A Comparative study of immunomodulatory activity of deer and cow milk proteins

Abstract

The aim of this study was to compare the immunomodulatory activity of peptides derived from deer and cow milk using in vitro lymphocyte proliferation assays and cytokine (IL-2 and INFγ) production by human peripheral blood mononuclear cells (PBMC). Bioactive peptides were produced using in vitro digestion and lactic acid bacteria (LAB) fermentation. Deer and cow milk were fermented using Lactobacillus delbrueckiisubspbulgaricus, Streptococcus salivarius subsp thermophilus and Lactobacillus casei strain Shirota at 37°C for 24 hours. Unfermented and fermented milk proteins were digested in two steps; imitating both the human stomach (Pepsin, pH 2.5, 30 min) and the duodenum (Corolase PP, pH 7.5, 30 min) digestion. The production of peptides was quantified using the O-phthaldialdehyde assay and the degradation patterns of the milk proteins were analysed by SDS-PAGE. Immunomodulatory activity of each hydrolysate was carried out for 0.125, 0.25 and 0.5 mg/ml protein concentrations. All unfermented and fermented deer and cow milk digestions were fractionated using Fast Protein Liquid Chromatography (FPLC) in order to separate and identify the most active fractions. Identification of peptides present in these immunostimulating fractions were carried out using Liquid Chromatography–Mass Spectrometry (LC-MS/MS) and database searching.

Deer milk contained 8.8 ± 0.13% protein which was twice the level found in cow milk (4.1 ± 0.02%). Peptide production was significantly higher in unfermented deer milk than unfermented cow milk (P< 0.05). The commercial proteolytic enzymes degraded milk proteins from deer more rapidly than those from cow. Deer milk fermentation gave higher peptide production than cow milk fermentation. Following in vitro digestion peptide production was significantly greater in deer milk ferment than cow milk ferment (P≤ 0.05).

Lymphocyte proliferation, interleukin-2 (IL-2) and interferon-γ (INFγ) production were significantly greater in deer milk digestions than cow milk digestions (P ˂ 0.001)at the same concentration. LAB fermentation of both deer and cow milk prior to in vitro digestion increased (P < 0.001) lymphocyte proliferation. Fermentation with lactobacillus strains increased lymphocyte proliferation and cytokine IL-2 and IFNγ production (P < 0.05) more than with streptococcus. The immunostimulating effect was significantly greater (P ˂ 0.001) at 0.125 mg/ml protein concentration than at higher protein concentrations.

The deer milk digests were more effective at increasing lymphocyte proliferation than the cow milk digests (P < 0.001) at the same protein concentration. Proliferation was higher (P < 0.001) at 0.125 mg/mL protein than at higher protein concentrations.

FPLC fractions (11, 22 and 30) with high immunostimulatory activity from deer digests (unfermented and fermented) showed higher lymphocyte proliferation and higher cytokine IL-2 and IFNγ production than the correspondening fractions of cow digests when measured in vitro with human PBMC.

Mixtures of different peptides with different protein origin have been identified in the above FPLC fractions in both deer and cow milk digests which could be responsible for theimmunostimulating effect. The main protein source was β casein for both deer and cow fractions. Some differences in amino acid composition of β casein, α1 and α2 casein were identified between deer milk than cow milk. Few previously identified immunostimulating peptide sequences were found in tested FPLC fractions in cow digests. Some different peptides were detected in deer fractions compared to corresponding cow fractions. No previously identified immunostimulating peptides were detected in deer digests. Further studies are required to further purify and identify unknown immunostimulating peptides. The activity could be confirmed with synthesised peptides.