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Evaluation of microbiological and sanitary quality of ewe's raw milk in Western of Algeria and detection of antibiotic residue by Delvotest.


Goat and sheep farming are of vital importance for the national economy in many countries in the Mediterranean region [37,32]. In Algeria, sheep herd predominate and represent 80% of the total livestock, with more than 10 million of sheep. [12]. According to the statistical date in 2007,Algeria has milk needs about 3.2 million liters per year but only 2 million liters are locally produced. Whereas, the sheep milk is an excellent raw material for dairy industry [44] so, in the western of Algeria, it is transformed by the flora naturally present in milk to cheese (Jeben), raw butter (Zebda Beldia) or ranced butter (Smen) and other dairy products [27]. However, the demand for sheep milk and its products is increasing [35]. In the first, it is tolerated by children suffering from allergy to cow's milk [1] and in the second consumers demand an original food and look of typical products especially in terms of origin and taste. Thus a several studies show that sheep milk is richer in fat, protein, ash, total solids, essential vitamins and minerals than cow's and goat milk [4,24]. Therefore, this particular composition facilitates the growth of microbial spoilage population [39]. The microbiological quality of milk can be affected by many factors such as adulteration, contamination during and after milking, the presence of mastitis, method of milking, race, animal health, stage of lactation, season [42], feeding [10] and the hygiene of farms [4]. Besides ovine milk has been implicated as an important source of infection by pathogenic bacteria associated with gastroenteritis such as Salmonella spp, Staphylococci, Enterococci and E. coli [1]. This indicates the possibility of bacterial contamination via the udder, by milking equipments or via the water used [14,7]. The objective of this study was conducted to evaluate the microbial and the sanitary quality of the raw ovine milk produced in western of Algeria and to detect the presence of antibiotics residue in this milk using the Delvotest, thus contributing on the research on improving milk safety.

Materials And Methods


A total of 105 samples of raw milk from clinically healthy sheep were collected from three different farms located in western of Algeria: 45 samples from Oran, 30 samples from Mascara and 30 samples from Relizane. Before a manual milking, teats were carefully cleaned with cotton wool impregnated with 70% of ethanol. After, the three first streams of milk were discarded; udders and mammary secretions were examined for macroscopic signs of abnormality. The samples were collected in sterile tubes and then placed in isotherm cool box (4[degrees]C) and transported to the laboratory of Applied Microbiology at Es-Senia university of Oran. The maximum time between sampling and bacteriological analysis of samples did not exceed 4h. Sample collection was done during the period from November 2011until July 2012. The samples were analyzed for their microbiological and hygienic quality as well as the prevalence of some pathogenic bacteria. A questionnaire was developed (Table 1) to obtain information on the general characteristics of farm management and hygiene practices.

Physicochemical analysis:

Immediately after milking, the milk temperature was measured using a thermometer, the pH was measured using pH meter. The Dornic acidity was titrated with a solution of NaOH(N/9) in the presence of phenolphthalein 1% as a color indicator, this acidity is expressed in Dornic degrees (decigram per liter of lactic acid) [22].

Detection of Antibiotic:

The presence of antibiotic was achieved by using the Delvotest SP-NT (DSM, Hollande), the method was carried out according to the instructions of the manufacturer. Thus, 100-Lil milk samples were added to individual Delvotest cups ready prepared containing Bacillus stearothermophilus var. calidolactis and pH indicator in solid medium, than cups were incubated at 64 [+ or -] 1[degrees]C for 3 h. Results were noted visually by interpreting the color change of a pH-indicator(Bromocresol Purple). This method was recognized by the Association of Official Analytical Chemists [28,29].

Microbiological analysis:

The total aerobic mesophilic flora (FAMT):

It is a good indicator of contamination which was enumerated on PCA agar medium and was inoculated in duplicate with 1 ml of diluted sample. All plates were incubated at 30[degrees]C for 24 h, based on the standard set by legislation.


Coliform bacteria were carried out on VRBL agar medium and incubated for 24 hours at 37 [degrees]C for total coliforms and 44 [degrees]C for fecal coliforms according to the standard (ISO 4832); E.coli was streaked onto eosine methylene blue (EMB) agar and then incubated overnight at 37[degrees]C. Typical isolates were confirmed based on their IMVIC pattern.


