiaw milk using the 60-day holding standard to achievesafety. In Europe, Protected Denomination of Origin (PDO)cheeses such as Camembert de Normandie are required tobe manufactured from raw milk to receive PDO status.Safety is not attained through aging (although these cheesesare aged a minimum of 21 days) but through regulationsspecified in European Union (EU) directives (e.g., no.2073/2005), which establish microbiological criteria forcheeses made from raw or thermally treated milk (9).

Although raw milk may contain L. monocyto genes, theoverall prevalence is low, with sporadic contamination andseasonal variability. When L. ?nonocvrogenes is present,

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levels in bulk tank milk are typically below 3 CFU/ml (27,30). Because L. monoctogenes is primarily an environ-mental pathogen, the primary risk to cheese safety is frompostpasteurization environmental contamination from thecheese making and/or aging environment (19). Pasteurizedor otherwise processed milk (4) and cheeses (8, 17, 19, 36)can contain L. monocvtOgefleS due to postprocessing con-tamination that can occur during manufacture, ripening, orwashing (19). Recontamination of foods with pathogenicbacteria may be a frequent and important cause of food-borne outbreaks in which postprocessing contamination isbelieved to be the cause of major outbreaks of listeriosisattributed to the consumption of soft cheese (12, 17). Theconcern with surface-mold-ripened soft cheese is not nec-essarily the survival of L. monocvtogenes but the growthpotential during aging and holding following increases inpH.

Unlike other pathogens, L. monocytogenes can surviveand continue to grow during refrigerated storage becauseof its psychrotrophic nature. The growth of L. monocvto-genes on the surface of Camembert-type cheese made frompasteurized milk has been previously investigated (1, 15,38). The use of relatively high inoculation levels (-500CFU/20 cm 2, 2 to 4 log CFU per sample) and the repeatedsampling and experimental aging conditions, including thesealing of samples in plastic containers or foil, do not rep-resent conditions commonly employed during commercialcheese manufacture. From a risk assessment perspective,realistic inoculation levels and conditions are important be-cause initial concentrations and conditions affect the be-havior of pathogens such as L. monocvto genes in cheese(13, 25, 32). Unlike pasteurized milk, raw milk may havea protective effect against pathogenic bacteria (6). Gay andAmgar (13) investigated the fate of L. monocytogenes add-ed to milk during the manufacture and ripening of Cam-embert cheese manufactured from either raw or pasteurizedmilk. These authors found that the lag phase and time to a10 CFU increase in L. monocytogenes levels were twiceas long in raw milk Camembert cheese than in its pasteur-ized counterpart, likely because of the microbiological com-position of raw milk, especially the presence of thermo-philic lactobacilli and yeasts.

Although L. monocytogenes has been studied exten-sively in both food and processing environments and ex-tensive risk assessments are available, limited data areavailable on the survival of L. monocytogenes introducedas a postprocessing contaminant on surface-mold-ripenedsoft cheese. Environmental contamination of food productsis an important factor to consider in future assessments suchas the Joint FDA—Health Canada Public Health Risk As-sessment regarding L. monocytogenes in soft ripenedcheese, which is currently underway. In the present study,the growth potential of L. monocytogenes introduced as apostprocessing contaminant on surface-mold-ripened softcheese was examined for the effects of (i) two initial in-oculation levels, (ii) the use of pasteurized versus raw milk,and (iii) ripening for 70 days.

