Determination of Elemental Contents and Microbiological and Chemical Properties of Çökelek Cheeses Consumed in Turkey

The Çökelek samples what 30 different were collected from randomly local bazaars to investigate heavy metal contaminant and mineral levels and some physicochemical and microbiological properties of samples. While the Pb was identified in 6 of the 30 samples, the As was only found in 4 of the samples. The mean major and trace element contents of Çökelek samples were ordered as Na > P > Ca > K > Mg and Al > Zn > Ni > Cu, respectively. The physicochemical properties indicated a high deviation among samples. The mean total solids, ash, salt, fat, protein waters soluble nitrogen contents, and sample ripening index were 29.83%, 1.88%, 0.68%, 4.31%, 19.84%, 0.33%, and 1.79%, respectively. The mean total aerobic mesophilic bacteria (TAMB) count of Çökelek samples was found as 8.26 log CFU g−1. The coliform bacteria and yeast-mold counts were detected in 11 and 27 of 30 samples, respectively. The mean coliform and yeast-mold counts were 1.82 log CFU g−1 and 7.11 log CFU g−1, respectively. Traditional cheeses are not mentioned in legal laws such as the Turkish Food Codex. So, there is no legal limit and standard production processes. This situation is a problem in terms of traditional cheese quality. For this reason, traditional cheese should perform further studied, and determine the legal limits.


Introduction
There are many kinds of produced traditional or industrial cheeses in Turkey. The most known cheese types were Turkish white, Kashar, Tulum, Çökelek, and Lor cheeses in Turkey [1]. Çökelek cheese has been known as the same as Lor. But Çökelek cheese is different from Lor in terms of production process and composition. Because Çökelek cheese is made from yogurt and buttermilk, Lor is produced from whey [2,3]. It has a vital role in Turkish cuisine, especially breakfast [4].
The Çökelek cheese is produced from the straining of yogurt or buttermilk which is acidified and heated. It has low-fat, low-calorie, and high-protein content and is cheap [2,5]. Especially, the Çökelek cheese is one of the most important dairy products for low-income families [2,5]. Dairy products could be a potential danger in terms of severe and common health problems when contaminated with heavy metals. Because dairy products are enjoyed by individuals of all ages all over the world [6].
The heavy metals entranceways to humans can be via the consumption of dairy products and meat. Because the plant absorbs and stores heavy metals from contaminated water or soil. The contaminated plants are eaten by animals and accumulate heavy metals in their tissue or are passed to milk. So, milk and dairy products have risks in terms of heavy metals contamination [7,8]. Some heavy metals could be ordered as lead (Pb), mercury (Hg), arsenic (As), and cadmium (Cd) [6,7]. These heavy metals have toxic and some harmful effects on humans. Heavy metals ingested by humans may lead to serious health problems such as kidney failure, genetic mutations, nervous system disorders, cardiovascular problems, many types of cancers, respiratory disorders, a weak immune system, and infertility [8][9][10]. Heavy metal content and microbiological quality of milk and dairy products could be accepted as indicators of hygienic quality and pollution [6,11]. On the other hand, one of the vital nutrients in milk and dairy products is minerals [8,12]. Minerals are essential for humans, and they have many physiological functions in the human body such as enzymes and hormonal structure, and regulative [9,12,13].
There is not a standard production process for the Çökelek cheese, and it is produced under poor hygienic conditions and in small firms [1]. So, its microbiological quality is very low. Milk and dairy products are a wonderful medium for pathogenic bacteria and other microorganisms [1,14]. The microbiological properties of products such as especially pathogenic and spoilage bacteria are affected in the determination of the shelf life of foods [15]. The high protein, moisture, lactose level, high-pH, low-salt content, and non-hygienic production process in cheese eased microbial contamination from the environmental [16][17][18]. The microorganisms can affect the sensory properties of cheese due to decomposing protein and fat and can lead to health problems and toxic compounds [19].
Some researchers investigate some properties of Çökelek cheese [1,2,14]. But the studies are not sufficient of Çökelek cheeses for the exhibition of their element content and microbiological properties. This research aimed determination of Çökelek cheese's heavy metal (Pb and As) and mineral (Ca, Na, Mg, K, P, Al, Ni) contents, microbiological and chemical properties, and in commercial sold Çökelek cheese in Trabzon city, Turkey.

