Recent advances in protein separation technology have allowed for the isolation of whey proteins and peptides of significant biological importance. In this study, we report a novel method for isolation and purification of the neuroprotective proline-rich polypeptides, also known as Colostrinin (CLN). Although CLN was first isolated from ovine colostrum and characterized as a complex of small molecular peptides, its constituents are present also in other mammal colostrums. The previous purification protocols are very tedious, time consuming, and, due to the diverse characteristics of colostrum, also very difficult to validate. Thus, the aim of this study was to develop a simple protocol with a maximum recovery rate for CLN peptides. Here we demonstrate the two-step extraction/purification method that consists of methanol extraction and ammonium sulfate precipitation as the general principles. When compared with the original material, CLN obtained by this method shows (1) similar pattern of peptides in SDS PAGE, (2) identical amino acid analysis, characterized by high content of proline (22%), a high proportion of nonpolar amino acids, a low percentage of glycine, alanine, arginine, histidine, and no tryptophan, methionine, and cysteine residues, (3) similar pattern of HPLC profiles, and (4) its ability to induce IFN gamma and TNF alpha. More importantly, the protocol for the production of high-quality CLN can be accomplished in less than a 48 h timeframe. In addition, avoidance of excessively harsh conditions preserves the structure and biological activity of the peptides.

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The increasing incidence of fungal infections together with the emergence of strains resistant to currently available antifungal drugs calls for the development of new classes of antimycotics. Naturally occurring antifungal proteins and peptides are of interest because of low toxicity, immunomodulatory potential and mechanisms of action distinct from those of currently available drugs. In this study, the potent antifungal activity of cystatin, affinity-purified from chicken egg white (CEWC), against the most frequent human fungal pathogens of the genus Candida was identified and characterised. CEWC inhibited the growth of azole-sensitive Candida albicans isolates with minimal inhibitory concentration (MIC) values ranging from 0.8 to 3.3 micromol l(-1), a potency comparable with those of fluconazole and histatin 5, the antimicrobial peptide of the human saliva. Similarly to histatin 5, CEWC activity was not compromised in azole-resistant isolates overproducing the multidrug efflux transporters Cdr1p and Cdr2p and did not antagonise fluconazole or amphotericin B. CEWC had candidacidal activity, as revealed by the time-kill assay, and, similarly to histatin 5, completely inhibited the growth at supra-MIC concentrations. This was in contrast to the fungistatic effect and trailing growth observed with fluconazole. CEWC inhibited the growth of Candida parapsilosis and Candida tropicalis at similar concentrations, whereas Candida glabrata was more resistant to CEWC.

Colostral proline-rich polypeptide complex (PRP) with immunoregulatory and procognitive activities shows beneficial effects in Alzheimer's disease (AD). As the laboratory method of isolating PRP is expensive, laborious, and time consuming, a new large-scale methanol method of PRP isolation was developed. The proline-rich polypeptide complex obtained by this new method (named methanol PRP-MPRP) from both ovine or bovine colostra shows psychotropic activity and inhibitory effect on amyloid β aggregation similar to those produced by the laboratory-scale method. The comparative study of the antioxidant properties, cytokine-inducing activity, and nitric oxide release of PRP and MPRP showed that preparations are biologically active, moreover MPRP should be used at concentration higher than 100 µg/ml to obtain results comparable to PRP.

Colostrum and milk are the initial mammalian nourishment and rich reservoir of essential nutrients for newborn development. Bioactive peptides isolated from natural sources, such as colostrum, serve as endogenous regulators and can be used as alternative therapeutic agents in the treatment of neurodegenerative diseases. One example is the previously unknown NP-POL nonapeptide isolated from Colostrinin. In the present study, we investigated a method of NP-POL nonapeptide isolation using Bio-Gel P2 molecular sieve chromatography. We showed the protective effect of NP-POL on 6-hydroxydopamine- (6-OHDA-) induced neurotoxicity using rat adrenal pheochromocytoma (PC12 Tet On) cells. Treatment of PC12 cells with NP-POL nonapeptide reduced 6-OHDA-induced apoptosis and caused transient phosphorylation of extracellular signal-regulated kinases (ERK 1/2), which were shown to promote cell survival. NP-POL nonapeptide also protected neuronal cells against oxidative injury induced by 6-OHDA. These results showed a potential use of NP-POL in the therapy of Parkinson's disease.