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Microbial quality and presence of moulds in Kuflu cheese
International Journal of Food Microbiology 115 (2007) 376 – 380 www.elsevier.com/locate/ijfoodmicro
Short communication
Microbial quality and presence of moulds in Kuflu cheese A.A. Hayaloglu a,⁎, S. Kirbag b a
Department of Food Engineering, Inonu University, 44280 Malatya, Turkey b Department of Biology, Firat University, 23119 Elazig, Turkey
Received 10 July 2006; received in revised form 30 November 2006; accepted 6 December 2006
Abstract The chemical and microbial qualities, including fungal flora, of 30 samples of Kuflu cheese randomly purchased from different markets in Turkey were investigated. The gross composition of the cheese samples ranged between 37.65–53.65% moisture, 6.21–40.09% fat-in-dry matter, 4.70–10.07% salt-in-moisture and 26.18–44.85% protein. The mean pH value of the cheeses was 6.29 ± 0.28 and pH values ranged from 5.52 to 7.22. Variations between the samples in terms of their gross composition suggested a lack of quality standards in cheesemilk, cheesemaking procedure and ripening conditions. The levels of main microbial groups including total mesophilic and coliform bacteria, yeasts and moulds and the presence of some potentially pathogenic microorganisms (E. coli, Salmonella spp. and Staphylococcus aureus) were determined. The high numbers of all microbial groups and presence of potentially pathogenic organisms in the cheese samples suggested that the production and maturation of Kuflu cheese should be improved by better hygiene. Moulds at the cheese surface were isolated and identified. A total of 24 different mould species were detected and the genus most frequently isolated was Penicillium spp. which represented 70.25% of total isolates. Penicillium commune, P. roqueforti and P. verrucosum were the most abundant species in the cheeses sampled. The other dominant fungal groups were Geotrichum candidum, Penicillium expansum and P. chrysogenum. Other genera isolated from the cheese were Acremonium, Alternaria, Aspergillus, Cladosporium, Geotrichum, Mucor, Rhizopus and Trichoderma. The potentially toxigenic species, including some Penicillum spp. and Aspergillus flavus, were also detected. © 2006 Elsevier B.V. All rights reserved. Keywords: Mould-ripened cheese; Blue cheese; Microbial quality; Fungal flora; Penicillium
1. Introduction Cheese ripening is a complex and dynamic biochemical process that includes protein breakdown, fat hydrolysis and lactose metabolism (El Soda et al., 1995; McSweeney and Sousa, 2000). These processes are catalyzed by agents such as residual coagulant, indigenous milk enzymes, starter or nonstarter microflora and secondary organisms. The secondary organisms include moulds and presence of moulds on the surface of mould-ripened cheese gives them a different appearance and flavor from other cheeses. The moulds have a more complex enzyme system than bacteria and their enzymes contribute to the maturation of the cheese, i.e., to proteolysis and lipolysis which are more extensive in these cheeses (Gripon, 1987). However, in other varieties mould growth on ⁎ Corresponding author. Tel.: +90 422 341 0010; fax: +90 422 341 0046. E-mail address: [email protected] (A.A. Hayaloglu). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.12.002
the cheese surface causes economic losses and quality problems (Kivanc, 1992; Kure et al., 2001). Some moulds are capable of producing toxic secondary metabolites (mycotoxins) and of producing undesirable flavor. Consequently, these organisms can pose a potential risk for public health (Kure et al., 2001; Montagna et al., 2004). Moulds growing on the surface of mould-ripened cheeses were isolated by several authors (Hasenekoglu, 1988; Kivanc, 1992; Kure and Skaar, 2000; Erdogan et al., 2003; Kure et al., 2004; Montagna et al., 2004) and they reported that Penicillium spp. were the dominating species on the cheeses. Kuflu cheese is a mould-ripened variety produced in the middle and eastern region of Turkey. Kuflu cheese has a semihard texture, rectangular shape and weighs between 300 and 500 g. The cheese is made from non-pasteurized, skimmed (or partially-skimmed) sheep's or goat's milk or their mixture. Cheesemilk is clotted with home-made calf rennet at 30–32 °C and, no starter culture or mould spores were added to the milk.
