Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Selection of raw materials for a consistent and high quality end product has been a
challenge for brewers globally. Various different factors may influence quality and although a
great number of methods for malt analysis exist today for the prediction of end product quality,
some still do not accurately represent malt performance in beer. This research focussed on
determining parameters in malts to predict two of the major beer quality determining factors
namely, foam- and flavour stability. Specific biochemical markers in barley malt such as lipid
transfer protein 1 (LTP1) lipoxygenase-1 (LOX-1), anti-radical/oxidant potential (AROP), free
amino nitrogen and intact protein were determined and used in beer quality prediction from malt
character. These biochemical quality predictions were then correlated with the end product beer
quality as assessed in sensory analysis trials on micro-brewed beers.
Being such a multi-faceted factor in beer, LTP1 have already become an attractive field
of study. LTP1 is primarily associated with stable beer foam, as a foam protein in its own right,
and acting as a lipid scavenger. This protein is also theorised to play a role in the stability of beer
flavour by possibly acting as anti-oxidant. Lastly LTP1 is known to have anti-yeast activity,
which could negatively impact fermentation. In this study LTP1 and its lipid bound isoform
LTP1b were successfully purified in an economical and easy five step protocol. Both isoforms
showed temperature stability at temperatures >90°C and prefer more neutral and basic pH
environments. Although the reported antioxidant activity was not observed, both purified LTP1
and LTP1b inhibited lipoxygenase-1 (LOX-1) activity, which is responsible for the enzymatic
breakdown of linoleic acid to form 2(E)-nonenal. This is a novel finding that links LTP1 also to
flavour stability. LTP1 exhibited anti-yeast activity whereas LTP1b lost most if not all the
activity. However, since most of the LTP1 is converted to LTP1b and glycosylated isoforms
during the brewing process fermentation will not be greatly influenced, while foam and flavour
stability could still be promoted by the presence of LTP1b.
Flavour deterioration of the final packaged product is partially due to the enzymatic
production of 2(E)-nonenal by LOX-1 and the presence of free oxygen radical species, limited
anti-radical/oxidant potential (AROP) and LTP1. The development of two 96-well micro-assays based on the ferrous oxidation-xylenol orange (FOX) assay for the determination of LOX-1 and
AROP was successfully accomplished and compared well with established assays. The LOXFOX
and AROP-FOX assays were specifically developed for the on-site, high throughput
comparative determination of LOX-1 and AROP in malt and other brewery samples.
The AROP-FOX and LOX-FOX micro-assays and a number of established assays were
used to categorise malts in different predicted quality groups, various biochemical markers were
measured which included LOX activity, LTP1 content, FAN values, intact protein concentration
and AROP. An excellent trend (R2=0.93) was found between FAN/LOX and LTP1/LOX which
also correlated with the novel observation that LOX-1 activity is inhibited by LTP1 at various
concentrations. These trends could assist brewers in optimal blending for not only high quality
end products but also fermentation predictions.
To determine whether these biochemical markers selected for screening in barley malt are
predictive of shelf life potential of the end product, sensory trials were performed. Three barley
malt cultivars were selected for LOX, AROP, LTP1, protein and FAN content and used in
micro-brewery trials at 0 and 3 months and evaluated using sensory analysis. Good correlation
was found between the biochemical predictors and sensory trial for the best quality malt and
beer. These parameters were therefore highly relevant for predicting shelf life potential, although
additional research is required to elucidate the effect of LTP1 and LOX-1 on each other during
the brewing process, since it seems that high LOX-1 concentrations could be leading to LTP1
decreases. With this study it is proposed that if more detailed protein or FAN characterisation is
used together with the screening of LOX-1, LTP1 and AROP, an more accurate shelf life
prediction, based on malt analysis, is possible and with the help of these parameters brewers can
simply blend malts accordingly. / AFRIKAANSE OPSOMMING: Die keuse van roumateriaal om 'n konstante eindproduk van goeie kwaliteit te lewer, was
nog altyd 'n uitdaging vir brouers wêreldwyd aangesien verskeie faktore 'n invloed het op die
kwaliteit van die produk. Alhoewel daar tans verskeie metodes vir moutanalise bestaan wat die
eindproduk–kwaliteit voorspel, is daar min wat werklik die eindproduk kwaliteit soos voorspel
deur moutanalise verteenwoordig. Hierdie navorsing fokus op die bepaling van mout-eienskappe
om twee van die belangrikste bierkwaliteitvereistes, naamlik skuim- en geurstabiliteit te
voorspel. Spesifieke biochemiese eienskappe in garsmout soos lipiedtransportproteien-1 (LTP1),
lipoksigenase-1 (LOX-1), antioksidant-antiradikaal potensiaal (AROP), vry aminostikstof (FAN)
is geïdentifiseer en gebruik in voorspelling van bierkwaliteit vanaf moutkarakter. Hierdie
biochemiese kwaliteit voorspellings is dan gekorreleer met die eindproduk soos ge-evalueer
d.m.v sensoriese analise op mikro-gebroude bier.
