Characterisation of starch from malting barley grown in South Africa

Balet, Sandra (2016-03)

Thesis (MSc Food Sc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: South African barley is not always suitable for malting purposes due to unpredictable weather conditions. Malted barley is imported from other barley producing countries such as Australia, the United States and Europe. It would be of great importance to characterise the local cultivars in order to lay a strategy for the local plant improvement. Different analyses were conducted to examine the functional properties of local cultivars for malting purposes. Pasting properties were determined using the Rapid ViscoAnalyser (RVA), amylose contents using size exclusion chromatography (SEC), phosphate contents using an iodine binding assay, particle size distribution using laser diffraction, granule morphology using scanning electron microscopy (SEM) and amylose and amylopectin fine molecular structure using SEC. The SSII gene was amplified using polymerase chain reaction (PCR) allowing allele specific SNP detection. Barley flour was used to examine pasting properties and starch molecular structure. Isolated starch was used for phosphate determination, granule size distribution and starch granule morphology. The DNA was extracted from barley leaves. PCA biplots were used to relate pasting properties with different cultivars. Multiple factors analysis (MFA) was done to relate different blocks of variables with one another, correlation circle graphs were used to graphically show relationship between the blocks of variables and an individual factor map was used to relate groupings of cultivars. The PCR amplicons were sequenced and analysed using Geneious software. From the pasting properties results, Metcalfe seems to be different from the rest of the cultivars, Baudin and Henrike shows similarity, Cristalia, Marthe and Cocktail showed similarities, as well as Erica, Nemesia, Disa and Houwink. Metcalfe, Baudin and Henrike seemed to be of good malting quality. There were no differences observed in phosphate contents, granule particle size distribution and amylose contents among the samples. The phosphate contents were between 0.21 to 0.9%, Houwink had the highest phosphate content and Henrike the lowest. Granule particle size was between 15.9 and 18.6 μm and amylose contents were between 23.8 and 25.5%. Baudin had a smaller granule diameter and Metcalfe the largest. Houwink had the lowest amylose content and Nemesia the highest. The phosphate content was low as expected in cereals. Granules were classified as large which is a good indication for malting purposes. Amylose content indicated that these cultivars had normal starch. There were no differences in the average degree of polymerisation (DPX), the samples showed average DPX between 3,420 and 4,330, Metcalfe had the highest DPX and Henrike the lowest. The amylose and amylopectin fractions in the three regions Amylopectin1, Amylopectin2 and Amylopectin 3 were analysed. There were no differences in AP1 and AP2 regions, however, a difference was observed in Am3 region among the ten cultivars. There was a clear variation in the chain length distribution (CLD) among the samples and also disruption of linearity in the chains length was observed especially in Marthe, Henrike and Cristalia. Eight set of primers were used to amplify portions of the SSII gene from the ten cultivars. Four amplified the expected fragment sizes 1055, 1059, 1096, and 1112 bp. The fragments were sequenced and analysed using NCBI blast program query ID 149533 and it was 99% identical to Hordeum vulgare SSII gene. From the sequenced results, all cultivars showed the same result and one SNP was identified on exon 5 which change the amino acid from leucine to proline.

AFRIKAANSE OPSOMMING: Weens onvoorspelbare weersomstandighede is Suid-Afrikaanse gars nie altyd geskik vir moutdoeleindes nie, en word moutgars ingevoer van ander lande wat gars verbou. Daarom is dit uiters belangrik om die plaaslike kultivars te tipeer ten einde ’n strategie vir plaaslike gewasverbetering te ontwerp. Plakeienskappe is met behulp van die snelle visko-ontleder (RVA) ondersoek; amilose-inhoud is met grootte-uitsluitingschromatografie (SEC) bepaal; fosfaatinhoud met behulp van ’n jodiumbindingstoets; styselkorrelgrootteverspreiding met behulp van laserdiffraksie; korrelmorfologie met behulp van skanderingselektronmikroskopie (SEM); en amilose- en amilopektien- fyn molekulêre struktuur is met behulp van SEC bestudeer. Die SSII-geen is deur middel van die polimerasekettingreaksie (“PCR”) versterk om alleelspesifieke SNP-opsporing moontlik te maak. PCA-bistippings is gebruik om plakeienskappe met verskillende kultivars te verbind. Meerfaktorontleding is onderneem om verskillende blokke veranderlikes met mekaar te verbind; korrelasiesirkelgrafieke is gebruik om die verwantskap tussen die blokke veranderlikes grafies voor te stel, en ’n kaart van individuele faktore is gebruik om kultivargroeperinge te verbind. Die PCR-amplikons is met behulp van die sagteware Geneious aan reeksbepaling en ontleding onderwerp. Uit die plakeienskapresultate blyk Metcalfe van die res van die kultivars te verskil, terwyl Baudin en Henrike ooreenkomste toon, Cristalia, Marthe en Cocktail gelyksoortig voorkom, en so ook Erica, Nemesia, Disa en Houwink. Die voorspelling kan gemaak word dat Metcalfe, Baudin en Henrike van ’n goeie moutgehalte is. Geen verskille in fosfaatinhoud, korrelgrootteverspreiding en amilose-inhoud word tussen die kultivars waargeneem nie. Die fosfaatinhoud was tussen 0, 21% en 0, 9%; Houwink toon die hoogste en Henrike die laagste fosfaatinhoud. Korrelgrootte was tussen 15, 9 μm en 18, 6 μm, en amilose-inhoud was tussen 23,8% en 25,5%. Baudin toon die kleinste korreldeursnee, en Metcalfe die grootste. Houwink het die laagste amilose-inhoud, en Nemesia die hoogste. Lae fosfaat is te verwagte by graan; die korrels is groot, wat ’n goeie aanwyser van moutgehalte is. Amilose-inhoud dui daarop dat hierdie kultivars oor normale stysel beskik. Die DPX was tussen 3,420 en 4,330; Metcalfe toon die hoogste en Henrike die laagste DP. Daar is geen verskille tussen die AP1- en AP2-streke nie; tog is verskille in die AM3-streek tussen die tien kultivars opgemerk. Daar is variasie in die kettinglengteverspreiding (“CLD”) tussen die monsters, en ontwrigting in lineariteit is veral by Marthe, Henrike en Cristalia opgemerk. Agt stelle aanvoorders is gebruik om gedeeltes van die SSII-geen van die tien kultivars te versterk. Vier daarvan het die verwagte fragmentgroottes 1055, 1059, 1096 en 1112 kbp versterk. Die fragmente is met behulp van ’n navraag (ID 149533) op die BLAST-program van die NCBI bevestig, en dit was 99% identies aan die SSII-geen van Hordeum vulgare. Alle kultivars toon dieselfde reeksresultate, en een SNP is op ekson 5 geïdentifiseer wat die aminosuur van leusien na prolien verander.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98539
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