Characterisation of Lactoferrin Isolated from Acid Whey Using Pilot-Scale Monolithic Ion-Exchange Chromatography

Abstract

<jats:p>The aim of this study was to characterize the properties of lactoferrin (LF) obtained in a process developed for its isolation from acid whey derived from the production of fresh curd cheese, using a unique technology of ion-exchange chromatography on CIM® monolithic columns. The freeze-dried lactoferrin samples produced on the pilot plant (capacity 1 m3) were examined for the purity, iron-binding capacity, antibacterial activity, and pH- and temperature-stability. Apo-LF inhibited several tested strains (enterobacteria, Staphylococcus, Streptococcus salivarius) except clostridia, lactic acid bacteria, and bifidobacteria. Sample of LF intentionally saturated with Fe3+ lost its antibacterial activity, indicating the involvement of mechanisms based on depriving bacteria of an iron source. All samples, regardless of the iron-saturation level, exhibited stability in pH range 4.0 to 11.0. LF with higher iron content (A-value = 41.9%) showed better thermal stability. Heat treatment up to 72 °C/3 s did not reduce antimicrobial activity against E. coli O157: H7 tox-. Higher purity (above 91%), higher iron-binding capacity and higher inhibitory activity against E. coli O157: H7 tox- compared to some similar products from the market was observed. These results demonstrate a high potential of monolithic ion-exchange chromatography for industrial processing of acid whey as a source of LF that can be used in new products with high-added value. The upscaling of the process is ongoing on a demonstration plant (10–30 m3/day capacity).</jats:p>

Stability-Indicating Analytical Approach for Stability Evaluation of Lactoferrin

“…The Arrhenius equation was linear in the temperature range from 60 to 80 • C. However, the rate constant at 50 • C significantly deviated from the correlation (Figure 4) indicating changes in the degradation mechanism of Lf between 50 and 60 • C. Our findings are following results obtained by other researchers who used the same Lf isolate samples to study the thermodynamic stability by UV-VIS spectroscopy. It was found that Lf is thermally stable up to about 53 • C with a denaturation temperature of 58.6 • C [21]. The activation energy calculated from the Arrhenius plot was found to be 78.4 kJ/mol, which is lower than values found in the literature.…”

“…High purity Milli-Q water was obtained through a Milli-Q A10 Advantage water purification system (Millipore Corporation, Bedford, MA, USA). Different lots of solid Lf isolate samples (1–3; A-value of 3.4–5.2% [ 21 ]), as well as liquid Lf isolate samples (elutions E1, E3, and concentrated E3) were obtained from ARHEL d.o.o. (Ljubljana, Slovenia).…”

“…Different spectroscopic techniques are also used for the evaluation of thermodynamic stability of Lf. Changes in Lf structure can be evaluated by fluorescence measurements [ 19 , 20 , 21 ]. UV spectroscopy is used for the determination of denaturation temperature [ 20 , 21 ], as well as for the determination of Lf content [ 22 ] and iron saturation [ 10 , 23 ].…”

Siğir Laktoferri̇ni̇n Çi̇ğ Sütün Mi̇krobi̇yoloji̇k Özelli̇kleri̇ne Etki̇si̇

“…However, no bacteriostatic effect was observed on Lactobacillus acidophilus. Matijašic et al, (2020) examined the antimicrobial effects of seven different lactoferrins isolated from whey and produced in the pilot plant by the disk diffusion method. Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica, Clostridium difficile, Klebsiella oxytoca, and Clostridium perfringens were selected as potentially pathogenic indicator bacteria.…”

“…Lactoperoxidase and lysozyme are the most abundant antimicrobial enzymes in milk, while lactoferrin is the most abundant antimicrobial peptide (Yılmaz and Tosun, 2012). Antimicrobial proteins and peptides are produced by the secretory fluids produced by many organisms as a primary defense mechanism (Matijašic et al, 2020).…”