Natural food preservatives extracted from plants are an emerging market in the food industry. Cowpea Vigna unguiculata L. Walp is a well-established crop around the world, and recent research has revealed that it contains multiple proteins, with potent antifungal and antiviral properties. Exploitation of the antifungal properties of cowpea protein is limited by the lack of characterisation of antifungal proteins, and the lack of knowledge about their efficacy when added to food. Furthermore, the mechanism of antifungal protein activity is not understood fully, and therefore requires further investigation. This study aimed to evaluate the antifungal potency of cowpea seed protein isolate (CPI) against bread yeasts and moulds using microbiological tests, and in a leavened bread application. The research investigated the effects of CPI on shelf life, and the sensory and textural acceptability of bread. Further aims included the characterisation of proteins in CPI with antifungal activity, and establishing an understanding of antifungal activity using computer modelling.
The first stage of the study (Chapter 2) involved the preparation of CPI, separation of high and low molecular weight protein fractions by ultrafiltration, and analysis of electrophoretic profiles using SDS PAGE. Analysis of CPI was performed using LCMS/MS, and the proteomics results reported for the first time. Twenty- three proteins with lowest hit number (HN) and highest score were selected from the first ninety-nine hits of matching of LC/MS/MS results to an existing database. The two most abundant proteins (with highest score number and lowest HN) were identified as vicilin (mw 49654), which is a storage protein, and then lipoxygenase (mw 97284) which is a metabolism protein.
The second stage of the study (Chapter 3) involved testing CPI activity against fungal growth using a micro spectrophotometric assay (micro plate method) and the spread plate method. Activity was tested against known bread spoilage moulds: Penicillium chrysogenum, Penicillium brevicompactum, Penicillium hirsutum, Aspergillus versicolor and Eurotium rubrum. The results showed CPI exhibited high antifungal activity against P. chrysogenum, P. brevicornpactum, P hirsutum and E. rubrum; no statistically significant effect was seen against A. versicolor. CPI exhibited different ranges of inhibition towards the same species at different concentrations demonstrating that the antifungal effect was concentration dependent. The antifungal activity of CPI was unaffected by heat treatment or protease treatment, indicating the antifungal components are heat stable and protease resistant. The antifungal activity of proteins in the CPI ultrafiltrate (10 kDa molecular weight cut off point) was increased by comparison with CPI and CPI retentate.
The third stage of the study (Chapter 4) involved testing the effect of CPI addition on shelf life, and the sensory and textural properties of leavened bread. A concentration of 2.3% of CPI showed the best resistance to fungal growth during the storage period. No growth was observed throughout the 8 day storage period at room temperature, whereas control samples began to show contamination on the fourth day of storage. The CPI filtrate (2.3%) showed better shelf life extension than CPI after 25 days, confirming the antifungal activity of low molecular weight proteins. Inclusion of 2.3% CPI did not significantly affect the sensory or textural acceptability of the bread. Although the hardness of bread containing CPI increased significantly after 3 days compared to the control, sensory acceptability was within acceptable limits.
The fourth stage of the study (Chapter 5) involved understanding the mechanism of action of defensins at the fungal cell wall. The hypothesis for defensin as a major antifungal protein in CPI was based on the results of the antifungal activity of the low molecular weight fraction of CPI, the heat stable and protease resistant antifungal properties of CPI, and confirmation of the presence of defensins in the proteomics results. The computer model detailing the simulation of defensin activity showed the adsorption of defensin molecules to the surface of a phospholipid bilayer membrane leading to a disordering of the membrane that would ultimately lead to disruption of cell metabolism
