Controlling Protein Stability (Clearing haze) Flashcards
What is Chitinases
An enzyme that breaks down chitin (a polysaccharide) but is a protein
What is unfolding
Unfolding is the process of the proteins unfolding leading to flocculation and precipitation.
What is flocculation?
a process by which colloidal particles come out of suspension to sediment under the form of floc or flake, either spontaneously or due to the addition of a clarifying agent.
Role of sulfur in protein stability
Disulphide bonds are important because they are a direct bond between two sulfur compounds. This can lead to protein that is more closed and folded due to the strong bond.
Isoelectric point
“iso” means the same, so when the discussing molecules, it can mean that they have the same charge. The charge is at zero when at the isoelectric point (same number of negative and positive charges.
3 instances when proteins are more stable
- With more disulphide bonds
- Within their optimal temperature ranges
- In conditions close to their isoelectric point
What is the isoelectic point range
4.5-7 pH
For protein stability do we want higher or lower pH
Lower pH is less stable for proteins so we want a higher pH
Temperature and protein stability
Lower temperatures are more stable
Which proteins are more and less heat resistant?
Chitinases and TLP are less heat resistant and LTP and invertases are more heat resistant
2 key things to remember with pH
- Melt temperature increases with pH
- Proteins are more stable at higher pH
What is ionic strength?
Minerality of the wine
Protein aggravation is driven by 2 things…
- Hydrophobic interactions
- Electrostatic interactions
More proteins =
usually more haze
Polysaccharides in regard to protein aggravation
They are always positive and favorable for stabilization
and
They contribute to maintain more hydrated and sulphated compounds
Protein unfolding is influenced by (2)
- protein composition
- storage conditions
Protein unfolding is promoted by (3)
- Higher temperature
- Lower pH
- Surface charge changes
Protein Aggregates are influenced by (2)
- Wine matrix components
- Storage conditions
Protein Aggregates are promoted by (2)
- More Chitinases, ions, acidity, phenolics, SO2
- Less polysaccharides, ethanol
Haze forming proteins (2)
and what they can depend on (2)
- Always present in grape skins
- Always considered PR proteins
- Viticultural factors
- Winemaking factors
Grape varieties that are more/less stable
High stability: Chard, Riesling, Glera
Medium: Pinot Grigio, sylvaner
Low stability: SB, Muscat, Gewurtz, Semillon
Aroma precursors are antimicrobial and we find more PRP in grapes with more aroma precursors
When does PRP formation start in the grape
Veraison
Is PRP more or less accumulated in stressed grapes
More accumulated (except for botrytis)
Water stress
Causes smaller berries and more PRP
Harvesting and formation of PRP
Hand picked grapes usually have less PRP than mechanized
Grape skin contact of formation of PRP
No maceration and free run juice will have less PRP than extended maceration and heavy pressing
How do fermentation conditions affect PRP
The longer ferm occurs the more the PRP decreases however it decreases more with lower pH and decreases even more with lower pH and higher ferm temperatures
Less than 50% decrease in protein with fermentation
Bentonite fining and PRP
Bentonite greatly reduces PRP
How can we check to see if there will be haze in the wine
Heat test. Heat the wine, then cool the wine and then measure
Trends with heat and heat test
Haze increases with heating time during the heat test
When does hazing actually occur
Hazing occurs during the cooling, and it increases with cooler temperatures
Critical threshold in wine
100mg/L of PRP
New Method heat test
Heat to 80 Degrees C for 2 hours, cool to 20 degrees C for 3 hours, there is no equilibrium time and total turnaround time is only 5 hours compared to 24 with the original method and 20 with the revised
What do we measure with the heat test
Turbidity. If the change in Turbidity is less than 2 NTU we have a protein stable wine
Does more haze = more bentonite
NO
Does more protein mean more bentonite?
Sometimes
Does the heat test predict real world haze
It way over compensates and predicts more than actual
Bentonite pros (5)
- Cheap
- Readily available
- Easy to use
- Non-toxic
- Effective
4 mechanisms of bentonite action
- Have a net negative charge
- Contains positive ions e.g. sodium or calcium
- Protein binds to bentonite and displaces smaller ions
- Proteins precipitate as bentonite lees
3 cons of Bentonite
- Wine lost as bentonite lees
- Non-selective: can remove aroma compounds
- Extra processing steps
How to reduce bentonite use?
Choose the right type of bentonite: different bentonites have different protein absorbing capacities.
Three things to consider:
1. Settling time
2. Dose
3. Lees Volume
Sodium based bentonite vs Calcium based
Na: Cheaper, use less, produces more lees
Ca: Less lees, use more, may promote tartrate instability
What can adding too much bentonite result in
It can result in pigment stripping (strip phenolics)
1 recycled alternative to bentonite
GSP Grape seed proteins. Grape seeds are roasted at 180 degrees C for 10 mins
Which bentonite is more reliable for clarifying the must
Calcium. There is a risk of calcium instability. Protein stability increases with Ca bentonite
Should we do one large dose of bentonite
NO. Its best to do a small dose of Ca bentonite for clarification after ferm, then some Na bentonite for fining
Role of GSP
For fining. It is able to remove chitinases. It very slightly changes the sugar, but can greatly increase the pH
Can GSP affect esters?
Yes, the seeds are full of fatty acids, which are brought out via ethanol and are then made into esters by yeast .
Can we use GSP alone?
No, as they are not purified they cannot remove hazing proteins alone, GSP is good to use when you want to use less bentonite
GSP benefits (3)
- can be a viable solution to reduce use of bentonite
- should be used with wine not requiring a large amount of fining
- May be a good sustainable and economic alternative
