Tulum peynirlerinde patojen bakteriler

Abstract

İstanbul piyasasından temin edilen 60 adet tulum peyniri örneğinde patojen bakterilerden Escherichia coli, Listeria monocytogenes ve Staphylococcus aureus ile mezofilik aerobik bakteri, koliform ve fekal koliform sayıları belirlenmiştir. Patojen bakteri gelişmesini etkileyen faktörlerden sıcaklık, pH, asitlik, nem ve tuz değerleri saptanmıştır. Tulum peyniri örneklerinin muhafaza edildiği sıcaklıklar (-1.0) - (+19.2) °C, pH değerleri 4.35 - 6.17, asitlik % 0.426 - 1.669, nem değerleri % 36.7 - 50.3, tuz değerleri % 1.79 - 4.35, kurumaddede tuz değerleri % 3.06 - 8.30 aralıklarında değişmiş ve ortalamalar sırasıyla sıcaklık 8.6 °C, pH: 4.97, asitlik: % 0.938, nem: % 43.7, tuz: % 2.72, KM'de tuz: % 4.87 olarak tespit edilmiştir. Tulum peyniri örneklerinde Listeria cinsi bakterilere rastlanmamıştır. Mezofilik aerobik bakteri sayıları 1.105 - 2.3. 108 cfu /g arasmda değişmiş ve ortalama 1.4. 107 cfu /g olarak tespit edilmiştir. Peynir örneklerinin % 96.67'sinde S.aureus bulunduğu, sayılarının 0-8.7. 104 cfu /g arasmda değiştiği ve ortalamanın 4.5. 102 cfu /g olduğu saptanmıştır. Tulum peynirlerinin % 72'sinde koliform bakterilerin % 70' inde fekal koliform bakterilerin ve E.coli Tip l'in bulunduğu, koliform ve fekal koliform sayılarının 3 - 103 /g arasında değiştiği belirlenmiştir.

Cheese is a milk product which has been used for over 4000 years. Meanwhile it is a nutritious commodity having various types which differ in flavor and texture. Tulum cheese is a special type of Turkish cheese. It is considered the most popular type following white pickled arid kashar cheeses. The tulum cheese is usually produced by small manufacturers utilizing very primitive production technology other then sophisticated techniques. It is generally manufactured from raw milk, preferably the ewe's milk. Rennet is added to warm milk (26-39 °C) to coagulate milk. Although there is no standard curd processing technique, principally the curd is robed and pressed several times. Salt is added during robing. The curd is then filled tightly to a special bag made from skin of the goat and is ripened at temperatures of 3-8 °C and relative humidities of % 70-85 for 3 to 4 months. The tulum cheese can also be consumed fresh (İnal and Ergiin, 1990; Kurt et al.,1991b). The use of raw milk and unsanitary handling conditions during its production may result in the presence of some pathogenic bacteria. Pathogens that can contaminate milk include various species of bacteria. Among these, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Salmonella are the hazardous bacteria causing illness. Based upon the epidemiological data and the characteristics of individual pathogens, enteropathogenic E.coli, Lmonocytogenes and Salmonella are categorized as high risk bacteria for the cheese industry. Enteropathogenic E.coli is a significant cause of gastrointestinal diseases. Listeria monocytogenes causes abortion, death of newborn, inflammation of the brain and surrounding tissues and serious infection at risk populations which include immunocompromised persons such as infants, pregnant women, elderly and patients. Staphylococcus aureus is characterized as low risk bacteria since its growth is easily controlled by the lactic culture technology (Johnson et al., 1990 b). However the frequent contamination by Staphylococci from dairy personnel and high incidence of staphyloccocal mastitis in dairy herds cause the staphyloccocal toxication to be the most commonly occuring type of cheese related outbreaks (IDF, 1980). The pathogenic effect of S. aureus is related to toxins produced by the organism itself. IX Pathogenic bacteria may contaminate milk and cheese by several ways. The principal factor determining the absence ör presence of pathogens in raw milk is the health condition of cow. Mastitis is an inflammatory disease of the udder and is mainly a result of the contamination of Staphylococcus and Streptococcus. Other pathogenic organisms that can cause mastitis and thus appear in milk include E.coli and Lmonocytogenes. The second factor is the environmental conditions where the cows are held. Environmental items such as feces, soil, feed and water may cause transmission of pathogens to milk. Other potential soıırces of contamination by pathogenic bacteria also include improperly cleaned and sanitized equipment and personnel who handles the milk (Marth, 1985). The sources of contamination by pathogenic bacteria also include the sanitary conditions in the processing area and hygienic practices and health conditions of personnel dealing with the production of cheese. The growth and survival of pathogenic organisms in cheese are influenced by several factors. These factors are the characteristics of pathogenic organism such as tolerance to heat, acid and salt conditions, count in milk and in factory environment and physiological conditions. The factors related to cheesemaking process include time-temperature profile, pH profile and metabolite generation of lactic acid bacteria, enzyme addidion, types of additives, cheese composition and curing conditions. The objective of this study was to