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International Journal of Dairy Science

Year: 2007 | Volume: 2 | Issue: 1 | Page No.: 33-41
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Research Article

Evaluation of the Hygienic Quality of Market Milk of Khartoum State (Sudan)

Nahid N.I. Mohamed and Ibtisam E.M. El Zubeir

ABSTRACT

This study was carried out between August 2003 and March 2004. One hundred and twenty milk samples were collected from supermarkets in Khartoum State. Sixty milk samples were collected during summer and other 60 during winter from the same supermarkets. The milk samples were examined for microbial quality that include total bacterial count, spore forming bacteria count, coliform count. Enumeration, isolation and identification of E. coli, S. aureus, Salmonella spp. and the presence of Brucella as detected by milk ring test were also estimated. The criteria used for identification of the isolates were reaction to Gram's stain, morphological appearance, cultural characteristics and biochemical tests. Similarly phosphatase test was done. The present study revealed that high average total bacterial count (5.63x109±2.87x1010 cfu mL-1) was found for the raw milk samples. Moreover, during summer the total bacterial count of milk (1.04x1010±4.01x1010 cfu mL-1) was higher than during winter (9x108±2.51x109 cfu mL-1). Staphylococcus aureus was detected in 46.7% of the milk samples with mean count of 4.9x104±1.29x106 cfu mL-1. During summer the mean count was 7.44x105±1.66x106 cfu mL-1, which was higher than the count during winter (1.61x105±2.3x105 cfu mL-1). Escherichia coli were detected in 2.5% of the milk samples with a mean of 6.0x105±7.94x105 cfu mL-1 and coliforms bacteria were detected in 82.5% with a mean of 3.32x106±1.43x107 cfu mL-1. Also during summer the mean was 5.15x106±2x107 cfu mL-1, which was higher than during winter (1.45x106±3x106 cfu mL-1). Spore forming bacteria were detected in 32.5% of the milk samples with a mean of 4.81x106±1.4x107 cfu mL-1. Also during summer the mean was 7.15x106±1.79x107 cfu mL-1, was higher than during winter (1.45x106±3x106 cfu mL-1). There was no Salmonella spp. in any of the milk samples collected during the present study, while milk ring test for Brucella showed that 44.1% of the milk samples were positive for Brucella. Moreover 54.4% of which were detected during winter and 45.61% were found during summer. Also during this study 44.17% of the milk samples were positive to phosphtase test. All the milk samples (100%) were found to be negative to the presence of formalin. Conclusions and recommendations for improvement of hygienic quality of marketed milk in Khartoum State were suggested. Sanitary standards should be established in this country so as to control milk production and marketing. Further studies should be done to evaluate safe and good supply of consumed milk.
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INTRODUCTION

Hygienic controls in raw milk are that the milk should be obtained from healthy animals and from animals not been treated with antibiotics or other veterinary drugs, which can be transferred to the milk (Murphy and Boor, 2000). Similarly milking routines should prevent the damage of tissues, which lead to infections of the udder and prevention of the contamination of milk by the stable environment and milking equipment as well as controlling temperature and time in order to minimize the growth of pathogens (IDF, 1994).

Measurement of bacterial numbers in milk is of interest because they are indicator of poor hygienic production or ineffective pasteurization of milk (Harding, 1999). Gran (2002) concluded that hygienic aspects are linked with transportation, preservation and handling of dairy product.

In Brazil, Beloti et al. (1999) mentioned an advertising campaign involved public health department was developed to stop the sale of unpasteurized raw milk and to show its risk because microbial studies revealed 4-7 positive samples in Brucella ring test out of 42 milk samples.

Marketing of milk and milk products is of greatest importance since successful operation of many farms is dependent upon the income from the milk sold, particularly when dairying is the major enterprise (El Aggab, 1996). Mustafa (1994) mentioned that the quantities that go into shops of Khartoum State in the morning are refrigerated for short time before selling, however refrigeration is a service of high efficiency in milk market system in Khartoum State and this service is not available for producers and for many selling centers which often result in losses due to perishability of the product.

In Sudan many researchers reported on the importance of milk in public health and stated that because of prevalence of some communicable diseases of milk and spoilage of this perishable product sanitary standards regulating milk production and marketing, should be established in Sudan and setting plans for improvement of milk hygiene and quality (Mustafa and Idris, 1976; Mohamed et al., 1999; Yagoub et al., 2005; Elmagli and El-Zubair, 2006). Hence the present study is a contribution to the evaluation the hygienic quality of market raw milk in Khartoum State. Enumeration, isolation and identification of some pathogenic bacteria in the market’s milk collected during summer and winter were also estimated.

