Evaluation of Listeria monocytogenes Populations during the Manufacture and Vacuum-Packaged Storage of Kashar Cheese

Cetinkaya F. , G.E. Soyutemiz: Evaluation of Listeria monocytogenes Populations during the Manufacture and Vacuum-Packaged Storage of Kashar Cheese. Acta Vet Brno 76, 2007: 143-148. Behaviour of Listeria monocytogenes was investigated during the manufacture and storage of Kashar cheese. For this purpose, pasteurized milk was inoculated at four different levels varying between 3 to 6 log cfu·ml-1 with the pathogen and its survival was determined in milk, coagulum, curd and cheese received from the production steps and at the 1st and 7th days of the storage. The heat treatment process applied after curd acidification step at 75 °C for 5 min was the most efficient barrier and greatly influenced the viability of the bacteria. Nevertheless this step was not enough to destroy all the bacteria especially when inoculated > 3 log cfu·ml-1 levels. For all the groups, this stage resulted in approximately 3 log decrease. Storage at 6 ± 1 °C had no influence on the viability and the count of pathogen remained nearly constant throughout storage. Results of this study indicate that neither production steps nor storage at 6 ± 1 °C for 7 days in vacuum packages was enough to eliminate L. monocytogenes in Kashar cheese particularly if the initial level was high. Thus, in addition to the pasteurization process, hygienic measures should be taken into account to avoid possible secondary contaminations. Kashar cheese, inoculum, Listeria monocytogenes, survival Kashar cheese is one of the most popular semi-hard cheeses produced in the Balkan countries since the 11th and 12th centuries (Topal 1991). It shows similarity with the ‘Pasta Filata’ type of cheese such as Mozzarella (Halkman K. and Halkman Z. 1991). It is produced from either sheep or cow’s milk, or a mixture of both, and subjected to the stretching process at 75 °C during manufacture. It contains an average of 24.2% protein, 4.2% ash, 41.9% moisture, 25.1% fat, 4.6% salt (Tekinsen 2000); and its pH and acidity (LA%) are between 4.9 5.4 and 0.8 2.3, respectively (Cet inkaya 2000; Halkman et al. 1994). While Kashar is the second most popular cheese in Turkey with production of around 49.000 tons/year (Anonymous 2003), there is no standardized technique for its manufacture. It is produced either from raw milk according to traditional technology which is still employed in some dairies or from pasteurized milk in modern facilities. In traditional techniques, milk used in Kashar manufacture was not subjected to pasteurization process. Therefore the unique way to eliminate undesirable bacteria is the heat treatment applied at 75 °C during curd stretching (Halkman et al. 1994; Soyutemiz et al. 2000). L. monocytogenes is a ubiquitous Gram-positive, facultative intracellular bacterium that causes severe infections in both humans and animals (Cabanes et al. 2002; Farber and Peterkin 1991). Several outbreaks and sporadic cases of listeriosis associated with the consumption of pasteurized milk, cheeses made from unpasteurized milk and other dairy products contaminated with L. monocytogenes have been reported (Dal ton et al. 1997; Schuchat et al. 1997; Schlech 2000; Makino et al. 2005; Maijala et al. 2001). While particularly soft-ripened cheeses were the most frequently incriminated dairy products in regard to listeriosis, hard and semi-hard cheeses had also potential risk for public health ACTA VET. BRNO 2007, 76: 143–148; doi:10.2754/avb200776010143 Address for correspondence: Dr. Figen Cetinkaya University of Uludag, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology, Gorukle, 16059 Bursa TURKEY Phone: +90 224 442 9200 ext. 278 Fax: +90 224 442 8025 E-mail: figcetinkaya@yahoo.com http://www.vfu.cz/acta-vet/actavet.htm (Back et al. 1993; Genigeorgis et al. 1991). In some cases, the incidence was even found higher in hard and semi hard cheeses surface compared to soft cheeses (Pak et al. 2002). Guerra and Bernardo (1997) indicated that 17 of the 68 traditional hard and semi-hard cheeses (25%) manufactured in Alentejo were contaminated with Listeria spp. and eight of them (11.8%) were identified as L. monocytogenes. In studies conducted in Turkey on Tulum (Colak et al. 2006) and Herby cheeses (Sagun et al. 2001), two traditional semi hard cheeses, the incidence of the pathogen was found 4.8% and 3.93%, respectively. To date, to our knowledge