30 Journal of Applied and Industrial Sciences, 2013, 1 (2): 30-37, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE )

Research Article

Some Bacterial and Physical Quality of Pasteurized Milk in Khartoum

Adil, M. A. Salman *1 and Eltaf, M. Hagar2
1

Department of Preventive medicine, College of Veterinary Medicine, University of Bahri 2 Preimer Company for dairy processing- Khartoum Sudan (Received: March 19, 2013; Accepted: May 29, 2013)

Abstract- This study was designed to monitor some bacteriological and physical quality of milk from two dairy factories before and after pasteurization, and also to assess the compliance of this quality detected with the standard set by the Sudanese Standards and Meteorology Organization (SSMO). Two hundred and Twenty raw and pasteurized milk samples were collected from dairy factories and sale points in Khartoum State. The average total bacterial counts before pasteurization was1105-9106 cfu/ml and after pasteurization it was 0-9104 cfu/ml. Most of the market samples in day 1,3 and day 6 showed range of 0-1104 cfu/ml. Staphylococci counts before pasteurization were found in range of less than 1104 cfu/ml and after pasteurization were in the range of 0-910 cfu/ml. The Coliform counts before pasteurization were in the range of 110 3-9103 cfu/ml and immediately after pasteurization the range was reduced to 51102 cfu/ml. E.coli was identified from24%of the samples before pasteurization. Before pasteurization acidity was found to be 0.17%, directly after pasteurization the acidity was 0.15-0.16% but it was 0.16-0.17% in market samples.

produced with a low bacterial count the temperature must be kept low until the point of processing ] 7]. Contaminations of raw milk and the consequent high bacterial count in milk originates from milking wet dirty udders, the milking system used, the cooling and storage temperature and the holding time ]8[The bacterial count is a useful method to measure milk quality, a bacterial count ranging between 9105 - 9106 cfu/ml is acceptable [ 9] , and the mean standard plate count of raw milk is 1.2910 6 cfu/ml , [10] but when milk was pasteurized it was reduced to 1.2104 cfu/ml. Grade A milk has a count less than 1105 cfu/ml and grade B milk has count with less than 3105 cfu/ml [11] , but the majority of pasteurized milk sample has a count of 110 3 cfu /ml. [12 ].

Milk is often contaminated by Escherichia coli under lack of sanitary conditions which can affect public health. The coliform count is related to the unsanitary milking process and INDEX TERMS: Acidity, Bacteria, Pasteurized milk, Khartoum, dirty cow`s environment [13]. E. Coli was isolated from 32% Sudan. of raw fresh milk with Coliform count of less than 100 cell/ml I. INTRODUCTION in Khartoum state [14]. Many studies indicated that the ilk is a nutritious food for human beings. It also serves as production of high quality liquid milk is dependent on the a good medium for the growth of many microorganisms microbiological quality of raw material. The spoilage of such as Staphylococcus and Coliform. Bacterial processed milk is primarily due to bacterial activity, presence contamination of raw milk can originate from different and activity of post-pasteurization contaminations and types sources, including low quality raw milk, improper refrigeration and activity of pasteurization resistant micro-organisms which and an inadequate packaging system [1,4]. Raw milk are the main limiting factors in extending the shelf life of high deteriorates in few days and pasteurized milk with high temperature short time (HTST) pasteurize milk [15,17].In temperature short time system (HTST) has a shelf life of about addition other factors which limit the shelf life of refrigerated pasteurized milk include the time and temperature of seven days ]5[. pasteurization, and storage temperature of milk after Measurement of bacterial numbers in milk is of interest pasteurization [2,18 ]. because they are indicator of poor milk hygiene production or In the Sudan milk and milk products play an important role ineffective pasteurization of milk. Some microbes such as gram negative Psychrotrophs, Coliforms and other pathogenic in the family diet. Most of the milk produced in the Sudan is bacteria such as Escherichia Coli ,Staphylococcus aureus may consumed as raw in both rural and urban areas, while the rest also be found in milk ]6[.The hygienic quality of milk at the is processed into milk products in urban areas [9] . Therefore, point of production is also of importance from both public certain requirements should be fulfilled in order to produce health and consumer perception points of view. For milk to be safe and clean milk.

