Parameters involved in the enzymatic deconstruction of the Wine Grape cell wall matrix during winemaking

Gao, Yu (2016-03)

Thesis (PhD)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Commercial enzyme preparations, consisting of pectinases and glucanases, are commonly used in the wine industry to; (1) enhance the extraction of beneficial compounds (e.g. anthocyanins, tannins, aroma etc.) from the grape berry, (2) to facilitate the clarification of the wine before the filtration, and (3) to maximize juice yield for alcoholic fermentation. All three factors are important in improving quality parameters (e.g. body, aroma, ageing potential) and market value for winemakers. The efficacy of any enzymes, including wine enzymes is dependent on their ability to target specific glycan linkages for catalysis, and thereby deconstruct polymeric architecture. However, in the case of grapes, very little information is known on grape berry cell walls, that can assist in a rational enzyme mixture design strategy for customizable applications (e.g. enhanced colour). This is partly due to the complex nature of fruit cell walls, factors such as grape maturity, inter‐vineyard variability in ripeness levels at harvest, potential cultivar differences and the crude nature of enzyme mixtures used by the wine industry. New tools are needed to understand cell wall architecture, and here the use of Carbohydrate Microarray Polymer Profiling (CoMPP) technology, which combines glycan microarrays with sets of characterised monoclonal antibodies (mAbs) and carbohydrate binding modules (CBMs) for plant cell wall polymer epitopes detection. CoMPP is extensively used with multivariate data analysis methods in this thesis to evaluate the complex nature of the grape cell walls and how enzymes are able to deconstruct them under winemaking conditions. This study consists of three parts focusing on a Cabernet Sauvignon vineyard: (1) The validation of cell wall profiling tools combined with fractionation approaches on berry pomace cell walls isolated from wine fermentations. This is to confirm the utility of the approach in the winemaking conditions, and provide a baseline reference dataset for later studies. The study revealed for that grape pomace consists of two main fractions: (i) a partially methyl‐esterified pectin‐rich outer layer and (ii) a highly methyl‐esterified pectin‐rich layer which ‘coats’ an inner layer of xyloglucan‐cellulose rich skin cells. (2) A study focusing on the inter‐vineyard variation of grape berry cell walls collected at harvest using a panel based sampling design. This was combined with how commercial enzyme preparations influenced the potential inter‐vineyard variation in harvested grape cell walls that were brought into the winemaking process. Here the study showed that the enzymes performed efficient de‐pectination, without de‐esterification, and significantly reduced cell wall variability in pomace and wines from treated panels. (3) In order to study the grape berry cell wall deconstruction process in more detail, the use of pure recombinant enzymes (singly and in combinations), were used in an extensive winemaking study for the first time that we are aware of. The use of cell wall profiling tools revealed: (i) that pectin lyase appears to be a core enzyme in probably many enzyme preparations, because it results in effective de‐pectination without de‐esterification (ii) other enzyme combinations that combine only endo‐polygalacturonase activity with de‐esterification appear to only unravel the walls and not degrade effectively. This study, plus the contexts provided by the first two, permitted us to develop a hypothetical model which illustrates the cell wall structures in different tissue layers of the grape berry. This new grape berry cell wall model apart from extending our scientific knowledge in this area, will now permit hypothesis testing in, fruit development, plant‐pathogen interactions, as well as helping designing the tailored enzymes for use in different winemaking scenarios.