investigate 60 samples of tulum cheese from retail markets for the presence of pathogenic bacteria including Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and of mesophilic aerobic bacteria, coliforms and fecal coliforms. The samples were also examined for physical and chemical properties such as shelf- temperature, moisture content, pH, acidity and salt concentration. Enumeration of S.aureus was açhived on mannitol salt ağar. Colonies circular, smooth, convex and forming yellow zones were selected for suspicious S.aureus colonies. These colonies were examined for gram reaction and coagulase test. Gram positive and catalase positive colonies were defined as S.aureus. Coagulase test was carried by using latex aggutination kit (Oxoid, Staphytect DR650M). Coliform counts were estimated according to FAO (1992) through most probable number (MPN) procedure based on gas formation in lauryl tryptose broth. Confirmation of coliforms were açhived by brilliant green bile broth. The presence of fecal coliforms were confirmed by EC broth. E.coli was isolated from gassing EC broth by streaking on eosin methylene blue ağar. Colonies appeared black with ör without metalic shining were selected and exposed to some biochemical tests such as gram reaction, motility with wet mouth method and IMViC patterns. Gram negative cocoid, motile cultures having IMViC pattern ++- were considered as E.coli. Listeria monocytogenes was examined by FAO (1992) method. This method includes preenrichment of sample in Listeria enrichment broth and streaking on lithium chloride phenyl ethanol moxalactam ağar. Colonies of Listeria species x appear to be sparkling blue ör white by Henry illumination technique. Listeria species forms black ör brown colonies on oxford ağar observed by ordinary illumination. Listeria species are identified according to their gram reaction, motility and biochemical tests. The contents of moisture, salt concentration and acidity were determined according to the method of the TSE (1989) for white cheese. Moisture content was evaluated by heating sample at 105 °C to a constant weight. Sodium chloride content was determined by titrating sample extract with 0. l N AgNO3. Acidity of samples was measured by titrating the sample extract with 0. l N NaOH. The pH of samples were determined by the method of Standart Methods for Dairy Products (Case et al., 1985) using Radiometer pH meter (PHM92) and combined electrode (GK 2401 C). Temperatures of the samples were examined using a digital thermometer (Ebsero, HI8054). Tulum cheese samples were taken from 6 different regions in İstanbul and were transported to the laboratory at 4-5 °C using a refrigeration chest. The pH values of samples varied between 4.35-6.17 with a mean of pH: 4.97 and 90 % of samples were in the range of pH: 4.5-5.5. These pH values are appropriate for survival and growth of pathogenic bacteria. The minimum pH value suitable for the growth of S.aureus is 4.0; for E.coli and Lmonocytogenes, this value is 4.3. Acidity of samples ranged in 0.426-1.669 % with an average of 0.938 %. The obligatory standard for tulum cheese allows a 1.5 % acidity for the first grade tulum cheese and a 2.0 % for the second grade tulum cheese (TSE, 1978). Accordingly 96.97 % samples having acidity lower than 1.5 % were accepted as first grade tulum cheese and the remaining (3.03%) as the second grade of tulum cheese. There was no linear correlation between pH and acidity of samples since correlation coefficent (r=0.313) was not significant at P< 0.05. This is partially related to the different buffering capacity of samples and partial dissociation of acids as stated by Bostan (1991 a). The moisture contents of samples were found to be in the range of 36.7-50.3 % with a mean of 43.7 %. The obligatory standard allovvs a maximum 40 % of moisture content for tulum cheese. Hence, about 88.33 % of samples did not meet the requirements in the standard. The salt contents of samples ranged betv/een 1.79-4.35 % with an average of 2.72 %. Similary salt in dry matter contents varied between 3.06 - 8.30 % with an average of 4.86 %. The obligatory standard for tulum cheese allovvs a maximum of 6 % salt in dry matter for the first grade and 8 % for the second grade. These results showed that 81.67 % of samples having salt in dry matter below 6 % were of first grade and the renıainings were of second grade. xi The refrigeration temperature is important to prevent the biochemical and microbiological changes. it is also legally prohibited to exceed 10 °C during transportation and marketing of cheese. Results showed that tulum cheeses were stored at temperatures of -1.0 to +19.2 °C with an average of 8.6°C. 41.67 % of samples were stored at temperatures higher than 10°C and consequently they did not meet the requirement of obligate standards. If cheeses were ripened by molds during storage above 7 °C, the molds can utilize lactic acid and thereby increases pH of cheese. This phenomenon is important for the keeping microbiological quality of cheese, since high pH values (alkaline) favor the growth of pathogenic organısın. Tulum cheeses contam high level of Penicillium