*Corresponding author: Adil Salman E-mail: adilsal4@yahoo.com

31 Journal of Applied and Industrial Sciences, 2013, 1 (2): 30-37, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE ) These requirements include: clean milking and cooling of milk immediately after transference of milk to processing in ideal plants with stick quality control. Handling of raw milk is a big problem in the Sudan as the dairy farms are located in different remote areas with minimum infra-structure in addition to the prevailing hot climate of the country with lack of cooling and transportation facilities, appreciable amount of milk is spoiled even while in transit. The main objective of this study is to estimate acidity and bacterial quality of 1- raw milk intended for pasteurization. 2- pasteurized milk immediately after processing in the factory 3- pasteurized market milk at different time interval . II. MATERIAL & METHODS Milk samples were collected from two factories and different sale points in Khartoum State- Sudan. The factories are located in Khartoum North, while market samples were collected from the three municipalities Khartoum, Khartoum North and Omdurman. Sample collection A total of 220 milk samples were collected from two factories (A &B). Factory A has its own dairy farms and use their own milk for processing, while factory B purchases milk from local market. Market samples were collected at three different levels in different batches as follows: 1- Fresh milk samples: Fifty samples were collected from raw fresh milk supplied to factory A and B in clean and sterile bottles. 2- After pasteurization samples: Fifty samples from the milk tanks of two factories A and B were collected immediately after pasteurization in clean and sterile bottles. 3- The market samples (shelf life): one hundred twenty samples were collected from pasteurized milk produced by the two dairy factories from different sales points in Khartoum state at days 1, 3and 6. Laboratory tests 1- Acidity: The test was performed according to the method described by [19] 2-Bacteriological analysis a) The Total bacterial count (T.B.C) was performed according to[20 ] b)The Coli form Count and E.Coli identification were carried out using Violet Red Bile agar (VRB) and Eosin Methylene Blue Agar (EMB agar) [21]. c)The Staphylococci count was done was done using the Baird Parker Agar [21 ] . Statistical analysis SPSS 12 analysis packages and One-Sample t test was used for comparison of milk samples III. RESULTS Most milk samples of both factory A (88%) and B (87.5%) showed a range of 1105-9106cell/ml of total bacteria count before pasteurization, 80% of the samples from factory A showed a range of more than 1107 cell/ml while 12.5% in factory B showed a range of 0-9x104cell/ml (Fig. 1a and Fig. 1b).

After pasteurization 96% of the samples were in the range of 09x104cell/ml. in factory A and 43.5% were in the same range in factory B, while a range of 1x105 -9x105 was 56.5% in factory B. (Fig.1a and Fig.1b). Statistically, the difference in Total Bacterial Count between factory A and B was significant for both factories before and after pasteurization at P<0.05 (Table 1).

32 Journal of Applied and Industrial Sciences, 2013, 1 (2): 30-37, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE ) Table (1). One-Sample Statistics (One-Sample Test) of Total Bacterial Count For milk samples from factory A&B before and after pasteurization. Mea Std. n Deviation Std. Error Mean 2.04 .35 7.02E-02 1.88 .34 6.90E-02 1.04 .20 4.00E-02 1.57 .51 .11

Before Pasteurization A Before Pasteurization B After Pasteurization A After Pasteurization B

N 25 25 25 25

Test Value = 0 95% Confidence Interval of the Difference T 29.04 27.19 26.00 14.81 Sig Mean Df (2-tailed) Difference 24 .000 2.04 23 .000 1.88 24 .000 1.04 24 .000 1.57 Lower 1.90 1.73 .96 1.35 Upper 2.18 2.02 1.12 1.78

Before Pasteurization A Before Pasteurization B After Pasteurization A After Pasteurization B