MATERIALS AND METHODS

Source of Samples
In this study 120 milk samples were collected from 60 supermarkets in Khartoum State. Sixty sample in summer (August- September 2003) and the other 60 in winter (December 2003-January 2004) at both season twenty milk samples were collected from each big city in Khartoum State.

Collection of Samples
Samples were collected from four shops in each selected area in each city from five supermarkets. The collection was done at the evening in sterilized labeled bottles. They were kept in the refrigerator at 4°C and transported in an ice box to the laboratory of the Department of Dairy Production, Faculty of Animal Production, University of Khartoum.

Microbial Examination of Milk Samples
Microbiological parameters studied include the counts of E. coli, Staphylococcus aureus, coliforms bacteria, Spore forming bacteria and Salmonella spp.

Types of cultural media used include plate count agar (S-D’s Product No. 7405) that was used for total bacterial count, Mannitol salt agar (Hi Media M118) a differential and selective medium used for Staphylococcus aureus. Eosin methylene blue agar (Oxoid CM69) a selective and differential medium used for Escherichia coli. MacConkey’s agar (Hi Media M081) was used for detection and enumeration of coliform bacteria. Salmonella-Shigella agar (Hi Media M108) is a selective medium for salmonella and shigella, Sterilization of equipment was done by autoclaving at 121°C for 15 min, while all the media were sterilized by autoclaving at 121°C for 15 min, except MR-VP media, sugar media and S.S agar media (Barrow and Feltham, 1993). Serial dilutions were made according to Richardson (1985). Preparations of samples for total spore count were done according to Harrigan and McCance (1976). The counting and calculation of the colonies were done manually. Isolation and identification were done according to Harrigan and McCance (1976) and Barrow and Feltham (1993).

Milk Ring Test
It is an agglutination reaction used to detect the presence of antibodies of Brucella in the milk. The tetrazolium stained antigen was obtained from the Central Veterinary Research Laboratory, Soba using the method described by Cruickshank et al. (1975).

Statistical Analyses
The analysis was carried out using SPSS program (Statistical Package for Social Sciences). All the data of this experiment were analyzed statistically by using complete randomized design and least significant difference test. One way ANOVA was used to determine the effect of season, city and area on milk samples.

RESULTS AND DISCUSSION

Results of total bacterial count obtained during the present study showed high bacterial count, which ranged between 1.13x105 to 2.45x1011 cfu mL-1 (Table 2). During summer the total mean count of market milk samples was 1.04x1010±4.01x1010 cfu mL-1, while during winter it was 9x108±2.51x109 cfu mL-1. This might be due to the fact that high temperature during summer enhances the growth and multiplication of bacteria. This agreed with Aleksieva and Krusher (1981) who stated that microbial contamination correlated strongly with the season and the highest numbers were noted during the warm months. The high bacterial counts for raw milk were expected under tropical condition like Sudan due to the fact that high temperature enhances growth and multiplication of bacteria (Barakat, 1995). Moreover, Dirar (1975) stated that under tropical condition many factors such as high temperature, absence of sanitary conditions for production of milk in the dairy farms and unavailability of cooling during handling and transportation of milk affect the quality of milk. When the milk samples collect from different cities and areas were compared it was observed that there were significant differences (p≤0.01), which indicated that the quality of market milk differ from city to another (Table 4). The total mean bacterial count of market milk collected from the different cities was found to be 5.63x109±2.87x1010 cfu mL-1 (Table 2). This was agreed with Khan et al. (2002) who found that the total viable count in raw milk ranged between 1.59x1010 to 2.59x1011 cfu mL-1. Also it was agreed with Mahari and Gashe (1990) who found that the total viable count was ranged between 4x107 and 1x109 cfu mL-1. Similarly Godefay and Molla (2000) found the mean total aerobic plate count in milk upon arrival at the processing plant was 1.9x108 cfu mL-1. However in Germany Jung (2002) stated that in recent years there has been a reduction in the bacterial content in raw milk due to improvement in milk hygiene. However there has been a significant increase in the milk content of cold tolerant bacteria in the last 10 years, due to an increase in the duration of milk storage and long distance transport of milk as he reported.

It was observed that the milk samples collected from Khartoum North had relatively high mean count (1.6x1010±4.84x1010 cfu mL-1) than those of Khartoum (5.9x108±1.28x109 cfu mL-1) and Omdurman (2.6x108±8.51x108 cfu mL-1). This might be attributed to the fact that 27 raw milk samples from Khartoum North were phosphatase positive compared to 8 and 18 were positive in Khartoum and Omdurman, respectively (Table 1). Also sanitary conditions for production of milk in the farm, unsanitary conditions associated with handling of the samples within supermarkets and inadequate cooling during transportation and storage of milk might be some factors, as indicated by the questionnaire from the marketing centers (not shown data).