spontanously. Consequently storage of tulum cheeses at high temperatures may result in the increase of pH, hence favors the growth of pathogenic organisms. it is seen that chemical and physical composition of tulum cheeses is different among samples. Differences in composition indicate the variation especially in production and ripening conditions. Therefore it is necessery to standardize production techniques and ripening conditions. Mesophilic aerobic counts varied from 1.105 cfu/g to 2.3.108 cfu/g with an average of 1.4.107 cfu/g. These results showed the count of non-lactic bacteria since nutrient ağar that contains no sugar. The absence of sugar in this medium prevents the growth of lactic acid bacteria. Davis (1986) classified hard cheeses according to their microorganism counts as follows: cheeses having mesophilic aerobic counts lower than 104 microorganism/g are satisfactory; cheeses with 104-105 microorganism/g counts are doubtful and cheeses with counts higher than 105 microorganism/g are unsatisfactory. Ali mesophilic aerobic counts were higher than 104 microorganism/g and 98.33 % of samples ha ve the counts higher than l O6 microorganism/g. High counts of mesophilic aerobic bacteria were mainly due to use of raw milk in the production of cheese. Kurt et al. (1991a) stated that omiting the cooling process of milk after milking stage and using unpasteurized milk may cause a high level of total bacterial count. There was significant correlation (r=0.334) between mesophilic aerobic counts and coliform counts (P < 0.05). This was because of the growth of coliform bacteria that cannot utilize lactose. Coliform counts of tulum cheese ranged from <3 to > 1.1. l O3 /g. About 23.33 % of samples contained l O2-103 /g coliform and 21.67 % of samples contained higher than 103 /g coliform. Davis (1986) classified hard cheeses according to the coliform counts as cheeses having coliform counts lower than l O3 /g are satisfactory, cheeses with 102-103 /g counts are doubtful and cheeses with counts higher than l O3 /g are unsatisfactory. 71.67 % of tulum cheese samples contained coliform bacteria. Almost ali of the coliform bacteria (97.7 %) were compromised of fecal coliform and E.coli. The presence of fecal coliform and E.coli showed the contamination of cheese by feces. Since almost ali of the total coliforms were fecal coliform and contain E.coli, this needs a special consideration. Although not ali the serotypes of E.coli is pathogenic, enteropathogenic E.coli control can be maintained by the control of coliforms in cheeses. xii The counts of S.aureus ranged between 0-104 cfu/g with a mean of 4.5.102 cfu/g. The S.aureus counts reached to a maximum level of 8.7.10* cfu/g. The highest contamination level of l O4 to l O5 cfu/g was observed in 6.67 % of samples. Also the concentrations of 103 to 104 cfu/g, 10M03 cfu/g and 10 to 102 cfu/g were detected in 30, 38.33 and 21.67 of samples, respectively. Davis (1986) classified hard cheeses according to the bacteria counts as; cheeses having coagulase positive S.aureus lower than 10 microorganism/g are satisfactory, cheeses with counts 10-100 microorganism/g are unsatistactory. Mor- Mur (1992) also reported the classification of the counts of potentially pathogenic Staphylococcus. According to this classification, foods containing potentially pathogenic organism at a level maximum of 1000 cfu/g are suitable f ör human consumption. Our results showed that about 36.67 % of samples were unsuitable for human consumption since the maximum level of 103 cfu/g was exceeded. None of samples contained Listeria species. Listeria monocytogenes can grow över pH : 4.3 and is known as salt tolerant organism. Tulum cheeses having an average pH of 4.97 and 2.72 % salt concentration appear to be a suitable medium for growth and survival of Lmonocytogenes. Absence of Listeria in tulum cheese sample may be due to the absence of this particular pathogen in milk. Although no Listeria was found in tulum cheeses, coliforms, fecal coliforms and coagulase positive S.aureus were frequently observed in use of microbiologically low quality milk and the lack of hygienic conditions during production. Therefore hygienic measures must be maintained to prevent pathogenic bacteria contaminating milk and cheese. Hence, the personnel must be trained for safe cheesemaking procedures and hygienic practices. Persons suffering from gastrointestinal ör inflammatory diseases must not be involved in milking ör cheesemaking processes. Equipments and utensils used in production must be properly cleaned and sanitized. High salt concentration may control the growth of pathogenic organisms in cheese. However milk must alvvays be pasteurized and active starter cultures must be used, instead of using high salt concentrations. The minimum temperatures for growth of Listeria and for E.coli are 4°C and 2.5°C respectively. Storage at refrigeration temperature may control the growth of some pathogenic bacteria such as S.aureus. But the temperature of storage may fluctuate during transportation and marketing; thus storage at refrigeration temperature must not be regarded as a way of controlling pathogenic bacteria.