The difference is significant in the bacterial count between the two factories before and after pasteurization at P<0.05 In sales point samples obtained from factory A 100%, 95% and 90% of the samples were in range 0-9104 cfu in day 1, 3 and day 6 respectively (Fig. 2a).While in sales point samples obtained from factory B 70%, 80% and 60% of T.B.C was found in the range of 0 - 9X104 cfu in day 1, 3 and 6 respectively. Only 10% of samples collected in day 6 showed a range between 110 59106 cfu from factory A (Fig.2a). The difference was also significant for TBC between day 1 and day 6 in factory A at P<0.05. (Table 2). Table (2): One-Sample Test of factory A for sale points in day 1 and day 6.

Test Value = 0 95% Confidence Interval of the Difference T 12.365 15.280 11.059 df 19 18 17 Sig. (2-tailed) .000 .013 .000 Mean Difference 1.30 1.11 2.39 Lower 1.08 .95 1.93 Upper 1.52 1.26 2.84

TBC coliform Staphylococci

The difference is significant in the bacterial count between day 1 and 6 in factory A after pasteurization, at 95% confidence interval. In factory A and factory B, 80% and 52% of the samples showed a Coliform count in the range of 1103 cfu before pasteurization. After pasteurization 80% and 40%of the samples in factory A and B were in the range of 1-102 cfu. (Fig 2a and Fig.2b).

33 Journal of Applied and Industrial Sciences, 2013, 1 (2): 30-37, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE )

In day 1, day3, and day 6, 50%, 40% and 20% were in the range of 1-102 cfu in factory A, and 40%, 50% and 20% samples from factory B respectively (Fig 2a and Fig. 2b). The difference in Coliform counts between day 1 and day 6 was statistically significant (Table 2) E. Coli were detected from 24%and 12% of Coliform positive milk samples from factory A and factory B respectively before pasteurization. In factory A and factory B 15% of the Coliform positive samples at the sales point in day 6 were E. Coli positive, but in day 3, 15% of the samples from factory B at the sales point were also positive (Tab Table (3). E.coli detection from coliform positive milk samples.

E.Coli test A Before pasteurization B A After pasteurization B A Day3 Day6 A

+ve 6 24% 3 12% 0

-ve 19 76% 22 88% 25 100%

Total number of Samples 25

25 25

0 0 3 15%

25 100% 20 80% 17 85% 17 85% 17 85%

25 20 20

Day3

3 15%

20

Day6

3 15%

20

34 Journal of Applied and Industrial Sciences, 2013, 1 (2): 30-37, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE ) The range of Staphylococci counts in all milk samples before pasteurization in both factories A and B were more than1104 cfu in about 76% and 88% in the two factories respectively. After pasteurization the counts were 0 - 910 cfu in about 72% and 100% in the two factories respectively. In day 3 almost all sales point samples from factory B showed a range of 0 - 9X10 cfu/ml while 61% of the samples were in the range of 1102 -9103 cfu/ml in the same factory in day 6 (Fig 3).

As for factory A the count of Staphylococci of range of 0 910 cfu was found to be 85%,50% and 40% in day 1,3 and 6, respectively , and the range of 1 102 -9103 cfu/ml was 10.5%,10.5% and 61.1% in days 1,3 and 6 respectively, statistically there was a significant difference in Staphylococci

count between day 1 and 6 in factory A at 95% confidence interval, (Table 2). At the sale points the acidity within the range of 0.14%-0.18%, were 40% and 20% in factory A and factory B in day six. (Table 4)

Acidity Range

Factory A

Factory B

Before pasteurization 15%- 18% 19% 20%- 21% Total 15(60%) 7 (28%) 3 (12%) 25

After pasteurization 20 (80%) 3(12%) 2 (8%) 25

Before pasteurization 14 (56%) 7 (28%) 4 (16%) 25

After pasteurization 20 (80%) 00 5 (20%) 25

Table (4): Acidity of milk samples of factory A&B before and after Pasteurization Table (5) Acidity range at sale points Acidity range Day one 15 (75%) 14%-18% 19%-21% Total 5 (25%) 20 Factory A Day Three 16 (80%) 4 (20%) 20 Day Six 8 (40%) 12 (60%) 20 Day One 18 (90%) 2 (10%) 20 Factory B Day Three 10 (50%) 10 (50%) 20 Day Six 4 (20%) 16 (80%) 20