AFRIKAANSE OPSOMMING: Kommersiële ensiembereidings, wat bestaan uit pektinases en glukanases, word algemeen gebruik in die wynbedryf om; (1) die ekstraksie van voordelige verbindings(bv. antosianiene, tanniene, aroma ens.) vanuit die druifkorrel te verbeter, (2) om die verheldering van wyn voor filtrasie te fasiliteer, en (3) die sap opbrengs vir alkoholiese fermentasie te verhoog. Al drie faktore is belangrik om kwaliteitseienskappe (bv.volheid, aroma, verouderingspotensiaal)en markwaarde vir wynmakers te verbeter. Die effektiwiteit van enige ensieme, insluitende wynensieme, is afhanklik van hulle vermoë om spesifieke glukaanverbindings te teiken vir katalise, en daardeur die polimeriese argitektuur af te breek. In die geval van druiwe is daar egter baie min inligting beskikbaaroor die druifkorrel selwande wat kan help met die ontwikkelingstrategie vir ‘n rasionele ensiemmengsel vir pasgemaaktegebruike (bv. kleurverbetering). Dit is deels die gevolg van die komplekse samestelling van vrugte selwande, faktore soos druif rypheid, verskille in rypheid binne ‘n spesifieke wingerd tydens parstyd, potensiële kultivar verskille en die ruwe aard van ensiembereidings wat in die wynbedryf gebruik word. Nuwe metodes word benodig om die selwandargitektuur te verstaan, en hier is die gebruik van dieomvattende mikrorooster polimeer profielbepalings tegnologie (Comprehensive Microarray Polymer Profiling; CoMPP), wat glukaan mikroroosterskombineer met gekarakteriseerde monoklonale teenliggaampies (mAbs) en koolhidraatbindende modules (CBMs) vir plant selwand polimeer epitope, baie waardevol.In hierdie tesis word CoMPP breedvoerig gebruik,tesame met multiveranderlike statistiese data analise metodes, om die komplekse aard van die druifkorrel selwand te evalueeer asook die vermoë van ensieme om dit te dekonstrueer onder wynmaak toestande. Die studie bestaan uit drie dele wat fokus op 'nCabernet Sauvignon wingerd: (1) Die bevestigingvan die selwand profielbepalingsmetodes tesame met fraksioneringstegnieke opselwande afkomstig vangepersde druiwedoppetydens die fermentasieproses. Hiermee is die nuttigheid van die benadering onder wynmaaktoestandebevestig, en ‘n basislyn verwysingsdatastel geskep vir verderestudies. Hierdie studie het gewys dat gepersde druiwedoppe bestaan uit twee hoof fraksies: (i) ‘n gedeeltelik metiel‐veresterde pektienryke buitenste laag en (ii) ‘n hoogs metiel‐veresterde pektienryke laag wat ‘n binneste laag van xiloglukaan‐ en selluloosryke dopselle bedek. (2) ‘n Studie wat fokus op die binne‐wingerd variasie van druifkorrel selwandsamestelling,wat bepaal is op driuwe wat versamel is tydens oestyd volgens ‘n paneelgebaseerde steekproefontwerp. Dit is gekombineer met 'n ondersoek na die invloed van kommersiële ensiembereidingsop die moontlikebinne‐wingerd variasie tydens wynbereiding. Die studie het gewys dat die ensieme effektiewe depektienase uitgevoer het, sonder de‐esterifikasie, en die variasie in selwande van gepersde druiwedoppe en wyn, vanaf behandelde panele, is aansienklik minder gemaak. (3) Gesuiwerde rekombinante ensieme (alleenof in kombinasie) is, sover bekend,vir die eerste keergebruik in ‘n breedvoerigewynmaakstudie om sodoende die druifkorrelselwand dekonstruksie proses in meer detail te bestudeer. Die selwand profielbepalings metodes het gewys: (i) dat pektien liase skynbaar ‘n kernensiem in baie ensiembereidings is, omdat dit effektiewe depektinase veroorsaak sonderde‐esterifikasie (ii) ander ensiemkombinasies, bestaande uit endo‐poligalakturonase en de‐esterifikasie aktiwiteite, kan skynbaar slegs die selwande ontrafel, maar nie effektiewe degradasie veroorsaak nie. Hierdie studie, asook die konteks wat deur die eerste twee verskaf is, laat ons toe om ‘n hipotetiese model te ontwikkel wat die selwandstrukture in die verskillende weefsellae van die druifkorrel aandui. Hierdie nuwe druifkorrel selwandmodel verbreed ons wetenskaplike kennis in die veld en maak hipotesetoetsing moontlik ten opsigte van vrugontwikkeling, plant‐patogeen interaksies, en help met die ontwikkeling van ontwerpersensieme vir gebruik onder verskillende wynmaak toestande.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98298
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