MATERIALS AND METHODS

Materials: Two composite fresh buffalo milk samples collected from different locations at Sharkia and Dakhlia (rural zone) were obtained from the Dairy Technology Unit, Dairy Science Department, Faculty of Agriculture, Cairo University. Another composite fresh buffalo milk sample was obtained from the herd of the Faculty of Agriculture, Cairo University (urban zone).

As for the conditions of herds it was as the following:

Methods of analysis: Buffalo milk samples were analysed for total solids, fat and acidity contents according to AOAC (1999) methods. Pesticide residues were determined. In the pesticides division of Principal Central Laboratory, Faculty of Agricultural, Cairo University, Pagliuca et al. (2005) method was used for organophosphorus pesticides determination in buffalo milk samples collected during the first week of August, 2011. However, the QUECHERS method described by Anastassiades et al. (2003) was used in the Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Food, Ministry of Agriculture, Agriculture Center for determination of different classes of pesteside residues in samples collected during the first week of December, 2011.

Statistical analysis: Data are expressed as the Mean±SD. Date were analyzed one-way analysis of variance (ANOVA), followed by assessment of differences by HSD post-hoc test. All statistical calculations were performed using by SPSS Inc. (2007) statistical package. Results were considered statistically significant at p<0.05.

Experimental procedures
Evaluation of pesticide residues in collected samples: Three composite samples of fresh raw buffalo milk (3 kg each) collected from the previously mentioned locations were obtained. Five hundred milliliters raw milk from each sample was mixed thoroughly for preparation of the fourth sample. All samples were analyzed for detection of some pesticide residues.

Effect of processing on some organophosphorus pesticides spiked in buffalo milk: Equal amounts of raw buffalo milk from each source were mixed thoroughly and treated to study the effect of pasteurization, sterilization, fermentation and coagulation on distribution of pesticides among milk. Briefly, the first part was spiked with malathion at level of 4 mg/100 g milk. While, the other part was spiked with dimethoat level of 4 mg/100 g milk. Spiked milk samples were then shake vigorously to ensure complete absorption and distribution among milk. Each part of contaminated samples was then divided into five portions. The first portion was left as control, the second portion was pasteurized (72°C/15 sec) and immediately cooled, the third portion was sterilized (121°C/15 min) in an autoclave and then rapidly cooled, the fourth portion was fermented by yogurt starter culture (0.05 g L^–1) at 43°C/2.5 h) and immediately cooled and the fifth portion was coagulated by animal rennet (2 mL L^–1) at 40°C till complete coagulation then the curd was cutting and the whey was draining. Finally, malathion and dimethoat were then extracted and determined in both treated and untreated samples.

RESULTS AND DISCUSSION

Organophosphorus and organochlorines are widely used in many agroindustrial areas of modern agriculture in Egypt as they are considered economically important for high yield production. Although pesticides provide effective and practical control of disease vector bearing and crop destroying insects, the necessity of using these toxic materials on human or his immediate environment has created potential hazards that are of great public health significance (Shaker and Elsharkhawy, 2015).

Therefore, preliminary trails were made in cooperation with the Pesticides Division of Principal Center Lab, Faculty of Agriculture, Cairo University and the Central Lab of Residue Analysis of Pesticides and Heavy Metals in Food, Ministry of Agriculture, Agriculture Center, in order to distinguish the occurrence of the predominant pesticide residues in Egypt, buffalo milk collected from different locations on one hand and follow the effect of some processing on some pesticides spiked in buffalo milk on the other hand.

Evaluation of naturally occurred pesticide residues in Egypt, buffalo milk collected from different locations: Fat, total solids and acidity contents of milk collected from Sharkia, Dakhlia and Giza provinces were determined. All samples have normal composition as shown in Table 1. Reports obtained from the Pesticides Division of Principal Center Lab, Faculty of Agriculture, Cairo University, showed organophosphorus pesticide contamination in buffalo milk samples collected from different locations in Egypt.

Dimethoate was found in the range of 0.000-0.493 mg/100 g, while malathion was in the range of 0.022-0.040 mg/100 g milk as shown in Table 2 and Fig. 1. In this respect, Mallatou et al. (1997) investigated thirty eight samples of bovine milk and found that eleven milk samples contained organophosphorus pesticide residues with contents below the maximum permitted amount and the contents in two samples were 43 and 280 mg kg^–1.

Table 1:	Composition of raw buffalo milk collected from different locations

Table 2:	Pesticide residues naturally occurred in egyptian buffalo milk collected from different locationsa
aDetermined in the Pesticides Division of Principal Central Laboratory, Faculty of Agriculture, Cairo University, bND: Not detected

Fig. 1:	Pesticide residues naturally occurred in Egypt, buffalo milk collected from different locations

Table 3:	Recovery of dimethoate and malathion pesticides spiked in raw buffalo milka
aDetermined in the Pesticides Division of Principal Central Laboratory, Faculty of Agriculture, Cairo University, bRecovery (%) = (mg determined/mg spiked)×100

Pagliuca et al. (2006) also found that among one hundred and thirty five raw milk samples analyzed, thirty seven samples were positive for traces and ten samples showed organophosphorus pesticide contamination in the range of 5-18 mg kg^–1.