A.A. Hayaloglu, S. Kirbag / International Journal of Food Microbiology 115 (2007) 376–380
After curd forming, the curd is transferred into a cotton bag for whey drainage, which takes about 24 h. Then, the curd is broken into pieces the size of pea grains by hand, dry-salted and retransferred into the bag. During the first 10 days, the curds are pre-ripened in the cotton bag; then tightly filled into tulum (a goat skin bag). The cheese is finally placed in natural caves where ripening takes place at 80–90% relative humidity and at 6–12 °C. After ripening of the cheese, the cheese is cut into blocks with a knife and the cheese blocks are ripened in the caves or rooms until blue-green moulds spontaneously grow on the surface of the cheese. The color of Kuflu cheese is white with blue or green veins; however, blue or green colors dominate its surface after the growth of moulds during mildewing. The aroma and flavor of Kuflu cheese are very intense due to its complex microflora which comes from the cheesemilk, home-made calf rennet extracts or environmental flora during ripening. Consumption of the cheese has increased due to its being low- or nonfat and because of its intense flavor. The cheese is consumed without discarding the mouldy part of the cheese. To the authors' knowledge, although there are no reports of health hazards for humans after consumption of Kuflu cheese, we aimed to investigate the fungal flora, hygienic quality and gross composition of the cheese. Therefore, we determined the chemical and microbiological composition of the cheese and identified the moulds at the genus and species level. 2. Materials and methods 2.1. Cheese samples A total of 30 Kuflu cheese samples were purchased from different markets during the autumn of 2004. The samples were taken using a sterile knife and packed in aseptic polyethylene bags and kept at + 4 °C until analysis. 2.2. Chemical analyses Cheeses were analyzed in duplicate for moisture, fat, total protein, salt, pH and titratable acidity as described by Hayaloglu et al. (2005). 2.3. Microbiological analyses For each cheese sample, 10 g was weighed and dispersed aseptically in 90 mL of citrate buffer (2%, w/v) and homogenized in a sterile polyethylene bag using a Stomacher (Seward Laboratory Blender Stomacher 400 Lab Blender UK) for 1.5 min. Serial dilutions were made in 0.1% sterile peptone water and all determinations were made in duplicate (Harrigan, 1998). The enumeration of total mesophilic bacteria (Plate Count Agar, Merck, Darmstadt, Germany) at 30 °C for 48 h (Messer et al., 1985), total coliforms (Violet Red Bile Agar, Merck, Darmstadt, Germany) at 37 °C for 48 h (Hartman and La Grang, 1985), yeasts and moulds (Potato Dextrose Agar, Merck, Darmstadt, Germany) at 21 °C for 7 days (Frank et al., 1993), Staphylococcus aureus (Baird-Parker Agar, Merck, Darmstadt,
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Germany) at 37 °C for 48 h (Harrigan, 1998) and Salmonella spp. (Tetrathionate Broth and Hecktoen Agar) at 35 °C for 24 h (Baumgart, 1993) were performed. 2.4. Isolation and identification of moulds After preparation of serial dilutions of the cheese samples, each was plated onto Malt Extract Agar (Merck) and the plates were incubated at 25 °C for 7 days. Next, isolates were subcultured on Yeast Extract Sucrose Agar (Oxoid), CzapekDox Agar (Merck), Potato Dextrose Agar (Merck) and Sabouraud Dextrose Agar (Merck). The media were inoculated three-point and incubated at 25 °C for 7 days, but Sabouraud media was incubated at 30 °C for 7 days. The isolated moulds were identified by their morphological characteristics and pigments as described by Barnett and Hunter (1987), Pitt (1988), Pitt and Cruickshank (1990) and Samson et al. (2000). 3. Results and discussion 3.1. Chemical composition The gross chemical composition (moisture, fat, protein and salt contents) and pH of Kuflu cheese samples were determined. Wide ranges in pH and chemical composition were observed. The pH values ranged from 5.52 to 7.22 and the average value was 6.29. The average pH value was lower than that of other studies on mould-ripened cheeses (Prieto et al., 1999; Schlesser et al., 1992). The pH in Blue cheeses increases during ripening due to catabolism of lactic acid and deamination of amino acids by moulds. Trends were similar to those of pH observed in acidity. The moisture concentration of the cheese changed between 37.65 and 53.65% and the average moisture contents were higher than in Tulum cheese. According to these results, Kuflu cheese can be categorized as semi-hard cheese in terms of its moisture content. Fat and fat-in-dry matter (FDM) contents of the cheese were considerably lower than those of other Blue cheeses. Average values for fat and FDM were 6.30 and 12.18%, respectively. Kuflu cheese is manufactured from skimmed or partially-skimmed milk and hence, the fat contents were low in comparison to other mould-ripened varieties. Total protein content of the cheeses was higher than that of other mould-ripened cheese. These differences can be attributed to the concentration effect on compositional parameters due to the low level of fat in Kuflu cheese. Salt-in-moisture (SM) contents of Kuflu cheese, which ranged from 4.70 to 10.07%, were higher than those of other mould-ripened cheeses (Prieto et al., 1999; Sousa and McSweeney, 2001). 3.2. Microbiological analysis The microbial counts of Kuflu cheese samples are shown in Table 1. Total mesophilic bacterial counts were high (ranged from 8.26 to 10.69 log cfu/g cheese) in the cheeses, probably due to the use of raw milk with high microbial load in their manufacture and uncontrolled ripening conditions. The cheese samples which were ripened open to the atmosphere might have
