19: ALCOHOL FERMENTATION
- Page ID
- 157087
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)- Define fermentation and explain how yeast converts sugars into alcohol and carbon dioxide.
- Monitor and record the fermentation process by observing visible signs such as bubbling or pH changes.
BACKGROUND
Wine making is an ancient form of fermentation, a metabolic process in which microorganisms convert sugars into alcohol and other by-products. This technique has been practiced by human civilizations for thousands of years. The process likely began by accident, when overripe or crushed fruit was left at room temperature and naturally occurring microbes began to ferment the sugars, producing alcohol and changing the fruit’s flavor and aroma. This accidental discovery introduced early humans to the intoxicating and preservative effects of fermentation.
Wine can be made from virtually any sweet fruit, including grapes, plums, cherries, currants, and strawberries. It can even be made from sweetened water. Mead, a traditional beverage made by fermenting honey and water, is one of the oldest examples of wine-like drinks and was especially popular in early Celtic and Nordic cultures.
In modern wine production, the yeast species Saccharomyces cerevisiae is commonly used because of its reliability and efficiency in converting sugars into ethanol (alcohol) and carbon dioxide. The amount of sugar in the starting fruit juice determines the potential alcohol content of the final wine: more sugar results in more alcohol. During fermentation, yeast continues to convert sugars until the supply runs out or the alcohol level becomes too high for the yeast to survive.
Since alcohol inhibits the growth of many other microbes, it acts as a natural preservative. This quality made wine especially useful in ancient times as a way to preserve the calories and nutrients of fruit. In fact, in some regions where clean drinking water was scarce or unreliable, wine and other fermented beverages were safer to consume than untreated water.
Although wild yeasts and bacteria are naturally present on the skins of grapes, not all of these organisms are beneficial for wine making. Some can spoil the wine by producing acetic acid or other unwanted by-products. In modern wineries, grapes are often treated with sulfur dioxide or gently pasteurized to eliminate wild microbes. After this step, a known culture of yeast is added to begin a controlled fermentation.
The visual haze seen on the surface of fresh grapes is often a layer of wild yeast. Historically, this natural yeast helped start the fermentation process. Grapes became a favored fruit for wine production in part because of this yeast-rich surface. However, controlling fermentation by using selected strains has led to more consistent and higher-quality wines.
The difference between red and white wine lies in how the grapes are handled before fermentation. Red wine is produced by fermenting the crushed grapes along with their skins and seeds. The skins contribute color, flavor, and tannins—compounds that influence the texture and taste of the wine. In contrast, white wine is made by removing the grape skins before fermentation. This results in a lighter color and different flavor profile, as the juice ferments without extracting compounds from the skin.
In some fermentation processes, very little carbon dioxide is retained. This produces still or non-sparkling wines. If the carbon dioxide is allowed to escape during fermentation, the wine remains still. However, when fermentation occurs in a sealed container, the carbon dioxide stays dissolved in the liquid. This process results in sparkling wine, such as champagne.
A significant milestone in understanding fermentation came in the mid-1800s, when Louis Pasteur demonstrated that Saccharomyces cerevisiae was responsible for the desirable fermentation in wine. He also introduced the process of pasteurization, which involves gently heating liquids to reduce or eliminate unwanted microorganisms. By reducing contamination and allowing the controlled addition of a known yeast culture, pasteurization greatly improved the quality and consistency of wine production.
In this laboratory exercise, you will create your own small batch of wine using pasteurized fruit juice and a commercial yeast culture. You’ll apply techniques developed over centuries to begin and monitor the fermentation process. Be sure to follow each step carefully—while fermentation is a natural process, small errors in handling can lead to contamination or spoilage.
MATERIALS (Per Group of 4)
3 large screw-cap glass tubes
Fresh grapes or other soft fruit
1 Scalpel or sterile cutting tool
1 Glass stirring rod
1 DI water battle
4 Sterile transfer pipets
2 pH test strips
1 Saccharomyces cerevisiae culture
1 500 mL Beaker
1 Hot plate
1 Thermometer
1 bottle of methylene blue
Microscope slides
METHODS/PROCEDURES
1. Label your tubes as follows:
a. Tube #1: Crushed fruit; unpasteurized; uninoculated
b. Tube #2: Crushed fruit; pasteurized; uninoculated
c. Tube #3: Crushed fruit; pasteurized; inoculated with Saccharomyces cerevisiae.
2. Wash your hands thoroughly with soap and water. Rinse your fruit under running water to remove any surface debris.
3. Using a scalpel, carefully cut the grapes (or other selected fruit) into quarters. Place 2–3 cut pieces into each of three large, screw-cap glass tubes.
4. Gently mash the fruit in each tube using a clean glass stirring rod. Be careful not to crack or break the glass. If your fruit does not release much juice (ask your instructor to confirm), add 1 mL of sterile water to each tube using a sterile pipet.
5. Measure and record the pH of each sample in Figure 3. Use a transfer pipet to remove a small amount of liquid—do not insert the pH strip directly into the tube.
6. Prepare a boiling water bath using a beaker with approximately 300 mL of water. The water level should be higher than the fruit level in the tubes, but not so high that water can enter the tubes. Do not allow water from the bath to spill into the samples.
7. Once the water reaches a boil, carefully place Tubes #2 and #3 into the water bath. Turn off the hot plate immediately after adding the tubes.
Pasteurize these samples by leaving them in the hot water bath for 5 minutes. Then, using test tube holders, remove the tubes and allow them to cool to room temperature in a test tube rack.
8. Inoculate Tube #3 with Saccharomyces cerevisiae using a transfer pipet (approximately 4–5 drops).
9. Once the tubes have cooled, recheck and record the pH of all three tubes. Also observe and note the color, smell, and any other distinguishing
characteristics of each sample at this time.
10. Tightly screw the caps onto all three tubes. Incubate them at room temperature or at 30°C for one week.
After one week of incubation:
11. Before opening the tubes, examine each for signs of gas production (bubbles may indicate fermentation).
12. Perform a simple stain on the contents of all three tubes. Record your observations.
13. Measure and record the final pH of each tube.
14. Record any signs of fermentation, such as a change in pH, gas bubbles, or the characteristic aroma of alcohol.
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Exercise #24 Alcohol Fermentation
NAME ______________________
EXPECTATIONS
What visible or measurable changes do you expect to observe in a tube where fermentation has successfully occurred? List at least two signs.
RESULTS
Fermentation Observations:
|
Tube |
Treatment Description |
pH Before Incubation |
pH After Incubation |
Bubbles Present? (Y/N) |
Alcohol Smell or aroma? (Y/N) |
Color / Appearance before incubation |
Color/ Appearance after incubation |
|
A |
Crushed fruit, uninoculated & unpasteurized |
|
|||||
|
B |
Crushed fruit, uninoculated & pasteurized |
|
|||||
|
C |
Crushed fruit, inoculated & pasteurized |
|
Simple Stain Observations:
CONCLUSIONS
1. Which tube showed the clearest signs of fermentation and alcohol production? Describe the evidence that supports your conclusion
2. Examine your simple stain results. What types of microbial cells did you observe? Were there differences in shape or abundance between the tubes? What can the differences tell you about the tubes?
3. Alcohol is known to inhibit the growth of many microorganisms. How do you think this property benefits wine production? What effect might it have on contamination?
4. Alcohol is known to inhibit the growth of many microorganisms. How do you think this property benefits wine production? What effect might it have on contamination?
5. Given that wine can be made from many types of fruit, how might the sugar content of the fruit you choose affect the fermentation process and final alcohol content?