MATERIALS AND METHODSCultures. Five L. inonocvtogenes strains were used in this

study: FSL R2-499, FSL N3-022, and FSL Jl- l 19 (Dr. MartinWiedmann, Department of Food Science, Cornell University, Ith-aca, N.Y.) (II) and F5069 and F5027 (Dr. Catherine Donnelly,Department of Nutrition and Food Sciences, University of Ver-mont, Burlington). Frozen (-78°C) stock cultures were inoculatedinto 9 ml of Trypticase soy broth with 0.6% yeast extract andincubated at 30 ± 1°C for 18 h for two subsequent transfers beforeuse. Viable numbers of L. inonocvtogenes in suspension for eachculture were determined after serial dilution from aerobic platecounts on Petritilm AC films (3M Microbiology, St. Paul, Minn.)incubated at 32 ± 1°C for 48 ± 2 h. Based on consecutive platecounts, equal proportions of cells from each culture were com-bined as a cocktail yielding — 3 X 109 cells per ml. Freeze-driedstarter cultures of Laciococcus lactis subsp. lactic, L. lacris subsp.cremoris, L. lacris subsp. lactis biovar diacetvlactis, and Strep-tococcus salic'arius subsp. thermophilus and ripening cultures ofKluvveromvces lactic, Geotrichum canthdum, and Penicilliwncandic/um (EZAL MAOI I and MAO 19. MD99, T050, KL71,GEOI7. and SAM3; Danisco, Copenhagen. Denmark) were storedat —35°C until used.

Milk collection. Approximately 100 liters each of raw andpasteurized (nonhomogenized) milk were obtained from a localdairy plant in ID-gal (37.8-liter) sanitized stainless steel milk cans.Raw milk samples were collected before entering the pasteurizer,and pasteurized milk samples were high-temperature short-timepasteurized (72°C for 15 s) and collected bypassing the homoge-nizer. Mean temperatures of pasteurized and raw milk duringtransport (-90 mm) were 4.83 ± 0.70 and 6.76 ± 0.26°C, re-spectively, with maximum values of 6.89 ± 0.87 and 7.78 ±0.87°C, respectively. Milk cans were refrigerated (3 ± 1°C) im-mediately upon arrival until milk was used the next morning (22± 2 h). L. monocytogenes was not detected in either raw or pas-teurized milk through selective enrichment in Lisieria enrichmentbroth at 30°C for 24 h and then plating of 0.1 ml on CHROMagarListeria plates (CHROMagar, Paris, France) incubated at 37°C for24 h.

Cheese manufacture. Surface-mold-ripened soft cheese wasmanufactured from 100 liters each of raw and pasteurized milk inseparate vats on three different days as three independent trials.Each trial yielded — 35 cheeses of each milk type for a total of— 70 cheeses. From the three trials, a total of — 210 cheeses weremanufactured in this study, 120 of which were used for micro-biological analyses. A 45-min delay between raw and pasteurizedmilk cheese manufacture was employed to allow enough time be-tween steps to avoid overlap and to allow thorough cleaning andsanitizing of utensils. Cheeses were manufactured with a slightlylower moisture content to endure the 60-day holding period, ex-tending the ripening so that cheeses were in a consumable stateon day 60. Temperature and acidification profiles were monitoredand recorded with a temperature and pH data logger (model DO9505, Delta Ohm. Padua, Italy). Milk samples for compositionaland microbiological analyses were taken from the filled vat,chilled and refrigerated (3°C), and used within 24 h. For cheesemanufacture, refrigerated raw and pasteurized milk (pH 6.7 to 6.8;titratable acidity, 0.16 to 0.17%) was added to separate sanitizedpilot scale double-O style steam-jacketed cheese vats (KuselEquipment Co., Watertown, Wis.) at the University of Vermontpilot plant. Rehydrated glucono delta-lactone (GDL; 30 g1100 li-ters; Jungbunzlauer S.A., Marckolsheim, France) and CaCl 2 (10mlIlOO liters) were added to filled vats. GDL is used extensively

