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20191
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Subject: Animal Biochemistry × Author: DEEPAK CHANDRAN × Start date: 2000 ×
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    ThesisItemOpen Access
    ASSESSMENT OF IMMUNOMODULATORY EFFECTS OF GOAT LACTOFERRIN AND ITS PEPSIN HYDROLYSATE
    (College of Veterinary and animal Science,Mannuthy, 2019) DEEPAK CHANDRAN; UMA R.
    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.