Omdat LTP1 soveel fasette in bier beïnvloed, het dit reeds 'n aanloklike studiefokus
geword. LTP1 word hoofsaaklik geassosieer met stabiele skuimkwaliteit in bier en tree op as 'n
lipiedmop (“lipid scavenger”). Die proteien speel teoreties ook 'n rol in die stabiliteit van bier
geur deur moontlik as 'n anti-oksidant op te tree. Laastens is LTP1 bekend vir sy antigis
aktiwiteit wat moontlik 'n negatiewe uitwerking op fermentasies het. Gedurende hierdie
navorsing is LTP1 en sy lipiedbinding isoform LTP1b suksesvol gesuiwer met 'n ekonomies en
eenvoudige 5-stap protokol. Beide isoforme het stabiliteit by temperature >90°C en meer
neutrale en basiese pH omgewings getoon. Alhoewel die voorheen gerapporteerde anti-oksidant
aktiwiteit vir LTP1 nie bevestig kon word nie, is daar wel gevind dat beide LTP1 en LTP1b,
LOX-1, wat verantwoordelik is vir die ensimatiese afbraak van linoleensuur na 2(E)-nonenal, se
aktiwiteit inhibeer. Dit is 'n unieke bevinding wat LTP1 ook koppel aan geurstabiliteit. LTP1 het
antigis aktiwiteit getoon, maar LTP1b het die meeste, indien nie alle antigis-aktiwiteit verloor.
Omdat die meeste van die LTP1's omgeskakel word na LTP1b's en geglikosileerde isoforme
tydens die brouproses, sal fermentasie nie beduidend beinvloed word nie, maar die skuim- en
geurstabiliteit sal steeds bevorder word deur die blote teenwoordigheid van die LTP1b. Geurverval van die finale verpakte produk is gedeeltelik a.g.v die ensimatiese produksie
van 2(E)-nonenal deur LOX-1 en die teenwoordigheid van vry suurstofradikaal spesies, beperkte
AROP en LTP1. Die ontwikkeling van twee 96-putjie mikroessaïs, gebasseer op die yster
oksidasie-xilenol oranje (FOX) essai vir die bepaling van LOX-1 en AROP, was suksesvol en
het goed vergelyk met reeds gevestigde essaïs. Die LOX-FOX en AROP-FOX mikroessaïs is
spesifiek ontwikkel vir die residente, hoë deurvloei vergelykende bepaling van LOX-1 en AROP
in mout en ander brouery-monsters.
Die AROP-FOX en LOX-FOX mikroessaïs en 'n paar gevestigde essaïs is gebruik om
moute te kategoriseer in die verskillende voorspelde kwaliteitsgroepe. Die biochemiese merkers
wat gemeet is het die volgende ingesluit: LOX aktiwiteit, LTP1 inhoud, FAN waardes, proteïen
konsentrasie en AROP. 'n Merkwaardige korrelasie (R2=0.93) is gevind tussen FAN/LOX en
LTP1/LOX wat ook ooreenstem met die waarneming dat LOX-1 aktiwiteit onderdruk word deur
LTP1 by verskeie konsentrasies. Hierdie korrelasies kan brouers help met optimale versnitting
van moute vir, nie net die hoogste kwaliteit eindproduk nie, maar ook vir fermentasie
voorspellings.
Om te bepaal of hierdie geselekteerde biochemiese merkers in mout die potensieële
raklewe van die eindproduk verteenwoordig, is sensoriese evaluerings uitgevoer. Drie gars-mout
kultivars is geselekteer o.g.v LOX-, AROP-, LTP1-, proteïen- en FAN-inhoud en gebruik in
mikro-brouery proewe en op 0 en 3 maande en is ge-evalueer deur sensoriese analise. Goeie
korrelasie is gevind tussen die biochemiese voorspellers en sensoriese evaluering vir die beste
kwaliteit mout en bier. Hierdie maatstawwe is daarom uiters relevant vir voorspelling van die
potensiele rakleeftyd, alhoewel addisionele navorsing nodig is om die effek van LTP1 en LOX-1
op mekaar gedurende die brouproses te bepaal. Dit blyk dat 'n hoë LOX-1 konsentrasies kan lei
tot 'n afname in LTP1. Met hierdie studie word dit voorstel dat, as meer gedetaileerde proteien
of FAN karakterisering saam met LOX-1, LTP1, en AROP analise uitgevoer word, 'n meer
akkurate raklewe voorspelling moontlik is en met behulp van hierdie parameters kan brouers
moute dienooreenkomstig versnit.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/86558 |
| Date | 04 1900 |
| Creators | Nieuwoudt, Melanie |
| Contributors | Rautenbach, Marina, Manley, Marena, Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 1 v. (various pagings) |
| Rights | Stellenbosch University |
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