Table 1: Incidences and frequencies of microorganisms in milk samples collected from different supermarkets in Khartoum State
Image for - Evaluation of the Hygienic Quality of Market Milk of Khartoum State (Sudan)

The rate of isolation of spore forming bacteria was 32.5% in the three cities (Table 1) with the total mean of 4.81x106±1.4x107 cfu mL-1 (Table 2). This agreed with Shamanova et al. (1998) who found that all samples contained aerobic spore forming bacteria with the average value of 9.0±0.4x102 cfu cm-3. Similarly Hahn et al. (1999) reported that B. cereus was detected in 8.1% of the samples. The presence of these organisms in raw milk are often related to poor farming practices for example feeding cows with poor quality silage and inadequate cleaning system (IDF, 1994). Also Giffel et al. (2002) reported that silage is considered to be a significant source of contamination of raw milk with spores. The present study showed that there were non significant differences of spore forming bacteria of milk samples collected from different cities, areas and seasons (Table 4). It was also observed that the milk collected from Khartoum North had the highest rate and mean of spore forming bacteria (76.92% and 6.23x106±1.58x107 cfu mL-1) than that collected from Khartoum (7.69% and 3.08x105±1.13x105 cfu mL-1) and Omdurman (15.39% and 5.51x103±5.2x103 cfu mL-1) as shown in Table 2. During summer the total mean of spore forming bacteria in market milk was found to be 7.15x106±1.79x107 cfu mL-1 and during winter it was 1.45x106±3.0x106 cfu mL-1 (Table 2). Also during both seasons the raw milk samples collected from Khartoum North had the highest mean count than that collected from Omdurman and Khartoum.

The total rate of isolation of coliform bacteria was found to be 82.5% (Table 1) and the mean count was 3.32x106±1.43x107 cfu mL-1. These results were agreed with Al-Taraz et al. (2003) who detected the coliform in 142 samples of 160 milk samples and the total coliforms ranged from 2.5x104 to 1.4x106 cfu mL-1. Similarly Aleksieva and Krusher (1981) found that in about 27% of the samples the coliform count was up to 1.4x104 cfu mL-1 and in about 42% it was about 1x105 cfu cm-3. However, these results were higher than those of Hassan and Al-Sanjary (1999) who found that the mean fecal coliform counts was 2.1x104 cfu mL-1. Similarly Godefay and Molla (2000) found the mean coliform count was 7.1x104 cfu mL-1 upon arrival at the processing plant. Also, Jayarao and Wang (1999) detected coliforms in 62.3% of the bulk milk samples with mean count of 3.4 log. 10 cfu mL-1. However Murphy et al. (2001) stated that coliforms are found in fecal and bedding materials and poorly cleaned milk handling and storage equipment. It was observed that when the means of the milk were compared there were non significant differences (Table 4), this might indicated that there was no different in coliforms numbers in the different cities and areas.


Table 2: Some microbiological quality of milk samples collected from different cities in Khartoum State during summer and winter seasons
Image for - Evaluation of the Hygienic Quality of Market Milk of Khartoum State (Sudan)
M = Mean, SD = Standard Deviation

Moreover, it was observed that the milk samples collected from Khartoum North had the highest mean coliform count (6.89x106±2.52x107 cfu mL-1) than that collected from Khartoum (2.39x106±3.69x106 cfu mL-1) and Omdurman (7.98x105±1.16x106 cfu mL-1) as shown in Table 2. During summer the mean count was 5.15x106±2x107 cfu mL-1 and during winter it was 1.45x106±3x106 cfu mL-1 (Table 4). It was found that during summer the mean was high than during winter. This might be due to the higher temperature, which increases the growth of coliform bacteria (Aleksieva and Krusher, 1981).

There were only 3 isolates of E. coli, which were found in the raw milk collected from Khartoum North during summer (Table 1) and the mean total count was 6x105±7.94x105 cfu mL-1 (Table 2). The presence of E. coli in raw milk indicated fecal contamination and/or poor hygienic practices (IDF, 1994). This result is agreed with Haj Mahmoud (2002) who found the percentage of E. coli in raw milk to be 7.2%. Similarly Ahmed (1995) found that the average rate of isolation of E. coli was 15.95%. On the other hand Deutz et al. (1999) examined a total of 133 cow bulk milk samples for the presence of E. coli O157 and they found that only 1.5% of the milk samples contained E. coli O.157.

The rate of isolation of Staphylococcus aureus was found to be 46.7% in the different cities of Khartoum State (Table 1) with total mean count of 4.94x104±1.29x106 cfu mL-1. It is probable that the high incidence and high counts of S. aureus was favored by lack of hygiene during milking and also unavailability of cooling during transportation and storage of milk (Asperger, 1994). He also reported that nasal areas, hands and skin of human are recognized as sites of contamination. It is observed during collection of the milk samples that some employees in supermarkets practiced bad habits during selling such as snuffing and taking. Also mastitic milk could be the source of this organism (Mohamed et al., 1997).