Food Prot., Vol. 71, No. 8SURVIVAL OF L. MONOCYTOGENES ON SURFACE-MOLD-RIPENED SOFT CHEESE1565

throughout the EU as an acidulant in the manufacture of surface-mold-ripened soft cheeses and was therefore added in the manu-facture of our experimental cheese to produce a food matrix oftypical chemical composition. In the United States, GDL is a gen-erally recognized as safe additive (21 CFR 184.1318) and is ap-proved for use as a pH control agent. as defined in 21 CFR170.3(o)(23) in some foods. The action of GDL helps graduallydecrease the pH during the lag period of the freeze-dried culture,providing consistent and controlled acidity development beforethe addition of rennet. Milk temperature was raised to between 36and 39°C and the lactic starter and ripening cultures were added:starter culture included 2.25 Danisco culture units (DCU) ofMAO1I and MAOI9. 0.25 DCU of MD99. and 1.5 DCU ofTA054 and TA050: ripening culture included 0.3 DCU of KL7I.0.2 DCU of GEOI7. and 0.6 of DCU SAM3. The temperaturewas maintained for 30 to 60 min until the target pH of 6.5 to 6.55was reached. Once proper p1-I was attained, calf rennet (20 ml!100 liters. 1:15,000: New England Cheesemaking Supply, Ash-field. Mass.) was added to the vat, and the milk mixture wasstirred for approximately 45 s. Cutting time was determined bymultiplying the time of flocculation by 3. Once desired firmnesswas reached, the coagulum was cut into curds measuring 2 by 2cm with sanitized stainless steel curd knives. Curds were allowedto settle for 5 mm, stirred for 5 mm, allowed to rest for 5 mm,and stirred for another 5 mm. After an additional 5 min of rest.3017c of the whey was removed. The curds were then gently stirredand transferred to plastic hoops (model M5-3864, Fromagex, Ri-mouski, Québec, Canada) to drain and turned regularly at 1.5. 3.and 8 h after hooping. The initial draining temperature of 27°Cwas slowly decreased to 21°C at the time of dehooping. The fol-lowing morning, once the target pH of 4.8 to 4.9 was reached,cheeses were removed from hoops and placed in saturated brine(270 g!liter NaCl, pH 4.9. 12 to 13°C) for 70 mm.

Drying, aging, and holding conditions. Upon removal fromthe brine, cheeses were transferred to racks that had been sanitizedwith 70% isopropyl alcohol for the drying phase. Drying and rip-ening were conducted in a lab scale aging chamber constructedfrom a modified wine refrigerator (model WC491BG, Avanti. Mi-ami, Fla.). A small ultrasonic misting humidifier (Sunbeam model697, Jarden Consumer Solutions, Boca Raton. Fla.) providedmoisture by atomizing deionized water into very fine cool mistparticles that are easily absorbed in the air without affecting tem-perature. Humidity levels were controlled with a repeat cycle timeswitch (Talento 121. Grasslin Controls Co., Mahwah. N.J.) set topredetermined on-off cycles to maintain desired humidity. For thedrying phase, the aging chamber was maintained at 14 to 15°C(57 to 59°F) and 80 to 85% relative humidity for 24 h. The rip-ening phase was conducted at 13 to 14°C (55 to 57°C) and 90 to95% relative humidity for 12 1 days. Once proper surface moldgrowth was achieved, cheeses were wrapped in 225- by 225-mmmultiplex wrapping paper (PVCF-23. White Mould. Fromagex)and transferred to a standard refrigerator at 4 ± 1°C until day 70.

Bacterial inoculation. Cocktails of L. monocyto genes wereserially diluted to obtain either low-level (10 to 20 CFU/ml) orhigh-level (100 to 200 CFU/ml) inocula. After the drying phase,cheeses were removed, and one surface (100 cm 2) was inoculatedwith I ml of the appropriate inoculum (high or low) using a sterileL-spreader to achieve low and high contamination levels of 0.1to 0.2 and I to 2 CFU/cm 2 , respectively. The cheeses from eachtrial (n = 40) were assigned to one of four treatments. Treatmentsconsisted of raw milk, low-level inoculum (RL. n = 10), rawmilk, high-level inoculum (RH, ii = 10), pasteurized milk, low-level inoculum (PL. n = 10), and pasteurized milk, high-level

inoculum (PH. n = 10). Inoculated cheeses were allowed to dryin a laminar flow hood for -10 mm. The process was then re-peated for the remaining opposite surface. Inoculated cheeses werethen placed in the lab scale aging chamber as previously describedfor ripening.

