Abstract
Slaughterhouse blood is a by-product of the meat production industry and represents a rich source of protein with potential beneficial properties. The results reported in this thesis describe the generation of hydrolysates and bioactive peptides from four commercial New Zealand slaughterhouse animals (deer, sheep, pig and cattle) using enzymatic hydrolysis and proteases from fungal and plant sources, and investigates their biological activities.
Initially, the properties of unhydrolysed slaughterhouse animal blood from deer, sheep, pig and cattle were investigated. The slaughterhouse blood was separated into the plasma, red blood cell and white blood cell fractions. Standard blood constituents were measured and fractionated blood was assessed for antioxidant activity, angiotensin converting enzyme (ACE) inhibitory activity and antimicrobial activity. Cattle blood had the highest total plasma proteins. The unhydrolysed red blood cell fractions of all four animal species contained higher antioxidant activity compared to the plasma fractions. Negligible levels of ACE inhibitory activity were found for all animal blood fractions. Antimicrobial activity was detected towards Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa with sheep white blood cells from which a crude neutrophil extract was obtained that demonstrated concentration dependent inhibitory effects on the growth rates of these bacterial strains.
Plasma separated from deer, sheep, pig and cattle abattoir blood was hydrolysed using protease preparations from plant (papain and bromelain) and fungal (FP400 and FPII) sources. Antioxidant and antimicrobial activities of the peptide hydrolysates obtained after 1, 2, 4 and 24 hours of hydrolysis were investigated. The release of trichloroacetic acid-soluble peptides over the hydrolysis period was monitored using the o-phthaldialdehyde (OPA) assay, and the hydrolysis profiles were visualised using SDS-PAGE. The major plasma proteins in the animal plasmas were identified using MALDI-TOF-TOF MS. Hydrolysates of plasma generated with fungal proteases exhibited higher 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, oxygen radical scavenging capacity (ORAC) and ferric reducing antioxidant power (FRAP) than those generated with plant proteases for all three animal plasmas. No antimicrobial activity was detected in the hydrolysates.
The red blood cell fractions (RBCF) from deer, sheep, pig and cattle blood obtained were also hydrolysed using the same protease preparations. RBCF hydrolysates generated with papain exhibited higher ferric reducing antioxidant power (FRAP), and oxygen radical scavenging capacity (ORAC) than those generated with bromelain, FP400 and FPII. Antimicrobial activity against E coli, S. aureus and P. aeruginosa was detected in the 24 hour hydrolysates of FP400 and FPII RBCF. Lower antioxidant properties were observed in hydrolysates of animal red blood cells compared to plasma hydrolysates. However, in contrast, antimicrobial activity was detected towards bacteria in the fungal treated RBCF hydrolysates but not in the plasma hydrolysates.
The hydrolysis of cattle plasma with fungal protease FPII generated a hydrolysate (FPII + CP24) with high in vitro antioxidant activities as tested using chemical based antioxidant assays (ORAC, DPPH and FRAP). This hydrolysate was also tested for its in vitro antioxidant activity in a cell based antioxidant assay using Vero cells treated with a peroxyl radical generator 2,2’-Azobis(2-amidinopropane) dihydrochloride (AAPH) and was found to have protective effects at the highest concentration tested.
The FPII + CP24 hydrolysate was fractionated using gel permeation, OFFGEL isoelectric focusing and RP-HPLC techniques to separate and identify the most active antioxidant peptide fractions. Fifteen novel peptide sequences were identified in the most active RP-HPLC fraction using Liquid Chromatography-Mass Spectrometry (LCMS) and database searches.
This identification and analysis of bioactive compounds with antioxidant and antibacterial activity in slaughterhouse blood makes a unique contribution to current knowledge on available bioactives from this resource and may lead to novel utilization of waste animal blood.