1193

Journal of Food Protection, Vol. 59, No. 11, 1996, Pages 1193-1199 Copyright ©, International Association of Milk, Food and Environmental Sanitarians

Growth of Listeria monocytogenes in the Myzithra, , and during Storage at 5, 12, and 22°C

DEMETRIOS K. PAPAGEORGIOU,* MINA BORI, and ANTONIOS MANTIS Downloaded from http://meridian.allenpress.com/jfp/article-pdf/59/11/1193/1666024/0362-028x-59_11_1193.pdf by guest on 24 September 2021

Department of Food Hygiene and Technology, School of Veterinary Medicine, Laboratory of Milk Hygiene and Technology, Aristotle University of Thessaloniki, 54006 Thessaloniki,

(MS# 95-304: Received 29 November 1995/Accepted 22 March 1996)

ABSTRACT times leading to a carrier state) to severe manifestations. The severe forms of human listeriosis present as meningoencepha- The fresh whey cheeses Myzithra, Anthotyros, and Manouri litis followed by septic infections and occasionally isolated were inoculated with Listeria monocytogenes Scott A or California organ involvement. Groups at highest risk are pregnant to contain ca. 5.0 X 102 CFU/g of and incubated at 5, 12, women, neonates, adults with underlying disease (cancer, and 22°C. The initial pH of the finished whey cheeses Myzithra, AIDS, diabetes, chronic hepatic disorder, transplant recipi- Anthotyros, and Manouri were 6.50, 6.41, and 6.30 respectively. Fat in dry matter was 16.3% in Myzithra, 65.9% in Anthotyros, and ents), the elderly (>65 years old) and other immunocompro- 71.7% in Manouri cheese; the moisture contents of the cheeses mised individuals. Death is rare in healthy adults but can were 68.4, 66.9, and 52.2% respectively. The pH of the cheeses occur at a rate as high as 30% in persons at highest risk (12, dropped gradually to between 5.30 to 4.97 during storage. Dupli- 28, 30, 32, 38, 41). The pathogen can be transmitted to cate samples of whey cheeses were tested for numbers of L. humans from infected animals (11, 23) or through consump- monocytogenes and pH. Listeria counts were obtained by surface- tion of listeria-contaminated foods. Dairy products are the plating on lithium chlorite-phenylethanol-moxalactam agar (LPM). most often incriminated in cases of human listeriosis (1, 2, 8, Selected Listeria colonies were confirmed biochemically. The 14,20,21,39). growth of L. monocytogenes in the whey cheeses was very rapid. Early in the 1960s milk and milk products were Generation times of L. monocytogenes at 5°C ranged between 16.16 and 20.16 h and were significantly longer than those suspected as possible vehicles for food-borne human listerio- observed at 12°C, which ranged between 5.07 and 5.81 h. sis in European countries (16, 29, 40), but little information Generation times at 22°C ranged between 1.68 and 2.70 h. The on the presence and behavior of L. monocytogenes in milk generation time of L. monocytogenes Scott A at 5°C in Anthotyros and milk products was available in the literature at that time. cheese (20.16 h) was significantly longer than those of the same Since 1981, several food-associated outbreaks and sporadic strain at 5°C in Myzithra cheese (16.16 h) and Manouri cheese cases of human listeriosis (1,2,8,14,22,32,39) prompted (17.81 h). Also, the generation time of L. monocytogenes California researchers to determine the incidence and behavior of L. at 22°C in Myzithra cheese (2.70 h) was significantly longer than monocytogenes in milk and dairy products and other foods. the generation time of strain Scott A (1.93 h). Maximum popula- L. monocytogenes has been detected in 2 to 6% of raw tions of L. monocytogenes were reached after 24 to 30 days at 5°C, milk samples from a number of countries including the after 5 to 12 days at l2°C, and after only 56 to 72 h at 22°C and ranged from 6.92 to 8.81 log CPU/g. Maximum populations were USA, Canada, Greece, , and (6, 7, 10, 13, significantly lower in Myzithra cheese at 5 and 12°C than 18, 19, 38) and in 2 to 10% of a wide variety of cheese maximum populations in Anthotyros and Manouri cheese at the samples. Soft cheeses are often contaminated (4, 5, 10, 19, same temperatures independent of the inoculated strain. 21,27). In Greece Listeria spp. have been found in 3.75% of raw ewes' milk samples, in 6% of raw goat's milk samples, Key words: Whey cheeses, Listeria growth; L. monocytogenes in 6% of mixtures of raw ewe's and goat's milk samples, in 16.2% of soft Myzithra cheese samples, in 20.8% of Listeria monocytogenes causes listeriosis, a disease of Anthotyros cheese samples, in 5.8% of Manouri cheese both humans and animals. In animals listeric infections often samples, in 2% of cheese samples and finally in 20% of result in encephalitis, abortion, and mastitis (40). In humans environmental samples from 8 cheese-processing plants. the illness can range from a mild flu-like sickness (some- The high prevalence in the products correlated well with the high prevalence in the dairy plant environment (10). The Council of the European Communities Directive 92/46 E.E.C., which has already been ratified by the Greek * Author for correspondence. Tel: 031 999806; Fax: 031 999803. government, concerning the health rules for the production 1194 PAPAGEORGIOU, BORI, AND MANTIS and marketing of raw milk, heat-treated milk, and milk- Inoculation and storage of whey cheeses based products, provides that L. monocytogenes must be Cheese from three different batches was used for three trials kept out of these products; otherwise the products must be for each of the whey cheeses resulting in nine trials (three products withdrawn from the market. Previous work indicated that and three incubation temperatures) for all the experiments. Cheeses for the first trial were prepared and stored at 5°C for 38 days; for the modest