the survival of L. monocytogenes and the effects of special manufacturing factors used in the production of Kashar cheese have not been studied. The aim of the present study was to investigate the influence of special manufacturing steps and vacuum-packaged storage of Kashar cheese on the behaviour and survival of L. monocytogenes, particularly when inoculated at different levels. Materials and Methods Bacter ia l cul tures and preparat ion of inoculum L. monocytogenes strain SLCC 2375 serotype 4b used in this study was kindly provided by Dr. Ch. Jacquet, Institut Pasteur, Center National de Reference des Listeria, Paris, France. Stock cultures were maintained on Tryptone Soya Agar (TSA) (no. CM131, Oxoid, UK) slants at 4 °C and subcultured monthly. For intermediate cultures, a loopful of the stock cultures was transferred into Tryptone Soya Broth (TSB) (no. CM129, Oxoid, UK) and incubated at 35 °C for 24 h to obtain a cell density of about 103 to 106·ml-1, and then cultured on Oxford Agar (no. CM856, Oxoid, UK) followed by incubation at 35 °C for 24 h. Star ter cul tures Commercial cultures G3Mix6 (Visbyvac, L 91115254) composed of Lactococcus lactis spp. lactis and Lc. lactis ssp. cremoris and SSK1 (Visbyvac, L 71812254) composed of Streptococcus salivarius spp. thermophilus, Lactobacillus delbrueckii spp. bulgaricus and Lb. helveticus which were obtained from Peyma-Chr. Hansen (Istanbul, Turkey) were used as the starter culture. Test tubes containing 9 ml reconstituted sterile skim milk (11%) were inoculated with the activated starter stock culture. This culture was subcultured for 24 h at 30 °C before use in cheese making. Cheese making was performed with 1% of starter culture combination including 1:1 rate of G3 Mix 6 and SSK 1. Cheese product ion Kashar cheeses were prepared according to the protocol given in Fig. 1. The pasteurized cow milk (at 72 °C for 15 s) to be used in cheese making was firstly tested for the presence of L. monocytogenes before inoculation. Each of the cheeses was prepared by using 40 litres of pasteurized milk in a stainless steel cheese vat. CaCl2 (0.025%) was added to the milk after cooling to 35 °C. L. monocytogenes, at the final concentration of approximately 3, 4, 5, and 6 log cfu·ml-1 for the groups 1, 2, 3 and 4, respectively and G3Mix6 and SSK1 starter cultures combination (1%) grown 24 h at 30 °C were inoculated into milk. A 4 ml volume of calf rennet diluted in cold water was also added to induce coagulation. The coagulum (usually 45 60 min after rennet addition) was cut in nearly 4 × 4 cm cubic forms and remained quiescent for ca 15 min. The temperature of the vat content was gradually raised to 41 °C under continuous stirring. Thereafter, the coagulum was collected in cheesecloth and pressed for syneresis (2 4 h). Subsequently, the curd was cut into blocks (25 35 cm) and kept at room temperature for 6 7 h. At the end of this period, the curd took an elastic form and its pH reached 5.10 5.20. At this stage, heat treatment of curd in brine containing 6% NaCl at 75 ± 1 °C for 5 ± 0.5 min was performed. For this purpose, the curd was cut into long strips and placed in a water bath in a stainless steel bucket containing several holes (5 mm in diameter). After heat treatment, the curd was manually kneaded for 5 min to eliminate air bubbles and moulded in plastic moulds (10 cm in diameter and 6 cm in height). Cheese wheels were kept at room temperature for a period of 20 h and turned over once in every two hours at this time. After 20 h, the cheese wheels (approximately 600 g weight) were removed from the plastic moulds. Thereafter, they were vacuum packaged in polyethylene bags and stored at 6 ± 1°C for 7 day. Sampling Duplicate samples were taken from the pasteurized milk, inoculated milk, coagulum, acidified curd, heat-treated curd and cheese after storage for 1 and 7 days. Microbiological analysis For enumeration of L. monocytogenes, the samples (10 ml or g) were transferred into individual sterile stomacher bags, mixed with 90 ml of 0.1% peptone water and homogenized in a Seward Stomacher 80 Lab System for ~ 2 min. Thereafter, a 0.1 ml portion of the homogenate was plated by spreading in duplicate on Oxford agar plates with supplement (no. SR140E, Oxoid, UK). To increase the detection level to 10 organisms/g, 0.2 ml of the initial 1 : 10 dilution was surface plated on each of 5 plates of Oxford agar, and resulting Listeria counts were combined. 144

