Characterization of the fruit proteolytic system of Bromelia serra Griseb. (Bromeliaceae) and its application in bioactive peptides release.

A crude extract with proteolytic activity was prepared from edible fruits of Bromelia serra, containing cysteine peptidases with molecular masses between 24.1 and 25.9 kDa. The extract presented an optimal pH range of 6.03-9.05, retained more than 80% of activity after thermal pre-treatments at 23, 37, and 45°C (120 min), but it was rapidly inactivated after 10 min at 75°C. These proteases were employed to hydrolyze soybean proteins, bovine casein and bovine whey, achieving degrees of hydrolysis of 18.3 ± 0.6, 29.1 ± 0.7, and 12.6 ± 0.9% (55°C, 180 min), respectively. The casein 180 min-hydrolysate (55°C) presented the maximum value of antioxidant activity (2.89 ± 0.12 mg/mL Trolox), and the whey protein 180 min-hydrolysate (55°C) showed the highest percentage of angiotensin-converting enzyme inhibition (91.9 ± 1.2%). This low-cost enzymatic preparation would be promising for the food industry because it requires mild working conditions and yields hydrolysates with biological activities useful as ingredients for functional food. PRACTICAL APPLICATION: Proteolytic enzymes are employed in the food industry in a wide variety of processes since they modify the properties of proteins causing beneficial effects such as improvement digestibility, diminution of allergenicity, and release of bioactive peptides. Fruits from Bromelia serra possess cysteine peptidases that could be used in food biotechnology because they are capable to hydrolyze soybean and milk proteins by mild working conditions and to provoke the release of bioactive peptides. These hydrolysates containing antioxidative and ACE-inhibitor activities would be useful as ingredients for functional foods or as nutraceuticals, which are nowadays two products highly required by consumers.

Peptidases from Maclura Pomifera for Preparation of Food Protein Hydrolysates: Purification by Single-Step Chromatography and Characterization of Pomiferin I.

Our objective was to isolate peptidases from the latex of Maclura pomifera fruits and use them to hydrolyze food proteins, as well as to purify and characterize the main peptidase. Two partially purified proteolytic extracts were prepared by ethanol (EE) and acetone (AE) precipitation from an aqueous suspension of exuded fruit latex. EE was used to hydrolyze food proteins with a ratio of 0.19 caseinolytic units (Ucas) per mg of substrate. Different values of hydrolysis degree were observed for hydrolysates of egg white, soy protein isolate, and casein at 180 min (9.3%, 31.1%, and 29.1%, respectively). AE was employed to purify a peptidase which exhibited an isoelectric point (pI) of 8.70 and whose abundance in AE was 28.3%. This enzyme was purified to homogeneity using a single-step procedure by cation-exchange chromatography, achieving an 8.1-fold purification and a yield of 16.7%. The peptidase was named pomiferin I and showed a molecular mass of 63,177.77 Da. Kinetic constants (KM 0.84 mM, Vmax 27.50 uM s-1, kcat 72.37 s-1, and kcat/KM 86.15 mM-1 s-1) were determined employing N-α-carbobenzoxy-L-alanyl-p-nitrophenyl ester as substrate. Analysis by PMF showed only partial homology of pomiferin I with a serine peptidase from a species of the same family.

Preparation of soy protein hydrolysates with antioxidant activity by using peptidases from latex of Maclura pomifera fruits.

A partially purified proteolytic extract prepared from Maclura pomifera latex was employed in hydrolyzing a soybean-protein isolate (4.2 mg/mL). The hydrolysis-product formation, monitored by tricine-sodium-dodecyl-sulfate-polyacrylamyde-gel electrophoresis and reverse-phase high-performance liquid chromatography, indicated that after 10 min of reaction the main soybean proteins disappeared. The maximum degree of hydrolysis was 36.2% after a 180-min digestion. The 90-min hydrolysate presented an IC50 of 31.6 ±â€¯0.2 µg/mL, and a trolox equivalent antioxidant capacity of 157.6 and 176.9 µmoles TE per g of peptide determined by two different methods. Analysis by matrix-assisted-laser-desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS), followed by the application of bioinformatics tools, enabled the deduction of fourteen theoretical peptide sequences containing antioxidant amino acids at >60%, none of which sequences had been previously reported as antioxidants. Finally, we consider that this 90-min hydrolysate would constitute a promising ingredient in the manufacture of functional foods.

ACE-inhibitory peptides from bovine caseins released with peptidases from Maclura pomifera latex.

In work reported here, a proteolytic extract prepared from Maclura pomifera latex was employed to hydrolyze bovine caseins. Densitograms of Tricine-sodium-dodecyl-sulfate-polyacrylamide-gel electrophoresis (SDS-PAGE) indicated that the caseins were considerably degraded after a 10-min reaction. The degree of hydrolysis determined by the 2,4,6-trinitrobenzenesulfonic-acid method was 17.1±0.7% after 180min of digestion. The concentration of small peptides increased with hydrolysis time, and analysis by reverse-phase high-performance liquid chromatography (RP HPLC) and mass spectrometry, revealed a virtually unchanged peptide profile. These results suggested that those proteases were highly specific, as only certain peptide bonds were cleaved. The hydrolysate of 180min displayed the highest inhibition of angiotensin-converting enzyme (ACE) showing an IC50 of 1.72±0.25mg/mL, and the analysis of the peptide fractionation in this hydrolysate by RP HPLC exhibited two peaks responsible for that activity. Fragmentation analysis through the use of iterated matrix-assisted-laser-desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF MS/MS) with the aid of bioinformatics tools enabled us to deduce two peptide sequences-one, YQEPVLGPVRGPFPIIV, having been previously reported as an ACE-inhibitor; the other, RFFVAPFPE, as yet undescribed. The presence of bioactive peptides in these casein hydrolysates argues for their potential use in the development of functional foods.