Are your wines Protein Stable?
Protein instability (also known as heat imbalance) occurs when "dormant" proteins in wine become insoluble and fall out of solution. This effect is known as protein precipitation, and it happens, most infamously after the wines are bottled. Apart from being visually unattractive (seeming hazy or "foggy"), these proteins are benign to the user, and it does not change the organoleptic features of the wine. However, from the consumer's perspective seeing such impurities in a wine indeed alters its primary impression and, likely, they ignore any of the wine's more positive attributes. Fortunately, these latent molecules are easily detectable and can be eliminated with minimal effort indeed. But before we explore how to extract & banish these unpunctual organic molecules into the dark depths of our septic tanks, let's first consider their origins. Proteins in wine. Wine contains many proteins. Winemakers have recognized proteins in wine for a very long time indeed. In modern vinification practices, winemakers source proteids from other products and incorporate them into their craft. Isinglass, a protein obtained from fish swim bladder, is used to clarify musts/juices. Egg albumen, the protein from egg whites is used to remove bitter/astringent parts, and Mannoproteins found in yeast cell-walls are used to "sweeten" or condition near finish wine. Proteids from external sources, as mentioned above, does not result in protein instabilities, (if indeed they are used correctly). More notably are the grape-related constituents that influence the juice-protein content and, ultimately, residual proteins in the wine. Fruit maturity at harvest, disease occurrence, crop load, grape variety, fruit processing procedures, fruit chemistry, and fining may all affect grape-juice protein content. The two primary haze-forming proteins are chitinases and thaumatin. They are the direct result of the grapevine's viral and fungal defensive mechanisms. The plants synthesize these polypeptides in response to environmental stresses & pathogenic attacks. (O'Kennedy, 2015) states, "Over the centuries, the grapevine has adapted to such an extent, however, that these proteins may form even if there is no pathogen that stimulates it (constitutive expression)." Those proteins survive the winemaking process because they are highly immune to proteolysis (the breakdown of proteins) and the low pH characteristic of wines (R. B Ferreira et el, 2001). They persist in mediums that are low in phenolic content (i.e. white, rose & light red wines), yet rarely in wines that are rich in phenolics, for instance, heavier reds. The reason for this is that proteins have an affinity to phenols. In a medium that is abundant in phenols, proteins bind to them, become insoluble and fall out of suspension during the advanced stages of winemaking. Merely venturing to quantify the fraction of proteins that may conclude in haze formation is an inferior metric because it is the wine's temperature & it's chemistry that ultimately defines the individual protein's solubility (Dr. B. Zoeckiein, unknown). What exactly happens when the wines turn opaque? The protein balance of any wine is affected by its temperature, pH, and alcohol volume. The mechanism by which temperature fluctuation causes instability I'll explain as follow: Suppose you have a young Pinot gris in the vat. It has been fermented, racked and filtered clear, but not treated with a protein-removing fining agent. The probabilities are that this wine bears haze forming proteins. Initially, passive proteins are soluble, and the wine exhibits no turbidity. Latent polypeptides in the wine maintain a positive (electrostatic) charge; therefore, two similar particles would not bind. A change in seasonal weather occurs, thus increases the ambient warmth and inadvertently the storage temperature of your Pinot gris. Suddenly the Pinot gris turns cloudy. What happened? The fluctuation in temperature initiates a process call protein unfolding. When proteins unfold, their "shape" changes. This modification in form alters the molecules' physical characteristics and the way it interacts with other, similar molecules. Whereas, before the uncoiling, the proteins in questions repulsed one another; thereupon, they adhere. Larger aggregates grow insoluble and noticeable to the naked eye, which we then perceive as haze. A chemical shift of the wine's constitution can also initiate protein flocculation. Subject to the wine pH and the varietal characteristics, proteins in suspension may have a negative charge. Variations in bulk wine composition & its chemistry can transpose protein charge. For example, mixing two individually stable wines may result in an unstable composite. In the latter case, an alteration in pH or alcohol is probable catalysts. How can we reduce troublesome proteins from a young wine? In most young white & rosé wines, bentonite is a dependable fining agent. Bentonite is a montmorillonite clay, and it has a negative charge. Positively charged proteins adsorb to the clay, their coupled weight causes precipitation and elimination of the protein-bentonite aggregate. Bentonite doesn't adsorb to negatively charged proteins, in which case alternative fining agents may prove more beneficial. Gelatine is one such fining ingredient that responds with negatively charged colloids. By executing methodical bench trials, the winemaker can determine the measure of fining compound required to protect the wine. Bench trials comprise administering a fining material solution in incremental quantities to small, representative samples to ascertain the adequate additions required for the bulk-wine treatment. Bench trial setup for bentonite. You need the following: - x5 25ml test-tubes, - 100ml volumetric flask, - 25ml volumetric pipette, - 1ml graduated pipette - Magnetic stirrer & stir-bar. - Betonite (representative), - Water (representative), - Wine sample (representative), - Items required for testing protein stability (see below heat vs chemical tests). Method: Prepare a 2.5% bentonite solution the day before testing. To do this, weigh 2.5g of bentonite and fill half of the 100ml