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Table 1 Log counts (log cfu/g cheese) of the microbial groups found in Kuflu cheese Statistical parameter Total mesophilic bacteria Coliform bacteria Staphylococcus aureus Yeasts and moulds E. coli Average Range ±SD
9.31 8.26–10.69 0.53
2.51 b1–3.04 0.48
2.77 b1–4.92 1.05
been contaminated by environmental flora. High levels of salt in the cheese did not adversely affect the growth of bacteria. Coliform organisms were detected in all except for 3 samples; the average values of coliform bacteria were 2.26 log cfu/g. By Turkish regulations the maximum allowable counts of coliform group bacteria are 2 log cfu/g in Turkish White-brined cheese (Anonymous, 2001). The intestinal origin of these bacteria and their presence in cheese should be considered as evidence of poor manufacturing conditions and lack of hygiene. Coliform group bacteria were also found in cheeses such as Kopanisti (Tzanetakis et al., 1987), Spanish blue (Lopez-Diaz et al., 1995), Karin kaymagi (Cakmakci et al., 1995) and Carra cheeses (Aygun et al., 2005) purchased from markets. The presence of these group bacteria in the cheese also suggests their presence in the milk. In addition, manual contact is usual during the marketing of this type of cheese. The pH and salt contents of Kuflu cheese samples were favorable for the growth of coliform. Yeasts and moulds were present all samples with values varying from 4.83 to 6.94 log cfu/g cheese. The mould counts were extremely high in Kuflu cheese and mould veins appeared in the centre of the cheese. In addition, piercing the cheese allows the growth and sporulation of moulds throughout its surface (Gripon, 1987). Some potentially pathogenic bacteria were found in Kuflu cheese samples (Table 1). E. coli (17 samples; 56.6% of total), Salmonella spp. (7 samples; 23.3% of total) and S. aureus (11 samples, 36.6% of total) were found in the samples. S. aureus were present in our samples at levels of 2.00 to 4.92 log cfu/g. Uncontrolled environmental conditions during manufacturing and ripening favored their presence and survival. These potentially pathogenic bacteria can multiply at refrigerated temperatures and are tolerant to salt. Normally, these pathogens are present in cheese at first stage of maturation; then they become inactivate as ripening proceeds. Catabolism of lactic acid by moulds and yeasts on the cheese surface results in an increase in pH, facilitating survival of pathogens (Sousa and McSweeney, 2001). Consequently, these organisms are able to be isolated at the stage of consumption. De Boer and Kuik (1987) and Lopez-Diaz et al. (1995) could not isolate E. coli and Salmonella from their samples of mould-ripened cheese. 3.3. Isolation and identification of moulds A total of 24 different species of mould were isolated and identified in Kuflu cheese samples. Penicillium commune, P. roqueforti, Geotrichum candidum and P. verrucosum were the mould species most frequently isolated (Table 2). These four species made up 36.7% of the total number of moulds. The genus most frequently isolated was Penicillium spp. which
6.36 4.83–6.95 0.53
Salmonella spp.
Detected in 17 samples Detected in 7 samples
represented 70.25% (111 isolates of Penicillium spp.) of total isolates. Other genera isolated were Alternaria, Acremonium, Aspergillus, Cladosporium, Geotrichum, Mucor, Rhizopus and Trichoderma. Alperden (1978) examined 85 cheese samples produced in the Marmara region of Turkey and showed that Penicillium spp. was the major contaminant representing 54% of total isolates. Penicillium spp. was also the predominant mould genus on blue-veined cheeses (Gripon, 1987), Picon Bejes-Tresviso (Prieto et al., 1999), Jarlsberg and Norvegia (Kure and Skaar, 2000; Kure et al., 2001), Gorgonzola (Gobbetti et al., 1997), Cabrales (Florez et al., 2006) and as a contaminant on other cheeses such as Cheddar (Bullerman and Olivigni, 1974), Teleme (Zerfiridis, 1985), Kasar (Kivanc, 1992) and goats' and sheep's milk cheeses sold in Italy (Montagna et al., 2004). P. commune (10.1%) and P. roqueforti (8.9%) were the dominant mould species in the cheeses. P. commune is a wild type of P. camemberti and frequently contaminates foods (Pitt, 1988). Kure et al. (2001) examined cheeses from four different factories and P. commune dominated the isolates in two of these factories. This mould was also found on Cheddar cheese as a spoilage agent by Hocking and Faedo (1992) and on Italian goats' and sheep's milk cheeses by Montagna et al. (2004). Table 2 Mould species isolated from Kuflu cheese samples Mould species
Number of isolates
Frequency (%)
Acremonium alternatum Alternaria alternata Aspergillus flavus A. fumigatus A. niger Cladosporium cladosporioides C. herbarium Geotrichum candidum Mucor himealis M. mucedo Penicillium brevicompactum P. chrysogenum P. citrinum P. commune P. crustom P. echinulatum P. expansum P. roqueforti P. solitum P. spinolusum P. verrucosum P. viridicatum Rhizopus nigricans Trichoderma harzianum Total: 24 species
2 3 1 7 3 3 2 13 3 2 7 9 8 16 6 8 10 14 8 5 15 5 5 3 158
1.3 1.9 0.6 4.4 1.9 1.9 1.3 8.2 1.9 1.3 4.4 5.7 5.1 10.1 3.8 5.1 6.3 8.9 5.1 3.2 9.5 3.2 3.2 1.9 100