Material and Methods
Çökelek cheeses (30 different samples) were collected from different districts and bazaars of Trabzon province, Turkey. The samples were conveyed with a sterile cup and under a cold chain (+ 4 °C) to the Şiran Mustafa Beyaz Vocational School Food Laboratory. And samples were kept in the refrigerator (+ 4 °C) until analysis.

Heavy Metal and Mineral Content
To determine heavy metal levels and the mineral composition of the Çökelek cheeses, we used an inductively coupled plasma-mass spectrometer (ICP-MS; Agilent 7700, Japan). The samples were prepared with a wet-burning method using a microwave oven. The defined by Güler [20] method was used.
For the wet-burning, 2 g cheese sample was weighed into the vessels of microwave (Start D, Milestone Inc., Sorisole, Italy), and then, 8 mL HNO 3 (65%) and 2 mL H 2 O 2 (30%) were added for digestion of the samples. The microwave operation parameters were heated up to 200 °C at 15 min and held at 45 min and 500 bar, and cooled to after. The heavy metal and mineral content of the cheese samples that were diluted to 100 mL with ultrapure water were determined by ICP-MS.

Chemical Analysis
Total solids (%), total nitrogen (TN) (%), titratable acidity (% LA), and salt (%) contents of the Çökelek cheese samples were determined according to AOAC methods [21]. To determine the protein contents of samples multiplied the total nitrogen amount by 6.38. The fat ratio was determined by the Gerber method [22]. A pH meter was used for pH measurements. The water-soluble nitrogen (WSN) of samples was determined with the method described by Bütikofer et al. [23]. The Khejdahl method was used to determine the WSN ratio [21]. The Ripening Index was calculated using equation (WSN/TN) × 100.

Microbiological Analysis
Weighted as 10 g from Çökelek cheese samples were placed into 90 mL sterile sodium chloride (NaCl) solutions (0.85% w/v). Then, other dilutions were prepared as decimals. Total aerobic mesophilic bacteria (TAMB) number was determined in Plate Count Agar (PCA; Merck, Darmstadt, Germany) after incubating at 30 °C for 72 h [24]. Coliform bacteria colonies were counted on the Violet Red Bile Agar (VRBA; Merck, Darmstadt, Germany) after incubating at 37 °C for 48 h [25]. Potato dextrose agar (PDA; Merck, Darmstadt, Germany) was used to count yeast and mold colonies. For this, PDA was incubated at 25 °C for 5 days [26].

Statistical Analysis
All obtained data were evaluated using SPSS 17 statistical program. Max, min values, and standard deviations of all data were indicated.

