الفهرس 
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Abstract
Thirty random samples of each raw milk, fresh cream and yoghurt were collected from different dairy farms, groceries, supermarkets and dairy shops and examined for enumeration of psychrotrophs by two methods; standard plate count method (SM) at 7°C for 10 days and modified rapid count method (MM) at 32°C for 2 days.
Gram-negative psychrotrophs were isolated and identified and their proteolytic and lipolytic activities were determined on specific growth media under different degrees of temperature with periodical observations up to 14 days.
Pseudornonas fluorescein; Aeromonas hydrophila subspecies hydrophila and Alcaligenes faecalis were selected as they constituted the highest frequency distribution within the isolated strains and the highest incidence within the examined samples. Their proteolytic and lipolytic activities on specific growth media were observed and the properties of their extracellular lipases were determined. Resistance of their lipases to different industrial thermal treatments was determined in the laboratory.
Due to lake of information about the role of Serratia grinzisii, which was isolated from raw milk, as a lipolytic Gram-negative psychrotroph, it was chosen to undergo some investigations on its lipolytic activity and purification and partial characterization of its extracellular lipase. Its crude lipase was purified to homogeneity by ion exchange chromatography and gel filtration by using CM-cellulose, DEAE-cellulose and Sephadex G-150. Finally, its purity, as well as, its molecular mass was estimated by one-dimensional discontinuous gel electrophoresis (Lammeli gel method) on a 10 % polyacrylamide gel containing 0.1 % SDS with staking gel. The study concerned with the proteolytic and lipolytic activities of the isolated Gram-negative psychrotrophs showed that the percentage of The study concerned with the proteolytic and lipolytic activities of the isolated Gram-negative psychrotrophs showed that the percentage of lipolytic strains in the total number of the strains was considered high (75.43%) and seemed to be equal to the percentage of proteolytic strains (75.86%). Considerably higher percentage of lipolytic strains (32.76%) can produce lipolytic activity at 6°C, while very low percentage (1.72%) of proteolytic strains was found and all of them isolated originally from yoghurt samples.
Results obtained from investigation of the effect of time and temperature on the beginning of the activity declared that at 6°C low percentage the strains could begin their proteolytic activity after 8 days, on the other hand, some strain showed their lipolytic activity after 0.5 day, while the highest percentage of the strains began their activity after 1.5 day. It is suggested that the efficient control of low storage temperature up to 8 days could control the proteolytic activity. The lipolytic strains seemed to be faster and began their activity under low temperature after very short time.
Observations of the beginning of the activity were conducted to detect the maximum activity of the strains. At 6°C, the most active lipolytic strains showed their maximum activity after 2 days, while the proteolytic activity was very weak during the 14 days of incubation.
Observation of the activities of the selected strains on specific growth media showed that the proteolytic activity of Pseudomonas fluorescens and Aeromonas hydrophila spp. hydrophila were high at higher incubated temperature. At 6°C Ps. fluorescens could not show its proteolytic activity, while that of Aer. hydrophila spp.. hydrophila was very weak and appears after 7 days of incubation. Alcaligenes faecalis could not show its proteolytic activity during the 14 days of incubation under different degrees of temperature. Protease production by the selected strains could be controlled by efficient cold storage up to 7 days.
The lipolytic activity of Ps. fluorescens appeared after 6 hours with large discoloration zone of activity, at 6°C the activity began weakly after 12 hours and reached its maximum activity after 7 days. Aer. hydrophila spp. hydrophila showed that at 30°C the lipolytic activity of the organism appeared after 12 hours and the organism could produce its lipase at 6°C after 1 day. Alcaligenes faecalis produced its lipase at 30°C after 6 hours with considerable large discoloration zone of activity, while at 6°C the activity begins very weak after 5 days. Lipase production by Alcaligenes faecalis could be controlled by efficient cold storage up to 5 days.
Determination of the properties of the extracellular lipases of the selected strains revealed an optimum substrate concentration of 2% tributyrin in the substrate emulsion with an optimum reaction time ranged from 8 to 10 minutes. Optimum pH was 8 for lipase of Ps. fluorescens and 8.5 for lipases of Aer. hydrophila spp. hydrophila and Alc. faecalis. The pH/activity curve declared that the lipases of Ps. fluorescens, Aer. hydrophila spp. hydrophila and Alc. faecalis, respectively had 63.63, 23.4 and 3.8% of their maximum activity at pH 6.6 (pH of milk). Lipases of Ps. fluorescens and Alc. faecalis showed high stability over a pH range 8-9. Under pH 6.6 these lipases could not resist holding at 4°C for 48 hours Lipase of Aer. hydrophila spp. Some of the industrial thermal treatments applied in this study could not completely inactivate lipases from the selected strains, however, the UHT l (ultra-high temperature treatments at 140°C for 4 seconds) and UHT2 (150°C for 1 second) succeeded in elimination of these lipases.
Results obtained from purification and partial characterization of Serratia grimisii extracellular lipase revealed that the purified lipase exhibited optimum pH at a range 8 - 9 and the enzyme was very stable over the pH range 7 - 9. The optimum temperature was observed at 37°C, however, it had a high activity at 5°C (80 to 90% of maximum). The molecular mass of the purified lipase was estimated to be 57 kDa. It showed heat lability, so it may be eliminated by efficient heat treatment of milk. At pH 6.6, it showed some activity and stability. Also, the organism can show its lipolytic activity at 6°C after 12 hours. Finally, some recommendations were suggested to control the presence of such microorganisms in milk and dairy products to avoid their undesirable changes that resulted in economical losses, beside the possibility of their public health hazard.