As discussed in Importance of Nitrogen in Winemaking, proteins represent a significant portion of wine's total nitrogen content. In juice/must, proteins usually represent less than 10% of total nitrogen content. In wine, levels are far higher and reach up to 40%. There are several viticultural and enological factors effecting wine protein content.
In the Vineyard
Proteins synthesized during berry development account for approximately half of total wine protein. After veraison, protein synthesis in grapes occurs at a similar rate as sugar level increase. Higher protein levels are associated with:
- Grapes sourced from warmer growing regions.
- Grapes grown at lower crop levels.
- Grapes harvest at higher maturity.
- Grapes harvested mechanically.
In the Winery
Winemakers tend to give more thought to wine protein levels during maturation of wines than earlier in the process. It is always important to consider down-the-line effects of any winemaking activity. Indeed, pre-fermentation processes have a larger impact on protein levels in the resultant wine than many winemakers realize.
- Whole cluster pressing will have lower protein uptake than grapes that are destemmed first. The logic behind this is grape stems act in limiting protein diffusion.
- Skin contact prior to pressing will typically increase protein concentration in the juice, though uptake and length of contact is a variety-specific relationship.
- Solids separation removes large amounts of juice nitrogen content, including proteins. The amount is dependent on the type of solids separation (settling vs. floatation) and the use of fining agents. For example, bentonite may remove 50% of total nitrogen content.
A small amount of protein is produced by yeast during fermentation, but this tends to not effect overall wine protein significantly. Post-fermentation processes have negative and positive effects regarding wine protein levels
- Extended lees contact and lees stirring increases wine proteins. This primarily due to yeast autolysis, the process of cell breakdown and destruction by its own enzymes.
- Maturation in oak barrels or tanks commonly decreases wine proteins, which will react with oak phenols and precipitate out of solution.
- Fining with benotonite after fermentation will remove large amount of nitrogen composed of over half of the protein.
- Fortification typically produces significant lees precipitation, containing a large quantity of proteinaceous lees.
The solubility of wine proteins is highly dependent on the ionic strength of the partiularly protein and the wine's alcohol concentration, temperature, and the pH.
An interesting relationship exists between the isoelectric point of proteins and wine pH. The isoelectric point is where positive and negative charges are equal: proteins have a negative charge when pH is above the isoelectric point, and vice versa. Typical wine pH is very close to its proteins' isoelectric point, when proteins are least soluble. This relationship makes removing unstable proteins tricky, requiring the correct type and rates of fining agents.
Winemakers are primarily concerned with proteins in regards to wine stability, which is still largely undetermined. A long list of factors, from grape variety and climate to protein molecular size and interactions with other wine components, effect the exact type and concentration of proteins in wine.
The phenomenon known as protein haze occurs when soluble proteins precipitate in bottled wines. Protein haze makes the wine appear cloudy or highly turbid, considered a defect by most producers and consumers. It is likely composed of several compounds: soluble proteins, polysaccharides, insoluble protein-polyphenol complexes, and metal-protein complexes (proteins act as nuclei for soluble iron, copper, etc.).
Wines with high phenol concentrations will rarely have issues with protein haze since phenols will react and remove sufficient amounts of protein to make the wine stable. This is the reason that protein levels are far more of a concern in white grape varieties, since most red varieties have sufficient phenol levels to stabilize proteins. Protein levels and color instability is highly correlated in red varieties such as Pinot Noir. Due to phenol content, oak maturation also increases protein stability in wines compared to those held in stainless steel vessels.
As the largest source of wine protein, grapes are also the largest source of protein instability. It is believed that proteins originating from yeast do not pose issues with stability.
Evaluating Protein Stability
Evaluation of protein stability should only be conducted after all other winemaking procedures have been completed. In other words, just prior to bottling. Any change to the balance of temperature, pH, and alcohol content from processes such as acidification, malolactic fermentation, fortification, and cold stabilization can lead to precipitation of wine protein complexes.
Protein stability evaluation is not an exact science, and thus involves predictive techniques. These can include heat testing, heat-and-cold testing, and bentonite testing. Most winemakers err on the side of caution, resulting in wines that will be over-fined to ensure stability in their finished product.
The following is a method common to many wineries that I have used with good success.
- Filter sample through a sterile filter (0.45 µm). If the sample is still cloudy (i.e. from tank sitting on lees), you may need to centrifuge it prior to filtering.
- Fill one test tube with filtered sample as a control.
- Fill a second test tube and heat to 80° C (180° F) for two hours (many wineries like to do so for six hours instead, while others heat at lower temperature for up to 24 hours; I believe two hours at this temperature sufficiently precipitate proteins).
- After sample is heated, allow it to return to room temperature. Giving the sample several hours or overnight is advisable to allow precipitation; if this is to be done, refrigerate both the control and heated sample but make sure to allow both to return to room temperature prior to reading.
- Compare the two samples. Ideally, this should be completed with a turbidity meter (nephelometer); protein stable samples are deemed those with an NTU <1.0. Visual comparison with a bright light can be completed in lieu of a turbidity meter but my not adequately assess stability.
If a haze does appear, the wine should be fined to remove excess proteins. There are several different types of bentonite available today, and most wineries have their favorite. A bentonite fining trial should be conducted by preparing samples at varying addition rates (addition rates will vary depending on varietal, location, type of bentonite, etc.).
Then, complete the heat stability test again. I often find that bentonite fining in a controlled environment like this results in over-fined wine in the cellar, so I advise choosing a slightly lower rate than determined in the trial. Of course, heat stability should be re-tested once bentonite fining is completed.