Heavy Metal Contents
Lead is among the most dangerous toxic elements for human health [7]. Water, soil, and air could be polluted by industrial actions, fertilizers, motor vehicles, and pesticides. Pb indirectly passes into plants and animals [27].
Pb contamination in cheese samples might be caused directly or indirectly by water, air, and soil. In addition to the environmental conditions, equipment that is used in the transportation and production of milk can also be another reason for Pb contamination [7,27].
In the present study, the Pb was determined on 5 of 30 samples. The levels of lead (Pb) in the samples were changed from 0.00 to 0.257 mg kg −1 (Table 1). Sidawi et al. [8] determined the mean Pb levels of Imeruli and Sulguni cheeses as 0.121 mg kg −1 and 0.258 mg kg −1 , respectively. Öksüztepe et al. [28] found that the samples which were sold in Elazığ have no Pb contaminant. Dağcilar and Gezer [7] investigated heavy metal concentrations of Halloumi, Kashar, White, and Lor cheese samples in Northern Cyprus. Pb was not detected in Kashar, White, and Lor cheese samples, but the mean Pb content of Halloumi samples was found as 0.035 mg kg −1 . Meshref et al. [29] found that the minimum and maximum Pb level of Kareish cheese samples which is one traditional cheese of Egypt was 0.194 mg kg −1 and 0.6495 mg kg −1 , respectively. The mean Pb level of Kareish cheese samples was found as 0.43 mg kg −1 . Ghafari and Sobhanardakani [10] found the mean Pb content of commercial cheese sold in Iran as 11.70 µg kg −1 .
The maximum level of Pb must be 0.02 mg kg −1 in milk and dairy products according to Alimentarius Commission Codex [30] and Turkish Food Codex [31]. The Pb levels of these samples did not comply with Alimentarius Commission Codex [28] and Turkish Food Codex [29] except for one sample ( Table 1).
As is a toxic heavy metal that widely exists in the environment, particularly in water, air, soil, and rocks. It causes contamination of plants, animals, and consequently edible food [27]. As concentration was determined in 4 of 30 cheese samples. The level of As detected in samples was changed between 0.01 mg kg −1 and 0.08 0.015 mg kg −1 (Table 1). Öksüztepe et al. [30] reported that no arsenic was detected in Çökelek and Kurut samples. Özlü et al. [32] did not detect any As contamination in fresh and ripened Kashar samples. Some researchers determined As levels of samples as 0.11 mg kg −1 (Kashar cheese, Turkey) [33], 0.00066 mg kg −1 (lor, Northern Cyprus) [7], and 0.002-0.020 mg kg −1 (Kashk, Iran) [9]. There is no information about the maximum level of As in milk and dairy products in legislation rules [30,31]. But according to World Health Organization [34], tolerable weekly intake (TWI) level for As must not be more than 0.015 mg kg −1 .
It has been stated that the As level could have been increased depending on the high use of pesticides, herbicides, and insecticides in agriculture [7]. Moreover, groundwater and drinking water may include a high-level of As and their use in livestock has led to an increase in As levels in dairy products [27]. In general, heavy metal levels of milk and dairy products can be affected by many factors such as milk production conditions and cheese production equipment.

