MICROBIOLOGICAL EVALUATION OF MOZZARELLA CHEESE

MICROBIOLOGICAL EVALUATION OF MOZZARELLA CHEESE

DINA. N. ALI and WALAA M.A. ELSHERIF

Animal Health Research Institute, Assiut Regional Laboratory

Email: dinanour2010@yahoo.com

INTRODUCTION

Mozzarella cheese is asemi hard, white, un-ripened has very lively surface sheen and has unique property of stretch-ability. It owes its characteristics mainly to the action of lactic acid on dicalcium-para-caseinate that may be consumed shortly after manufacture. Its melting and stretching characteristics are highly appreciated in the manufacture of pizza, where it serves as a key ingredient (Jana and Mandal, 2011).

Mozzarella has been the ubiquitous increase in the popularity of pizza, in which mozzarella is the main cheese used. The functional attributes of importance for pizza include the desired degrees of flow and stringiness on baking. The cheeses best endowed with these characteristics, especially stretch-ability, are members of the pasta-filata group (Fox et al., 2000).

Milk used for cheese manufacture is required to be pasteurized at 72°C for 15 s or its equivalent, according to US Food and Drug Administration (FDA) regulations (FDA, 2011), which destroys most common pathogenic or spoilage bacteria. However, heat-resistant pathogenic and spoilage bacteria may be present in raw milk or equipment surfaces, and even non-heat resistant bacteria may gain access during cheese manufacture and storage (Kikuchi       et al., 1996). The presence of these bacteria is detrimental to cheese shelf life and quality (El-Gazzar and Marth, 1992; Schlesser et al., 2006). Although Mozzarella cheese might be contaminated with pathogens, such as Salmonella serovars (De Felip and Toti 1984), Staphylococcus aureus (Bowen and Henning, 1994) and Listeria monocytogenes (Buazzi et al., 1992). Similarly, the Stx2 shiga toxin produced by enterohemorrhagic Escherichia coli O157:H7 is resistant to milk pasteurization and other equivalent heat treatments and is destroyed only by 100°C for 5 min (Rasooly and Do, 2010).

Microbial contamination, causing approximately one-fourth of the world’s food supply loss, has become an enormous economic and ethical problem worldwide (Huis in‘t Veld, 1998). Dairy products are an excellent growth medium for a wide range of microorganisms and, thus, display a reduced shelf life (Ruegg, 2003). The microbiological quality of dairy products is influenced by the initial flora of raw milk, the processing conditions, and post-heat treatments. Spoilage bacteria and various bacteria of public health concern can be found in these products and their concentrations should be kept as low as possible (Varga, 2007).

Therefore, the aim of this work was to evaluate the quality of mozzarella cheese and compare the results with Egyptian Organization Standards and International Standards.

MATERIALS and METHODS

A total of 50 mozzarella cheese samples were taken for analyses aseptically after few hours of collection from supermarkets in Assiut City, Egypt. The samples were collected in clean plastic bags as marketed to the consumers and transported, as soon as, possible to be examined for:-

1- Total viable count according to A.P.H.A. (1992).

2- Yeasts and molds counts according to Harrigan and MeCance (1976).

3- Total coliform counts (MPN) according to FAO (1992).

4- Isolation of Staph. aureus according to A.O.A.C. (2000).

5- Isolation of E.coli: Samples were prepared to isolate the E. coli according to FAO (1992).          

6- Isolation of Salmonella according to Wallace et al. (2009).

A- Serodiagnosis of E.coli:

This part has been done in the Food Hygiene Lab in the Faculty of Veterinary Medicine of Moshtohor, Banha Univ., Egypt.

The isolates were serologically identified according to Kok et al. (1996) by using rapid diagnostic E. coli antisera sets for diagnosis of the Enteropathogenic types. The technique applied as recommended by the manufacture (DENKA SEIKEN Co., Japan).

B. Serological identification of Salmonellae:

Serological identification of Salmonellae was carried out according to Kauffman – White scheme (Kauffman, 1974)for the determination of Somatic (O) and flagellar (H) antigens using Salmonella antiserum (DENKA SEIKEN Co., Japan).

C. Identification of total Staphylococci species:

Morphological examination (Cruickshank et al., 1975).

Biochemical identification:

Catalase activity test (MacFaddin, 1976).

Detection of haemolysis (Collins and Lyne, 1984).           

Mannitol fermentation test (Bailey and Scott, 1978).

Coagulase test (Baron et al., 1994).

Thermostable nuclease test "D-Nase activity" (Lachia et al., 1971).

D-Nase agar plates were inoculated with loopfuls of suspected colonies by spotting them on small areas of the plates which incubated at 37°C for 18 hours. Moreover, the incubated plates were flooded with normal hydrochloric acid which precipitated DNA resulting in cloudiness of the plates. Accordingly, Appearance of a clear zone around the colony indicated the production of D Nase and recorded as positive result.