. In some cases, the incidence was even found higher in hard and semi hard cheeses surface compared to soft cheeses (P a k et al. 2002).Guerra and Bernardo (1997) indicated that 17 of the 68 traditional hard and semi-hard cheeses (25%) manufactured in Alentejo were contaminated with Listeria spp.and eight of them (11.8%) were identified as L. monocytogenes.In studies conducted in Turkey on Tulum (Colak et al. 2006) and Herby cheeses (Sagun et al. 2001), two traditional semi hard cheeses, the incidence of the pathogen was found 4.8% and 3.93%, respectively.
To date, to our knowledge the survival of L. monocytogenes and the effects of special manufacturing factors used in the production of Kashar cheese have not been studied.The aim of the present study was to investigate the influence of special manufacturing steps and vacuum-packaged storage of Kashar cheese on the behaviour and survival of L. monocytogenes, particularly when inoculated at different levels.

Bacterial cultures and preparation of inoculum
L. monocytogenes strain SLCC 2375 serotype 4b used in this study was kindly provided by Dr. Ch. Jacquet, Institut Pasteur, Center National de Reference des Listeria, Paris, France.Stock cultures were maintained on Tryptone Soya Agar (TSA) (no.CM131, Oxoid, UK) slants at 4 °C and subcultured monthly.For intermediate cultures, a loopful of the stock cultures was transferred into Tryptone Soya Broth (TSB) (no.CM129, Oxoid, UK) and incubated at 35 °C for 24 h to obtain a cell density of about 10 3 to 10 6 •ml -1 , and then cultured on Oxford Agar (no.CM856, Oxoid, UK) followed by incubation at 35 °C for 24 h.

Starter cultures
Commercial cultures G 3 Mix6 (Visbyvac, L 91115254) composed of Lactococcus lactis spp.lactis and Lc.lactis ssp.cremoris and SSK1 (Visbyvac, L 71812254) composed of Streptococcus salivarius spp.thermophilus, Lactobacillus delbrueckii spp.bulgaricus and Lb.helveticus which were obtained from Peyma-Chr.Hansen (Istanbul, Turkey) were used as the starter culture.Test tubes containing 9 ml reconstituted sterile skim milk (11%) were inoculated with the activated starter stock culture.This culture was subcultured for 24 h at 30 °C before use in cheese making.Cheese making was performed with 1% of starter culture combination including 1:1 rate of G3 Mix 6 and SSK 1.

