Chemical Composition and Screening of Aatoxin M1 in Cows’ Milk in Gadarif Town, Sudan

Background: Milk is a perfect complete perishable food that could be adulterated or contaminated by wide varieties of substance throughout the chain of its production. Of these aatoxins M1 was considered as an important health risk associated with the milk. The objectives of this study are to analyse milk samples, for chemical composition and detection of aatoxin M1. Method: One hundred milk samples were collected from different sources randomly; 35 samples from farms, 35 samples from sale points and 30 samples from groceries in Gedarif town. Milk-Kana was used for determination of chemical composition (fat, solids not fat, lactose and protein and analysis of freezing point, density and added water) and Unisensor kits were used as screening method for detection of aatoxin M1 in milk. Results: The chemical composition of milk samples collected from farms, sales points and groceries showed lower content of the fat (3.4±1.3%, 3.4±1.3% and 3.5±1.25%), solids not fat (7.7±1.1%, 8.1±1.7% and 8±1%), lactose (4.2±0.6%, 4.4±0.8% and 4.3±0.5%) and protein (3±0.4%, 3±0.5% and 3±0.4%). The freezing point of milk samples revealed -.503±.079, -.524±.007 and -.522±.099 °C, while the density showed 0.025±0.003, 0.026±0.005 and 0.026±0.003 gm/cm 3 , respectively. Moreover the added water was found as 10.6±11.6%, 10.6±10.4% and 8.7±8.2% in the samples collected from farms, sale points and groceries, respectively. The occurrence of aatoxin M1 in raw milk samples were found in 22(27.50%) of the samples. The presence of aatoxin contamination was high in milk samples collected from sales points (15.0%) followed by farms (11.25%) compared to those obtained from groceries (1.25%). Conclusion: The lower chemical composition of milk ns the high added water suggested adulteration of milk. Moreover the presences of aatoxin M1 in the milk samples might indicate that the cow milk was contaminated with aatoxins through feed. Hence this study recommended that good hygienic practices should be considered in Gadarif town. Similarly regulations and lows for selling the milk should be implemented by the ocial authorities.


Introduction
Fresh milk is considered as a complete diet because it contains all the essential nutrients such as lactose, fat, protein, minerals and vitamins in balanced ratio rather than the other foods (Hossain and Dev 2013). The composition of cows' milk is of the greatest importance for the dairy industry. Since its processability is highly in uenced by composition. Knowing the composition of milk also helps to assess adulteration and the quality of the milk for consumers and milk processing industries (Gurmessa and Melaku 2012). On average, bovine milk is composed of 87% water, 4-5% lactose, 3% protein, 3-4% fat, 0.8% minerals, and 0.1% vitamins (Haug et al. 2007; Link mark-Mansson 2003).
A atoxins are toxic secondary metabolites of moulds; Asperigillus avus and A. parasiticus are the main mycotoxin which could be associated with milk ( Zinedine 2007). Moreover Ali et al. (2014) found that the concentration of AFM1 in Khartoum State was affected by the source of concentrated feed but not the farm size. This might be because most of important milk producing areas in Sudan has no rigid systems of inspection on the farms and most of the products of these farms are sold through venders and groceries (Ahmed and El Zubeir 2007). Therefore, the incidence of AFM1 contamination in milk from dairy cow must be considered a risk, and raw milk is continually investigated and surveyed with respect to AFM1 contamination worldwide (Ketney et al. 2017).
The incidence of a atoxin M1 (AFM1) contamination in milk and milk products is a serious health hazard for human worldwide.
Thus, the global monitoring of AFM1 in raw milk has been ongoing for decades (Min et al. 2020). Several surveillance and studies showed the occurrence of AFM1 in milk samples from France (Boudra et al. 2007), Portugal ( Fadlalla et al. 2020; Yousof and El Zubeir 2020). Thus good agricultural practices are needed, which include appropriate drying techniques, maintaining proper storage facilities and taking care not to expose grains or oil seeds to moisture durig transport and marketing (Magan and Aldred 2007). Hence the present study was conducted with the obectives of investigating and comparing the chemical composition of milk from farms, sale points and groceries offered for consumption in Gadarif town. Also the detection of a atoxin M1 occurrence in milk from different sources in Gadarif town was examined.

Study area
The study was conducted in Gadarif State (Baladia Locality) situated between latitude 12-17 • North and longitude 34-36 • East, geographically it has a wide variety as well as heavy rains, which range between 600-900 mm during the year. The livestock is the most important renewable resource in the state and some of the state's population depend on it, and comes secondly after agriculture. The state is very rich in livestock, which is estimated to 7% of the total census of livestock in Sudan. The number of livestock in Gadarif State is approximately ve million heads of different species, this increase to seven million heads in the rainy season as the result of movement of animals from the traditional pastoral system in the prevailing system. According to the Ministry of Animal Resources and Fishery of Gadarif State (MARF 2011), the total number of animals is estimated to be 3.896.134 head. Sheep herds comprise about 48% of the total animal number followed by goats (24%), cattle (24%) and camels are about 4%.

