Assessment of Aflatoxin M1 Concentrations During Production and Long Storage of Salted (Tuzlu) Yogurt

Bu calismada, yuksek performansli sivi kromatografisi (HPLC) kullanilarak tuzlu yogurdun uretimi ve depolanmasi sirasinda sutteki aflatoksin M 1 (AFM 1 ) konsantrasyonunun belirlenmesi amaclanmistir. Y apay yolla iki farkli duzeyde (0.05 μg/l ve 0.1 μg/l) aflatoksin M 1 (AFM 1 ) ile kontamine edilmis sutlerden tuzlu yogurt uretilmistir. 0.05 µg/l AFM 1 ve 0.1 µg/l AFM 1 ile kontamine edilmis sutlerden yogurt ve suzme yogurt uretimi sirasiyla % 65, % 70.25 ve % 73.75, % 81.12 duzeyinde AFM 1 kaybina neden olmustur. Ayrica tuzlu yogurda uygulanan depolama isleminin (90 gun), tuzlu yogurdun AFM 1 icerigini sirasiyla 0.019 ve 0.027 µg/l degerlerine azalttigi tespit edilmistir (0.05 μg/l ve 0.1 μg/l AFM 1 ). Depolama periyodundaki zamanlar arasindaki fark istatistiksel olarak onemli bulunmustur (P<0.01). Tuzlu yogurt uzun raf omrune ve yuksek isil isleme sahiptir ve AFM 1 her iki seviyede de tamamen kaybolmamistir.


Introduction
Aflatoxins are synthesized, especially by the Aspergillus parasiticus, Aspergillusnomius and Aspergillus flavus species [1,2], and rarely by other Aspergillus, Penicillium and Rhizopus species [3,4]. Up to now, almost 19 different toxic differentiation of aflatoxins have been declared [5]. A. parasiticus produces both B and G aflatoxins, while Aspergillus flavus only produces B aflatoxins [6]. International agency for research on cancer (IARC) categorized AFB1 as Group I of carcinogen and AFM1 as Group 2B of carcinogenic compounds [1,7]. AFB1 is thought to be the most potent toxic aflatoxin and metabolically produces the monohydroxy derivative AFM1 [8,9,10]. AFM1 is almost as acutely toxic as AFB1, while its mutagenic and carcinogenic potential seems to be lower [11,12,13]. Aflatoxins metabolized to the 8-9-epoxide connect macromolecules and cause cancer, hepatopathy and immunosuppression [9,14].
The United States Food and Drug Administration has defined a limit of 500 ng⁄l for AFM1 in milk and dairy products [15], while the European Commission has defined a limit of 50 ng⁄l for AFM1 in these products *Correspondingauthor: zehraalbay32@gmail.com [16]. The Turkish Food Codex legal limits for AFM1 in milk is 0.05 µg⁄kg [17]. Aflatoxins cause cancer, slow down child development, suppress the immune system, and may cause death [18]. Therefore, it is significant to assessment of aflatoxin M1 concentrations in milk and dairy products since it poses a potential health hazard.
The most significant problem caused by milk and dairy products in terms of AFM1 is that it is stable against heat process such as UHT, sterilization and pasteurization. This is the reason why AFM1 does not decrease in amount during the manufacture of dairy products [19]. Salted (tuzlu) yogurt, which is prepared by heating (second pasteurized at 90°C) of strained yogurt, is a traditional milk product that has a high amount of dry matter and long shelf-life [20,21]. 1-5% of salt is added into the salted (tuzlu) yogurt during the heating process to eliminate microbial development, and to decrease water activity [22,23,24].
The aim of this study was to investigate the dispersion and stability of AFM1 during the manufacture and the storage of salted (tuzlu) yogurt. In this study, by adding AFM1 in two doses (0.05 µg/l and 0.1 µg/l) into the milk used for producing salted (tuzlu) yogurt, the effects of straining, heat treatments (applied to milk and strained yogurt) and storage on the change of the initial concentration of AFM1 were investigated. The changes in the AFM1 content during manufacturing and storage were determined by the immunoaffinity column, High Pressure Liquid Chromatographic method.

