Growth stimulating effect of bovine milk lactoferrin on dermal cells and probiotic bacteria

*Corresponding author: Ivan F. Gorlov, Volga Region Research Institute of Meat-and-milk Manufacture and Processing, 400131, Rokossovskogo St., 6, Volgograd, Russia. Tel. : +78442391101. Fax. : +78442391142. E-mail: niimmp@mail.ru. Received: 27 June 2016; Revised: 23 April 2016; Accepted: 24 April 2016; Published Online: 29 April 2016 Titov, et al.: Growth stimulating effect of bovine lactoferrin Emir. J. Food Agric ● Vol 28 ● Issue 8 ● 2016 541 destructive for the cell membrane to start: A bacterium without coat, unable to withstand the external pressure, “bursts”. This resembles the operating principle of antibiotics, but the drug-induced antimicrobial agents¢ damage both the bacteria and the cells of the human body. Antimicrobials also cause the death of beneficial microflora. Intestinal dysbiosis develops, the digestibility of nutrients, vitamins, trace elements decreases, the immune protection weakens, as large number of immunocompetent cells are killed at struggle with microbes. Bovine lactoferrin is free of such deficiencies. Due to the bovine lactoferrin’s multifunctionality aimed at maintaining homeostasis of a living body, it has recently attracted attention of researchers. Furthermore, analysis of the data suggests that bLF refers to a group of proteins that regulate receptor mechanism for implementing many cellular processes, including proliferation of both animal and microbial cells, and especially of the cells of beneficial intestinal microflora (Legrand et al., 2008; Tomita et al., 2009). At the same time, the problems of the structural and functional role of the receptors, their affinity activity against the proliferation inducing factor, the signal passage in the cell have not been learned that determines the relevance of further research. The choice of an adequate model of the cell target, which regulates the cell proliferation through the receptor mechanism of the factor, is of particular importance in the experiment planning. Given that the up-to-date sector in the surgical transplantation is the search for new natural growth regulators of cells and human tissues in vitro, the study of the bLF applicability in this field of knowledge has been of special significance. Taking into account the above factors, the team of authors set the task to study the effect of bLF, isolated from cow’s milk, on dermal fibroblasts and human keratinocytes. The bLF Peptides, the most active in reference to stimulating the bifidogenic property of beneficial microorganisms, proved to be more effective in protection the GI tract from ulceration of the gastric mucosa and dysbiosis. The purpose of this work was to solve the regulation problems of protection processes through biologically active proteins in the animal body, in particular, to study the role of the receptor mechanism in the effect of lactoferrin and its derivatives (peptides) on the cell proliferation. MATERIALS AND METHODS Preparation and identification of bLF Due to the fact that bLF is contained in the milk in considerable amounts (the highest concentration is found in colostrum: In the female – up to 6 g/l, in the cow – up to 300 mg/l), in our studies, bLF was isolated from cow milk and purified by the ion exchange chromatography using cation exchanger Macro-Prep HQ Support (Bio-Rad Laboratories Inc., USA). The resin washed with distilled water was placed into in a potassium phosphate buffer (0.05M, pH 6.7) with constant stirring for 1.5 hours to charge the functional groups. Then, the charged ion exchanger was separated from the buffer and placed in the skim milk (Zhao et al., 2015). It was left with constant stirring at 4±2oC for 24 hours. The exchanger with bound protein was separated by decantation and washed with potassium phosphate buffer (0.05M, pH 6.7). The resin was packed into column by gravity. Elution of proteins was performed by potassium phosphate buffer (0.05M, pH 6.7) with concentration gradient of NaCl (0.1-1.0 M) at a flow rate of 1.5 ml/min. The isolated protein fractions with the absorbance value more than 0.1 (at 280 nm) were dialyzed through a semipermeable membrane against distilled water at 4±2oC for 24 hours. The identification of bLF was carried out by the methods of iron-binding capacity determination, electrophoresis, enzyme-linked immunosorbent assay (ELISA). The amount of bLF in chromatographic fractions was determined by ELISA (competitive option) based on polyclonal antibodies (Fotschki et al., 2015). The iron-binding capacity was assessed by saturating capacity of protein with ferric iron in the presence of carbonate ions. Change of bLF into apoand holo-form was achieved by dialysis of its solutions against citric acid (pH 2), and iron carbonate, respectively (Shimazaki, 2000). The iron saturation degree of the protein was determined spectrophotometrically according to the color change at 465 nm (Shimazaki et al., 1998). Electrophoresis was performed by Laemmli method (Laemmli, 1970). For comparison, some experiments were carried out using commercially available bLF (Sigma, USA).