Source Of Milk Samples
One hundred samples of milk were collected randomly from Gadarif locality from different locations; 35 samples of raw milk were collected from dairy farms; 35 samples of raw milk was collected directly from sellers point and 30 samples of raw milk were collected from groceries.

Collection Of Milk Samples
Raw bulk milk samples were collected in the afternoon during May to June 2018. After collection, the milk samples were kept in ice box till the next morning, and then they were taken to the laboratory of the Faculty of Agricultural and Environmental Sciences for analysis of chemical composition and detection of a atoxin.

Laboratory Examination Of Milk Samples
The raw milk samples were subjected to chemical composition analysis and detection of a atoxin.

Chemical Examination
The chemical composition including fat, total solids, density, protein, freezing point, lactose and adding water using Milkana (Milkana…. 8_10_2 Serial no. 701097 Express plus, Bulgaria).

Test For A atoxin M1 Detection
The A asensor kit 041 and its associated accessories (Heatsensor, negative and positive standards, deionized water) that were used in the present study were produced by the Unisensor Company (Liege, Belgium). It is a rapid (10 minutes at 40°C) test that use for the quanti cation of a atoxin M1 (AFM1) in raw milk samples. The limit of quanti cation (LOQ) of the a sensor is 20 ppt Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js with a range of quanti cation up to 150 ppt. A sensor test requires the use of microwells that containing a predetermined amount of antibody lined to gold particles and a dipstick made up of a set of membranes with speci c capture.
The lines for a valid test include the upper red (control line) that should be visible after second incubation time (7 minutes). The test was done by suspending 200 µ1 of milk sample with the reagent from microwell. A speci c antibodie will bind the analyses; if present; during the rst incubation time (3 minutes). When the dipstick is dipped into the sample, the liquid starts running vertically on the dipstick and passes through capture zones.
The development of a color at the test line indicates that the sample is free of a atoxin M1. On the opposite, the presence of a atoxin M1 in the sample will not cause the appearance of the colored signal at the test capture line. The concentration of a atoxin M1 present in the milk sample will be based on the intensity of the line color that should be started from the bottom line of a atoxin M1. The results were directly interpreted by visual observation. When the test line was darker in color like the control line, the result was negative, which means that at the given sensitivity of the test, the milk samples contain no a atoxin M1 or a atoxin M1 at a lower level than the value stated in the enclosed a atoxin M1 limit of detection. When the line was as the same intensity or lighter in color than the control line, the result is considered positive (+) and the sample should contain higher concentration than 100 ppt. When there was no test line at all, the milk sample should contain higher concentrations of a atoxin M1 residues and considered as full positive (++) as was described by UNISENSOR (2013).

Statistical analysis
The data of milk composition was subjected to statistical analysis using SPSS program (SPSS 2008). The analysis has carried out after obtaining results using ANOVA table of analysis and presentation the results.

