ASSESSMENT OF IMMUNOMODULATORY EFFECTS OF GOAT LACTOFERRIN AND ITS PEPSIN HYDROLYSATE
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Date
2019
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College of Veterinary and animal Science,Mannuthy
Abstract
Lactoferrin, well known as a minor whey protein, is an 80kDa iron-binding
glycoprotein primarily present in milk. It exhibits an array of biological activities
including antioxidant, antimicrobial, anticancer, metal binding and
immunomodulatory properties. Many studies point out that whey proteins contain
various bioactive peptides in their primary sequences whose immunoregulatory and
therapeutic potentials have not been much explored. The present study focussed on
the isolation and characterisation of lactoferrin (gLf) from the colostrum/milk of
Malabari goats, followed by the preparation of its pepsin hydrolysate (gLPH) so as to
assess their in vitro immunomodulatory potentials on peripheral blood mononuclear
cells (PBMCs). Colostrum samples collected from Malabari goats maintained at
University Goat and Sheep Farm, College of Veterinary and Animal Sciences,
Mannuthy were processed and treated with ammonium sulphate to remove globulins
from the samples. Fractions containing albumin and remaining proteins including
lactoferrin after dialysis were loaded on to CM- Sephadex C-50 cation exchanger
column and eluted with a step gradient of 0.4, 0.6 and 0.8M NaCl. The presence of
gLf in the high OD280 value fractions eluted with 0.8M NaCl was confirmed by 12 per
cent SDS-PAGE and Western blotting. The concentration of gLf as estimated by
Lowry’s method was found to be 15.103 mg/L of colostrum. The gLf was hydrolysed
by treatment with three per cent porcine pepsin under acidic conditions at 37 °C for
four hours to form gLPH. The immunomodulatory potentials of gLf and gLPH were
studied on bovine PBMCs which were cultured along with specific combinations of
mitogen, phytohaemagglutinin (PHA)/gLf/gLPH at 37°C for 72h in five per cent CO2
humidified air. A wide range of concentrations of both gLf and gLPH were utilized to
assess their cytoproliferative effect on PBMCs with or without mitogens. In the
presence and absence of mitogen, higher concentrations of lactoferrin were found to
significantly inhibit the proliferation of PBMCs whereas lower concentrations
brought about significantly active proliferation of the cells. Maximum proliferation
with or without mitogen was observed with 1.5 µg/mL of culture. The cell
proliferation potentials of gLPH was higher than that of gLf. With gLPH, in the
presence and absence of mitogen, maximum proliferation of PBMCs could be
detected with the highest concentration of 50 µg/mL. The cells cultured with gLf and
gLPH showing maximum cell proliferation were harvested and their RNA were
isolated to study the expression of pro-inflammatory cytokines by real time PCR. It
was demonstrated that gLf and gLPH showed potentiated anti-inflammatory activity
by significantly inhibiting the expression of the pro-inflammatory cytokine genes IL1β, IL-6 and TNF-α.