RESULTS

Table 1: Statistical analytical results of microbiological examination of Mozzarella cheese samples.

Table 2: Incidence of Staph.aureus, E.coli and Salmonellae recovered from Mozzarella cheese samples.

Table 3: Serological identification of E.coli strains isolated from Mozzarella.

• EHEC: - Enterohaemorrhagic E. coli, ** EPEC: - Enteropathogenic E.coli, *** ETEC: - Enterotoxigenic E. coli.

Table 4: Serological identification of Salmonellaestrains isolated fromMozzarella.

Table 5: Identification of S.aureus strains isolated from Mozzarella.

Table 6: Summarized results of microbiological examination of Mozzarella cheese samples compared with the International Standards (IDF, 1984).

Table 7: Summarized results of microbiological examination of Mozzarella cheese samples compared with the Egyptian Standards (E.O.S.Q.C., 2005).

DISCUSSION

The total bacterial count gives a quantitative idea of the presence of mesophilic aerobic microorganisms of animal origin. It serves as an important criterion to evaluate the microbial quality of various foods and also the degree of freshness of food (Nanu et al., 2007). Data presented in Table 1 illustrated that total bacterial count in examined mozzarella cheese samples ranged from 4x10³ to1.8X10⁷ with an average count of 3.84X10⁶ bacteria /g,  Francesca      et al. (2014) reported nearly similar results which indicated that the TBC  in mozzarella cheese was in the range of  10³ to 10⁵ organisms/g, similar results were  also obtained by Asperger (1991) who reported that the total bacterial count in mozzarella cheese stored at 4 °C increased and was >10⁷ cfu/g after 1 week of storage. Also, Tanweer (2011) detected the initial TBC in mozzarella cheese samples 1 x10⁶ cfu/g and increased to 8 x10⁶ cfu/g after 5 weeks of storage and reported that presence of atmospheric air and O₂ affect the overall quality of mozzarella cheese during storage, Also the highly nutritious nature of dairy products makes them especially good media for the growth of microorganisms. Milk contains abundant water and nutrients and has a nearly neutral pH. The major sugar, lactose, is not utilized by many types of bacteria, and the proteins and lipids must be broken down by enzymes to allow sustained microbial growth (Loralyn and Robert, 2009).

Also, in Table 1 coliforms were present in 96% of samples with an average count of 8.5×10 cfu/g. Higher results were obtained by Tanweer (2011) who found that  the initial coliform count was 6x10² cfu/g in Mozzarella cheese increased to 1000 cfu/g after 5 weeks of storage. The presence of coliforms or yeasts is indicative of low processing temperature, especially at filling or negligent sanitation. The major microbiological problem with these products is growth of yeasts and molds, especially if free moisture is available at the surface (Marth and Steele, 2001). Moreover some cheese defects may be caused by poor milk quality (late lactation milk, milk from mastitic animals, high in enzymes of animal origin, i.e. lipase and protease), inappropriate rate of acid development by the starter, or poor manufacturing and storage regimens (Fawaz et al., 2011).

In cheese production, slow lactic acid production by starter cultures favors the growth and production of gas by coliform bacteria, with coliforms having short generation times under such conditions. In soft, mold-ripened cheeses, the pH increases during ripening, which increases the growth potential of coliform bacteria (Frank, 2001).

Yeasts were presented in 94% of samples with an average count of 3.2x10⁶ cells/g. while, molds were present in smaller amount only in 32 % of samples with an average count of 6.8x10⁴ cells/g (Table 1). Our results were slightly similar to (Tanweer, 2011) who reported that the initial count of yeast and mold counts of mozzarella cheese increased from 5x10² to 2x10⁵ and 5x10⁵ cfu/g, respectively after 3 weeks of storage at 7±1 °C.

Molds can grow well on the surfaces of cheeses when oxygen is present, with the low pH being selective for them. In packaged cheeses, mold growth is limited by oxygen availability, but some molds can grow under low oxygen tension. Molds commonly found growing in vacuum-packaged cheeses include Penicillium spp. and Cladosporium spp. (Hocking and Faedo, 1992). Penicillium is the mold genus most frequently occurring on cheeses.

The results listed in Table 2 indicated that Staph.aureus was existed in 24%, of all examined samples. Staph. aureus is a ubiquitous bacterium, both human and animal commensal (Jay, 2000). Consequently, many foods can be contaminated by this species thus representing hazard for human health.

It is interesting to observe that the heating phase of pasteurization (until milk reached 65ºC) produced an important lethal effect on Listeria sp., Staphylococcus sp. and especially Salmonella sp. but not on Mycobacterium spp. (Raimundo et al., 2013), While contamination occurs mainly post pasteurization contamination or because of insufficient pasteurization.

