Efficient mechanical disruption of Lactobacillus helveticus, Lactococcus lactis and Propionibacterium freudenreichii by a new high-pressure homogenizer and recovery of intracellular aminotransferase activity.
Microbiological studies often involve bacterial cell fractionation, which is known to be difficult for Gram-positive as compared to Gram-negative bacteria. Our purpose was to test the breaking efficiency of a new high-pressure pilot homogenizer for three Gram-positive species involved in dairy technology and to assess the activity of an intracellular aminotransferase. Varied pressures (50, 100 and 200 MPa) were applied to concentrated bacterial suspensions (1.2 mg dry weight/ml) of Lactobacillus helveticus, Lactococcus lactis and Propionibacterium freudenreichii. Breaking efficiency was estimated by decreases in optical density at 650 nm, cellular dry weight and viability. The proteins released were quantified and the residual intracellular aminotransferase activity was estimated using leucine as substrate. One run at 50 MPa was sufficient to break 80% of lactobacilli cells whereas 200 MPa were required for the same efficiency for L. lactis and P. freudenreichii. Whatever the pressure, leucine aminotransferase activity was recovered in the supernatant after cell breaking. This new high-pressure pilot homogenizer can allow rapid (20 s/run), easy, continuous and highly efficient cell breaking for intracellular enzyme recovery or other purposes. As the species tested were not phylogenetically related, and had different morphologies and cell wall compositions, we conclude that most Gram-positive bacteria may be broken efficiently by this new device.
Saboya
L V
LV
Escola Superior de Agricultura "Luiz de Queiroz" (ESALQ/USP), Av. Padua Dias 11, Caixa Postal 9, SP13418-900, Piracicaba, Brazil.
Maillard
M-B
MB
Lortal
S
S
D001431:Bacteriological Techniques; D002458:Cell Fractionation / Q000295:instrumentation* / Q000379:methods*; D006094:Gram-Positive Bacteria / Q000201:enzymology* / Q000502:physiology*; D007778:Lactobacillus / Q000201:enzymology / Q000502:physiology; D013294:Lactococcus lactis / Q000201:enzymology / Q000502:physiology; D051276:Leucine Transaminase; D011312:Pressure; D011424:Propionibacterium / Q000201:enzymology / Q000502:physiology; D000637:Transaminases / Q000378:metabolism*
D000637:Transaminases; D051276:Leucine Transaminase