Table 3: Incidences and load of S. aureus and E. coli counts of milk samples collected from different cities in Khartoum State during summer and winter seasons
Image for - Evaluation of the Hygienic Quality of Market Milk of Khartoum State (Sudan)
M = Mean, SD = Standard Deviation

Table 4: Comparison of microbial content of milk samples collected from different areas and cities of Khartoum State using one way ANOVA analysis
Image for - Evaluation of the Hygienic Quality of Market Milk of Khartoum State (Sudan)
NS = Non Significant (p>0.05), * = Significant at p≤0.05, ** = Significant at p≤0.01, *** = Significant at p≤0.001

Inadequate refrigeration and long storage before use are some factors which enhances the presence of S. aureus (Asperger, 1994). This high mean counts of S. aureus was agreed with Al-Taraz et al. (2003) who found Staphylococcus were detected in 80% of the milk samples with the count ranging from 1x103 to 1.3x106 cfu mL-1. Similarly, Khan et al. (2002) found that mean Staphylococcus count was 4.7x108 cfu/100 mL of raw milk. Also Shamanova et al. (1998) found the average count of coagulase positive staphylococcus was 6.0±0.2x102 cfu cm-3.

It was found that the highest rate of isolation was found in raw milk collected from Khartoum North (58.93%) than that of Khartoum (8.93%) and Omdurman (32.14%). Comparison of the means of the milk samples collected from the different cities and areas (Table 4) revealed significant differences (p≤0.001). Moreover, the milk collected from Khartoum had the highest mean count (3.25x106±3.4x106 cfu mL-1) than those of Omdurman (2.63x105±3.38x105 cfu mL-1) and Khartoum North (2.02x105±3.10x105 cfu mL-1) as shown in Table 3. Higher total mean S. aureus count was obtained during summer (7.44x105±1.66x106 cfu mL-1) for the milk samples collected from the three cities compared to that collected during winter (1.61x105±2.3x105 cfu mL-1) as shown in Table 3.

Results of the present study revealed that 44.17% of the milk samples showed ring test positive reactors for Brucella (Table 1). Moreover, this indicated that nearly half of market milk is contaminated with Brucella spp. It was observed that the milk collected from Khartoum North had the highest percentage of Brucella (50.94%). It was also observed that any positive phosphtase test is Brucella ring test positive. This is indicated that some of the Brucella negative sample might be due to the effect of heating milk as the phosphatase enzyme was destroyed by heating. The detection of Brucella antibodies in the milk samples might be due to excretion of the antibodies by the infected or carrier cows (Ahmed, 1995). This result agreed with Haj Mahmoud (2002) who found 45.9% of the samples showed milk ring test positive reactors. Moreover, Ibrahim (1973) found that nearly all herds around Khartoum city were reactors. Suliman (1987) found 15.2% of the samples were positive for Brucella by the milk ring test. Beloti et al. (1999) found 4.76% of the samples were positive in the Brucella ring test. Alves et al. (2001) found four positive samples and 6 suspicious samples of 79 milk samples sold in Brazil. Similarly Brucella positive isolation from milk have been reported in Turkey (Iihan et al., 1999), in India (Hussein et al., 2000), in Egypt (Abdel-Hakiem, 1999) and Oman (El-Amin et al., 2001).

In all samples Salmonella spp. were not found and this might be due to the fact that Salmonellae are rarely shed into the milk and natural infection of bovine mammary gland is possible but relatively rare (Vlaemynck, 1994). He also stated that contamination of raw milk with Salmonella usually takes place from external sources. This result is agreed with Deutz et al. (1999), Shamanova et al. (1998) and Stephan and Buhler (2002) who didn't find Salmonella in their surveys.

The present study concluded that the hygienic quality of milk is low and it supported Akasha and Al-Jibori (2003) who recommended that milking should be done under hygienic conditions and milk should be cooled immediately after milking and should be heat treated to control bacteriological quality. Hence milk must be produced, distributed, handled and marketed under the control of milk commission and the commission must have a sanitary inspector and veterinarian to enforce its methods and standards. Employees in farms and shops should be inspected at periodical intervals and they must be free from communicable diseases. Establishment and applications of Sudanese standards and grades for marketing milk and pricing structure should include the grade and the quality of milk. Further studies are needed for detection of toxins that produced by S. aureus, E. coli and spore forming bacteria (Bacillus spp. and Clostridium spp.) and other harmful microorganisms.

ACKNOWLEDGMENT

The author would like to acknowledged the financial support of the Islamic Development Bank (ISDB), Jadah Sudia Arabia with thank.

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