Casein fermentate of Lactobacillus animalis DPC6134 contains a range of novel propeptide angiotensin-converting enzyme inhibitors.
This work evaluated the angiotensin-converting-enzyme (ACE)-inhibitory activities of a bovine sodium caseinate fermentate generated using the proteolytic capabilities of the porcine small intestinal isolate Lactobacillus animalis DPC6134 (NCIMB deposit 41355). The crude 10-kDa L. animalis DPC6134 fermentate exhibited ACE-inhibitory activity of 85.51% (+/-15%) and had a 50% inhibitory concentration (IC50) of 0.8 mg protein/ml compared to captopril, which had an IC50 value of 0.005 mg/ml. Fractionation of the crude L. animalis DPC6134 fermentate by membrane filtration and reversed-phase high-performance liquid chromatography (HPLC) generated three bioactive fractions from a total of 72 fractions. Fractions 10, 19, and 43 displayed ACE-inhibitory activity percentages of 67.53 (+/-15), 83.71 (+/-19), and 42.36 (+/-11), respectively, where ACE inhibition was determined with 80 microl of the fractions with protein concentrations of 0.5 mg/ml. HPLC and mass spectrometry analysis identified 25 distinct peptide sequences derived from alpha-, beta-, and kappa-caseins. In silico predictions, based on the C-terminal tetrapeptide sequences, suggested that peptide NIPPLTQTPVVVPPFIQ, corresponding to beta-casein f(73-89); peptide IGSENSEKTTMP, corresponding to alpha(s1)-casein f(201212); peptide SQSKVLPVPQ, corresponding to beta-casein f(166-175); peptide MPFPKYPVEP, corresponding to beta-casein f(124133); and peptide EPVLGPVRGPFP, corresponding to beta-casein f(210-221), contained ACE-inhibitory activities. These peptides were chosen for chemical synthesis to confirm the ACE-inhibitory activity of the fractions. Chemically synthesized peptides displayed IC50 values in the range of 92 microM to 790 microM. Additionally, a simulated gastrointestinal digestion confirmed that the ACE-inhibitory 10-kDa L. animalis DPC6134 fermentation was resistant to a cocktail of digestive enzymes found in the gastrointestinal tract.
Hayes
M
M
Teagasc, Biotechnology Centre, Moorepark, Fermoy, County Cork, Ireland.
Stanton
C
C
Slattery
H
H
O'Sullivan
O
O
Hill
C
C
Fitzgerald
G F
GF
Ross
R P
RP
D000595:Amino Acid Sequence; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002216:Captopril / Q000494:pharmacology; D002364:Caseins / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005285:Fermentation; D005374:Filtration; D020128:Inhibitory Concentration 50; D007778:Lactobacillus / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism
D000806:Angiotensin-Converting Enzyme Inhibitors; D002364:Caseins; D010455:Peptides; D002216:Captopril; D007703:Peptidyl-Dipeptidase A