LTP1 and LOX-1 in barley malt and their role in beer production and quality

Nieuwoudt, Melanie (2014-04)

Thesis (PhD)--Stellenbosch University, 2014.

Thesis

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.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/86558
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