All species of enterobacteriaceae were counted on Mac Conkey agar medium and incubated for 24 h at 37[degrees]C, they form violet colonies, surrounded or not by a violet halo of bile salts precipitates. After a morphological description, a biochemical confirmation test of bacteria growing on culture medium was performed by using the following tests: Gram, catalase, oxidase, TSI, urease, indole, ONPG, VP and H2S production.

Detection and enumeration of fecal streptococci:

1 ml of the dilutions were inoculated on Roth broth (presumptive) and incubated at 37[degrees]C for 24h, the positive tubes were streaked in Litsky broth (confirmatory). The tubes were incubated at 37[degrees]C for 24 hours. Tubes showing a disorder and a pellet violet were considered positive and were counted. This gives the characteristic number which corresponds to the most probable number determined from the Mc Grady table [45].

Staphylococci count:

[8]: 0.1 ml from the previously prepared dilutions of the examined samples was introduced and spread on the surface of Mannitol salt agar medium plates (Institut Pasteur, Algeria). Inoculated plates were incubated at 37[degrees]C for 48 h and Staphylococci count cfu/ml was calculated and recorded. The identification of staphylococci was performed by determining the catalase, coagulase and biochemicalprofile using the kit API Staph (Biomerieux, France).

Detection of Salmonella:

For salmonella, it performs a pre-enrichment in peptone water, followed by enrichment in selenite broth for 24 hours at 37[degrees]C, then the isolation and enumeration were performed on the SS medium (Salmonella-Shigella) and incubated for 24h at 37[degrees]C.

Enumeration of sulphite-reducing Clostridium (CSR):

Tubes containing [10.sup.-2] and [10.sup.-1] dilutions were heated for 10 min at 80[degrees]C to destroy vegetative forms, 0.5 ml of a 5% solution of sodium sulfite and 2 to 3 drops of citrate solution iron 5%were added, 7 ml of agar Beef Liver (VF) (Institut Pasteur, Algeria) was added to ensure anaerobic. Incubation was performed at 37 [degrees]C for 24 to 48 hours. The large black colonies are considered sulphite-reducing clostridia [26].

Count of lactic acid flora:

Lactic acid bacteria were enumerated on MRS medium [9]. Prepared dilutions were inoculated on Petri dishes with MRS medium and incubated at 30 [degrees]C for 48 h. isolated and purified bacteria were characterized using conventional methods of microbiology (Gram, catalase and biochemical tests).

Enumeration of fungal flora (yeasts and moulds):

The enumeration of yeasts and moulds was carried out on Malt extract agar medium and incubated at 25[degrees]C for 72 h for yeasts and 5 to 7 days for fungi.

Somatic cells:

CMT (California Mastitis Test), also known as the Teepol Test used to evaluate the level of inflammation of the udder (number of cells per ml of milk) was performed on all samples as described by Schalm et al., (1971). Depending on the viscosity of the gel formed by mixing CMT-reactive milk, the CMT results coded 2 or 3 (+or++) were considered related to subclinical mastitis.


During the study period, 105 samples of raw ewe milk were collected from three different farms in the Algerian west. All results are reported in Tables 1, 2, 3, 4 and 5. The analysis of these results reveals the presence of significant variations between all the different farms studied.

From the questionnaire it was revealed that flock size was ranging from 40 to 150, 70 to 180 and 20 to 65 animals in Relizane, Mascara and Oran farms respectively. Grazing was adopted in Relizane and Mascara's farms. All the animals belonged to Ouled-Djelal race, no mastitis case was observed. (Table 1)

The temperatures values were ranged between 36.8 and 37.8 in all the milks, the pH value of the milks, which is also an indicative of the sanitary state of the sheep and of milking hygiene was ranged between 6.31and 7.14, 6.53 and 7.08, 6.38 and 7.06 in Relizane, Oran and Mascara respectively. The milk acidity was similar in all samples for the maximum value (22[degrees]D) (Tab. 2).

The antibiotic test revealed that 4.76% of the samples were positive for antibiotic residues (Tab.3).

Concerning the total aerobic mesophilic flora (FMAT) and by region, the samples collected at Oran farm showed a highest average value with 117 x [10.sup.3] cfu/ml, followed by Mascara farm with mean values of 116 x [10.sup.3] cfu/ml.