Cheese production
Kashar cheeses were prepared according to the protocol given in Fig. 1.The pasteurized cow milk (at 72 °C for 15 s) to be used in cheese making was firstly tested for the presence of L. monocytogenes before inoculation.Each of the cheeses was prepared by using 40 litres of pasteurized milk in a stainless steel cheese vat.CaCl 2 (0.025%) was added to the milk after cooling to 35 °C.L. monocytogenes, at the final concentration of approximately 3, 4, 5, and 6 log cfu•ml -1 for the groups 1, 2, 3 and 4, respectively and G 3 Mix6 and SSK1 starter cultures combination (1%) grown 24 h at 30 °C were inoculated into milk.A 4 ml volume of calf rennet diluted in cold water was also added to induce coagulation.The coagulum (usually 45 -60 min after rennet addition) was cut in nearly 4 × 4 cm cubic forms and remained quiescent for ca 15 min.The temperature of the vat content was gradually raised to 41 °C under continuous stirring.Thereafter, the coagulum was collected in cheesecloth and pressed for syneresis (2 -4 h).Subsequently, the curd was cut into blocks (25 -35 cm) and kept at room temperature for 6 -7 h.At the end of this period, the curd took an elastic form and its pH reached 5.10 -5.20.At this stage, heat treatment of curd in brine containing 6% NaCl at 75 ± 1 °C for 5 ± 0.5 min was performed.For this purpose, the curd was cut into long strips and placed in a water bath in a stainless steel bucket containing several holes (5 mm in diameter).After heat treatment, the curd was manually kneaded for 5 min to eliminate air bubbles and moulded in plastic moulds (10 cm in diameter and 6 cm in height).Cheese wheels were kept at room temperature for a period of 20 h and turned over once in every two hours at this time.After 20 h, the cheese wheels (approximately 600 g weight) were removed from the plastic moulds.Thereafter, they were vacuum packaged in polyethylene bags and stored at 6 ± 1°C for 7 day.

Sampling
Duplicate samples were taken from the pasteurized milk, inoculated milk, coagulum, acidified curd, heat-treated curd and cheese after storage for 1 and 7 days.

Microbiological analysis
For enumeration of L. monocytogenes, the samples (10 ml or g) were transferred into individual sterile stomacher bags, mixed with 90 ml of 0.1% peptone water and homogenized in a Seward Stomacher 80 Lab System for ~ 2 min.Thereafter, a 0.1 ml portion of the homogenate was plated by spreading in duplicate on Oxford agar plates with supplement (no.SR140E, Oxoid, UK).To increase the detection level to 10 organisms/g, 0.2 ml of the initial 1 : 10 dilution was surface plated on each of 5 plates of Oxford agar, and resulting Listeria counts were combined.
All plates were incubated aerobically at 37 °C for 48 h and characteristic colonies (exhibiting a grey-black colour with a black halo) were counted.Representative colonies (five per counted plate) were confirmed as L. monocytogenes, based on morphology, catalase production, betahaemolysis, tumbling motility, oxidase test, xylose, rhamnose and mannitol fermentation.For samples, in which L. monocytogenes was not detected by direct plating, enrichment procedure was applied.This included incubation of 25 g cheese samples in 225 ml Tryptone Soya Broth + 0.6% Yeast Extract (TSB, no.CM129, Oxoid, UK; YE, no.L21, Oxoid, UK) at 35 °C for 48 h.Enrichment culture was then plated on Oxford agar medium and plates were examined for typical Listeria colonies after 48 h incubation at 37 °C.Following above mentioned confirmation tests, the characteristic colonies were identified as L. monocytogenes (Hitchins 1995).

Determination of pH
The pH of each sample was measured with an Orion pHmeter (Ionalyzer model 399A/F, Cambridge, Mass., USA) at 25 ± 1 °C (Kosikowski and Mistry 1997).

Statistical analysis
The experiments were established in a completely random design with three replications.All data were subjected to analysis of variance using MSTAT-C (Version 2.1 -Michigan State University 1991) and MINITAB (University of Texas at Austin) software.The significance of differences between groups was determined at the 0.01 probability levels, by the F-test.The F-protected least significant difference (LSD) was calculated at the 0.01 probability level.