growth of L. monocytogenes occurred primarily in second trial, at l2°C for 16 days; and for the third trial at 22°C for 7 Cheddar, Feta, and Blue cheese during the first stage of days. For each of the nine trials two separate experiments were production. Growth ceased when the pH of the cheeses done using cheese of the same batch; one for L. monocytogenes dropped to below 5.0 and numbers of the pathogens California and one for Scott A. The results in Table I show that decreased markedly during ripening and storage of the cheeses made on three different days were almost identical in their cheeses (25, 26, 34). Substantial growth occurred in Brick composition. and Camembert cheese during the ripening process when the For each trial, ca. 1.8 kg of fresh cheese was taken from the pH of cheeses was >5.75 (35, 37). Also a significant vat just after cooking at 82 to 92°C for 20 min and correlation of Listeria growth, pH values (>5.5), and placed into a steam-sanitized hoop for drainage for ca. 5 h. The Downloaded from http://meridian.allenpress.com/jfp/article-pdf/59/11/1193/1666024/0362-028x-59_11_1193.pdf by guest on 24 September 2021 absence of starter cultures during cheese manufacture has hooped cheese was placed in an ice chest, transported to the been observed in market cheeses (9). The whey cheeses laboratory within ca. I h, and stored at 5°C until draining was complete (5 h total). The temperature of the cheese after draining Myzithra, Anthotyros, and Manouri are made without starter was 5°C. culture; they may contain up to 70% moisture and exhibit pH Fifty grams of cheese were inoculated with a calculated values between 6.0 to 6.5. Anari whey cheese (the Greek- volume of Listeria cultures and mixed thoroughly in a 250-g Cypriot name for Myzithra whey cheese) was incriminated capacity yoghurt container with a sterile metal spoon. Later, a in cases of listeriosis in women in February 1988 in England second 50-g portion of cheese was added into the same yoghurt (2, 21). Thus the purpose of this research was to determine container and the sum of 100 g of cheese was again mixed the rate of growth of L. monocytogenes in the fresh whey thoroughly. The same schedule of inoculation was repeated in cheeses Myzithra, Anthotyros, and Manouri. Whey cheeses another container. Finally both portions were combined into one are traditionally produced in Greece from whey recovered 200-g sample which was placed into 250-g capacity yoghurt during Feta cheese production, a cheese-making procedure container with a well-fitted plastic cap, preventing loss of moisture. in which ewe's and goat's milk is used. This inoculation procedure ensured adequate distribution of Liste- ria cells in the cheese. To have enough cheese for sampling for each trial, cheese in 6 containers of 200 g each was inoculated with L. MATERIALS AND METHODS monocytogenes California (3 containers) and Scott A (3 contain- ers). Inoculated cheese in containers was refrigerated until all 6 Cultures of L. monocytogenes containers of 200 g each were prepared and then were stored at 5°C L. monocytogenes Scott A and California were used in this (first trial), l2°C (second trial), and 22°C (third trial). To minimize study. Stock cultures were maintained at 4°C on tryptose agar (TA) any contamination, the yoghurt containers used were unsealed just (Difco Laboratories, Detroit, MI) slants and were transferred before the inoculation and the cheese was sampled with a sterile monthly. Listeria cultures used to inoculate fresh whey cheeses metal spoon. were prepared as previously described (24, 34, 43). Working Listeria cultures were decimally diluted in sterile 2% citrate solution and a calculated volume of the third decimal dilution was Sampling schedule and enumeration of L. monocytogenes added to 200 g of fresh Myzithra, Anthotyros, or Manouri cheese so The frequency of sampling to enumerate L. monocytogenes that the number of L. monocytogenes in the inoculated cheese was depended upon temperature of incubation. After a preliminary ca. 5.0 X 102 CFU/g. experiment, sampling intervals were adjusted to observe the lag, logarithmic, and stationary phases of growth and the maximum populations of L. monocytogenes in the whey cheeses. Duplicate Manufacture of whey cheeses: Myzithra, Anthotyros, and Manouri samples for enumeration of L. monocytogenes and determination of Whey cheeses Myzithra, Anthotyros, and Manouri used in the pH were taken at 2-day, I -day, and 8-h intervals for the cheeses experiments were made in a commercial milk production facility in incubated at 5, 12, and 22°C respectively. central , Greece. Whey cheeses are prepared by heating Ten-gram cheese samples were used for the initial (l: 10) whey, resulting in the denaturation and coagulation of the protein. dilution in warm (42 ± 2°C) sterile tryptose broth with 2% sodium Myzithra cheese was prepared by heating 1,000 liters of whey in a citrate in sterile Stomacher bags of ca. 500-ml capacity (44). steam-jacketed whey cheese vat to 92°C. The emerged on the Samples were blended in a Stomacher 400 for 3 min. Appropriate surface when the temperature raised to 82°C. The curd was cooked serial decimal dilutions were made in a warm (42 ± 2°C) sterile at 82°C to 92°C for 20 min for the complete denaturation of whey solution of 2% sodium citrate, followed by duplicate surface proteins. Finally, the cheese was drained without pressing for 3 to 5 plating of 0.1 ml of appropriate dilutions on lithium chloride- h and stored at 5°C (15). phenylethanol-moxalactam agar (LPM) (17). The agar plates were To prepare Anthotyros whey cheese the above procedure was incubated aerobically for 48 h at 35°C. Colonies typical of those modified to include the addition of 60 kg of