The indicator of hygiene practices such as coliforms, were presented with rather high rates exceeding the Algerian standard. The analysis revealed a contamination of samples with total and fecal coliforms mainly in Mascara region with mean values of 113 x [10.sup.1] cfu/ml and 109 x [10.sup.1] cfu/ml respectively (Tab.4).The E.coli was the predominant bacteria, it was isolated from 43.80% of the tested samples. Enterobacteriaceae were detected in most of the samples with high levels mostly in Oran's farm with a mean value of 108 x [10.sup.1] cfu/ml followed by Relizane farm with 99.4x [10.sup.2] cfu/ml.

Presence of the fecal Streptococcus was detected in 19,6% of the samples analyzed with a mean count of 286, 601 and 898 cfu/ml, in Relizane, Oran and Mascara farms respectively.

Staphylococcus were detected in 37.14% of the samples, Oran farm revealed a significantly higher Staphylococcus count than other region with 179 x [10.sup.1] cfu/ml, while the lowest level count was detected in milk of Mascara farm with 617 cfu/ml (Tab.4).

The isolated Staphylococci strains belong to the four following species: Staphylococcus aureus, Staphylococcus xylosus, Staphylococcus epidermidis and Staphylococcus lentus at percentages of 52.28%, 25.71%, 14.28% and 5.72% of total isolates respectively (Tab.5). Also, It should be noted a total absence of salmonella and clostridia, in all milk samples.

Table 4 shows that mean count of the lactic acid bacteria were prevalent in all milks with higher levels, the mean value was 21,4 x [10.sup.3], 110x [10.sup.3] and 108x [10.sup.3] in Relizane, Oran and Mascara respectively,96% of the isolated strains were Lactococcus. The average concentration of yeasts and molds in milk was 39.2 x [10.sup.2]; 80.2 x [10.sup.2] and 58.3 x [10.sup.2], in Relizane, Oran and Mascara respectively.

The somatic cell count of milk depends on the sanitary state of the udder, on milking hygiene and on various stressors. For ewe's milk, reference values have yet to be established, the CMT results revealed that the prevalence of subclinical mastitis was detected in 37.14% of the sheep herd; Oran milks have a highest value of positive cases (Tab. 3).


Microbiological quality control of milk is a very delicate task, given the great variety of conditions to be found in the Sheep sector; also it is important for its conservation and even for its transformation [18]. The results of this study showed the variability of the quality of raw ewe's milk from one region to another and indicate a poor quality of this milk regard to accepted standards.

The mean values of pH of sheep milk studied in all the regions were in accordance with the finding by Asif et al., [6], Gasmi-Boubaker et al., [16] and Rouissi et al., [40]. It was higher in mastitis milk than in normal milk. Mathieu (1998) indicate that normal milk pH range between 6,2 to 6,8. The pH value can be used as an indicator to the hygienic quality of milk, while the acidity mean values were similar to the data finding by Gasmi et al., [16]. Asif et al., [6] reported a higher acidity mean values 0.23 % in raw ewe's milk in Pakistan. The pH and acidity values depends on the casein content, minerals and ions, the hygienic conditions during milking, the total microbial flora and its metabolic activity and handling of milk [23].

The mean values of positive cases of antibiotics residues in Algerian sheep milk were very low but higher than that found by Yamaki et al., [46] with 1.7%. These results revealed a little use of antibiotic in the treatment of mastitis sheep infections in the west Algerien farms. The Delvotest method have a good detection limits for a wide range of betalactam antibiotic, sulphonamide, and tylosin residuals in ewe milk, but do not detect streptomycin, gentamycin, neomycin, erythromycin, oxytetracycline, tetracycline, chloramphenicol and trimethoprim [5]. Milk and its products destined for human consumption must be free of inhibiting substances [38].

Contamination by FAMT was relatively high mainly in Oran farm with mean value of 117 x [10.sup.3] cfu/ml, 52.38% of samples included flora which exceed the standard recommended by the Algerian official journal. A low total bacteria count in ewe milk were reported by Abd El Aal et al., [1] with 19 x [10.sup.2] cfu/ml and the lowest mean value were found by. The total aerobic mesophilic flora informs us on hygienic quality of raw milk. This is the most sought flora in microbiological analysis. From this results, it was observed that the high values of FAMT was correlated with the poor hygiene conditions in the farms and the probable transmission of microorganisms from the animal itself.