35 Journal of Applied and Industrial Sciences, 2013, 1 (2): 30-37, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE ) IV. DISCUSSION Milk processing was practiced since the early sixties in Sudan by the government sector but still most of the milk is consumed as raw. Recently the private sector contributed to this business by establishing many milk plants in Khartoum state. In this study a range of 1105-9106 cfu/ml TBC for raw milk was found to be 87.5%, and 88% in factory B and A respectively. This range was almost in agreement with the range required by the Sudanese Standards and Metrology Organization, ]22[ for raw milk which was 5X1051X106cfu/ml. The range is comparable to that obtained by similar studies where a range of 1106 -9106 cfu/ml in Khartoum State was reported ]9,23[. The acceptable limit of TBC of the European Union ]24[ of raw milk was reported to be 1x104 cfu/ml . The findings in this study were comparable to that of ]25[ who reported a range of 4105cfu/ml. High bacterial count is expected under tropical conditions such as the Sudan due to the fact that high temperature enhances growth and multiplication of bacteria in addition to absence of sanitary conditions and unavailability of cooling facilities during handling and transportation of milk ]9[. In pasteurized milk the total bacterial count of less than or equal to 9104cells/ml was the lowest range, 43.5% and 96% of the samples in factory B and A were in this range respectively. This range was in compliance with what was recommended by the Sudanese Standards and Metrology Organization, ]22[ for pasteurized milk which was 1104 9104 cfu /ml, and higher than was reported by the European Union which was 3x104. The data obtained during this study showed that 56.4% of the pasteurized milk samples were in the range of 1105-9106 cfu /ml in factory B while it was 4% in factory A. This higher count might be due to higher TBC count of the raw milk used in factory B, or recontamination from equipment during and after pasteurization. The findings in this study were in agreement to that reported by ]26[ who reported that about 70% of the pasteurized milk samples tested had aerobic bacteria count exceeding the regulatory limit of 2104cfu/ml for grade A pasteurized milk set by the United States Food and Drug Administration (FDA) PMO ]27[ . Statistically the difference in the count before and after pasteurization was significant, a high count of TBC before pasteurization may affect the count of the bacteria after pasteurization. The data obtained from the sales point showed discrepancy between various samples. This may be attributed to inadequate cooling systems at the sale points, improper pasteurization or recontamination after pasteurization. In factory A, 90% of the samples from different batches at day 6 conform with the limits set by SSMO (910 4 cells/ml), while only 20% from factory B were within these limits. Although in factory B, milk was collected from their own farm, in cans, but their cans may not be well washed and disinfected regularly. The load of bacteria in raw milk in transporting cans and residues of milk in tanks is one of the important reasons for recontamination of raw milk and this affect the load of pasteurized milk ] 9[. Also this high total bacteria count might be due to improper pasteurization and contamination of packaging materials. Coliform in milk is one of the best indices for judging sanitation ]28[ .In this study, 76% and 32% of raw milk samples in factory A and B were in the range of 1103-9104 respectively. This range is higher than that reported by American Public Health Association, less than 102]29[. The higher Coliform count in raw milk used in factory B may be due to the unsatisfactory milking practices in the farm from which the milk was collected. A similar count of 9103 cfu /ml in Khartoum state was reported ]14,30[.In Jordan a higher range of 2.5104-1.4106 was reported ]31[. In this study lower TBC value was obtained for pasteurized milk compared to what reported by]32[ who showed a range of 6.5105 to 6.51014 cfu. The official limits set by the SSMO for pasteurized milk for Coliform bacteria was 1x5 -1x102 cfu/ml ]22[ . The result of this study showed that, 80%of the samples from factory A and 40% from factory B conform with this limits. The results were in agreement with[ 27[who suggested a range of less than 102 cfu/ml. in Algeria [ 33[ stated that (31.5 %) and (6.5 %) samples of milk from the two sources of samples at sales point were not in compliance with the acceptability threshold fixed at 10 cfu/ml. Coliform bacteria counts of pasteurized milk showed lower numbers than these reported by ]32[ . The lower Coliform counts might be due to hygienic quality of raw milk, proper pasteurization process, good packaging and good storage conditions. This agreed with] 27[who reported that the total bacterial standards for grade A pasteurized milk should be < 10 Coliform/ ml. In this study, at the sale points samples of pasteurized milk, 50%, 40%,49% and 20% of samples from factory A comply with the official standards. Towards the end of the validity, both factories showed a lower compliance (20%). This might be due to poor cooling efficiency at these points and variations in samples that were collected from different batches. Several workers isolated E.coli from milk and stated that it might cause a potential risk particularly for children ]34,37[. However, in this study, before pasteurization 24% of the samples from factory A and 12% from B were positive , comparable to what was reported by ]14[ who reported about 32% of the raw bulk milk were E. coli positive in Khartoum state. After pasteurization there was no growth of E.coli in the samples obtained from both factories. The sale points samples of factory A showed no E coli growth at day 3 while 15% of the samples were positive in day 3 in factory B, but in day 6 both factory A and B showed positive E.coli results (towards the end of shelf life).