cream (63% fat formed by L. monocytogenes were counted, and selected colonies content) to 1,000 liters of whey when the temperature of the whey were confirmed as L. monocytogenes according to the procedure of was 68°C to 70°C. Also 10 kg of NaCI was added when the Ryser and Marth (34), which is based on a positive catalase temperature of the whey was 73°C to 75°C. To prepare Manouri reaction, tumbling motility, the appearance of blue-green colonies cheese, 80 kg of cream (63% fat content) was added to 1,000 liters on tryptose agar, and results of biochemical tests found with API of whey when the temperature of the whey was 68°C to 70°C and 20S test strips (API System, Montalieu Vercieu, France). Ten-gram 15 kg of NaCI was added when the temperature of the whey was cheese samples from all nine lots used in the experiments were 73°C to 75°C. tested for L. monocytogenes before they were inoculated with the GROWTH OF LISTERIA MONOCYTOGENES IN WHEY CHEESES 1195 pathogen. Initial (I: 10) dilutions made in sterile tryptose broth in addition of salt to whey cheeses is allowed in this regulation sterile stomacher bags were reexamined after 2, 4, 6, and 8 weeks (42). The whey is heated in whey cheese vats, capacity 1,000 of cold enrichment at 4cC. Cold-enrichment samples were surface liters at the most, by using steam, which is either directed to plated on LPM agar and the plates were incubated aerobically for the double wall of the cheese vat or infused directly in the 48 h at 3SOC (34). mass of whey. The rate of heating is such as to attain the temperature of 88 to 90°C in 40 to 45 min. This time- Analysis of cheeses temperature condition also has a desirable influence on the Duplicate moisture and fat determinations were made for each yield of cheeses (15). Commercial whey cheeses produced trial of cheeses used as described in reference (31). Duplicate NaCI in Greece usually contain about 1 to 2% salt. Manouri and determinations were made on IO-g cheese samples using the Quantab chloride titrator test strip (Miles Laboratory, Inc., Elkhart, Anthotyros are consumed as table cheeses. Myzithra is used IN) method. The pH of the cheeses was determined at the time of as a table cheese, but mainly for the preparation of certain testing for L. monocytogenes with a pH meter (type 522, foods and cheese pies. Low-fat fresh Myzithra cheese also is wisseschaflich- Technische Werk. Statten, Germany). produced in Greece without addition of salt. Some people Downloaded from http://meridian.allenpress.com/jfp/article-pdf/59/11/1193/1666024/0362-028x-59_11_1193.pdf by guest on 24 September 2021 used to add sugar or honey to eat this unsalted whey cheese. Calculation of growth rate and statistical methods Salted and dried Myzithra whey cheese is also produced in Growth curves were constructed for each combination of Greece (15). The whey cheeses used in this study were made Listeria strain and incubation temperature of each cheese. Genera- in a commercial milk-processing facility in central Macedo- tion times for L. monocytogenes were calculated using the follow- nia, Greece. The Myzithra cheese was of the unsalted ing formula (33): Generation time = 1I10g P - log P /0.301 2 j low-fat type usually produced in Greece. (T - Tj), where Tj = time of Pj, T = time of P , Pj = CFU/g at 2 2 2 The whey cheeses Myzithra, Anthotyros, and Manouri hand P = CFU/g at T . 2 2 used in this study complied with the Greek standards for All data on generation times and maximum populations were analyzed by use of the SPSS statistical program. The effects of moisture and fat content for all nine trials, except for the strain, kind of cheese and incubation temperature on generation FDM in Myzithra cheese, which was distinctly lower time and maximum population levels were analyzed using multi- because low-fat fresh Myzithra was used in these experi- way analyses of variances (ANOVA). Fisher's LSD (least signifi- ments. This was done purposely because the low-fat fresh cant difference) test used to determine significant differences product is most commonly produced in Greece. Also the salt between means. For all analyses a level of significance CY. = 0.05 content for Anthotyros and Manouri cheeses was that was used. usually found on the market. The average NaCI content for Anthotyros and Manouri cheeses was 1.42% (S.D. ± 0.08) RESULTS AND DISCUSSION and 2.28% (S.D. ± 0.08) respectively. The average pH values of the Myzithra, Anthotyros, and Composition of cheeses Manouri cheeses were 6.50, 6.41, and 6.30 respectively. The results for moisture, fat in dry matter (FDM), and These pH values dropped gradually to between 5.30 to 4.97 salt content of the whey cheeses Myzithra, Anthotyros, and during the entire experiment for seven trials. In two trials of Manouri used in the experiments are summarized in Table 1. the Manouri cheese (stored at 12°C for 16 days and 22°C for All results are means of two measurements. The Greek 7 days), the pH values remained relatively constant and regulatory standards for whey cheeses provide that Myzithra above 6.10. For all nine trials, the pH values were not below and Anthotyros cannot contain more than 70% moisture or 5.7 during the growth of L. monocytogenes. Only when less than 50% and 65% FDM respectively. Also, Manouri Listeria populations reached the stationary phases of growth cheese cannot contain more than 60% moisture or less than did pH values decrease below 5.7. These high pH values are 70% FDM. No standard exists for the amount of salt, but the favorable for Listeria growth. This has been noted for L. monocytogenes in other cheeses having pH values >5.5 (9, TABLE I. Moisture, fat in dry matter, and salt content of Myzithra, 35,37). Anthotyros and Manouri cheeses

