Microbes in the wine bottle: Sources, manifestation, and suppression tactics

If a bottled wine is microbially unstable, it most likely will reveal itself to the consumer in a number of unpleasant ways. In a Practical Winery and Vineyard Journal article (Monitoring microbes during cellaring/bottling, practicalwinery.com, January/February 2010), Lisa Van de Water identified the danger signals for microbes in the bottle as follows:

• Change in color, aroma, or flavor
• CO₂ production
• Clouding.

Van de Water says that to assess the microbial stability of a wine before bottling, four questions need to be asked:

• Does the wine have residual fermentable sugar?
• Does the wine have residual malic acid?
• Does the wine have Pediococcus?
• Does the wine have Brett.

If there is residual fermentable sugar in the wine, the problem could either be a re-fermentation of the sugar by yeasts or metabolization of the sugars by lactic acid bacteria (LAB). The author recommends filtering 250 ml of wine for yeast and 100 ml for bacteria and culturing the membranes to assess bottle stability. PCR tests can help monitor bottled wines for microbes that are at high levels. As it relates to the question of residual malic acid, Ms. Van de Water says that LAB can cause a number of problems if they metabolize residual malic acid in the bottle. Wines with residual malic acid should be filtered with a 0.45µ membrane. 

The most common microbes to manifest in the bottle are presented in the table below.

Microbe	“Danger” Metric	Manifestation in Bottle
S. cerevisiae	5 - 10 cells/bottle; > 2 g/l RS;  cannot grow without sugar	Cork-pushing carbonation at 1400 mg/l CO₂; perceivable spritz at 800 mg/l CO₂ (would require 1.6 g/l refermentable fructose or glucose); visible haze above 1000 mg/l RS
Zygosaccharomyces	1 cell/bottle; cannot grow without sugar
Surface film yeasts	Cannot grow after 1 week in bottle
Dekkera/Brettanomyces	1 to 50/ml; 0.2 g/l RS	100 mg/l can lead to visible Brett yeast haze; Small amount of CO₂; horse sweat and Band Aid® odors; bitter, metallic finish
Oenococcus	500/ml if malic acid present; ok if no malic acid; 0.2 g/l RS	Cork-pushing carbonation at 1400 mg/l CO₂; perceivable spritz at 800 mg/l CO₂ (would require MLF of 2.4 g/l malic acid); a rise in pH; cloudiness, a fine sediment
Lactobacillus	If a substrate is present; 0.2 g/l RS	Cork-pushing carbonation at 1400 mg/l CO₂; perceivable spritz at 800 mg/l CO₂ (would require MLF of 2.4 g/l malic acid); a rise in pH; cloudiness; a fine sediment
Pediococcus	Any visible under a microscope; 0.2 g/l RS	Cork-pushing carbonation at 1400 mg/l CO₂; perceivable spritz at 800 mg/l CO₂ (would require MLF of 2.4 g/l malic acid); biogenic amines (sometimes); unpleasant aftertaste (sometimes); cloudiness; a fine sediment
Acetobacter	Present only if the closure is compromised

Sources: Lisa Van de Water, Monitoring microbes during cellaring/bottling, Practical Winery and Vineyard Journal, practicalwinery.com, January/February 2010); Christian Butzke, Preventing Refermentation, extension.purdue.edu.

As it relates to the question of what causes microbial instability to manifest in the bottle, the answer is “a cascade of events.” First, the environment has to be hosting microbes. The sources of the infection could be (i) spoilage yeasts transiting from the vineyard in sour-rot grapes; (ii) Brettanomyces bruxellensis,  which can make "the winery itself a primary habitat surviving in the walls ... interior surfaces of presses and fermentation tanks, or on the wood of barrels"; or (iii) residual sugar or malic acid, which could serve as the substrate for fermentative action by S. cerevisiae or LAB. The second event, which builds on the first, is an insufficiently sanitary regime to allow defeat/suppression of the microbes prior to bottling. And third,  a non-robust testing regime that allows the bottling and sale of wines that are microbially unstable. 

The following steps are recommended in order to minimize the opportunities for bottle-manifested microbial instability :

1. Test the wines in the cellar to determine exactly what types of microbes, if any, are resident in the wine. To detect Brettanomyces, Ms. Van de Water recommends culturing the wine on media containing 50 ppm of the antibiotic cycloheximide (to inhibit growth of other yeasts) and, if present, the culture will manifest “white, hemispherical colonies in three to seven days.” The Brettanomyces culture also produces a strong acetic acid smell. Levels of 100 CFUs/ml is cause for concern. Conduct enzymatic tests to determine whether fructose and glucose are above the level of 0.5 g/l. The ratio of glucose to fructose is important so the sugars should be tested separately (According to Ms. Van de Water, “Saccharomyces is reluctant to use fructose if the glucose/fructose ratio is less than 1:6 to 1:10). Use enzymatic tests to determine whether LAB is present in the wine. Malic acid levels should come in below 30 mg/l. If the wine is shown to be microbially stable upon completion of the tests, then go to Step 3. If not, go to Step 2.
2. Filter the wines with a .45 µ membrane
3. Take preventative steps to combat oxidation. According to Enology Note, “excessive oxygen pickup during filtration and bottling reduces the sulfur dioxide concentration notably.” Preventative steps to combat oxidation include: SO₂ additions during or just prior to wine movement, nitrogen blanketing; CO₂ sparging; and the flushing of lines and receiving tanks.

In addition to the steps above, the following actions should be taken to insure that the wine retains its stability during the bottling process (Lansing, Managing Bottling Operations, winebusiness.com, May 2011): 

• Purge the cellar tank and blanket with CO₂ before filling with transferred wine
• When releasing wine from cellar tanks to bottling line filters, verify that the tank wine meets target parameters for alcohol %, DO, CO₂, and other chemical metrics
• Verify that in-line wine filtration upstream of the line filter is in place and meets target parameters for sterile wine filtration
• Perform clean-in-place for wine lines and follow at least every 72 hours to ensure microbial stability
• Purge filter bowls with nitrogen to minimize DO intrusion
• Perform all necessary QC for the rinses, filter, corks, and other line components to maintain the highest quality.

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