A preliminary study on milk kefir proteomic characterization.

Kefir is a South Caucasian drink, belonging to traditional fermented milk, it is made by inoculating cow, goat, or sheep milk with kefir “grains” which is a starter made up of proteins, lipids, bacteria and yeasts kept together by a liposoluble polysaccharide called "Kefiran". From a nutritional point of view, the proteins in milk kefir are partially digested so that they can be easily used by the organism, it is a font of essential amino acids, calcium and magnesium, important minerals for the maintenance of a healthy nervous system, phosphorus and vitamins. Milk kefir is also considered a probiotic drink thanks to the large numbers of microorganisms that are present in this beverage, it is suitable for lactose-intolerant individuals since the “grains” shows a -galactosidase activity. A very recent study, performed in high-fat diet mice, reported that kefir consumption modulates gut microbiota and mycobiota and prevents obesity and fatty liver disease by promoting fatty acid oxidation [1]. However, until now, there is a gap in literature on the proteomic composition of this fermented drink. Therefore, the objective of this study was a proteomic analysis of kefir milk (derived from bovine milk) by two-dimensional electrophoresis (2-DE). Before to be analyzed, kefir milk was centrifuged (13000g for 20 min) and then filtered with 0.22 filter in order to remove bacteria. The total protein present in the filtered kefir were concentrated by precipitation with 90% ammonium sulphate. The precipitated proteins were resuspended in 50mM tris/HCl pH 7.5 and before 2-DE were treated with 2D-Clean-Up (GE Healthcare). 0,5 mg of total proteins were resuspended in a rehydratation buffer containing urea and detergents and subjected to isoelectric focusing on an immobilized pH gradient 3-10 (Immobiline DryStrip gel, 18cm, GE Healthcare). The second dimension consisted of a 15% SDS-PAGE. After electrophoresis, the proteins were stained by Coomassie blue. The gel was analyzed by the software PDquest (Bio-Rad) for spot quantitation, determination of isoelectric point and molecular weight. As a result, were found two broad protein regions, one in the pH range of 3.4-6.2 and Mr of about 31.0 kDa, another one in the pH range of 3.4-5.3 and Mr of about 21.0 kDa which could derive from bovine milk casein and -lactoglobulin, respectively. Interestingly, there are several low molecular weight peptides, which may derive from the fermentative processes. The presence of low molecular weight peptides is one of the characteristics that determines the digestibility of kefir milk with respect to whole bovine milk. These low molecular weight proteins may be the bioactive peptides, defined as specific protein fragments that have a positive impact on body functions and conditions and may ultimately influence health [2]. Bioactivities described for milk-derived peptides includes opiate, immunomodulatory, antimicrobial, antioxidative, antithrombotic, anticancer, mineral carrying and growth-promoting properties. These milk-derived peptides are mainly obtained from milk casein, but some classes of peptides are also derived from whey proteins and may show one or more synergic activities [3]. The data obtained in this preliminary study are very encouraging and, up to now, there aren’t data on the kefir proteomic characterization in the literature. The spot proteins separated by 2-DE will be extracted from the gel and will be identified by mass spectrometry. The results that will be obtained from this study will clarify the protein composition of kefir milk and also identify and characterize the peptides (of which this matrix is particularly rich) that could have an important nutraceutical function and hence a positive impact on human health.