MATERIALS AND METHODS

Samples Collection
A total of 90 samples, 30 each of raw cows milk directly post-milking (250 mL), potable water (100 mL) and cow`s manure (50 g) were aseptically collected from different dairy farms at Sharkia provinces, Egypt during winter season. In addition to, 100 swabs (2x5 cm) were collected from hands of milker, dairy equipments and surface of udder and teats (25 each). All Samples were put in sterile bag and kept in ice-bag whereas directly transferred to food hygiene laboratory, Suez Canal University for microbiological analysis.

Sample Preparation
Milk samples divided into two equal parts (125 mL each), one part used for activation of Lactoperoxidase System (LPS) and other kept as control. The samples were prepared according to the methods of by Gennari and Dragotto (1992) as follows: the original dilution 1:10 dilution from the collected samples was prepared by adding 25 mL of milk or water sample to 225 mL of 0.1% sterile buffer peptone water (Oxoid, UK) in sterile stomacher bags of approximately 500 mL capacity. Meanwhile, 5 g of manure samples was added to 45 mL of sterile solution of 0.85% NaCl and 1% tryptone (Difco, UK), then, samples were blended in a Seward Stomacher (400^R/UK) for 2 min. On the other hand, each swab was put in sterile test tube containing 9 mL of 0.1% sterile buffer peptone water (Oxoid, UK). One milliliter of the original dilution was transferred serially into sterile test tubes containing 9 mL of 0.1% sterile peptone water to obtain a final dilution of 10⁷.

Activation of Raw Milk Lactoperoxidase System
Activation of LP was carried out within 2 h after milking according to International Dairy Federation (IDF, 1988) by adding sodium thiocyanate (14 mg L^-1) to the milk samples. Then, plunging milk for about one minute followed by sodium bicarbonate (30 mg L^-1). The milk sample was then stirred for another 2-3 min. The enzymatic reactions were completed in the milk within 5 min after addition of H₂O₂ donor. Another same volume of raw cow`s milk samples were kept as control. Both samples were kept at room temperature 25 ± 1 °C and prepared for microbiological evaluation as former then examined after 0, 2, 4, 6, 8 and 10 h post-activation.

Enumeration of Pseudomonas spp.
0.1 mL from each serial dilution and was previously incubated swabs were plated on pseudomonas selective agar base plates containing CFC supplement (Oxide, cod. SR 102 E) then, incubated for 48 h of at 30 °C ± 1. All colonies growing on the plates were counted and the total numbers of pseudomonas count were expressed as cell forming unit (cfu) mL^-1 for milk and water samples, gram for manure samples and cm² for swabs.

Identification and Characterization of Isolates
The different colonies were selected from each plate and after purification, they streaked into Tryptose Soya Agar (TSA) slant and incubated for 24 h at 30 °C. Identification of isolates was carried out on bases of morphological, cultural and biochemical characteristics as described by Krieg and Holt (1984) and Kwan and Skura (1985).

RESULTS AND DISCUSSION

Incidence of Pseudomonas spp.
Microbial contaminations become critical for raw milk especially from the time between milking until reach to consumers. Pseudomonadaceae are among the most important spoilage bacteria in raw milk which constitutes for up 78% of psychrotrophic microflora (Muir et al., 1979; Vela, 1997). In this study, 114 (60%) out of 190 samples were positives for the presence of pseudomonas, whereas 20 (66.7%) out of 30 milk samples were recorded positive (Table 1). In concerning to contamination sources, higher recorded positive numbers for the prevalence of pseudomonas were obtained from water (100%), followed by dairy equipments (92%), then manure (83.3%) samples. Contamination of raw milk by Pseudomonas is mainly from soil and water (Zaki et al., 1996). Milker, udder surfaces and teat were reported the lower incidence for the presence of pseudomonas that may be due to their drying conditions and the predominance of other skin microflora. Many investigators in other study recorded various results for the incidence of Pseudomonas in raw milk and their pertinences (Eman, 1992; Zaki et al., 1996; Dinsmore et al., 2001).

Pseudomonas can gain access to milk via manure, polluted water, dairy equipments and dairy workers (Khalil, 1992). Such microbes as other psychrotrophes can multiply in milk stone deposits on equipment surfaces during shutdown periods and contaminate milk when started (Shah, 1994). The obtained results confirmed there are no controlled conditions during milk production in dairy farms at Sharkia province, Egypt.

Total Pseudomonas Counts in the Examined Samples
The mean values of total pseudomonas counts in the collected samples from dairy farms at Sharkia provinces, Egypt were shown in Table 2. It is not doubtful that manure samples had shown the highest pseudomonas counts (log_–10 6.22 cfu g^-1) among the evaluated samples. There were a considerable pseudomonas counts in the samples collected from water, udder and teat, hand`s milker and equipments.

The major contamination sources of pseudomonas in raw milk are from improperly cleaned equipments and faeces, soil, water (Erskine et al., 1987). Consequently, the obtained results revealed that the minimum, maximum and mean values of total pseudomonas counts in raw milk samples were 2.14, 6.21 and 3.54 log_–10 cfu mL^-1, respectively. The results obviously reflected the inadequate sanitary and hygienic measures applied during milking processes at those dairy farms. Many investigators at different institutes have recording various results concerning the total pseudomonas counts in raw milk depending on the hygienic statutes of the farms (Ahmed, 1995; Zaki et al., 1996; Desmasures et al., 1997).

Table 1:	Numbers (%) of positive and negative samples for the incidence of Pseudomonas spp.

Table 2:	Log mean counts of pseudomonas in the examined samples from dairy farms
* Counts expressed as log–10 cfu mL-1, ** Counts expressed as log–10 cfu g-1, ~ Counts expressed as log–10 cfu cm-2, *** SE Means Standard Error

Table 3:	Total pseudomonas count (log–10 cfu mL-1) of control and activated milk samples at storage room temperature (25 °C)
*SE with difference letter(s) in the column were significantly difference (p>0.05)

Activation of Milk Lactoperoxidase System (LP)
The activation of milk lactoperoxidase system constitutes an alternative method of milk preservation in rural areas and its values where cooling facilities or chemical preservatives are unavailable. From the conducted study, the total pseudomonas count (log_–10 cfu mL^-1) of control and activated milk samples at storage room temperature (25 °C) were shown in Table 3. There were a significant difference between control and activated samples regarding the pseudomonas counts up to 8 h under room temperature storage. LP activation delayed or even caused complete inhibition of psychrotrophic microorganisms` growth in activated milk samples (Sondhi et al., 1992; Wolfson and Sumner, 1993). In addition, LP activation has a bactericidal effect against pseudomonas (Marks et al., 2001).

CONCLUSION

It is clear that manure water, dairy equipments and hand`s milker were the most contaminated sites for pseudomonas. Enumeration of pseudomonas in raw milk gives a good evaluation of the farm hygiene. Activation of milk LP within 2-3 h after milking by using sodium thiocyanate (14 g L^-1) and sodium bicarbonate (30 mg L^-1) could be used for controlling the pseudomonas growth in the raw milk. Thus, improving the keeping quality of raw milk during transportation until suitable method of storage is applied by consumers or the factory.