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P. roqueforti is used in the manufacture of Roquefort, Stilton and Gorgonzola (Gripon, 1987; Fox et al., 2000) and thus is also isolated from these cheeses (Aran et al., 1986; Aran and Eke, 1987; Tsai et al., 1988; Kivanc, 1992; Erdogan et al., 2003). Crumbly texture and small air holes in Kuflu cheese allow the growth of Penicillum spp., in particular P. roqueforti. In addition, P. roqueforti is capable of growth in the air with high levels of carbon dioxide (Kure and Skaar, 2000). P. verrucosum, Penicillium expansum and P. chrysogenum were the Penicillium species most frequently isolated from Kuflu cheese. P. verrucosum was found by Northolt et al. (1980) as a contaminant on visibly mouldy cheeses in shops, households, and warehouses. Its mycotoxin-reducing capability was reported by Pitt (1987) and Larsen et al. (2001). P. verrucosum was found to be the predominant mould on surface and inner parts of Kasar cheese by Kivanc (1992). Separately, P. expansum and P. chrysogenum were isolated at a low frequency in Jarlsberg and Norvegia cheeses (Kure and Skaar, 2000; Kure et al., 2001). Apart from Penicillium spp., the mould species most frequently isolated from Kuflu cheeses were G. candidum, Aspergillus fumigatus and Rhizopus nigricans. These three species made up 15.8% of total mould isolates from the cheese. G. candidum represented 8.2% of the moulds on the cheese and its appearance was a white-velvety coat on the surface. G. candidum also affects the cheese biochemistry during ripening; it releases lipases and proteases into the cheese matrix, reduces bitterness, contributes cheese aroma, neutralizes the curd acidity produced by lactic acid bacteria and stimulates the growth of surface bacteria such as Brevibacterium spp. (Marcellino et al., 2001). G. candidum was not isolated frequently from Kasar cheese (Kivanc, 1992) and Jarlsberg and Norvegia cheeses (Kure and Skaar, 2000; Kure et al., 2001); however, Kure et al. (2004) reported that G. candidum was frequently isolated from one of four cheese factories in Norway. A few isolates of other genera including Acremonium, Alternaria, Cladosporium, Mucor and Trichoderma spp. were also found on Kuflu cheese. The toxigenic properties of these five species in cheeses were not reported by Samson et al. (2000) and CAST (2003). The study describes the chemical and microbial composition and fungal flora of Kuflu cheese. Results showed that many problems appeared to originate in the production, ripening and marketing stages of the cheese in terms of its bacteriological quality and the presence of some potentially pathogens including E. coli, S. aureus and Salmonella. Moulds isolated and identified from the cheese, if they produce mycotoxins, may represent a health hazard. Penicillium spp. was the dominant fungal flora in Kuflu cheese and made up about 70% of the total mould isolates. Erdogan et al. (2003) studied the mycotoxinproducing capability of P. roqueforti isolates from mouldy Tulum cheese and they found that P. roqueforti isolates produced some mycotoxins including patulin, penicillic acid, roquefortine or PR toxin. Uncontrolled mildewing of the cheese must be considered as a problem and provides no guarantee of product safety for human consumption. For the ripening of Kuflu cheese, non-toxigenic strains of moulds should be selected.
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Acknowledgements This work was financially supported by the Inonu University Scientific Research Projects Unit (Project No: 2005/34). Prof. Paul McSweeney (University College, Cork, Ireland) is acknowledged for critical reading of the manuscript. References Alperden, I., 1978. Hayvansal urunlerde mikotoksin arastirmalari ve kalite kontrol esaslari. Tubitak-Mam Bilimsel End. Arast. Enst., vol. 31, p. 129. Aran, N., Eke, D., 1987. Mould mycoflora of Kasar cheese at the stage of consumption. Food Microbiology 4, 101–104. Aran, N., Eke, D., Alperden, I., 1986. Yari sert karakterdeki Turk peynirlerinde kuf florasi. Ege Universitesi Muhendislik Fakultesi Dergisi 4, 12–15. Anonymous, 2001. Turk Gida Kodeksi. Mikrobiyolojik kriterler tebligi, No. 2001/19. Resmi Gazete, 02.09.2001, Sayi: 24511. Aygun, O., Aslantas, O., Oner, S., 2005. A survey on the microbiological quality of Carra, a traditional Turkish cheese. Journal of Food Engineering 66, 401–404. Barnett, H.L., Hunter, B.B., 1987. Illustrated Genera of Imperfect Fungi. Mcmillan Publishing Co., London. Baumgart, J., 1993. Microbiologische untersuchungen von lebensmitteln. Behr's Verlag, Hamburg. Bullerman, L.B., Olivigni, F.J., 1974. Mycotoxin producing-potential of molds isolated from Cheddar cheese. Journal of Food Science 39, 1166–1168. Cakmakci, S., Sengul, M., Caglar, A., 1995. The microbiological and chemical quality of Karin Kaymagi cheese produced in Turkey. Milchwissenschaft 50, 622–625. CAST, 2003. Mycotoxins: Risks in Plant, Animal, and Human Systems. Council for Agricultural Science and Technology, Ames, Iowa, USA. De Boer, E., Kuik, D., 1987. A survey of the microbiological quality of blueveined cheeses. Netherlands Milk and Dairy Journal 41, 227–237. El Soda, M., Farkye, N., Vuillemard, J.C., Simard, R.E., Olson, N.F., El Kholy, W., Dako, E., Medrano, E., Gaber, M., Lim, L., 1995. Autolysis of lactic acid bacteria: impact on flavour development in cheese. In: Charalambous, G. (Ed.), Food Flavours: Generation, Analysis and Process Influence. Elsevier Sciences B.V., Amsterdam, The Netherlands, pp. 2205–2223. Erdogan, A., Gurses, M., Sert, S., 2003. Isolation of moulds capable of producing mycotoxins from blue mouldy Tulum cheeses produced in Turkey. International Journal of Food Microbiology 85, 83–85. Florez, A., Ruas-Mediedo, P., Alonso, L., Mayo, B., 2006. Microbial, chemical and sensorial variables of the Spanish traditional blue-veined Cabrales cheese, as affected by inoculation with commercial Penicillium roqueforti spores. European Food Research and