Characterisation of zein from South African maize of varying endosperm texture

Thesis (MSc Food Sc)--University of Stellenbosch, 2011.

Includes bibliography.

ENGLISH ABSTRACT: Maize is an important crop for both human and animal consumption. Maize kernel texture (kernel hardness) is an important quality trait for many sectors in the South African maize industry, where a harder texture is desired. Both total protein content and the main storage proteins, zein, have been associated with kernel texture. The zein profiles of South African white maize hybrids, from a breeding program, grown at three localities together with their respective inbred parent lines were evaluated to determine the difference in zein expression. For only the hybrids, total protein content, zein content and degree of hardness (kernel texture) was determined to establish possible relationships. Zein consists of four main classes, α-, β-, γ-, and δ-zein, which can further be divided into sub-classes. Zein was characterised using matrix-assisted laser desorption ionisation time-offlight mass spectrometry (MALDI-TOF MS) after optimisation of the zein extraction and matrix preparation procedures. Two matrices [2-(4-hydroxyphenylazo)benzoic acid (HABA) and α- cyano-4-hydroxy-cinammic acid (CHCA)] and three pH levels (<1.5, 1.7 and 2.9) for matrix solutions were investigated. Two solvent concentrations (50% and 70% acetonitrile (ACN) were also investigated. Parallel to this investigation, a zein extraction procedure was optimised. Zein was extracted from non-defatted (NDF) and defatted (DF) maize meal at ambient temperature and 60°C and meal with different particle size distributions. Particle size of maize meal did not influence the quality of spectra. Regardless of matrix conditions used, zein extracted at 60°C from NDF meal produced spectra with a low signal-to-noise (S/N) ratio. When only HABA was included (pH<1.5), not all γ-zein sub-classes were observed. This was also true for zein extracted at ambient temperature from NDF and DF meal. Good S/N ratios for all zein classes were obtained when zein was extracted from NDF meal at ambient temperature using a matrix solution containing both matrices in 70% ACN at pH 2.9. MALDI-TOF MS provided accurate detection and good profile resolution for zein characterisation. Spectral profiles of hybrids were a combination of their respective inbred parent lines. Zein proteins were quantified using reverse-phase high performance liquid chromatography (RP-HPLC). Total protein content was determined and kernel texture assessed using a particle size (PSI) method. Significant (P<0.01) differences were observed for total protein content and degree of hardness between certain hybrids as well as average values for localities. Correlations and principal component analysis indicated that hybrids with a higher protein and total zein content had a harder kernel texture. The β- and γ-zein contents also correlated positively with kernel hardness. However, when expressed as percentage of total zein the opposite was observed. Collectively the α-zeins and most individual α-zeins correlated positively with hardness. Scanning electron microscopy micrographs showed differences between the floury endosperm of harder and softer maize kernels; illustrating starch types (amylose and amylopectin) should also be analysed in future hardness studies. The correlations obtained were not strong (r<0.6) and the variation explained by first two principal components was low. Other maize constituents, more hardness measurements should be considered in future studies to improve this.

AFRIKAANSE OPSOMMING: Mielies is a belangrike gewas vir beide mens- en dierlike inname. Mieliepittekstuur (pithardheid) is ‘n belangrike kwaliteitseienskap vir baie sektore in die Suid Afrikaanse mielieindustrie, waar ‘n harder tekstuur verlang word. Beide totale proteïeninhoud en die hoof opbergingsproteïen, zein, is al geassosieer met pittekstuur. Die zein profiele van Suid- Afrikaanse witmielie basters, van ‘n teel program, wat by drie lokaliteite verbou is sowel as hul onderskeie ingeteelde ouerlyne is geevalueer om verskille in zein uitdrukking te bepaal. Die totale proteïeninhoud, zeininhoud en graad van hardheid is bepaal om verhoudings vas te stel. Zein bestaan uit vier hoof klasse, α-, β-, γ-, en δ-zein, wat verder onderverdeel word in subklasse. Zein is gekarakteriseer met matriks-ondersteunende laser desorpsie ionisasie tyd-vanvlug massa spektrometrie (MBLDI-TVV MS) na die zein ekstraksie en matriks voorbereidingprosedures geoptimaliseer is. Twee matrikse [2-(4-hydroksiephenylazo)benzoë suur (HABA) en α-cyano-4-hydroksie-kaneelsuur (CHCA)] en drie pH vlakke (<1.5, 1.7 and 2.9) vir matriksoplossings was ondersoek. Twee oplossingkonsentrasies [50% and 70% asetonitriel (ACN)] is ook ondersoek. Zein ekstraksie kondisies is ook geoptimiseer. Zein is geëkstraheer van nie-ontvette (NOV) en ontvette (OV) mieliemeel by omgewings temperatuur en 60°C. Die partikelgrootte van die mieliemeel het nie die kwaliteit van spektra beïnvloed nie. Ongeag watter matrikskondisies gebruik is het zein wat van NGV meel by 60°C ge-ekstraheer is, spektra met ‘n lae sein-tot-geraas (S/G) verhouding geproduseer. As die HABA alleenlik gebruik (by pH<1.5) is, is nie alle γ-zein klasse waargeneem nie. Dit was ook waar vir zein wat by omgewings temperatuur van NGV en OV mieliemeel ge-ekstraheer is. Goeie S/G verhoudings is waargeneem met zein, ge-ekstraheer van NGV mieliemeel by omringende temperatuur, met die bebruik van beide matrikse in 70% ACN by pH 2.9. (MBLDI-TVV MS) het akkurate en goeie resolusie van profiele vir zein karateriseering verksaf. Spektrale profiele van basters was ’n kombinasie van hul onderskeie ouer lyne. Zeinproteïene is met omgekeerde-fase hoë prestasie vloeistofchromatografie gekwantifiseer. Totale proteïeninhoud is bepaal en die hardheidsgraad was bepaal met ‘n partikelgroote indeks (PGI) metode. Beduidende verskille tussen proteïeninhoud en hardheidsgraad is waargeneem tussen sekere basters sowel as gemiddelde waardes by die verskeie lokalieteite. Korrelasies en hoofkomponent analise het aangedui dat basters met ‘n hoër proteïeninhoud en totale zein ‘n harder tekstuur gehad het. Die β- en γ-zeininhoud het ook positief gekorreleer met hardheid. Alhoewel, wanneer die proteïene as persentasie van totale zein uitgedruk is, is die teenoorgestelde waargeneem. Gesamentelik het α-zein asook die meeste indivduele α-zeins positief gekorreleer met hardheid. Skandeer elektron mikroskopie mikrograwe het verskille tussen die meelerige endosperm van harder en sagter pitte aangedui; meer proteïenliggame was aanwesig in die harder pitte en die stysel was digter gepak. Dus moet stysel tipes ook in ag geneem word in toekomstige hardheidsstudies. Korrelasies wat verkry is, was nie hoog (r<0.6) nie en die variasie verduidelik deur die eerste twee hoofkomponente was laag. Ander miielie samestellende dele as ook meer hardheidsmetings moet in toekomstige studies in ag geneem word om dit te verbeter.

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