Bacterial enumeration. The following samples were ana-lyzed: milk, cheese after brining and drying step (19 h), andcheese during ripening on days 7 and 14. Cheese samples alsowere taken during holding at 4°C on days 21, 28, 35, 42, 49, 56,63. and 70. Milk standard plate counts (SPCs) for both raw andpasteurized milk were determined using Petrifllm aerobic countplates (3M Microbiology) that were incubated for 48 ± 3 h at 32± 1°C (official method 986.33. AOAC International. Gaithers-burg, Md.). Resulting counts were rounded off to two significantfigures at the time of conversion to SPCs. Cheeses were analyzedout 10 day 70 to reflect growth during distribution and retail. Thetop and bottom surfaces of each cheese were analyzed separatelyto increase the sensitivity of detection. Results were then com-bined for analysis. For example. a cheese was considered positivefor Listeria when either a top or bottom sample was positive;when both surfaces were negative, the cheese was considered neg-ative for Lisicria. Whole top and bottom 100-cm 2 surfaces (firstI to 1.5 cm, 100 g) of each cheese were removed using a sterilecheese cutter, placed in separate sterile Whirl-Pak bags (Nasco.Fort Atkinson. Wis.). appropriately diluted in sterile Butterfield'sphosphate buffer (BPB), and stomached for 3 min in a Stomacher400 circulator (Seward Ltd., Worthington, UK). The resulting ho-mogenate was serially diluted (10_ I ) in BPB to facilitate enu-meration. To reach a detection limit of ^:5 CFU/g, large (150 byIS mm) petri dishes (25384-326 VWR) containing 30 ml ofCHROMagar Listeria were inoculated with I ml of homogenatein duplicate. Refrigerated plates were allowed to dry in a laminarflow hood for -10 min before inoculation. After incubation at37°C for 24 h, turquoise colonies surrounded by a white halo werecounted. Sugar tubes were utilized to further discriminate L. mon-ocvtogenes from Lisreria ii'anot'ii. Random presumptive L. mon-

ocvto genes colonies were confirmed with an automated PCR assay(BAX DuPont Qualicon. Wilmington, Del.).

Physicochemical analysis of cheese. Physicochemical anal-yses were conducted on the milk before cheese manufacture, afterdehooping, and after the drying step. The following analyses wereperformed in duplicate: protein (Kjeldahl method), pH (AccumetResearch AR 150 with a flat tip electrode: Accumet Reference 13-620-46, Fisher Scientific International Inc., Hampton, N.H.), totalsolids (TS) after drying to constant weight at 102°C, and chloride(Chloride Analyzer 926, Nelson Jameson. Marshfleld, Wis.). Saltin moisture phase (SMP), moisture nonfat substance (MNFS), andfat in dry matter (FDM) were determined using the formulas

SMP = [salt/(100 - total solids)] X 100

MNFS = 100 - TS/(100 - fat)] X 100

FDM = (fat/TS) X 100

Titratable acidity (0.1 M NaOH. phenolphthalein indicator) wasdetermined for the milk before and throughout manufacturing.Ripening surface and cheese interior p1-I measurements also weretaken at each microbiological sampling interval.

Statistical analyses. The resulting data were analyzed usingthe SPSS for Windows (version 15.0.1: SPSS Inc.. Chicago. Ill.).The general linear models procedure was used to perform univar-iate analyses of variance (ANOVA5) to determine the effect ofmilk type on the rate of change in mean log CFU per gram and

87.5

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a.5.5

54.5

4

1566D'AMICO ET AL. J. Food Prot., Vol. 71. No. 8

TABLE 1 . Moisture nonfat substance (MNES), salt in moisturephase (SMP), kit in dry matter (FDM), and protein values for raw(R) and pasteurized (P) surface-mold-ripened soft cheese

TrialMNFS (%)SMP (%)FDM (%)Protein (%)

1R64.813.3452ND"IP65.73.950ND2R67.133.35117.372P69.653.15117.813R63.743.855420.743P66.153.535320.39

a ND. not determined.

the effect of time on changes in both pH and mean log CFU pergram. SPCs, cheese composition, temperature, and humidity dur-ing drying, aging, and holding were compared between raw andpasteurized milk and cheeses using t tests. Nonparametric com-parisons of mean log counts of L. mnonocytogenes on individualdays were made with Mann-Whitney tests. Results with P values<0.05 were considered significant. Data are expressed as meanSEM of the three independent trials.