Technology 222, 250–257. Fox, P.F., Guinee, T.P., Cogan, T.M., McSweeney, P.L.H., 2000. Fundamentals of Cheese Science. Aspen Publishers, Inc., Gaithersburg, MD. Frank, J.F., Christen, G.L., Bullerman, L.B., 1993. Tests for groups of microorganisms. In: Marshall, R.T. (Ed.), Standard Methods for the Examination of Dairy Products, 16th edn. American Public Health Association, Washington DC, pp. 271–286. Gobbetti, M., Burzigotti, R., Smacchi, E., Corsetti, A., De Angelis, M., 1997. Microbiology and biochemistry of Gorgonzola cheese during ripening. International Dairy Journal 7, 519–529. Gripon, J.C., 1987. Mould-ripened cheeses. In: Fox, P.F. (Ed.), Cheese: Chemistry, Physics and Microbiology. Major Cheese Groups, vol. 2. Elsevier Applied Science, London, pp. 121–149. Harrigan, W.F., 1998. Laboratory Methods in Food Microbiology. Academic Press, San Diego, USA. 532 pp. Hartman, P.A., La Grang, W.S., 1985. Coliform bacteria. In: Richardson, G.H. (Ed.), Standard Methods for the Examination of Dairy Products. A.P.H.A., Washington, D.C., USA, pp. 173–186. Hasenekoglu, I., 1988. Erzurum ve cevresinde uretilen Kuflu peynirlerin mikrofungus florasi uzerinde bir arastirma. Kukem Dergisi 11, 35–42. Hayaloglu, A.A., Guven, M., Fox, P.F., McSweeney, P.L.H., 2005. Influence of starters on chemical, biochemical, and sensory changes in Turkish Whitebrined cheese during ripening. Journal of Dairy Science 88, 3460–3474.
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Short communication
Microbial quality and presence of moulds in Kuflu cheese A.A. Hayaloglu a,⁎, S. Kirbag b a
Department of Food Engineering, Inonu University, 44280 Malatya, Turkey b Department of Biology, Firat University, 23119 Elazig, Turkey
Received 10 July 2006; received in revised form 30 November 2006; accepted 6 December 2006
Abstract The chemical and microbial qualities, including fungal flora, of 30 samples of Kuflu cheese randomly purchased from different markets in Turkey were investigated. The gross composition of the cheese samples ranged between 37.65–53.65% moisture, 6.21–40.09% fat-in-dry matter, 4.70–10.07% salt-in-moisture and 26.18–44.85% protein. The mean pH value of the cheeses was 6.29 ± 0.28 and pH values ranged from 5.52 to 7.22. Variations between the samples in terms of their gross composition suggested a lack of quality standards in cheesemilk, cheesemaking procedure and ripening conditions. The levels of main microbial groups including total mesophilic and coliform bacteria, yeasts and moulds and the presence of some potentially pathogenic microorganisms (E. coli, Salmonella spp. and Staphylococcus aureus) were determined. The high numbers of all microbial groups and presence of potentially pathogenic organisms in the cheese samples suggested that the production and maturation of Kuflu cheese should be improved by better hygiene. Moulds at the cheese surface were isolated and identified. A total of 24 different mould species were detected and the genus most frequently isolated was Penicillium spp. which represented 70.25% of total isolates. Penicillium commune, P. roqueforti and P. verrucosum were the most abundant species in the cheeses sampled. The other dominant fungal groups were Geotrichum candidum, Penicillium expansum and P. chrysogenum. Other genera isolated from the cheese were Acremonium, Alternaria, Aspergillus, Cladosporium, Geotrichum, Mucor, Rhizopus and Trichoderma. The potentially toxigenic species, including some Penicillum spp. and Aspergillus flavus, were also detected. © 2006 Elsevier B.V. All rights reserved. Keywords: Mould-ripened cheese; Blue cheese; Microbial quality; Fungal flora; Penicillium
1. Introduction Cheese ripening is a complex and dynamic biochemical process that includes protein breakdown, fat hydrolysis and lactose metabolism (El Soda et al., 1995; McSweeney and Sousa, 2000). These processes are catalyzed by agents such as residual coagulant, indigenous milk enzymes, starter or nonstarter microflora and secondary organisms. The secondary organisms include moulds and presence of moulds on the surface of mould-ripened cheese gives them a different appearance and flavor from other cheeses. The moulds have a more complex enzyme system than bacteria and their enzymes contribute to the maturation of the cheese, i.e., to proteolysis and lipolysis which are more extensive in these cheeses (Gripon, 1987). However, in other varieties mould growth on ⁎ Corresponding author. Tel.: +90 422 341 0010; fax: +90 422 341 0046. E-mail address: [email protected] (A.A. Hayaloglu). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.12.002
the cheese surface causes economic losses and quality problems (Kivanc, 1992; Kure et al., 2001). Some moulds are capable of producing toxic secondary metabolites (mycotoxins) and of producing undesirable flavor. Consequently, these organisms can pose a potential risk for public health (Kure et al., 2001; Montagna et al., 2004). Moulds growing on the surface of mould-ripened cheeses were isolated by several authors (Hasenekoglu, 1988; Kivanc, 1992; Kure and Skaar, 2000; Erdogan et al., 2003; Kure et al., 2004; Montagna et al., 2004) and they reported that Penicillium spp. were the dominating species on the cheeses. Kuflu cheese is a mould-ripened variety produced in the middle and eastern region of Turkey. Kuflu cheese has a semihard texture, rectangular shape and weighs between 300 and 500 g. The cheese is made from non-pasteurized, skimmed (or partially-skimmed) sheep's or goat's milk or their mixture. Cheesemilk is clotted with home-made calf rennet at 30–32 °C and, no starter culture or mould spores were added to the milk.