Mineral Contents
Milk and dairy products contain some major trace elements [35]. Although the trace elements which are vital in the functions of humans are present at low levels in the human body, their high doses are harmful and cause health problems [8,9]. Especially milk and dairy products have potentially more at risk than other foods due to consumption by infants and elder people [8]. In the presented study, major elements such as Ca, Na, Mg, K, P, and trace elements such as S, Al, Ni, Cu, and Zn were found in Çökelek samples ( Table 1).
The levels of Ca, Na, Mg, K, and P in samples were between 19.3-714, 22.8-9035, 14.9-247, 52.2-299, and 719-5735 mg kg −1 , respectively (Table 1). Öksüztepe et al. [30] determined that mean contents of Ca, Na, Mg, K, and P in Çökelek cheeses that are sold in Elazığ were 327.76, 620.40, 136.11, 1142.24, and 67.46 mg kg −1 , respectively. Yasar et al. [36] found that the mean contents of Ca, Na, Mg, K, and P in brined and dry salted traditional Malatya cheeses were between 8981.60-12,230.32-12,033.12, 8981.60-9878, 4742.18-4844.54, 922.85-1151.37, and 3903.8-4263.7 mg kg −1 , respectively. Canozer and Köse [37] investigated some properties of Örgü cheese that are produced traditionally and industrially. They determined Ca content as 1736.64 mg 100 g −1 , Na content as 2491.7964 mg 100 g −1 , Mg content as 2209.0464 mg 100 g −1 , K content as 2261.7664 mg 100 g −1 , and P content as 9541.0064 mg 100 g −1 in traditional cheese samples. The mineral contents of milk have been affected by the feeding, age, and lactation stage of animals [32]. Al contents of samples were between 0.000 and 134 mg kg −1 (Table 1). Öksüztepe et al. [30] who study heavy metal and mineral contents of Çökelek and Kurut samples (n = 25) sold in Elazığ markets, Turkey, found the mean Al content as 3.05 mg kg −1 . Al is one of the most substantial and plentiful minerals in the world, and it is used in many different industries such as food (packing, additives), painting, and the cosmetic industry. There is no evidence related to its toxic effects on humans [34].
The high Ni concentration might cause neurotoxicity, oxidative stress, and breakdown of the male reproductive system [8]. The Ni content of the samples was changed between 0.021 and 0.849 mg kg −1 , and the mean value in the samples was 0.307 mg kg −1 ( Table 1). Öksüztepe et al. [30] did not detect Ni content in any Çökelek and Kurut samples sold in Elazığ.
The Ni level of traditional cheese samples produced in Georgia was determined as 0.011 mg kg −1 (Imeruli cheese) and 0.026 mg kg −1 (Sulguni) by Sidawi et al. [8]. In the presented study, Ni levels were higher than those allowable levels which are reported by Öksüztepe et al. [30] and Sidawi et al. [8]. The limiting level of nickel was reported as 0.027 mg L −1 in milk and cheese [8]. But, there is no information about the Ni level in legal rules.
Cu is one of the important elements for many human physiological events such as sufficient growth, cardiovascular system, the elasticity of the lungs, neuroendocrine function, and iron metabolism [8,29,34]. However, excessive Cu intake may lead to some health problems (gastrointestinal disorders, immunity problems, liver cirrhosis, neurological disorders, and dermatitis) [29]. The permissible level of Cu has been mentioned for milk and dairy products in the legal rules. The highest and the lowest Cu concentrations in Çökelek samples were determined as 2.40 mg kg −1 and 0.089 mg kg −1 , respectively (Table 1). Öksüztepe et al. [30] found the lowest Cu level as 0.41 mg kg −1 , the highest level as 1.81 mg kg −1 , and the mean Cu level as 1.13 mg kg −1 in Çökelek samples. Dağcilar and Gezer [12] reported that the mean Cu contents of Halloumi, Kashar, White, and Lor cheese samples sold in Northern Cyprus were 0.591 mg kg −1 , 0.270 mg kg −1 , 0.300 mg kg −1 , and 0.246 mg kg −1 , respectively. Meshref et al. [26] determined that the Cu content of traditional Kareish cheese sold in Egypt was between 0.002 and 0.53 mg kg −1 . The obtained highest Cu content was higher than those reported by Öksüztepe et al. [30], Meshref et al. [26], and Dağcilar and Gezer [12].
Zn is one of the essential minerals which is vital for humans. Zn has important effects on many physiological functions such as sexual maturation, the immune system, and insulin secretion. In addition, it is beneficial for the metabolism of protein, carbohydrates, lipid, and nucleic acid. All these functions of Zn are caused by the many enzymes' structures [8,27,29]. Excessive Zn intake may lead to anemia, gastrointestinal diseases, vomiting, fever, and lethargy [8,34]. Zn values of the Çökelek samples were between 0.934 and 11.9 mg kg −1 , respectively ( Table 1). The mean Zn level of Çökelek and Kurut samples were found as 4.19 mg kg −1 and 9.66 mg kg −1 , respectively, by Öksüztepe et al. [30]. The researchers determined the Zn levels of some traditional cheese as 8.59 mg kg −1 (Kareish cheese, Egypt) [29] and 0.07586-0.1248 mg kg −1 (Imeruli and Sulguni, Georgia) [8]. Ghafari and Sobhanardakani [10] reported that Zn contents of commercial cheeses were between 212 and 198 µg kg −1 . It is thought that the mineral content of Çökelek samples may change depending on factors of the animal reasons, used equipment in all processes, and packaging material.