RESULTS

Milk. SPCs for raw and pasteurized milk were signif-icantly different (P < 0.001), with mean counts of 6,900and 20 CFU!ml, respectively. L. mnonocvtogenes was notdetectable in any raw or pasteurized milk samples beforecheese manufacture.

Cheese composition. According to U.S. federal stan-dards of identity (43), soft ripened cheeses produced fromraw milk must be held for at least 60 days at no more than35°F (1.67°C) to ensure safety. To produce a 60-day-oldcheese with a stabilized paste typically found in a youngersurface-mold-ripened soft cheese, cheeses were manufac-tured with a slightly lower moisture target at dehooping.The federal standards of identity also specify that soft rip-ened cheese must contain at least 50% FDM. Despite de-creased moisture, all trial cheeses from both raw and pas-teurized milk contained >50% FDM with a mean (--SD)of 52% ± 0.01% for raw milk cheese and 52% ± 0.02%for pasteurized milk cheese. Overall, no significant differ-ences in general composition were observed between milktypes (Table 1).

Drying, aging, and holding conditions. Raw and pas-teurized milk cheeses were placed in separate chambers fordrying and aging and therefore were subject to variationsin conditions. However, no significant differences were ob-served in average temperature or average relative humiditybetween cheese types.

Changes in pH. In all trials, the target pH of 4.80 to4.85 was achieved at dehooping. Figures 1 and 2 illustratethe changes in pH observed on the surface and in the in-terior of cheeses, respectively, over time at each microbi-ological sampling interval. The univariate ANOVA re-vealed that the linear change in interior pH and the surfacepH over time was not significantly different between thetwo cheese types at both inoculation levels (interior highlevel, P = 0.373: interior low level, p = 0.586: surface

0 7 14 21 28 35 42 49 56 63 70 77Time (day)

LII

0 7 14 21 28 35 42 49 56 63 70 77Time (day)

FIGURE 1. Change in mean (iSD) surface pH of raw () andpasteurized ( ) milk cheese over time at the low (A) and high(B) L. monocytogenes inoculation levels.

high level, P = 0.423; surface low level, P = 0.480). Dif-ferences in inoculation level did not significantly affect therate of pH change (P = 0.691). However, there was a verysignificant overall change in pH over time (P < 0.001) atboth inoculation levels.

Survival of L. monocytogenes during drying, aging,and holding. After inoculation, L. monocvtogenes was un-detectable in all samples tested and remained below thedetection limit (^5 CFU/g) until growth began on day 21(RL) or day 28 (remaining treatments) with correspondingmean pH values of 5.16 (RL), 5.76 (PL) (Fig. 3A), 5.63(RH), and 5.91 (PH) (Fig. 313). In some treatments at thelow inoculation level, L. monocvto genes remained unde-tectable throughout aging and holding even at 70 days ofage (Table 2). Because of the very low level of inoculantand the subsequent undetectable nature of cells during theinitial 3 to 4 weeks, data from only the initiation of growthto day 70 were used for the univariate ANOVA. Log valuesof mean counts were used for analysis because of the pres-ence of zeros (null values) in the data. Figure 4A showsthe growth of L. monocvto genes over time on both raw andpasteurized milk cheese at the low inoculation level. Thelinear change in log mean CFU per gram over time fromthe onset of detectable growth was not significantly differ-ent between the two cheese types (P = 0.206). Mean countson individual days did not differ significantly betweencheese types. Figure 4B shows the growth of L. monocy-togenes over time on both raw and pasteurized milk cheese

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J. Food Prot.. Vol. 71. No. 8 SURVIVAL OF L. MONOCYTOGENES ON SURFACE-MOLD-RIPENED SOFT CHEESE

1567

B.A.

o 7 14 21 28 35 42 49 56 63 70 770 7 14 21 28 35 42 49 56 63 70 77

Time (day) Time (day)

FIGURE 2 Change in mean (±SD) interior pH of raw (+) and pasteurized ( ) iizilk cheese over time at the low (A) and high (B)