Fat in dry Growth ofL. monocytogenes in the whey cheeses at SoC Moisture, % matter, % Salt, % All nine lots of whey cheeses used for all nine trials Cheese Trial (Avg.::t: SD) (Avg.::t: SD) (Avg.::t: SD) were free of L. monocytogenes (measured by direct plating and cold enrichment up to 8 weeks (34) before they were Myzithra I 68.3 15.7 o inoculated with the pathogen. Growth curves of L. monocy- 2 67.8 17.0 o togenes in whey cheeses stored at 5°C are shown in Fig. 1 3 69.2 16.2 o (68.4 ::'::0.57) (16.3::':: 0.53) (0) and Fig. 2 for strains California and Scott A respectively. All Anthotyros I 67.1 66.8 1.37 results are averages of duplicate samples. A lag phase of ca. 2 67.8 65.2 1.37 2 days was noted at first, and then the generation times of L. 3 65.8 65.7 1.54 monocytogenes California in the whey cheeses Myzithra, (66.9 ::'::0.82) (65.9 ::'::0.66) (1.42::':: 0.08) Anthotyros, and Manouri were 16.89, 18.19, and 18.48 h Manouri I 50.6 72.8 2.23 respectively. The generation times of L. monocytogenes 2 52.6 71.7 2.40 Scott A in the same whey cheese products were 16.16, 20.16, 3 53.4 70.8 2.23 and 17.81 h respectively (Table 2). Statistical analysis of all (52.2 ::'::1.17) (71.7::':: 0.81) (2.28 ::'::0.08) data on generation times at 5°C indicates that the generation 1196 PAPAGEORGIOU, BORI, AND MANTIS