Chemical Properties
The physicochemical properties of Çökelek cheese are shown in Table 2 [37]. The obtained results in the study were lower than those reported by Kavaz et al. [2], by Çardak [1], and by Canozer and Köse [37]. According to the Turkish Food Codex, Lor cheese must contain at least 25% of the total solid [38]. The total solid content of the 6 samples among the 30 samples (Fig. 1) did not comply with the Turkish Food Codex [38].
The minimum, maximum, and mean values of belongings to ash were 0.612%, 8.46%, and 1.88%, respectively. The researches were determined the mean ash content of Çökelek samples as 2.60% [39], 2.14% [14], 4.04% [2], and 1% [5]. The reported results by other researchers were higher than those of the present study except for that reported by Celem et al. [7]. The lowest and highest salt values of samples were 0.105% and 6.67% (Table 2), respectively. Celem et al. [5] examined some properties of 20 Çökelek samples that were collected from markets in Malatya, Turkey. They found the minimum, maximum, and mean salt values of samples as 0.1%, 1.8%, and 0.3%, respectively. Öksüztepe et al. [14] determined the salt content of Çökelek samples between 0.21 and 0.76%. The Turkish Food Codex reported that the highest salt content of Lor cheese must be 6.00%. Only one sample's salt level was not in harmony with the Turkish Food Codex [38] (Fig. 2).
The fat values of Çökelek samples are indicated in Table 2. The mean fat value of samples was determined as 4.31%, while fat values were between 0.250 and 13.5%. In the performed studies regarding Çökelek, the mean fat levels of samples were 1.2% [39], 1.38 [14], 15.43 [1], and 1.6% [5]. The fat values reported by Ağaoğlu et al. [39], Öksüztepe et al. [18], and Celem et al. [7] were lower than the herein results, whereas the values that were determined by Çardak [1] were higher.
The protein and WSN values of the samples changed between 11.3-27.4 and 0.095-1.18%, respectively. Sagdic et al. [40] found protein and WSN values between 11.36 and 14.99% and 0.15 and 0.21%, respectively, when studying
The RI of the samples was between 0.610% and 5.57% ( Table 2). The RI values of Çökelek and Lor cheese samples were determined as 2.11% and 2.63%, respectively, by Kavaz et al. [2]. Sagdic et al. [40] determined the RI values of surk samples changed from 6.45 to 9.48%. Dervisoglu et al. [41] found the RI values of Kes cheese samples between 1.79 and 28.53%. It has been stated that the proteolysis level of the Çökelek cheese could be low. Because any enzymes have not been used in the production or low enzymatic activity of natural microbiota and enzymes in the product due to heat in the production process [5].
The acidity and pH are important parameters that affect the Çökelek cheese quality [42]. As seen in Table 2, the acidity values of samples changed between 0.185 and 1.34%. Celem et al. [7] determined the lowest and highest acidity values in fresh Çökelek samples as 0.4-0.8%, in skinripened Çökelek as 0.4-0.9%. Kavaz et al. [2] found that the mean acidity level of the Çökelek sample was 0.28%. It has been reported the lowest and highest acidity values of the traditional Örgü cheese samples were 0.18% and 0.52% by Canozer and Köse [37].
The pH values of Çökelek samples were determined as the lowest at 3.43, the highest at 6.49, and the mean at 4. 40 [42].
The physicochemical properties of samples demonstrated high changes in the present study. The changes might have been caused by the absence of a standard production process and legal rules about Çökelek cheese. There is no information or legal rules about the chemical and microbiological properties of Çökelek cheese in the Turkish Food Codex [38]. But, some properties of Lor cheese are determined in the Turkish Food Codex [38]. The Çökelek and Lor cheese are similar, but not the same [2,3].