Experimental design and preparation of salted (tuzlu) yogurt samples
During the manufacturing of the salted (tuzlu) yogurt, a total of 24 liters of raw cow milk was used. The milk was divided into three equal 8 liter measurements. A standard aflatoxin M1 (Sigma-Aldrich, CAS 6795-23-9; C17H1207; FW 238.3; Co., 3050 Spruce Street, MO 63103, St Louis, USA) was spiked to raw milk at the levels of 0.05 µg/l (A) and 0.1 µg/l (B). The last measurement of milk was taken as the control group (C) and no aflatoxin M 1 was added. After the samples were pasteurized at 95°C for 20 min, the 3 groups of milk were cooled to 43±1°C [23]. The yogurt culture was inoculated into the milk (2.5%) (YO-MIX, Real 500 and 600 series, DANISCO), and the samples were incubated at 42±1°C for 2.5-3 hours at pH 4.7. The yogurt samples were cooled by keeping them at room temperature for 15 min. Then, whey of the yogurt was drained. The strained yogurt was pasteurized at 90°C for 90 min (second pasteurization). At this point, salt was added about 1 g/100 g to sample A and B. The samples were cooled to 20°C. The salted (tuzlu) yogurt samples were placed into plastic-originated vacuum bags [25]. The samples were then transferred into a refrigerator, and stored at 4°C for 90 days (Figure 1). The samples were analyzed at storage days 1, 30, 60, and 90, at 4°C. All the analyses were replicated three times.

Aflatoxin M1 analyses
The aflatoxin M1 analysis was realized by High Performance Liquid Chromatography (HPLC) and using an immuno-affinity column (Afla M1 HPLC, Vicam, USA) [26]. The AFM1 standard was supplied from the Sigma company (Sigma-Aldrich, CAS 6795-23-9; C17H1207; FW 238.3; Co., 3050 Spruce Street, MO 63103, St Louis, USA), and prepared according to Anonymous [26]. The aflatoxin M1 concentrations of samples was determinated in Shimadzu HPLC system. C18 Lichrospher column (25x4.6 mm, 5 μm, Waters Spherisorb ODS-2, Germany) were used as analytical columns. The chromatographic separation composition was carried out using a fluorescence detector (an excitation wavelength of 360 nm and an emission wavelength of 430 nm) with a mobile phase (at a flow rate of 1 ml / min) containing acetonitrile: water (25:75, v / v). Under these circumstances the AFM1 was eluted from the column at around 5 minutes.
The pure aflatoxin M1 standard in a crystal form was dissolved in chloroform to prepare the stock solution. A series of calibration solutions were prepared at different concentrations (µg /ml) AFM1 using the prepared stock solution (Figure 2). Calibration curves are arranged by plotting the peak area for each calibration solution against the mass of injected AFM 1 . The detection limit of AFM 1 was 0.01 μg/kg. Their AFM1 contents were calculated and the recovery of the AFM1 was found to be 99.72%. The analytical results were not corrected for the recovery (Figure 2). Sample preparation: The milk samples were heated to 37 o C, and then filtered through Whatman No 4 filter paper [27]. The filtered milk (50 ml) was passed through an immuno-affinity column (3 ml/min). After, 1.25 ml methanol: acetonytrile (20:30) was collected in a vial by passing it through a column. 100 µl of prepared vial content was injected into the HPLC.
This process was conducted by modifying the method given by Govaris et al. [25], and Martins and Martins [28]. The yogurt samples were homogenized by stirring, and a 20 g sample was weighed. Chloroform (75 ml), saturated NaCl solution (1 ml) and diatome soil (5 g) were homogenized at a high speed for 2-3 minutes. Hexane (50 ml), distillated water (30 ml) and methanol (1 ml) were added into the evaporated sample. The bottom phase was passed through an immunoaffinity column (3 ml/min). 100 µl of prepared vial content was injected into the HPLC.

Statistical analyses
The obtained data were evaluated by the variance (ANOVA). The Tukey Tukey's multiple range test in the general linear model of the SPSS statistical package (SPSS 15.0 SPSS Ltd. Working UK) test was applied to see the difference between the samples. The differences between the averages were regarded significant at P<0.05 and P<0.01.