9 9 8 bO bO 8 7 -=! -=! 7 C+-c 6 C+-c 6 c) -+- Myzithra 5 -+- Myzithra 5 ci -0-- Anthotyros bO -O--Anthotyros bO 0 4 -'-Manouri o 4 -.-Manouri ~ 3 ~ 3 2 2 2

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6 6

5 5 -+-Myzithm -0-- Anthotyros -0-- Anthotyros -'-Manouri ---.- ManOIJrl

4 4 2 2 Days Days

FIGURE 1. Growth of L. monocytogenes California and changes FIGURE 2. Growth of L. monocytogenes Scott A and changes in in pH in the whey cheeses Myzithra, Anthotyros, and Manouri at pH in the whey cheeses Myzithra, Anthotyros, and Manouri at 5°C. 5°C. time of L. monocytogenes Scott A in Anthotyros cheese (20.16 h) was significantly longer than those of the same strain at 5°C in Myzithra cheese (16.16 h) and Manouri TABLE 2. Generation times and maximum populations of two cheese (17.81 h). Maximum populations were reached by strains of L. monocytogenes in the whey cheeses Myzithra, the two strains after 24 to 30 days at 5°C and ranged from Anthotyros. and Manouri at 5,12, and 22°C 7.84 to 8.62 log CFU/g (Table 2). Maximum populations were significantly lower in Myzithra cheese than in Anthoty- Generation Maximum L. time (h) population ros and Manouri cheeses independent of the Listeria strain Storage monocytogenes temperature strain Cheese Mean ± SD (log CPU/g) used. Results of previous work indicated that L. monocyto- genes achieved similar maximum populations of 107 to 108 5°C Scott A Myzithra 16.16::':: 2.48 7.97 CFU/g or ml in broth, 6% salted whey, and skim milk (24), Anthotyros 20.16::':: 4.90 8.32 in unsalted, filter-sterilized whey (36), in cheese (9), Manouri 17.81::':: 1.75 8.62 and in surface samples from fully ripened Camembert California Myzithra 16.89 ::'::1.33 7.84 cheese (35). Anthotyros 18.19::':: 4.24 8.14 18.48 ::'::3.88 8.07 Psychrotropic growth of L. monocytogenes also has Manouri 12°C Scott A Myzithra 5.65::':: 1.36 7.65 been reported to occur in unfermented dairy products (3, 24, Anthotyros 5.17 ::'::0.86 8.14 33, 36) and market cheeses with pH values >5.5 (9). The Manoun 5.68::':: 0.68 8.81 results of this work are in agreement with those reports. California Myzithra 5.35::':: 0.83 7.11 Anthotyros 5.07::':: 0.50 8.69 Growth ofL. monocytogenes in the whey cheeses at ]2°e Manoun 5.81 ::'::1.32 8.77 It has been observed that the transportation temperature 22°C Scott A Myzithra 1.93 ::'::0.41 8.49 for cheeses often deviates from 5°C and products may Anthotyros 1.95::':: 4.90 8.07 occasionally encounter temperatures up to 10 to l20e. Also, Manouri 1.79::':: 0.46 8.59 whey cheeses are usually packed at 12 to l4°C, a tempera- California Myzithra 2.70::':: 0.61 6.92 Anthotyros 2.55::':: 1.00 7.23 ture that retards multiplication of many bacteria and at the Manouri 1.68 ::'::0.16 8.54 same time is tolerable for the workers. GROWTH OF LISTERIA MONOCYTOGENES IN WHEY CHEESES 1197

