Production of Liquid Milk Protein Concentrate with Antioxidant Capacity, Angiotensin Converting Enzyme Inhibitory Activity, Antibacterial Activity, and Hypoallergenic Property by Membrane Filtration and Enzymatic Modification of Proteins

Abstract

Liquid milk protein concentrate with different beneficial values was prepared by membrane filtration and enzymatic modification of proteins in a sequential way. In the first step, milk protein concentrate was produced from ultra-heat-treated skimmed milk by removing milk serum as permeate. A tubular ceramic-made membrane with filtration area 5 × 10−3 m2 and pore size 5 nm, placed in a cross-flow membrane house, was adopted. Superior operational strategy in filtration process was herein: trans-membrane pressure 3 bar, retention flow rate 100 L·h−1, and implementation of a static turbulence promoter within the tubular membrane. Milk with concentrated proteins from retentate side was treated with the different concentrations of trypsin, ranging from 0.008–0.064 g·L−1 in individual batch-mode operations at temperature 40 °C for 10 min. Subsequently, inactivation of trypsin in reaction was done at a temperature of 70 °C for 30 min of incubation. Antioxidant capacity in enzyme-treated liquid milk protein concentrate was measured with the Ferric reducing ability of plasma assay. The reduction of angiotensin converting enzyme activity by enzyme-treated liquid milk protein concentrate was measured with substrate (Abz-FRK(Dnp)-P) and recombinant angiotensin converting enzyme. The antibacterial activity of enzyme-treated liquid milk protein concentrate towards Bacillus cereus and Staphylococcus aureus was tested. Antioxidant capacity, anti-angiotensin converting enzyme activity, and antibacterial activity were increased with the increase of trypsin concentration in proteolytic reaction. Immune-reactive proteins in enzyme-treated liquid milk protein concentrate were identified with clinically proved milk positive pooled human serum and peroxidase-labelled anti-human Immunoglobulin E. The reduction of allergenicity in milk protein concentrate was enzyme dose-dependent.

Bioactive Peptides from Liquid Milk Protein Concentrate by Sequential Tryptic and Microbial Hydrolysis

“…The membrane was placed in a stainless steel-made crossflo module. A static turbulence promoter was placed inside the membrane descriptions of the membrane module, tubular membrane and static turbul have been mentioned in a previous publication [23]. The membrane filtratio performed with trans-membrane pressure at 3 bar, a retention flow rate of a volume reduction factor of 2 at RT.…”

“…where J = permeate flux during filtration (L•m −2 •h −1 ), V = volume of permeate (L), A = active membrane filtration area (m 2 ), and t = filtration time (h) [23]. Furthermore, after the volume reduction factor of 2, the reduction of permeate flux was calculated, based on Equation (2):…”

“…After the filtration experiment, membrane cleaning was performed with Ultrasil P3-11 (Ecolab-Hygiene Kft, Budapest, Hungary), citric acid (≥99%, Merck, Darmstadt, Germany) and Milli-Q ultrapure deionized water (18.2 MΩ•cm) in sequence. The detailed methodology is described elsewhere [23]. Milli-Q ultrapure deionized water was obtained from the Milli-Q Synergy/Elix water purification system (Merck-Millipore, Molsheim, France).…”

“…After 10 min of the tryptic hydrolysis reaction, the bioreactor was immediately placed in a thermostat for the deactivation of trypsin activity. It was performed at a temperature of 70 • C for 30 min of incubation in a water bath [23]. Continuous scanning during trypsin-catalyzed hydrolysis was performed within a wavelength range of 900-1700 nm (NIR spectra), every minute, for the aquaphotomics analysis.…”

“…Milk is a source of an abundant number of proteins with unique biological properties, and is available around the globe, throughout the year. The hydrolysis of milk proteins, such as micellar casein, α-lactalbumin, β-lactoglobulin, immunoglobulin, bovine serum albumin, lactoferrin, lactoperoxidase and glycomacropeptide, with proteases producing amino acids and dietary peptides that offer several functional capabilities [23]. On the other hand, milk proteins are listed among the "big 8" allergens, due to the presence of linear and conformational epitopes [24].…”