Microbiological Properties
TAMB counts in dairy products demonstrate low hygienic quality in milk, insufficient pasteurization, and cross-contamination. The TAMB counts are a serious problem for food safety [17,19]. TAMB counts of samples were a minimum of 6.74 log CFU g −1 , a maximum of 8.99 log CFU g −1 and a mean of 8.26 log CFU g −1 (Table 3). Öksüztepe et al. [18] investigated the microbiological properties of Çökelek cheese sold in Elazığ at markets. They found that the TAMB mean count was 8.46 log CFU g −1 . Çardak [1] determined that the TAMB count of Çökelek samples collected from Aydın province was between 4.49 and 6.89 log CFU g −1 . Şanlı and Anlı [43] reported that the TAMB count of Çökelek samples changed from 3.31 to 3.58 log CFU g −1 during 28 days of storage. The obtained results in the study are similar to those determined by Öksüztepe et al. [18] but were higher compared to those found by Çardak [1] and Şanlı and Anlı [43].
Coliforms are one of the important indicator microorganisms the during cheese production process and then after [19]. As seen in Table 3, coliform bacteria counts were between < 2 and 5.92 log CFU g −1 . The Coliform bacteria mean count (1.82 log CFU g −1 ) of samples was higher than those reported by Öksüztepe et al. [18], Kavaz et al. [2], and Celem et al. [7], but it was lower than those of found by Çardak [1] and Önganer and Kırbağ [11].
The yeast and molds are important microorganism species in dairy technology as contaminated [16,44]. The yeast and mold counts are demonstrated in Table 3. Yeast and mold counts of samples ranged from < 2 to 8.57 log CFU g −1 , and the mean count was 7.11 log CFU g −1 . The yeast and mold mean counts were determined by Celem et al. [7] (fresh Çökelek 7.26 log CFU g −1 and ripened Çökelek 8.13 log CFU g −1 ) and by Çardak [1] (7.49 log CFU g −1 ) are similar to the mean yeast and mold counts of the samples analyzed in the presented study, while the mean yeast and mold counts in the studies by Ağaoğlu et al. [39] (5.11 log CFU g −1 ), Öksüztepe et al. [18] (3.10 log CFU g −1 ), and Kavaz et al. [2] (5.62 log CFU g −1 ) were higher.
The microbiological properties of cheese could have been changed depending on the cheese production process, storage conditions, type of cheese, and physicochemical characteristics [45]. There is no legal standard in the Turkish Food Codex about the quality criteria of Çökelek cheese. Additionally, Çökelek cheese has been generally produced traditionally and sold in local bazaars. So, Çökelek has not the standard production process. This situation could be caused problems with hygienic conditions and microbial contaminations. The differences in the microbiological properties might have been caused by mentioned above conditions.

Conclusion
The present study investigated the elemental content and physicochemical and microbiological properties of commercial sold Çökelek cheese samples in Trabzon, Turkey. The Pb in 6 samples and As in 4 samples contamination were determined. Heavy metal contaminants that have harmful effects on human health are determined in some samples. The mineral composition of samples indicated a certain change depending on many factors. The mean Ca, Na, Mg, K, P, Al, and Ni concentrations of samples were 157, 941, 72.6, 140, 2137, 12.9, and 0.307 mg kg −1 , respectively. The physicochemical properties of Çökelek samples showed high differences. The samples had low WSN and RI values due to limited proteolysis. The coliform and yeast-mold count of samples were determined as < 2 log CFU g −1 in 19 and 2 of 30 samples, respectively. The TAMB counts of most of the samples were > 8 log CFU g −1 .
Çökelek cheese is a variety of traditional cheese. And, there are no legal rules regarding the chemical and microbiological properties of Çökelek cheese. So, there is not a standard production process and composition. As a result, if legal rules regarding traditional cheese are made, the chemical and microbiological properties of traditional cheese such as Çökelek will be improved.
Data Availability Research data are not shared.