9 9 8 bQ8 ~7 ': 7 ;::i 6 ==! ~ ct-t 6 U 5 --+- Myzithra U 5 -D- Anthotyros bQ4 bQ --+- Myzithra --air- Mano uri o 3 o 4 ...:I -D- Anthotyros ...:I 3 2 -'-Manouri I 2 o I 2 3 4 5 6 7 8 9 10 II 12 13 14 IS 16 17 o I 2 3 4 5 678 9 ill" U U W ~ ffi n

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5 5 -D- Anthotyros -D- Anthoty ros -.-Manouri -'-Manouri 4 4 o I 2 3 4 5 6 7 8 9 10 II 12 13 14 IS 16 17 o I 2 3 4 5 6 7 8 9 10 II 12 13 14 IS 16 17 Days Days

FIGURE 3. Growth ofL. monocytogenes California and changes FIGURE 4. Growth of L. monocytogenes Scott A and changes in in pH in the whey cheeses Myzithra, Anthotyros, and Manouri at pH in the whey cheeses Myzithra, Anthotyros, and Manouri at ]2ae. ]2ae.

Myzithra cheese in this experiment dropped to 5.7 after Increasing the storage temperature from 5 to 12°C only 5 days at 12°C and finally to 4.97 after 16 days at 12°C. decreased the lag phase and reduced by ca. 3.3-fold the generation time of L. monocytogenes in all three whey Growth ofL. monocytogenes in the whey cheeses at 22°e cheese products. Fig. 3 and Fig. 4 are the growth curves for Storage at 22°C simulates room temperature. Everyone L. monocytogenes California and Scott A respectively in involved in food microbiology should know how critical whey cheeses stored at 12°C. All results are average values time and temperature are to food safety. The question for a of duplicate samples. After a lag phase of 12 to 15 h (data soft whey cheese made without starters, having up to 70% from preliminary experiments not shown in Fig. 3 and Fig. moisture and pH between 6.0 and 6.5, is: How long can it 4), generation times of 5.07 h to 5.81 h were observed (Table remain at room temperature? As expected, L. monocyto- 2) for both strains California and Scott A in all three whey genes grew most rapidly at 22°C with maximum populations cheeses. No significant differences in generation times were of 6.92 to 8.59 log CFU/g being observed after only 56 to 72 observed at 12°C independent of strains or products. These h of storage. Fig. 5 and Fig. 6 present the growth curves of L. findings on generation times of L. monocytogenes in whey monocytogenes California and Scott A respectively in whey cheeses stored at 12°C are consistent with reports of cheeses stored at 22°C. All results are averages of duplicate Rosenow and Marth (33), who determined generation times samples. The lag phase lasted only 6 to 8 h. Generation times of L. monocytogenes between 5.16 hand 6.03 h in whole, for L. monocytogenes California and Scott A in all three skim, and chocolate milk and whipping cream stored at whey cheeses ranged between 1.68 and 2.70 h (Table 2). The l30C. Maximum populations reached by the two strains in generation time of strain California in Myzithra cheese (2.70 whey cheeses after 5 to 12 days at 12°C ranged from 7.11 to h) was significantly longer than that of strain Scott A (1.93 h) 8.81 log CFU/g (Table 2) and were significantly lower in in the same product. Rosenow and Marth (33) also deter- Myzithra cheese than in Anthotyros and Manouri cheeses mined that the generation times of four strains of L. independent of strains. This is more evident in Fig. 3 where monocytogenes in whipping cream, whole, skim, and choco- the growth curve for L. monocytogenes California in Myzithra late milk stored at21 °C ranged between 1.72 and 1.92 h. cheese attains lower levels. This might be due to the lower Numerous reports concerning the heat resistance and pH values that were noted in this experiment. The pH of thermal inactivation of L. monocytogenes can be found in 1198 PAPAGEORGIOU, BORI, AND MANTIS