L. monocytogenes inoculation levels.

at the high inoculation level. The linear change in log meanCPU per gram over time from the onset of detectablegrowth again was not significantly different between thetwo cheese types (P - 0.962). Mean counts on individualdays did not differ significantly between cheese types (P <0.05) except on day 42. where mean counts of L. mono-

cytogenes were moderately higher in the pasteurized milkcheese (P = 0.1). For both treatments, there was a verysignificant change in log mean CFU per gram over time (P< 0.001). From the low inoculation level, populationsreached maximums of 2.96 t 2.79 and 2.33 ± 2.10 logCFU/g after 60 days of holding in raw and pasteurized milkcheeses, respectively. More extensive growth was observedin high-level inoculated cheese, where populations reached4.55 ± 4.33 and 5.29 ± 5.11 log CFU/g after 60 days ofholding for raw and pasteurized milk cheeses, respectively.Populations of L. monocvto genes grew at significantly high-er rates from the high inoculation level than from the lowinoculation level (P = 0.032) despite similar changes insurface and interior pH. This finding seems to suggest pop-ulation growth potential is dependent on initial contami-nation levels. However, when cheeses negative for L. inon-

ocytogenes were eliminated from the analysis and only thepositive cheeses were included (Fig. 4), there was no sig-nificant effect of initial inoculation level on the rate of

A.

growth (P = 0.096), although there was still no differencein rate of growth between cheese types (P = 0.963).

DISCUSSION

Although no standards exist in the United States forcompositional characteristics of soft ripened cheese beyondFDM. the Codex Alimentarius (21) classifies our experi-mental cheese as a semisoft full-fat surface-mold-ripenedcheese. The Codex Alirnentarius international individualstandard for Camembert (22) states that with a minimumFDM between 45 and 55%, moisture must not exceed 57%with a corresponding minimum dry matter of 43%. All con-ditions were met in our experimental cheese. With no dif-ferences in compositional characteristics or in drying, ag-ing, holding conditions, or initial inoculation level, the as-sumption was made that the only variable between cheeseswas milk type. Variance between cheeses within a vat can-not be controlled for and may contribute to any error thatoccurred when whole individual cheeses were sampled forbacteriological analysis. According to the FDA Center forFood Safety and Applied Nutrition Food Compliance Pro-gram for Domestic and Imported Cheese and Cheese Prod-ucts (44), samples from both domestic and imported chees-es must come from intact units from the same lot. In thepresent study, individual cheeses from the same lot were

B.

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JriIi 11 0 ¶.

-1- 1 -1 — —-0 7 14 21 28 35 42 49 56 63 70 770 7 14 21 28 35 42 49 56 63 70 77

Time (day) Time (day)

FIGURE 3. Change in log mean counts (SEM) of L. monocytogefleS at the low (A) and high (B) inoculation levels on the surface

of raw () and pasteurized ( ) milk cheese during aging and holding. 11 Data represent estimated inoculation levels (not actual counts).

4249

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TABLE 2. Number of cheese samples positis'efrr L. monocytogenes b y sampling clay"

No. of positive samples on day:

Cheese type714212835

Raw high 000IPasteurized high000IRaw low 001IPasteurized low000I

a Three samples were tested for each cheese type on each sampling day.

tested at each sampling point rather than taking multiplesamples from the same cheese over time to overcome theheterogeneous pattern of pathogen distribution in cheeseand because the fluid and soft texture of soft ripened chees-es make them difficult to cut and rewrap without alteringthe product.

During the initial 3 to 4 weeks of aging, populationsof L. ,nonocvto genes were below our detection limit. Theinability to enumerate also was observed by Ryser andMarth (38) when pasteurized milk Camembert-type cheesewas surface inoculated. One strain failed to grow but wasdetected after cold enrichment. However. Ryser and Marth(38) applied L. mnonocvto genes on top of the Pen icilliumncamemberti lawn on the surface of the cheese, meaning thatthe organism had to penetrate the rind to enter the cheese

111098

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a

07 14 21 28 35 42 49 56 63 70 77Time (day)

B.