Reports obtained from the Central Lab of residue analysis of pesticides and heavy metals in food, Ministry of Agriculture, Agriculture Center indicated that buffalo milk samples collected from different locations (December, 2011) were free from four hundred and two of different classes of pesticides including dimethoate and malathion. The Limited Of Quantification (LOQ) of the different classes of pesticides was in the range of 0.01-0.05 mg kg^–1 milk. Egyptian standards in 2010 indicate that the Maximum Residue Limits (MRL) of the different classes of pesticides for raw milk should be in the range of 0.01-0.1 mg kg^–1.

Data obtained are agreed with those illustrated in the report of FAO and WHO (2008). Abou Donia et al. (2010) on the other hand analyzed buffaloes and cows milk for pesticide residues and found that, none of the raw milk revealed the presence of any organophosphorus pesticides including dimethoate and malathion.

Comparing the results of the Pesticide Division of Principal Central Laboratory, Faculty of Agriculture, Cairo University and the Central Laboratory of Residue Analysis of Pesticides, one can say that the differences between the results obtained may be due to the sampling time or the sensitivity of the method of analysis. These results are in accordance with those of Pagliuca et al. (2006) who reported that the organophosphorus resedues in milk may influence by the season.

Effect of processing on some organophosphorus pesticides spiked in buffalo milk: Fresh buffalo milk was spiked with the desired pesticides to follow the effect of pasteurization, sterilization, fermentation and coagulation on the degradation and disappearance of pesticide residues. The pesticides used were dimethoate and malathion.

The recovery of dimethoate and malathion was 96.3 and 84.75% as shown in Table 3. Data obtained showed some loss in the added pesticides. This loss may be due to the loss during handling, extraction or purification of the pesticides. Pesticides loss during the extraction or purification steps was previously recorded by Braun and Tstanek (1982), Abou-Arab (1987) and Abd-Rabo et al. (1989).

Regarding the effect of different processes on dimethoate and malathion degradation, it could be note that all treatments had significant effects on the reduction of pesticides (Table 4 and Fig. 2). Table 4 shows that the degradation of dimethoate was 73.42% as a result of pasteurization, while malathion was completely destructed and not detected in milk sample. Sterilization process on the other hand, had a significant effect on the dimethoate and malathion content.

Fig. 2:	Effect of different processes on degradation of dimethoate and malathion spiked in buffalo milk

Table 4:	Effect of different processes on degradation of dimethoate and malathion spiked in buffalo milk
aDegradation (%): 100 (mg treated mg–1 untreated×100), Means in the same column with different superscript letters differ significantly

The percent of reduction was 75.72% for dimethoate and 95.99% for malathion. As shown in Table 4 it could be note that fermentation had a considerable effect. The concentration of dimethoate reduced from 3.852-0.520 mg/100 g and that of malathion from 1.695-0.048 mg/100 g. In other words, the pesticides degradation percentage was improved being 86.5% for dimethoate and 97.17% for malathion as compared with the other processes.

Data obtained in Table 4, also show that the degradation of malathion was greater than that of dimethoate. These results are in agreement with those found by Bo and Zhao (2010) and Zhao and Wang (2012), they mentioned that dimethoate and methyl parathion were more stable but malathion was the most labile.

Regarding the effect of heat treatments and fermentation process, Bogialli et al. (2005) and Ozbey and Uygun (2007) mentioned that the organophosphorus pesticides in foods were affected by fermentation, heat treatment and drying. The work of Kaushik et al. (2009) revealed that the food processing leads to large reduction in pesticide levels in most cases. On the other hand, Zhang et al. (2006) mentioned that the manufacturing technology of yogurt, including heat treatments (pasteurization and sterilization) and the action of microorganisms during storage can result in reduction of pesticide levels in yogurt. The results of Bo and Zhao (2010) revealed that the growth of lactic bacteria in bovine milk could reduce the residue levels of some organophosphorus pesticides and might decrease the safety risk of the products. The work of Bo et al. (2011) also revealed that two direct vat set starters enhanced organophosphorus degradation in whole milk in different extents.

As for the effect of coagulation process, data obtained showed that the degradation percentage of dimethoate and malathion of the curd was decreased as compared with the other treatments which may be attributed to binding of small amount of pesticide to the rennet enzyme (Abdou et al., 1983) or to the chemical nature of the pesticide (Pagliuca et al., 2006).

CONCLUSION

The Egypt government had banned the use of organophosphorus and organochlorine pesticides since 1980s. Although, our reports showed residues of dimethoate (0.000-0.493 mg/100 g) and malathion (0.022-0.040 mg/100 g) in buffalo milk samples collected from Sharkia, Dakhlia and Giza province in Egypt to be less than maximum limit accepted by the egyptian standards in 2010 for malathion but higher for dimethoate. As the milk processing treatment, we can conclude that the fermentation was the most effective process, which reduced the concentration of dimethoate and malathion pesticides to a great extent. Pasteurization however removed malathion completely and reduced the concentration of dimethoate.