9 9 8 8 ~7 ~7 ~ 6 ~6 c$ 5 c$ 5 ~Myzithra tlO tlO -O--Anthotyros .3 4 .34 -+-Manoyri 3 3 2 2 o 1 234 5 6 7 o 234 5 6 7 Days Days Downloaded from http://meridian.allenpress.com/jfp/article-pdf/59/11/1193/1666024/0362-028x-59_11_1193.pdf by guest on 24 September 2021

7 7

6 6 ~ ..~ ~Myzithra 0. ~Myzithra 0. = = -0-- Anthotyros -0-- Anthotyros 5 5 -+-Manoyri -+-Manoyri

4 4 o 234 5 6 7 o 234 5 6 7 Days Days

FIGURE 5. Growth ofL. rnonocytogenes California and changes FIGURE 6. Growth of L. rnonocytogenes Scott A and changes in in pH in the whey cheeses Myzithra, Anthotyros, and Manouri at pH in the whey cheeses Myzithra, Anthotyros, and Manouri at 22°C. 22°C.

the literature. Results of these reports indicate that current during 1989 were found to be contaminated with L. monocy- minimum vat (61.7°C/30 min) and HTST (71.7°C/15 s) togenes and had to be withdrawn from the market (38). pasteurization requirements are probably adequate to de- These facts prove that if present in whey cheeses, L. stroy expected levels of L. monocytogenes in raw milk (38). monocytogenes can generate enormous problems for both During whey cheese production the final time-temperature the dairy industry and public health. conditions for denaturation of whey proteins are 82 to 92°C The results of this work indicate how easily Listeria can for 15 to 30 minutes. Thus, even if the whey was contami- grow in Myzithra (Anari), Anthotyros, and Manouri .whey nated with very high levels of L. monocytogenes, the cheeses even at refrigeration temperatures. Pathogens hke L. bacteria would not survive processing. Fresh whey cheeses monocytogenes must be kept out of susceptible cheeses. To are in excellent microbiologic condition because of this accomplish this goal, the use of adequate hygienic practices high-temperature and long-time processing. However, post- is necessary during handling and packing of those products. processing contamination may occur during subsequent Also, postpacking pasteurization of whey cheeses is recom- handling and packing, reducing cheese quality. The high pH mended to minimize the risk. and high moisture content of whey cheeses are very favorable for bacterial multiplication. ACKNOWLEDGMENTS The growth and survival of L. monocytogenes at 4 to 30°C in 49 market cheese samples representing 24 types of Research supported by the General Secretariat of Research and cheeses was studied by Genigeorgis et al. (9). Ricotta cheese Technology of the Greek Ministry of Industry and Commerce, ~heAristotle (pH 5.9 to 6.1) was the best substrate for Listeria growth University of Thessaloniki, Greece, and the Macedoman Milk Industry among these types of cheeses. A woman diagnosed with MEVGAL S.A. We thank Dr. Chr. Batzios, Assistant Professor of Econom- ics and Statistics, for his helpful assistance with statistical analysis of the listeriosis contracted from whey cheese was reported in data. February 1988 in England (2, 21). Anari whey cheese was incriminated and L. monocytogenes was found in the cheese REFERENCES at levels of 3.0 X 107 to 5.0 X 107 CFU/g (21). Anari is the Greek-Cypriot name for Myzithra whey cheese. In another I. Anonymous. 1988. Reports of outbreaks of foodborne illness. Food- incident, 209 cases of imported into the USA borne listeriosis-Switzerland. J. Food Prot. 51:424-425. GROWTH OF LISTERIA MONOCYTOGENES IN WHEY CHEESES 1199

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