36 Journal of Applied and Industrial Sciences, 2013, 1 (2): 30-37, ISSN: 2328-4595 (PRINT), ISSN: 2328-4609 (ONLINE ) This result was higher than what was reported by SSMO for pasteurized milk of E.coli ]22[ which was Zero. In factory B the early detection of E.coli may indicate flocculation in the pasteurization process, since the samples were from different batches. In this study 76% and 88% of raw milk samples from factory A and B had a Staphylococci count of more than 1104 . These results are higher than those reported by ]38[ and ]39[ which were 45% and 68%,respectively. All these counts are beyond bacterial safety limit recommended by ]40[ not to exceed 103. After pasteurization 72% and 100% of the samples form factory A and B had a count of 0-910. Several studies showed a wide range of bacterial count,] 33[ found that 20% of the samples had a count of more than 10cfu/m. Laszlo ]12[reported the count of more than 103 cfu/ml . While ]39[ found that 30% of the samples were contaminated with Staphylococcus aureus. The Staphylococci can contaminate milk after milking or during processing ]9[ . Raw milk acidity reported in this study was in the range of 0.15-0.18% lactic acid in 60% and 56% of samples from factory A and B respectively. Which was better than what was reported by ]9[ who reported acidity of less than 0.20% in raw milk. It was also lower than that obtained by ]41[ who reported raw milk acidity between 0.18-0.2 % lactic acid, but in agreement with the result reported by ]42[ who reported an acidity range of 0.14 -0.18%, and they reported that a high acidity implies a high lactic acid content which, in turn, implies a high bacteria count in the milk. But after pasteurization, the acidity was in the range of 0.15-0.18% in 80% of the samples in both factories A and B which is in accordance with SSMO standard ]22[ . These findings are comparable to what is reported by ]23[ who showed that the mean acidity of pasteurized milk was 0.143 in Sudan and it is lower than 0.219% reported by ]43[ . The decrease of acidity after pasteurization was also reported by ]44[. In sale points pasteurized milk samples the acidity was stable till day 3, but later on day 6 (towards the end of shelf life ) only 40% and 20% of the samples in factories A and B conform with SSMO standards. Higher acidity reported here may be due to lack of cooling facilities during transportation of raw milk or improper storage. V. CONCLUSION 1. The quality of pasteurized milk was not stable, different batches were variable in their quality. 2. Albeit the good hygienic and controlled farm resulted in acceptable bacterial quality of pasteurized milk in factory A, the market samples showed unacceptable limits of bacteria. 3. Modern dairy farming coupled with efficient milk plants are of importance for production of safe, clean and wholesome milk.
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