Despite of the extensive public health measures over the past century, Salmonella remains the second most commonly identified cause of bacterial foodborne disease in the developed countries and a signficant cause of morbidity and mortality in the developing world (WHO, 2002 and Amin, 2004). 12% of examined samples were contaminated with Salmonella (Table 2) Although, there arerelatively low numbers of positive samples in thisstudy, the pathogen represent a potential risk toconsumers on the basis that all salmonellae arepotentially pathogenic (Zansky et al., 2002).

Also, Jay (2000) said that the greater efficiency of stretching could be explained by the fact that microorganisms were already injured by curd acidity but this explanation does not elucidate the behavior of Salmonella sp. and Staphylococcus sp. under the same conditions. It is possible that these microorganisms are less sensitive to acid injury than the others. Staph. aureus showing more resistance to stretching than the other microorganisms analyzed.

Escherichia coli are commensal organisms that reside within the host gut, but some pathogenic strains are recognized as a cause of gastroenteritis (Callaway     et al., 2003). Table 2 represents the occurrence of    E. coli in 20% of samples. Contamination from human and animal waste is traditionally indicated by the presence of commensal E. coli. Although these organisms are essentially nonpathogenic, their presence warns of the possible concurrent existence of pathogenic microbes (Sherfi et al., 2006).

Table 3, verified serological phenotypic identification of different E.coli isolated from all examined samples. The result represented that O157:H7, O111:H4 and O26 were identified as EHEC. The ETEC strain recognized in serogroups O128:H2 while, EPEC represented in O55:H7.

Foodborne outbreaks of Escherichia coli O157:H7 infection have been associated with a wide range of food products, including raw and pasteurized milk and milk products, such as cheese (Honish et al., 2005 and Strachan et al., 2005). In the late 1990s, both the U.S. Food and Drug Administration (FDA) and Health Canada proposed bans on the use of raw milk in cheese making.

E. coli O157:H7 can readily contaminate raw milk on the farm because dairy cattle are a known reservoir of Shiga toxin– producing E. coli, including enterohemorrhagic strains such as serotype O157:H7 (Wells et al., 1991). Similarly, the Stx2 shiga toxin produced by enterohemorrhagic E.coli O157:H7 is resistant to milk pasteurization and other equivalent heat treatments and is destroyed only by 100°C for 5 min (Rasooly and Do, 2010). Although the presence of Stx2 in foods is not known to cause illness upon direct consumption, Stx2 directly fed to mice caused mortality (Rasooly et al., 2010), and may hence be a human health risk.

Salmonellosis is a foodborne infection of major economic importance. According to information gathered from 84 countries responding to a global survey conducted by the World Health Organization (WHO), S. enteritidis and S. typhimurium accounted for 70% of all human and nonhuman isolates of salmonella reported worldwide between 1995 and 2008 (CDC, 2009). Corresponding to Table 4, it is persisted that the different identified strains of salmonella via sero-typing technique were 6%          S. typhimurium and 2% S. virchow, S.Haifa and S.enteritidis, respectively.

Some strains of staph.aureus are capable of producing many kinds of enterotoxins (SEs) which are currently being studied deeply and get much attention by the scientific community (Balaban and Rasooly 2000). Enterotoxigenic strains of Staph. aureus have been reported to cause a number of diseases or food poisoning outbreaks because of the ingestion of contaminated dairy products or milk (Asao et al., 2003). Also, Coagulase Negative Staph (CNS) is emerging as important minor mastitis pathogens in Egyptian dairy animals. Such species can cause substantial economic losses resulting in decreased milk production. This reflects the environmental hazard and therefore, udder health must be followed up and control of intra-mammary infections is consequently of the greatest importance for dairy farms (Jakeen et al., 2013). So, further identification was done to Staph. aureus strains, 10, 8 and 6% of  samples were Coagulase +ve / DNase –ve, Coagulase +ve / DNase +ve and Coagulase -ve / DNase –ve, respectively (Table 5).

Comparing the obtained results of examined mozzarella cheese samples with the International Standards (IDF, 1984) (Table 6), 98, 96, 94, 32, 20, 12 and 24% of samples failed to comply with the limits of the standards which indicated the poorer sanitary practices during cheese production according to TBC, coliforms, yeast, mold count, E.coli, Salmonella and Staph.aureus isolates, respectively.

According to the limits proposed by the E.O.S.Q.C. (2005), only 10, 6, 68, 80, 88 and 76% of samples were acceptable according to yeast, mold, coliforms count, E.coli. Salmonella and Staph.aureus isolates, respectively (Table 7). This result indicated the negligible sanitary control measures during production and handling of the products.

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