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After curd forming, the curd is transferred into a cotton bag for whey drainage, which takes about 24 h. Then, the curd is broken into pieces the size of pea grains by hand, dry-salted and retransferred into the bag. During the first 10 days, the curds are pre-ripened in the cotton bag; then tightly filled into tulum (a goat skin bag). The cheese is finally placed in natural caves where ripening takes place at 80–90% relative humidity and at 6–12 °C. After ripening of the cheese, the cheese is cut into blocks with a knife and the cheese blocks are ripened in the caves or rooms until blue-green moulds spontaneously grow on the surface of the cheese. The color of Kuflu cheese is white with blue or green veins; however, blue or green colors dominate its surface after the growth of moulds during mildewing. The aroma and flavor of Kuflu cheese are very intense due to its complex microflora which comes from the cheesemilk, home-made calf rennet extracts or environmental flora during ripening. Consumption of the cheese has increased due to its being low- or nonfat and because of its intense flavor. The cheese is consumed without discarding the mouldy part of the cheese. To the authors' knowledge, although there are no reports of health hazards for humans after consumption of Kuflu cheese, we aimed to investigate the fungal flora, hygienic quality and gross composition of the cheese. Therefore, we determined the chemical and microbiological composition of the cheese and identified the moulds at the genus and species level. 2. Materials and methods 2.1. Cheese samples A total of 30 Kuflu cheese samples were purchased from different markets during the autumn of 2004. The samples were taken using a sterile knife and packed in aseptic polyethylene bags and kept at + 4 °C until analysis. 2.2. Chemical analyses Cheeses were analyzed in duplicate for moisture, fat, total protein, salt, pH and titratable acidity as described by Hayaloglu et al. (2005). 2.3. Microbiological analyses For each cheese sample, 10 g was weighed and dispersed aseptically in 90 mL of citrate buffer (2%, w/v) and homogenized in a sterile polyethylene bag using a Stomacher (Seward Laboratory Blender Stomacher 400 Lab Blender UK) for 1.5 min. Serial dilutions were made in 0.1% sterile peptone water and all determinations were made in duplicate (Harrigan, 1998). The enumeration of total mesophilic bacteria (Plate Count Agar, Merck, Darmstadt, Germany) at 30 °C for 48 h (Messer et al., 1985), total coliforms (Violet Red Bile Agar, Merck, Darmstadt, Germany) at 37 °C for 48 h (Hartman and La Grang, 1985), yeasts and moulds (Potato Dextrose Agar, Merck, Darmstadt, Germany) at 21 °C for 7 days (Frank et al., 1993), Staphylococcus aureus (Baird-Parker Agar, Merck, Darmstadt,
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Germany) at 37 °C for 48 h (Harrigan, 1998) and Salmonella spp. (Tetrathionate Broth and Hecktoen Agar) at 35 °C for 24 h (Baumgart, 1993) were performed. 2.4. Isolation and identification of moulds After preparation of serial dilutions of the cheese samples, each was plated onto Malt Extract Agar (Merck) and the plates were incubated at 25 °C for 7 days. Next, isolates were subcultured on Yeast Extract Sucrose Agar (Oxoid), CzapekDox Agar (Merck), Potato Dextrose Agar (Merck) and Sabouraud Dextrose Agar (Merck). The media were inoculated three-point and incubated at 25 °C for 7 days, but Sabouraud media was incubated at 30 °C for 7 days. The isolated moulds were identified by their morphological characteristics and pigments as described by Barnett and Hunter (1987), Pitt (1988), Pitt and Cruickshank (1990) and Samson et al. (2000). 3. Results and discussion 3.1. Chemical composition The gross chemical composition (moisture, fat, protein and salt contents) and pH of Kuflu cheese samples were determined. Wide ranges in pH and chemical composition were observed. The pH values ranged from 5.52 to 7.22 and the average value was 6.29. The average pH value was lower than that of other studies on mould-ripened cheeses (Prieto et al., 1999; Schlesser et al., 1992). The pH in Blue cheeses increases during ripening due to catabolism of lactic acid and deamination of amino acids by moulds. Trends were similar to those of pH observed in acidity. The moisture concentration of the cheese changed between 37.65 and 53.65% and the average moisture contents were