Microbiological Quality of Soft, Semi-Hard and Hard Cheeses During the Shelf-Life

Abstract Cheeses as ready-to-eat food should be considered as a potential source of foodborne pathogens, primarily Listeria monocytogenes. The aim of present study was to determine the microbiological quality of soft, semi-hard and hard cheeses during the shelf-life, with particular reference to L. monocytogenes. Five types of cheeses were sampled at different time-points during the cold storage and analyzed for presence of Salmonella and L. monocytogenes, as well as lactic acid bacteria, Escherichia coli, coagulase-positive staphylococci, yeasts, molds, sulfite-reducing clostridia and L. monocytogenes counts. Water activity, pH and NaCl content were monitored in order to evaluate the possibility of L. monocytogenes growth. Challenge test for L. monocytogenes was performed in soft whey cheese, to determine the growth potential of pathogen during the shelf-life of product. All analyzed cheeses were compliant with microbiological criteria during the shelf-life. In soft cheeses, lactic acid bacteria increased in the course of the shelf-life period (1.2-2.6 log increase), while in semi-hard and hard cheeses it decreased (1.6 and 5.2 log decrease, respectively). Soft cheeses support the growth of L. monocytogenes according to determined pH values (5.8-6.5), water activity (0.99-0.94), and NaCl content (0.3-1.2%). Challenge test showed that L. monocytogenes growth potential in selected soft cheese was 0.43 log10 cfu/g during 8 days at 4°C. Water activity in semi-hard and hard cheeses was a limiting factor for Listeria growth during the shelf-life. Soft, semi-hard and hard cheeses were microbiologically stable during their defined shelf-life. Good manufacturing and hygienic practices must be strictly followed in the production of soft cheeses as Listeria-supporting food and be focused on preventing (re)contamination.


INTRODUCTION
Microbiological stability and safety of food during storage is related to many factors. Ready-to-eat food products, including cheeses, are intended for consumption without any treatment between final production step and consumption. The course of microbiological changes in different cheeses during storage and shelf-life depends on the production technology and cheese type (pasteurization, starters, acidity, ripening, etc.), physico-chemical properties In Croatia, microbiological surveys in recent years were mainly performed on traditional dairy products from non-pasteurized milk, and L. monocytogenes was frequently present (7,12). Thus, the aim of the present study was to evaluate the microbiological quality of pasteurized-milk cheeses during the shelflife with particular reference to L. monocytogenes prevalence and counts. The growth potential of L. monocytogenes was also evaluated in soft whey cheese during the shelf-life.

MATERIAL AND METHODS
Soft, semi-hard and hard cheeses were produced according to standard procedure in a local cheese producing plant and sampled at the end of production. Ten units of each cheese were sampled, stored in laboratory at 4°C and periodically analyzed during defined shelf-life (Table 1).

Cheese type Cheese name Shelf-life Time-point sampling
Microbiological parameters Chemical parameters For microbiological analyses, 25 g of sample was diluted in 225 ml of appropriate media (Buffered Peptone Water, Half-Fraser Broth or Peptone salt water) and homogenized for 2 min (Stomacher, Sedward, UK). Serial decimal dilutions were prepared and 0.1 ml or 1 ml of selected dilution were used for evaluation of lactic acid bacteria count (MRS, Merck, Darmstadt, Germany) at 30°C for 48 h, Staphylococcus aureus (Baird Parker agar, Merck, Germany) at 37°C for 48 h, Escherichia coli (Rapid E. coli, Bio-Rad, France) 37/44°C for 24 h, and sulfite-reducing clostridia (Iron Sulphyte Agar, bioMerieux, Marcy l'Etoile, France). Salmonella spp. was determined following a standard ISO 6579 method and Molecular Detection System (MDS, 3M, USA). L. monocytogenes presence and count was determined using ISO 11290-1 and 11290-2, respectively.
Water activity (a w ) was determined by means of HygroPalm AW1 (Rotronic, Switzerland), Table 1. Sampling scheme and parameters monitored in cheese during shelf-life pH with pH-meter (pH 510, Eutech instruments, Netherlands) and NaCl following Mohr's method. Challenge test was performed in whey cheese "skuta" (shelf-life 8 days) inoculated with mixed culture of L. monocytogenes (two strains of cheese origin and L. monocytogenes ATCC 7644). The strains were grown in Brain Hearth Infusion Broth (bioMerieux, France) at 37ºC for 24 h. One ml of each culture was centrifuged at 10000 g for 10 min, followed by supernatant removal and washing the cells with sterile saline water. Cells were diluted in 1 ml of sterile saline water and serially diluted to determine the cell number, using PALCAM agar (Merck, Germany) incubated at 37ºC for 24-48 h.
Appropriate dilutions were taken for cheese portion inoculation to obtain an inoculation level of 30-50 cfu/g. Cheese samples were inoculated in triplicate, vacuum-packed and stored at 4ºC for 8 days. L. monocytogenes count, pH and water activity were determined at day 0 and at the end of shelf-life.