Chemical composition
A total of hundred milk samples were collected from three sources; namely farms (35), sale points (35) and groceries (30) in Gadarif town. The means and standard deviations of the fat content for the milk samples collected from farms, sale points and groceries revealed 3.4±1.3%, 3.4±1.3% and 3.5±1.2%, respectively (Table 1). However the minimum and maximum values results were 1.12 and 6.45%, 1.03 and 6.90% and 1.44 and 6.59%, respectively as shown in Table 1. The means for total solids content of milk samples were found as 7.7 ±1.1%, 8.1±1.7% and 8±1%, respectively. The minimum and maximum values revealed 5.4 and 10.7%, 5.97 and 15.5% and 6.05 and 10.5%, respectively (Table 1). Milk lactose showed 4.2±0.6%, 4.4±0.8% and 4.3±0.5% for means and standard deviations, while the minimum and maximum values were 2.81 and 5.84%, 3.29 and 6.73% and 3.33 and 5.72%, for the milk samples collected from farms, sale points and groceries, respectively ( Table 1). The means for protein content of milk were 3±0.4%, 3±0.5% and 3±0.4% and the ranges were 1.97-4.05%, 2.86-4.68% and 2.29-3.97%, respectively (Table 1). The freezing point for milk samples collected from farms, sale points and groceries in Gadarif town revealed was found to range from -.666 to -.328° C (-.503±.079°C), -.726 to -.373°C (-.524±.007°C) and (-.654 to -.383°C (-.522±.099°C), respectively ( Table 2). The results of positive detection of a atoxin in milk samples collected from farms, sale points and groceries in Gadarif town showed that about 27.50% of the milk samples were contaminated with a atoxin M1 ( Table 3). The highest occurrence of a atoxin was found in the sale points (15.0%) followed by those obtained from the farms (11.25%) compared to those obtained from groceries (1.25%) as shown in Table 3. The milk samples showing strong positive (level 1) contamination of a atoxins were 6.25% and they belong to the samples obtained from farms. However the positive milk samples contaminated with a atoxins from level 2 were 21.25%, of which 5.0% from farms, 15.0% from sale points and 1.25% from groceries (Table 4).  Shuiep et al. (2016) reported that variations between milk fat content could be due to different management, feeding regimes, production systems and breed of cattle. They indicated that the local cows are signi cantly (P<0.05) capable to produce higher milk fat throughout their lactations. On the other hand, Mirzadeh et al. (2010) found that fat content was 3.90±0.97% in Iran, while Eckles and Combs (2004) reported that the average percent of fat in milk was 3.8% in India and concluded that the milk fat is the most valuable constituent of milk and should be considered as the food value of the milk. Also Pavell and Gavan (2011) reported that nutrition, climatic conditions and regional differences can be regarded as important sources of variation in the composition of milk.
The solids not fat (SNF) of milk samples collected from farms (7.7±1.1%) showed lower values than those obtained from sale points (8.1±1.7%) and groceries (8±1%) as sown in Table 1 2015) found that the total solids were 12.87% The average protein content of milk samples (3±0.4%) collected from the three sources in Gadarif town (  (Warsama et al. 2017). The lactose content was found in a range of 5.21 to 5.15% and 5.33 to 5.02%, in local and crossbred cows, respectively (Shuiep et al. 2016). This might be due to the fact that the lactose content of milk is affected by different locations and feedstuff that animals utilized (Kittivachra et al. 2007). On the other hand, Eckles and Combs (2004) reported that lactose has an important relation to the manufacture. However there is some evidence that lactose is the least cariogenic of the common dietary sugar. In addition, various other components of milk have been considered to be protective against dental caries (Bánóczy et al. 2009).
There was no signi cant difference between the results of milk sources in Gadarif town as this study revealed that the freezing point in sale points were -0.524±-0.007° C (Table 2). Similarly Ahmed El Zubeir (2007) reported that the freezing point of raw milk were -0.519±-0.0251° C and -0.533±-0.013° C in the samples collected during summer and winter, respectively and the average was -0.535±0.033° C. Marshall (1992) stated that a freezing point of -0.517° C is considered normal for milk and milk that freezes at or below this value is presumed to be free of added water.
In the present study the mean density was 0.026±0.005 g/cm 3 (  3 . This indicated addition of water or subtraction of fat as was shown in Table 1 that lower values were found for fat, lactose and solids not fat. Similarly El Zubeir et al. (2008) reported lower levels for the chemical content (fat, protein, lactose, SNF, and total solids) of the pasteurized milk compared to the raw milk samples obtained from different milk producing companies in Western Cape, South Africa.
The added water in the milk samples collected from the farms was estimated as 10.6±11.6% (Table 2). When comparing the present results, it was observed that the level of the added water was relatively high. This may be due to adulteration by adding water to milk in Gadaref town. The raw milk samples tested during this study (Table 3)  . Hence regular monitoring of AFM1 is necessary for evaluating their contamination and improvement status. Simultaneously, more precautions could be implemented on hygiene controls in order to limit AFM1 contamination in dairy products (Min et al. 2020

Conclusions
The results of the chemical composition of milk showed lower content of fat, solids not fat, protein and density. Moreover the added water was high. It was also observed that the highest percentage of most chemical composition was occurred in sale points. On the other hand, the presence of a atoxin in raw milk samples need further monitoring and control, although the present frequencies are low compared with the previous studies. The presences of a atoxin in milk samples might indicate that the cow milk was contaminated with a atoxin through feed because all samples showing strong positive were detected in the milk samples collected from the farms. The present study recommended that good practices should be adopted for dairy cows' feeding, and the sale point of milk should be improved. Also education and awareness should be conducted especially among farmers and livestock producers on the health hazards of a atoxins. Moreover strict lows and legislations should be implemented for the milk producers in order to minimize occurrence of a atoxins and to ensure the quality of milk and dairy products in country. This study was partially funded by a project received from Sudanese Standard and Metrology Organization, Sudan. The support of Ministry of Animal Resources and Fishery of Gadarif State is also acknowledged. The help offered by the dairy owners and the labours in the dairy farms is appreciated with thanks. Thanks are also extended to Animal Production Research Center (Kuko, Khartoum) staff for providing the Milkana device to be used in Gadarif.

Abbreviations
Authorship contribution KMAA, IEME and OHMA conceptualized the research proposal and study design; KMAA, ESS and IEME were involved in data acquisition including the collection of the milk samples; KMAA and ESS were involved in laboratory analysis, statistical analysis and interpretation of the results; KMAA, ESS, IEME and OHMA involved in the preparation of the rst original draft. IEME nalized the reviewing and editing of the manuscript. All the authors read and approved the nal version for submission. Funding: Not applicable.
Availability of data and material: All the generated data obtained during this study are included with the submission of the paper.
Ethics approval and consent to participate Not applicable. However the Ministry of Animal Resources and Fishery of Gadarif State are involved and the result of the present data was re ected to them in an o cial workshop organized by the extension department.