Results and Discussion
Influence of manufacturing steps and vacuum packaged storage of Kashar cheese produced from pasteurized milk that was inoculated with different L. monocytogenes levels was investigated.Data regarding the populations of L. monocytogenes during manufacturing stages and storage at 6 °C in vacuum-packages of Kashar cheese are shown in Table 1.Pasteurized milk samples were free (by direct plating and enrichment process) of L. monocytogenes before inoculation.It is a general consensus that L. monocytogenes does not survive at proper pasteurization (Rudolf and Scherer 2001;Farber and Peterkin 1991), nonetheless postprocess contamination is possible.The numbers of pathogens at different stages or days were significantly different between the groups (p < 0.01).When the coagulum was heated at 41 °C for approximately 30 min, the numbers of L. monocytogenes remained nearly constant.This can be explained by the prolonged lag phase of the bacteria under these conditions.A previous study showed that L. monocytogenes has a lag phase of approximately 2 h when grown in sterile whole milk incubated at 35 °C (Rosenow and M arth 1987).Therefore, growth during the early stages of cheese manufacture would not be expected.Following  Buazzi et al. 1992) and Cameros cheese (Olarte et al. 2002).This increase would also have resulted from a combination of favourable conditions such as high moisture of curd, high temperature and the initial pH, which was around 6.0 at the beginning and decreased to 5.0 during curd acidification.
In the 1 st , 2 nd , 3 rd and 4 th group cheeses, the numbers of L. monocytogenes decreased by an average of 3.94, 3.12, 3.42 and 3.32 log units after the heat treatment process, respectively.In the first group, where the initial inoculation level was approximately 3 log cfu•ml -1 , almost totality of the bacteria was eliminated.However, this heat treatment was not sufficient to eliminate the pathogen where its final count varied between 2.35 -4.09 log 10 cfu•g -1 in the 2 nd , 3 rd and 4 th group cheeses (ca 4, 5, 6 log cfu•ml -1 ).This can be explained by the fact that even though the temperature of brine used in heat treatment of curd is quite high to kill all the bacteria, heat transfer towards the curd core might be impeded by the massive structure of the curd block.In studies with Mozzarella cheese, the authors reported that even at high inoculation levels L. monocytogenes could be eliminated during stretching process at 77 °C for 1 -4 min (Buazzi et al. 1992;K i m et al. 1998).On the other hand, Villani et al. (1996) working on the elimination of L. monocytogenes during stretching of traditional Mozzarella cheese reported a decrease of about 2 log at 95 °C.
During the storage at 6 ± 1 °C for 7 days in vacuum packages, no bacteria was isolated on the 1 st and 7 th days in the 1 st group cheese.On the other hand, there was no remarkable change in bacteria counts of the 2 nd , 3 rd and 4 th groups after 7 days of storage.Previous studies reported the ability of L. monocytogenes to survive and even grow in the cheeses packed under modified atmosphere during refrigerated storage (Olarte et al. 2002;Whitley et al. 2000).Rogga et al. (2005) reported that L. monocytogenes cannot grow but may survive during storage at 4 °C and 12 °C in Galotyri cheese despite its low pH below 4.0.Contrarily, in a study realized on Cottage, Feta and Kasseri cheeses wrapped up by cellophane, Genigeorgis et al. (1991) observed that the pathogen numbers decreased to 1.87, 2.04 and 2.04 log cfu•g -1 after storage of 8, 8 and 6 days at + 4 °C, respectively.
Results of this work indicate that the heat treatment of curd at 75 °C for 5 min during Kashar cheese production was not sufficient to eliminate the pathogen especially when it is at > 3 log cfu•ml -1 level.Therefore, correlation between heating temperature and heating treatment time should be readjusted to inactivate L. monocytogenes if post-pasteurization contamination is at high levels (> 3 log cfu•ml -1 ).Nevertheless, L. monocytogenes can survive in Kashar cheese stored in vacuum packed conditions.This demonstrates that if Kashar cheese is made from raw or heat treated milk containing L. monocytogenes, its 146 storage at 6 ± 1 °C for 7 days will not render the cheese free from viable pathogens.The presence of L. monocytogenes at levels of 2.74 -4.53 log cfu•g -1 in Kashar cheeses at the end of storage indicates a possible public health hazard.According to the microbiological criteria for milk-based products in the European Union, L. monocytogenes should not be present in 25 g of cheeses (European Council Directive 1992).To avoid health risks and to offer fresh and safe Kashar cheese to the consumers, the use of raw milk with high hygienic quality is indispensable.Moreover, strict sanitary production steps and proper HACCP procedures including efficient pasteurization and post-pasteurization handlings constitute an important barrier to obtain good quality final products.