higher than in Tulum cheese. According to these results, Kuflu cheese can be categorized as semi-hard cheese in terms of its moisture content. Fat and fat-in-dry matter (FDM) contents of the cheese were considerably lower than those of other Blue cheeses. Average values for fat and FDM were 6.30 and 12.18%, respectively. Kuflu cheese is manufactured from skimmed or partially-skimmed milk and hence, the fat contents were low in comparison to other mould-ripened varieties. Total protein content of the cheeses was higher than that of other mould-ripened cheese. These differences can be attributed to the concentration effect on compositional parameters due to the low level of fat in Kuflu cheese. Salt-in-moisture (SM) contents of Kuflu cheese, which ranged from 4.70 to 10.07%, were higher than those of other mould-ripened cheeses (Prieto et al., 1999; Sousa and McSweeney, 2001). 3.2. Microbiological analysis The microbial counts of Kuflu cheese samples are shown in Table 1. Total mesophilic bacterial counts were high (ranged from 8.26 to 10.69 log cfu/g cheese) in the cheeses, probably due to the use of raw milk with high microbial load in their manufacture and uncontrolled ripening conditions. The cheese samples which were ripened open to the atmosphere might have
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Table 1 Log counts (log cfu/g cheese) of the microbial groups found in Kuflu cheese Statistical parameter Total mesophilic bacteria Coliform bacteria Staphylococcus aureus Yeasts and moulds E. coli Average Range ±SD
9.31 8.26–10.69 0.53
2.51 b1–3.04 0.48
2.77 b1–4.92 1.05
been contaminated by environmental flora. High levels of salt in the cheese did not adversely affect the growth of bacteria. Coliform organisms were detected in all except for 3 samples; the average values of coliform bacteria were 2.26 log cfu/g. By Turkish regulations the maximum allowable counts of coliform group bacteria are 2 log cfu/g in Turkish White-brined cheese (Anonymous, 2001). The intestinal origin of these bacteria and their presence in cheese should be considered as evidence of poor manufacturing conditions and lack of hygiene. Coliform group bacteria were also found in cheeses such as Kopanisti (Tzanetakis et al., 1987), Spanish blue (Lopez-Diaz et al., 1995), Karin kaymagi (Cakmakci et al., 1995) and Carra cheeses (Aygun et al., 2005) purchased from markets. The presence of these group bacteria in the cheese also suggests their presence in the milk. In addition, manual contact is usual during the marketing of this type of cheese. The pH and salt contents of Kuflu cheese samples were favorable for the growth of coliform. Yeasts and moulds were present all samples with values varying from 4.83 to 6.94 log cfu/g cheese. The mould counts were extremely high in Kuflu cheese and mould veins appeared in the centre of the cheese. In addition, piercing the cheese allows the growth and sporulation of moulds throughout its surface (Gripon, 1987). Some potentially pathogenic bacteria were found in Kuflu cheese samples (Table 1). E. coli (17 samples; 56.6% of total), Salmonella spp. (7 samples; 23.3% of total) and S. aureus (11 samples, 36.6% of total) were found in the samples. S. aureus were present in our samples at levels of 2.00 to 4.92 log cfu/g. Uncontrolled environmental conditions during manufacturing and ripening favored their presence and survival. These potentially pathogenic bacteria can multiply at refrigerated temperatures and are tolerant to salt. Normally, these pathogens are present in cheese at first stage of maturation; then they become inactivate as ripening proceeds. Catabolism of lactic acid by moulds and yeasts on the cheese surface results in an increase in pH, facilitating survival of pathogens (Sousa and McSweeney, 2001). Consequently, these organisms are able to be isolated at the stage of consumption. De Boer and Kuik (1987) and Lopez-Diaz et al. (1995) could not isolate E. coli and Salmonella from their samples of mould-ripened cheese. 3.3. Isolation and identification of moulds A total of 24 different species of mould were isolated and identified in Kuflu cheese samples. Penicillium commune, P. roqueforti, Geotrichum candidum and P. verrucosum were the mould species most frequently isolated (Table 2). These four species made up 36.7% of the total number of moulds. The genus most frequently isolated was Penicillium spp. which
6.36 4.83–6.95 0.53
Salmonella spp.