RESULTS
The results of the microbiological and chemical analyses of cheeses during the storage are summarized in Table 2  L. monocytogenes counts were below 10 cfu/g in all cheese samples during storage. Population of lactic acid bacteria increased during the storage of soft cheeses, reaching 7-8 log cfu/g at the end of shelf-life. The pH values, water activity and NaCl content in soft cheeses were in the range of Listeriasupporting values. Semi-hard and hard cheeses were characterized by decrease of lactic acid bacteria counts during the storage and a slight pH increase.
The pH values and NaCl content found in semihard and hard cheeses were not a limiting factor for Listeria growth. However, it is evident that water activity was below values that enables the growth of most foodborne pathogens.
Since soft cheeses showed to be supporting of Listeria growth according to their physico-chemical characteristics, the challenge test was performed with L. monocytogenes. Results of L. monocytogenes growth potential in whey cheese skuta are shown in Table 3, as well as pH and a w values (Table 4). Despite high water activity and optimal pH, L. monocytogenes inoculated in low numbers (30-50 cfu/g) didn't reach a critical limit of 100 cfu/g in cheese stored at 4 ºC for 8 days.

DISCUSSION
Microbiological quality and interpretation of microbiological findings of different kind of cheeses depends on the sampling points during the production or retail phase. In present study, the phase of post-processing was monitored in order to evaluate compliance with microbiological criteria, with particular reference to L. monocytogenes counts. In relation to overall microbiological quality, results showed that soft, semi-hard and hard cheeses were microbiologically stable during their defined shelf-life. Microbiological stability of semi-hard and hard cheeses relies on several hurdles including starter cultures competitiveness, increased salt content and, most importantly, low water activity (2,9). Physico-chemical values and their changes during the storage of semi-hard and hard cheeses in our study are in line with previous reports (11,13,15). L. monocytogenes was not present in viable counts, however it should be stressed that secondary contamination is possible during the cutting of the cheese into quarters or halves, followed by packaging. Prevention of contamination with Listeria at this point should be based on Good Hygienic Practice and verification of sanitation programs.
Soft cheeses in general are of limited durability, because of high moisture content and high pH which support the proliferation of some spoilage microorganism like Enterobacter spp.  Table 3. Growth potential of Listeria monocytogenes in whey cheese skuta  (1,10,16).
During the storage of whey cheese skuta the pH decreased and lactic acid bacteria count increased which is in accordance with other studies (10,16). Lactic acid bacteria are known to be beneficial and functional microbes in many different dairy products (20), however their outgrowth in this kind of products (whey cheese) could contribute to spoilage (8). Soft cheeses are ready-to-eat products that support the growth of L. monocytogenes based on their physic-chemical characteristics (14,18), which is also presented by the results in current survey (pH values 5.8-6.5, water activity 0.99-0.94 and NaCl content 0.3-1.2%). Challenge test showed that L. monocytogenes growth potential in whey cheese was 0.43 log 10 cfu/g during 8 days at 4°C, meaning that the proposed shelf-life is acceptable for products stored under defined conditions. Many studies from last decades emphasized that L. monocytogenes represents a serious public-health problem due to high prevalence in soft cheeses (7,17,19). Recent studies are more focused on bacterial kinetics in cheeses made from pasteurized milk during their shelf-life at different storage conditions, and related application of bio-protective strategies (3).

CONCLUSION
Compliance with microbiological criteria at the end of cheese production (final product) doesn't guarantee that microbiological hazards are excluded. Listeria monocytogenes is a ubiquitous bacteria and secondary contamination of products is possible under poor hygienic conditions. Despite the fact that the growth of the pathogen is limited in semi-hard and hard cheeses by low water activity, the secondary (surface) contamination could result in hazardous products. The significance of following strict hygienic procedures is evident even more in soft cheese production, since they support the growth of L. monocytogenes.