Total and fecal coliforms indicator of fecal contamination frequently contaminate milk (cow, goat and sheep), the levels of these organisms was high in Mascara farm exceeding the charges allowed by the national and international standards. This contamination of milk is a consequence of mammary infections in livestock. The total and fecal coliform values found were higher than that reported by and were in accordance to that reported by Gasmi et al., [16].

Staphylococci count observed in the present study (1.79 x [10.sup.3], 1.24 x [10.sup.3], 6.17 x [10.sup.2], in Oran, Relizane and Masara respectively) were lower than that reported by Guasmi et al., [16] in Mai month, but higher than the findings by in Rabat region. In a recent study in Swiss survey S. aureus was detected in 33% of sheep bulk-tank milk samples [36] with a maximum mean of 3.60 x [10.sup.3] cfu/ml. This significant contamination of our milks by Staphylococcus spp is a consequence of subclinical mammary in livestock; it can gain access to milk by direct excretion from infected udders or by contamination. The control of these pathogens in milk requires the development of control systems and determination of critical points at the farm level to prevent staphylococcal epidemics.

From the obtained results we found that Enterobacteriaceae were found in 79,04% of examined ewe's milk samples, lower finding were reported by Pilar Gaya et al., [17] with mean value of (4.4 x [10.sup.2] cfu/ml).These results can be interpreted as a consequence of the considerably higher pH value. The predominant isolates of Enterobacteriaceae strains were Escherichia coli, Klebsiella oxytoca and Proteus vulgaris at percentage of 77.5%, 10 % and 12.5% respectively.

The study revealed that fecal Streptococcus was presented in the sheep milk with a rate exceed the standards set by the national standard. Indeed, it was reported that the presence of fecal Streptococcus is the consequence of contamination of environment.

Lactic acid bacteria (LAB) were prevalent in all the samples, higher levels were found in normal milks comparing with the mastitis milk, the lactococci present the most population of LAB with 96% of the total strains isolates. Some lactic acid bacteria can be accommodated on the surface of the udder [11].

The results revealed that yeasts and fungi were present in the sheep milk without exceeding the standards set by ISO and AFNOR, The average concentration of yeasts and molds in milk were lower than finding by Gasmi et al., [16], it was reported that low humidity is responsible for low mold and yeast loads.

The conducted study also showed a complete absence of the most dangerous pathogens to human beings (Salmonella, Shigella, Clostridium sulfite-reducers).Similar results have been advanced by Maurer and Schaeren [35] in Suisse and Morocco, respectively, in raw sheep and cow milk.

Somatic cell:

It should be noted that Positive CMT scores of ewes were detected in 37.14% of the analyzed samples. Maisi et al., [33] found lower values 16.7 % of subclinical mastitis. It is estimated that 5-50% of glands in bovine, caprine and ovine dairy herds are subclinically infected with bacteria [15]. In present study E.coli is reported to be the predominant microorganism in ovine mastitis milk with Staphylococcus aureus. Somatic cell count (SCC) is widely used for evaluating milk quality [41,20]. It reflects the health of the mammary gland, mastitis can induce major changes in the sheep milk composition [21,3] So the hygiene measures of local farms and rational use of anti-infectives for the treatment of subclinical mastitis should be applied.


In conclusion, the results of this study highlight the poor microbiological and sanitary quality of sheep milk in western of Algeria, the data revealed a variety in the pH and acidity of milks, low detection of antimicrobial residue and a high count of FAMT, coliforms bacteria, E coli, enterobacteriaceae and staphylococci. The presence of these bacteria in raw milk is influenced by environmental factors, such as indoor housing of cattle and poor quality feed, such as silage, which are less likely to occur on Algerian farms. Therefore, It is necessary to minimize microbial contamination which can be achieved through healthy animal and milkers and hygienic practices followed in farms by training and guidance of farms' owners and their workers. Finally we must give more interest to the local ewe milk, because there are no sheep milk industries in Algeria it just use for family consumption.


