Enzymes in Wine Production Flashcards
(25 cards)
What are the main sources of the commercial winemaking enzymes?
- Most are from Aspergillus niger
- Trichoderma spp.
- egg white (lysozyme)
What regulatory bodies approve winemaking enzymes for use?
- EU/OIV International organisation of vine and wine
- US FDA
all are GRAS generally regarded as safe
What are the four main pectolytic enzymes?
- Pectin esterase
- Polygalacturonase
- Pectin lyase
- Pectate lyase
- What is the action of Pectin esterase?
De-esterifies galacturonic acid units. i.e. cleaves off methyl group. Produces Methanol from this reaction.
- What is the action of Polygalacturonase (PG)?
Cleaves (hydrolyses) links between non-esterified galacturonic acid units. consumes H2O
- What is the action of Pectin Lyase?
breaks NON-ESTERIFIED galacturonic acid units into single units by conducting a β-elimination (results in a new pi bond on adjacent atoms).
- What is the action of Pectate Lyase?
Cleaves ESTERIFIED pectin into single galacturonic acid units via β-elimination
Why are Pectinase enzymes used in white wine production?
- Maximise juice extraction by breaking down pectin in grape cell walls. increases free run.
- Aid in clarification and filterability. Pectins lead to premature filter clogging.
What are the benefits of pectolytic enzymes in red winemaking?
- increased colour and tannin extraction.
- increased free run
- easier settling
What is Cinnamyl Esterase and where is it from?
-from A. niger. can be a contaminant of pectolytic enzyme preparations.
-Performs the first step in the formation of volatile phenols- cleavage of the ester linkage between tartaric acid and hydroxycinnamic acid.
POF+ strains can then do the 2nd step- decarboxylation of the hydroxycinnamic acids.
This yields the precursor that Brettanomyces can reduce to 4EP or 4EG.
What is β-glucan and what is its source?
Why is this something we may want to break down?
- High molecular Wt polysaccharide. β-1-3 linkages along main chain and β-1-6 in side chains.
- Botrytis cinerea produces it during growth.
- Causes filterability issues in botrytized wines and may prevent sedimentation of fine hazes.
What can be done to remove β-glucan? when can this be done?
β-glucanase preparations can be added to wine.
They generally contain a β-1-3-glucosidase that can break up individual monomers on the main chain and a β-1-6-glucosidase that can break monomers off the side chains.
This is usually added during clarification or post-fermentation as they can inhibit yeast growth and are inhibited by tannin binding in red wine.
Why might we add β-glycosidase enzyme preparations to wine?
- A large amount of flavour and aroma molecules are glycosidically bound and have no sensory properties. e.g. monoterpenes, sesquiterpenes, norisoprenoids. releasing them enhances varietal aroma
- usually a β link to a glucose residue which is α linked to another sugar residue such as rhamnose or arabinose.
- β-glycosidase enzyme preparations release these flavour and aroma molecules.
β-glycosidase enzyme preparations release glycosidically bound flavour and aroma molecules (for example linalool. What are the steps to do this?
When are these enzymes added and what inhibits them?
- an α-arabinosidase or α-rhamnosidase cleaves the 1-6 linkage between the 2 sugar residues.
- β-glycosidase cleave the β linkage, releasing the flavour molecule (e.g. linalool) from the glucose unit.
These are added towards the end of fermentation as glucose inhibits the action of these enzymes.
What use for Glucose Oxidase is currently being investigated?
What are the drawbacks?
-lowering the alcohol conc of wine by converting glucose to gluconic acid (unfermentable).
- raises the acidity of wine. usually treated wines are used for blending.
- produces hydrogen peroxide so a catalase enzyme is needed to convert this to water to reduce oxidation.
What use for Acid Protease is currently being investigated?
Stabilizing wines against protein haze. replacing bentonite which removes flavour and aroma (also racking losses).
Hydrolyses linkages between amino acids, breaking down structure and colloidal nature.
Commercial scales of the enzyme not yet isolated.
What use of Urease is currently being investigated?
- Breakdown of Urea into ammonium and CO2.
- Urea formed degradation of arginine.
- Diverts urea away from ethyl carbamate pathway (a carcinogen).
What is the use of lysozyme?
-Prevents the growth of gram-positive organisms as it can break down peptidoglycan cell walls. Therefore is beneficial in inhibiting MLF and spoilage organisms such as Lactobacillus and Pediococcus, particularly in low acid must where SO2 is not greatly effective.
Particularly useful for stuck ferments. The activity of lysozyme diminishes after several days.
What use are oxidase and tannase enzymes being investigated for?
- Oxidase (such as laccase from B. cinerea) for the reduction of phenolic compounds.
- Tannase to reduce tannin. neither of these enzymes is currently in commercial use
What enzymes are produced by Botrytis cinerea?
- Laccase- phenolic oxidation. converts phenolic compounds to quinones that polymerise to form brown pigments. Resistant to low pH, SO2 and alcohol. Not easily removed with bentonite.
- Pectolytic enzymes and proteases, specifically pectinesterase and polygalacturonase. Pectin degradation products are β-glucan (see earlier card) and galacturonic acid. Galacturonic acid can be converted to calcium mucate (via mucic acid) which is an insoluble precipitate.
- Protease- linked to the production of pectolytic enzymes.
What are the enzymes that yeast produces during fermentation? What is the effect of their production?
- β-glycosidase. Hydrolyses link between sugar and flavour/aroma molecule. Releases sugar for yeast. The expression is limited as this is inhibited by glucose. Other strains such as Kloeckera and Hansenula produce higher amounts.
- extracellular proteases. Future upregulation may play a role in preventing protein haze. Production of these enzymes is generally weak. more important in sur lies ageing.
Describe briefly the process of yeast autolysis
Following death of the cell, hydrolytic enzymes that would usually be separate in the cell by internal membranes come into contact with other intracellular components as these membranes denature.
These hydrolytic/autolytic enzymes degrade cellular macromolecules and release breakdown products into the wine.
These breakdown products include amino acids, oligosaccharides, glycerides, fatty acids, and nucleotides.
What are the autolytic enzymatic activities during yeast autolysis?
- Proteases- break down proteins
- β- glucanases- breakdown polysaccharides into oligosaccharides.
- Lipases- breakdown lipids into diglycerides, monoglycerides and fatty acids.
- Nucleases- Breakdown nucleic acids into nucleotides and oligonucleotides.
How do the different autolytic breakdown products of yeast impact on wine aroma and flavour?
- Proteins and polysaccharides- foam formation, smaller bubble size and bead persistence in sparkling wine. (Primary breakdown product)
- Amino acids may go on to form compounds that contribute to aroma (Secondary breakdown product).
- Fatty acids (from lipid breakdown) may undergo esterification with ethanol to yield volatile ethyl esters that impact on aroma (Secondary breakdown product).