volumetric flask with representative water. Place the flask on the stirrer and include the stir-bar. Agitate at medium-fast and gradually add the 2.5g of bentonite until all of the granules have dissolved. Remove the stir bar and top-up the flask to 100ml. Return the stir-bar, stir for an hour and let it rest for at least 8 hours. Pipette 25ml of unfiltered wine sample into each test-tube and mark them (a) 50g/hl, (b) 100g/hl, (c)150g/hl, (d) 200g/hl, and (e) 250g/hl respectivley. Pipette (a) 0.5ml, (b) 1.0ml, (c) 1.5ml, (d) 2.0ml and (e) 2.5ml 2.5% bentonite solution respectively into each test-tube. Invert the test-tube three times to mix the sample amply. Let the sample sit for 20 minutes while the bentonite descends. You are now ready to test the protein stability of each sample. Testing for protein stability. There are two analyses that the winemaker can function to determine if, indeed, the wine or bench trial sample is heat stable. Both tests involve hastening the inevitable insolubility of inactive proteins. A sort of acceleration of protein precipitation. The methods are the (1) heat and (2) chemical precipitation test. 1) The heat test: You can conduct the heat test with or without a turbidity-meter; however, the former method presents more certainty. You need the following articles if testing with a flashlight: - 2 x 25ml test tubes, - a warm water bath, - a syringe filter, - 0.45-micron filter sheets, - a 50ml syringe, - and a small flashlight If you are using a turbidity-meter to accompany this test, you need: - 2 x 25ml test tubes, - Turbidity-meter vails, - Turbidity-meter, - Turbidity-meter calibrating solutions (4 point), - a warm water bath, - a syringe-filter, - 0.45-micron filter sheets, - a 50ml syringe. Method: Preheat the warm water bath to 80 degrees centigrade. Collect a 20-25ml wine sample that is representative of the batch or bench trial sample. If testing bulk-wine, the sample must come from a single vessel that has been blended and homogenized at least a week ere testing. Using the syringe and syringe-filter, pass the wine sample through the 0.45-micron filter into the two 25ml test tubes. The samples in both test tubes should be brilliantly clear. If the samples are not transparent, then either the filter sheet was compromised, or the syringe-filter gasket failed. In either scenario, you have to redo this step with a new 0.45-micron filter-sheet. Place one of the test tubes comprising the filtered wine sample into the pre-heated water bath and leave it in for a minimum of 3 hours at 80°C. Omit the other sample and keep it at an ambient 20°C. If you are employing a turbidity-meter to conduct this test, calibrate the meter. After a successful four-point calibration, use the sample that is not warming and fill the turbidity-meter vail with it. This sample must be room temperature. Take a turbidity reading and record the value as sample #1 (S1). The reading must be below 2NTUs. If the reading is not below 2NTUs, something is awry. It might be that the filter failed &releasing particles, your sample is too chilled, and condensation is forming on the vail, or the test tube is scratched/stained. After three hours in the warm water bath, transfer the test-tub and let it cool for 3 hours. The samples are ready to be examined. Testing without turbidity meter: Using the flashlight, cast light through the acclimatized sample. If you observe a continuous light bean crossing through the sample or if you notice any cloudiness, your sample is heat-unstable. Conversely, if the light enters & exits the tube with no beam, and the sample is brilliantly clear, then your sample is heat-stable. Experimentation with a turbidity-meter: Transfer acclimatized wine into a clean, scratch-free turbidity-meter vail. Take reading and record the NTU value as sample #2. Subtract the value of sample #2 (S2) from sample #1 ( S2 - S1). If the subtracted value is less than 2, your wine is heat-stable. 2) Chemical precipitation test: This experiment is more expeditious than the heat-test. It demands all the same equipment save for the warm water-bath. Additionally, you need a commercially prepared chemical reagent like Bentotest® or Bentocheck solution. You need the following items if testing with a flashlight: - 1 x 25ml test-tube, - a syringe filter, - 0.45-micron filter-sheets, - a 50ml syringe, - and a small flashlight, - reagent (Bentocheck/Bentotest®). If you are utilizing a turbidity-meter to conduct this test, you need: - 1 x 25ml test tubes, - Turbidity-meter vail, - Turbidity-meter, - Turbidity-meter calibrating solutions (4 point), - a syringe-filter, - 0.45-micron filter-sheets, - a 50ml syringe, - reagent (Bentocheck/Bentotest®). Method: Filter 25ml of a representative wine sample (see heat-test method) through a 0.45-micron filter-sheet. The sample must be brilliantly clear. When using a flashlight: Add the specified measure of a chemical reagent to the sample and let it sit for two minutes. Using the flashlight, cast light through the acclimatized sample. If you observe a continuous light bean passing through the sample or if you notice any haze, your sample harbours haze forming proteins. If the light enters & exits the tube with no beam, and the sample is brightly clear, then your sample is protein stable. When using a turbidity-meter: Calibrate the turbidity meter & transfer a volume of filtered sample into the turbidity-meter vail. Take an NTU reading and record the value (V1). Using the same sample, add a specified amount of chemical reagent to the vail's contents and let it sit for two minutes. Take a second NTU reading and record the value (V2). Subtract V2 from V1 (V2-V1). If the difference is less than 2, the sample is stable. If the subtracted value is equal to or more than 2, then further fining of your bulk-wine is necessary. References: 1. Dr. B. Zoeckiein & professor Emeritus (Unknown). Wine proteins & protein stability: Virginia Tech. 2. K. O' Kennedy (2015). The cause of protein instability in white wine: Oenology research, Winetech Technical. 3. R. B. Ferreira et al. (2001). The wine proteins: Trends in food science & technology, Volume 12, Issue 7, July 2001, Pages 230-239