This project was conducted to study the activity of secondary metabolites produced by one of a human microflora which is Lactococcus lactis in addition to its cell wall proteins and their effect on tumor cell lines in vitro. The study regarded the following:
First: Twenty samples of dairy products (bucolic sour yoghurts, pasteurized milk and raw milk) were collected, nine isolates of Lactococccus were isolated by propagating in MRS broth medium followed by subjecting the isolates to microscopic, cultural, physiological and biochemical tests which included the ability to ferment carbohydrates sources, catalase and gelatinase production, the ability to produce ammonia from arginine and the ability to grow at (10, 40 and 45°C), 4% NaCl and 0.1% methylene blue, in addition to the ability to grow at different pH (4, 4.5, 9 and 9.5). The results showed the presence of four isolates belonged to the genus Lactococcus sp. lactis.Second: Growing the bacteria in three broth production media, which were MRS, VVM and BHI at 37ºC for 6 hr followed by extraction of crude extracts using three ammonium sulphate saturation ratios which were 20%, 50% and 80% for each medium confidentially. The overall result was nine crude extracts (three from each medium), as well as, growing the bacteria in M17 broth media at 37ºC for 24 hr to extract cell wall proteins using sonication method followed by estimation the concentrations of extracted proteins depending on standard curve of bovine serum albumin. The concentrations of extracts varied from one to another depending on the type of production medium and saturation ratio.Third: Study the cytotoxic activity of prepared concentrations for each crude extract (10, 5, 2.5, 1.25, 0.625 and 0.3125 µg/ml) on two tumor cell lines (AMN-3 and Hep-2) for three incubation periods (24, 48 and 72 h) in addition to normal rabbit embryo fibroblast (REF) cell line for 72 h. only. The result illustrated a clear cytotoxic activity of these crude extracts with high significances on both tumor cell lines during the three incubation periods, suggesting that the cytotoxic effect of these crude extracts is a dose and time dependant, but on REF cell line, there is no significant effect of these crude extracts was reported, suggesting also, that may be the active compound of L. lactis posses some specificity in cytotoxicity on cancer cells but not on normal cells. The reflection coefficient for each of the four crude extracts at each of the two tumor cell lines (AMN-3 and Hep-2) was measured during the incubation period (72) h. and the relation between the concentrations of each crude extract, the intensity of their cytotoxic effect was reversible since the values of the reflection coefficient were negative.