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Beldjil Ali Asmaa Fatima, Benlahcen Kheira, Guessas Bettache, Aggad Habib, Kihal Mebrouk

Department of Biology, Faculty of Sciences, Laboratory of Applied microbiology, University of Es-Senia of Oran, BP 16, Es-Senia 31100, Oran, Algeria

Beldjil Ali Asmaa Fatima, Benlahcen Kheira, Guessas Bettache, Aggad Habib, Kihal Mebrouk: Evaluation of microbiological and sanitary quality of ewe's raw milk in Western of Algeria and detection of antibiotic residue by Delvotest

Corresponding Authors

Dr Beldjilali Asmaa Fatima, Department of Biology, Faculty of Sciences, Laboratory of Applied microbiology, University of Es-Senia of Oran, BP 16, Es-Senia 31100, Oran, Algeria E-mail: Tel:0775333631
Table 1: Different information of the sheep herd in
the three farms found in western Algeria

Questions Oran Relizane Mascara

Flock size(animals) 50-65 40-150 70-180
Milking No No No
Race of sheep OuledDjelal Ouleddjelal Ouleddjelal
Grazing No Yes Yes
Declaration of No No No
 mastitis cases

Table 2: Physico-chemical characteristics of Algerian
raw ewe's milk (pH, acidity and temperature).

Farm pH Acidity ([degrees]D)

 Mean Max Min Mean Max Min

Oran 6.84 7.08 6.53 17.87 22 14
Relizane 6.66 7.14 6.31 18.13 22 14
Mascara 6.68 7.06 6.38 18.7 22 15

Farm Temperature

 Mean Max Min

Oran 36.9 37.3 36.8
Relizane 37.2 37.4 36.9
Mascara 37.6 37.8 36.9

Table 3: Prevalence of mastitis in Algerian ewe's herd and the
presence of antibiotic residue in raw ewe milk samples.

Prevalence Mastitis CMT

 Number (+) Cases 0 + ++

Relizane 30 9 00 01 04
Oran 45 20 06 06 04
Mascara 30 10 03 03 02
Total 105 39 09 10 10
% 100 37,14 23,07 25,64 25,64

Prevalence CMT Antibtics

 +++ (-) Cases (+)

Relizane 04 21 01
Oran 04 25 02
Mascara 02 20 02
Total 10 66 05
% 25,64 62.85 4.76

Table 4: different microorganisms groups count in ewe's
raw milk samples of different western Algeria farms.

 FMAT CT CF Enterobacteria
 cfu/ml cfu/m cfu/ml cfu/ml

Relizan 90.2 107 97.3 99.4 x[10.sup.1]
e x[10.sup.3] x[10.sup.1] x[10.sup.1]
Mascara 116 130 109 85.6 x[10.sup.1]
 x[10.sup.3] x[10.sup.1] x[10.sup.1]
Oran 117 128 96.4 108 x[10.sup.1]
 x[10.sup.3] x[10.sup.1] x[10.sup.1]

 FeacalStreptoc Staph Clostridi Salmonell
 occscfu/ml cfu/m um a
 l cfu/ml sp.cfu/ml

Relizan 28.6 x[10.sup.1] 124 Ab Ab
e x[10.sup.1]
Mascara 89.8 x[10.sup.1] 61.7x Ab Ab
Oran 60.1 x[10.sup.1] 179 Ab Ab

 Yeast Lactic
 and acid
 fungi bacteri
 cfu/ml a

Relizan 39.2 21.4
e x[10.sup.2] x[10.sup.3]
Mascara 58.3 108
 x[10.sup.2] x[10.sup.3]
Oran 80.2 110
 x[10.sup.2] x[10.sup.3]

Table 5: Identification of bacterial species of different
isolates obtained from Western Algerian raw ewe's milks

Group of isolates Type of isolates Number Percentage

Staphylococci Staphylococcus 19 52,28
 Staphylococcus 9 25,71
 Staphylococcus 5 14.28
 Staphylococcus 2 5.71
Enterobacteria Escherichia coli 31 77.5
 Klebsiella 4 10
 Proteusvulgaris 5 12.5
Acidlacticbacteria Enterococci 50 96
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Title Annotation:Original Article
Author:Fatima, Beldjil Ali Asmaa; Kheira, Benlahcen; Bettache, Guessas; Habib, Aggad; Mebrouk, Kihal
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:6ALGE
Date:Jun 1, 2013
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