Results
It was determined that there was 0.047 and 0.098 µg AFM1 per liter of pasteurized milk respectively ( Table   1). In this study, it was found that 0.05 µg/l AFM1 (A) and 0.1 µg/l AFM1 (B) added to the milk decreased to 0.020 µg and to 0.034 µg through the yogurt production. AFM1 was not detected in the sample C (Control). After the filtration of yogurt serum, the AFM1 content of the strained yogurt samples (A and B) was found to be 0.030 µg and 0.043 µg, respectively. AFM1 was determined as 0.005 µg and 0.012 µg in the serum of yogurt. To emphasize the AFM1 losses, the total AFM1 content of the raw milk contaminated with AFM1 was considered as 100%. The total aflatoxin M1 losses in the products produced from this contaminated milk were shown in Table 1. In this study, it was determined that the AFM1 content in the strained yogurt was higher than yogurt samples, due to an increase of the dry matter.
In fermentation of yogurt, pH decreases, organic acids and some other fermentation by-products (such as volatile fatty acids, amino acids, peptides or aldehydes) occur. These compounds formed in yogurt and decreased pH may cause a reduction in the amount of AFM1 [29]. In addition, it is reported that the lactic acid bacteria used in fermentation reduce the amount of AFM1. In a recent study, AFM1 binding ability of lactic acid bacteria (Lactobacillus plantarum, Lactobacillus helveticus and Lactococcus lactis) and Saccharomyces cerevisiae strain were investigated in milk samples containing AFM1 at concentrations of 0.05 µg/l and 0.1 µg/l. As a result of these research, Lactobacillus helveticus and Saccharomyces cerevisiae strains were found to be 100% bound to AFM1. In addition, it was determined that Lactobacillus helveticus had higher binding potential than other lactic acid bacteria [30]. Some researchers reported levels of AFM1 in four milk samples ranging from 53.7 to 123.8 ng/ kg were found to exceed the EU MRL of 50 ng/ kg, whereas levels of AFM1 in 214 samples of processed UHT milk ranged from 2.29 to 21.4 ng/kg were found to all below the LOQ value [31]. In another recent study from China also reports AFM1 content of UHT milk samples in 2014 and 2015 found to be 88.6% and 59.6%, respectively [32]. AFM1 in the milk is comparatively stable, and it is not exterminated by pasteurization or heat treatments, therefore causes a serious health risk [10]. Nadira et al. [33] declared that 4/53 of dairy product samples had the contamination level greater than the European Commission (EC) limit (>50 ng/l). Iqbal and Asi [34] reported that AFM1 was detected in 61% of yogurt samples. Approximately 47% of these yogurt samples were found above the EU recommended limit. A recent study in Iran also reported that the rate of cow milk and cheese samples exceeding the EU limit were 35.9% and 10%, respectively and also explained that there is a relationship between the season and aflatoxin M1 content [35]. The reason for the decrease of the AFM1 content after production of the yogurt could be based on a low pH, by-products of fermentation or lactic acid bacteria and organic acids. The change in the structure of the casein during the yogurt production and the by-products occurring after the fermentation such as aldehydes, amino acids and volatile fatty acids may play a role in the degradation of AFM1.  A decrease in the aflatoxin concentration has also been defined in some acidified milk [36]. Hassanin [37] reported lactic acid that develops in yogurt during fermentation could cause the degradation of AFM1. The AFM1 levels of yogurt samples were found to be 0.043 and 0.075 µg/l, respectively and these AFM1 values become 0.052 and 0.088 µg/l after filtration have been reported by Govaris et al. [25].
It was determined that AFM1 content became 0.026 µg at 30 th day and 0.022 µg on the 60 th day in salted (tuzlu) yogurt (A). Also, the AFM1 content of the sample A was reported to decrease to 0.019 µg in 90 days of storage. It was found that the AFM1 content of salted (tuzlu) yogurt (B) was 0.034 µg/l, 0.027 µg/l, 0.027 µg/l on the 30 th , 60 th , 90 th day, respectively. The difference among samples was found to be significant statistically (P<0.01) ( Table 2).
Iha et al. [38] decated that the effects on AFM 1 of yogurt production and storage were minimal and total AFM1 mass in milk was reduced by 6% in yogurt. Another research that aflatoxin M1 in yogurt was reduced to around 59% of the level in milk during refrigerated storage at 4°C [37]. During the production and storage of yoghurt, changes in aflatoxin M1 levels may be caused by factors such as the pH, the concentrations of aflatoxin M1 in the milk [39]. The other most important reason for that decrease may besecond pasteurized and salting of strained yogurt during salted (tuzlu) yogurt manufacturing.
Motawee [40] reported that the losses of AFM1 were 20.5%, 21.4%, 22% for cheese curd prepared with 6%, 8%, and 10% salt. However, the salt ratio (about 1%) of salted (tuzlu) yogurt is lower than cheese curd. It is believed that the effect on amount of AFM1 of salt ratio is very little.

Discussion and Conclusion
In this study, two different levels of AFM1 (0.05 µg/l and 0.1 µg/l) were added in to milk during the manufacturing process of salted (tuzlu) yogurt. It was found to decrease the initial amount in both two concentration levels in all of the samples. This indicates that the production phases of the salted (tuzlu) yogurt and the 90 day storage decreased the initial AFM1 contents. The factors that are effective in the reduction of AFM1 are on the following; pasteurizing the milk, the filtering of the yogurt serum, the pasteurizing of the yogurt. However, even though some processes such as heat treatment (first pasteurization at 95°C for 20 min in milk and second pasteurization at 90°C for 90 min in strained yogurt), salt addition (about 1 g/100 g) in the production of salted (tuzlu) yogurt and 90 days of storage had been carried out, none of the samples had been completely removed from the AFM1. According to these findings, contamination should be prevented for the safe consumption of milk and milk products.