Detected in 17 samples Detected in 7 samples
represented 70.25% (111 isolates of Penicillium spp.) of total isolates. Other genera isolated were Alternaria, Acremonium, Aspergillus, Cladosporium, Geotrichum, Mucor, Rhizopus and Trichoderma. Alperden (1978) examined 85 cheese samples produced in the Marmara region of Turkey and showed that Penicillium spp. was the major contaminant representing 54% of total isolates. Penicillium spp. was also the predominant mould genus on blue-veined cheeses (Gripon, 1987), Picon Bejes-Tresviso (Prieto et al., 1999), Jarlsberg and Norvegia (Kure and Skaar, 2000; Kure et al., 2001), Gorgonzola (Gobbetti et al., 1997), Cabrales (Florez et al., 2006) and as a contaminant on other cheeses such as Cheddar (Bullerman and Olivigni, 1974), Teleme (Zerfiridis, 1985), Kasar (Kivanc, 1992) and goats' and sheep's milk cheeses sold in Italy (Montagna et al., 2004). P. commune (10.1%) and P. roqueforti (8.9%) were the dominant mould species in the cheeses. P. commune is a wild type of P. camemberti and frequently contaminates foods (Pitt, 1988). Kure et al. (2001) examined cheeses from four different factories and P. commune dominated the isolates in two of these factories. This mould was also found on Cheddar cheese as a spoilage agent by Hocking and Faedo (1992) and on Italian goats' and sheep's milk cheeses by Montagna et al. (2004). Table 2 Mould species isolated from Kuflu cheese samples Mould species
Number of isolates
Frequency (%)
Acremonium alternatum Alternaria alternata Aspergillus flavus A. fumigatus A. niger Cladosporium cladosporioides C. herbarium Geotrichum candidum Mucor himealis M. mucedo Penicillium brevicompactum P. chrysogenum P. citrinum P. commune P. crustom P. echinulatum P. expansum P. roqueforti P. solitum P. spinolusum P. verrucosum P. viridicatum Rhizopus nigricans Trichoderma harzianum Total: 24 species
2 3 1 7 3 3 2 13 3 2 7 9 8 16 6 8 10 14 8 5 15 5 5 3 158
1.3 1.9 0.6 4.4 1.9 1.9 1.3 8.2 1.9 1.3 4.4 5.7 5.1 10.1 3.8 5.1 6.3 8.9 5.1 3.2 9.5 3.2 3.2 1.9 100
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P. roqueforti is used in the manufacture of Roquefort, Stilton and Gorgonzola (Gripon, 1987; Fox et al., 2000) and thus is also isolated from these cheeses (Aran et al., 1986; Aran and Eke, 1987; Tsai et al., 1988; Kivanc, 1992; Erdogan et al., 2003). Crumbly texture and small air holes in Kuflu cheese allow the growth of Penicillum spp., in particular P. roqueforti. In addition, P. roqueforti is capable of growth in the air with high levels of carbon dioxide (Kure and Skaar, 2000). P. verrucosum, Penicillium expansum and P. chrysogenum were the Penicillium species most frequently isolated from Kuflu cheese. P. verrucosum was found by Northolt et al. (1980) as a contaminant on visibly mouldy cheeses in shops, households, and warehouses. Its mycotoxin-reducing capability was reported by Pitt (1987) and Larsen et al. (2001). P. verrucosum was found to be the predominant mould on surface and inner parts of Kasar cheese by Kivanc (1992). Separately, P. expansum and P. chrysogenum were isolated at a low frequency in Jarlsberg and Norvegia cheeses (Kure and Skaar, 2000; Kure et al., 2001). Apart from Penicillium spp., the mould species most frequently isolated from Kuflu cheeses were G. candidum, Aspergillus fumigatus and Rhizopus nigricans. These three species made up 15.8% of total mould isolates from the cheese. G. candidum represented 8.2% of the moulds on the cheese and its appearance was a white-velvety coat on the surface. G. candidum also affects the cheese biochemistry during ripening; it releases lipases and proteases into the cheese matrix, reduces bitterness, contributes cheese aroma, neutralizes the curd acidity produced by lactic acid bacteria and stimulates the growth of surface bacteria such as Brevibacterium spp. (Marcellino et al., 2001). G. candidum was not isolated frequently from Kasar cheese (Kivanc, 1992) and Jarlsberg and Norvegia cheeses (Kure and Skaar, 2000; Kure et al., 2001); however, Kure et al. (2004) reported that G. candidum was frequently isolated from one of four cheese factories in Norway. A few isolates of other genera including Acremonium, Alternaria, Cladosporium, Mucor and Trichoderma spp. were also found on Kuflu cheese. The toxigenic properties of these five species in cheeses were not reported by Samson et al. (2000) and CAST (2003). The study describes the chemical and microbial composition and fungal flora of Kuflu cheese. Results showed that many problems appeared to originate in the production, ripening and marketing stages of the cheese in terms of its bacteriological quality and the presence of some potentially pathogens including E. coli, S. aureus and Salmonella. Moulds isolated and identified from the cheese, if they produce mycotoxins, may represent a health hazard. Penicillium spp. was the dominant fungal flora in Kuflu cheese and made up about 70% of the total mould isolates. Erdogan et al. (2003) studied the mycotoxinproducing capability of P. roqueforti isolates from mouldy Tulum cheese and they found that P. roqueforti isolates produced some mycotoxins including patulin, penicillic acid, roquefortine or PR toxin. Uncontrolled mildewing of the cheese must be considered as a problem and provides no guarantee of product safety for human consumption. For the ripening of Kuflu cheese, non-toxigenic strains of moulds should be selected.
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