0 7 14 21 28 35 42 49 56 63 70 77Time (day)

FIGURE 4. Change in log beam, counts (±SEM) of L. mono-cytogenes at the low (A) and high (B) inoculation levels on thesurface of raw ()and pasteurized( ) mnilk cheese during agingand holding using on/v non-zero(positive) data. " Data representestimated inoculation levels (not actual counts).

before growth could occur. Decreases in populations on ornear the surface of Camembert-type cheese during the ini-tial stages of ripening also have been reported (1, 26). Somecells were likely transferred to the aging racks during theinitial stages of ripening in our study, viable cells weredetected through environmental sampling of the aging racks(data not shown). We were unable to determine quantita-tively the extent of this transfer.

When examining the survival of L. 'nonocvtogenes inCamembert-type cheese at different pH values, Ryser andMarth (38) found populations decreased at pH 4.6 andfailed to grow at pH 5.5. Cheese pH at dehooping (4.8 to4.9) increases during assimilation of lactate by K. lactis andG. candidu,n, decreasing the rind acidity, and because ofincreased ammonia concentration within the cheese. Sur-face pH increases more rapidly through the proteolytic andpeptidase activity of ripening fungi, notably P. camemberti(23). In our study, growth initiated at pH values between5.16 and 5.91, and once detectable the L. monocvrogene.rpopulation growth paralleled the gradual increase in pH, asfound in other studies (31, 33, 38). The delayed onset ofgrowth is likely due to microbial injury incurred from thepresence of salt and the low pH environment of the freshcheese surface (14). When L. monocytogenes (strains ScottA. OH, and CA) was inoculated into milk used for Cam-embert-type cheese manufacture, counts in wedge and in-terior samples decreased 10- to 100-fold during the first 17days of ripening, an observation attributed to the low pH(<5.5) and low storage temperature (IS to 16°C) (38). Gayet al. (14) found that L. Jnonocvtogenes Scott A populationsdeclined and were unable to grow in Richard's broth ad-justed to pH 4.8 used to simulate Camembert cheese at thebeginning of ripening.

Increased lag times as a result of microbial injury havebeen previously reported for L. rnonocvtogenes (3, 7, 18,46). The duration of the lag phase of L. monocvtogenesunder stresses such as high salt (35) or conditions simulat-ing soft cheese ripening (14) also is affected by inoculumsize. In our study. in some replicates at the low inoculationlevel, cells remained undetectable through day 70. Undervery unfavorable growth conditions, cells will die duringthe extended lag phase (32). With a very small inoculumsize, heterogeneity in cellular stress responses within thepopulation can result in the death of all the cells in theinoculum and thus no growth (32), or a few cells couldsurvive and thus initiate growth, as was observed in otherreplicates within the same inoculum set. The presence ofnull values and the variability in the duration of lag periods

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affected the growth potential for the two types of cheeses.thus contributing to the high level of variation in the logmean counts reported. The effect of inoculation level onthe growth—no growth barrier was evident when cheesesnegative for L. monocytoge/les were eliminated from theanalysis and included counts from only the positive chees-es. After elimination of the no-growth data, there was nosignificant effect of initial inoculation level on the rate ofgrowth suggesting that once population growth commenc-es, growth rates will be similar regardless of the initial in-oculum. Storage of these cheeses at 4°C also could haverestricted growth (1, 39). Little and Knochel (25) found thatonly the psychrotrophic pathogen Yersinia enterocoliticawas able to grow when introduced as a postprocessing con-taminant on stabilized Brie, whereas populations of bothSalmonella and Bacillus cereus declined at 4 and 8°C. Be-cause of the difficulty and variability in the detection of L.monocvtOgene.s from mold-ripened soft cheeses in general(34, 40), especially in raw milk cheeses with very low in-oculation levels (20, 34), samples that were negative bydirect plating did not undergo standard enrichment proce-dures.

After 60 days of ripening, maximum population levelsdiffered between cheeses of the two initial inoculation lev-els. Ryser and Marth (38) observed 2- to 3-log increases inthree strains of L. ,nonocvtO genes surface inoculated onto10-day-old Camembert-type cheese at 6°C. Maximum ob-served populations of approximately 3 to 5 log CFU/g weresimilar to the 4.55 and 5.29 log CFU/g observed in thepresent study (high level) despite differences in initial in-oculation level between these two studies. These contami-nation levels also are consistent with L. monocvto genes lev-els found in contaminated surface-mold-ripened soft chees-es at retail (17) and Listeria j000cua levels under similarconditions of inoculation and ripening (39). Back et al. (1)found an approximately 2-log increase in L. monocvto geneson the surface of laboratory manufactured Camembert-typecheese after 40 days at 3 and 6°C, although their resultswere not reported. Comparisons with data reported by Gen-igeorgis et al. (15) also are difficult because of differencesin methodology, storage conditions, and the units reported(log CFU per sample). Based on our results, the use of rawmilk rather than pasteurized milk does not seem to provideenhanced protection when L. inonocyto genes is introducedas a postprocessing contaminant in surface-mold-ripenedsoft cheese. Other investigators have reported that thegrowth of L. monocvto genes during the manufacture andripening of Camembert is unaffected by the use of a nisin-producing starter or raw milk when compared with the useof pasteurized milk (33).

Based on our data and results previously reported, sur-face-mold-ripened soft cheeses support the growth of L.rnonocvto genes when introduced postprocessing at levels^0.2 CFU/g, independent of the milk type used for cheesemanufacture. The use of pasteurized milk in the manufac-ture of surface-mold-ripened soft cheese does not provideprotection from L. monocvto genes introduced as a postpro-cess contaminant. Despite low pH, lower moisture, and thepresence of salt, L. lnonocvto genes introduced as a post-

processing contaminant survived and grew on soft ripenedcheeses held for ^:--60 days. The 60-day aging rule encour-ages extended refrigerated holding of these cheeses, whichcould inadvertently contribute to risk through the prolifer-ation of psychrotrophic pathogens. The safety of cheeses ofthis type must be achieved through control strategies otherthan a 60-day holding period, and revision of current fed-eral regulations are warranted.

Under U.S. Department of Agriculture (USDA) FoodSafety and Inspection Service rules, manufacturers ofready-to-eat products at high risk of L. monocvto genes con-tamination must operate under hazard analysis critical con-trol point (HACCP) systems, where products are consideredadulterated if they contain L. monocyto genes and if theyhave been in direct contact with a surface contaminatedwith L. monocvto genes. Producers must address control ofL. monocvtogenes in their HACCP plan, sanitation standardoperating procedures, or other prerequisite programs whoseeffectiveness is verified through frequent environmental andend product testing (41). Similar systems also may be ef-fective for preventing the contamination of high-risk chees-es.

Canada is currently seeking to develop a policy to re-place the 60-day aging requirement for soft and semisoftcheeses after the occurrence of outbreaks of listeriosis inCanada in 2002 that were linked to cheeses made frompasteurized or heat-treated milk. Although not in line withdomestic policy, the Canada Food Inspection Agency hasallowed the importation of certified raw milk soft and semi-soft cheese from France. The accompanying certificate ver-ifies that the cheese was manufactured in compliance withFrench national standards and verifies that the productsmeet the microbiological criteria of both Canada andFrance.

Epidemiologic evidence and the results of scientific in-vestigation make it clear that neither pasteurization nor 60days of aging are sufficient to ensure the safety of surface-mold-ripened soft cheese. Cheese safety can be better as-sured through a combination of stringent raw milk produc-tion and microbiological quality, improved process con-trols, the use of performance criteria, and aggressive envi-ronmental monitoring and finished product testing.

ACKNOWLEDGMENTS

This study was supported by a USDA Animal and Plant Health In-spection Service grant (05-9650-0196GR) to C. W. Donnelly. D. J.D'Amico was the recipient of second place in the Des eloping ScientistCompetition for his presentation of this work at the 2006 annual meetingof the International Association of Food Protection. The authors of thisstudv acknowledge Alan Howard for his assistance with statistical anal-vses and Monument Farms Dair\ for pros ding the milk.

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