Plants & Human Affairs (BIOL106)  -  Stephen G. Saupe, Ph.D.; Biology Department, College of St. Benedict/St. John's University, Collegeville, MN 56321; ssaupe@csbsju.edu; http://www.employees.csbsju.edu/ssaupe

Fermentation Products

 I.  Definitions
    The term fermentation is used in two ways:  (1) as a general term to describe any activity where microorganisms digest organic materials.  For example, during the processing of chocolate, the seeds are left in piles surrounded by pulp that is “fermented” by microorganisms; and (2) to refer to a particular type of chemical process in which sugar is metabolized without oxygen.

II.  Glycolysis – the first step of glucose breakdown      

• Refers to the breakdown of sugar (glucose – C6) to 2 molecules of pyruvic acid (C3). 

• Occurs in the cytoplasm of the cell

• Occurs in virtually all organisms – suggests it is an ancient biochemical pathway.

• Initial stage of sugar breakdown

• Function is to supply the cell with:  (1) energy (2 ATP's) and (b) building blocks, other molecules, that the cell can use for its activities (i.e., pyruvic acid can be used for making certain amino acids).

• Glycolysis doesn't require oxygen – occurs in the presence or absence

• Results in the production of only a small amount of ATP.

• If oxygen (aerobic conditions) is present, the pyruvic acid is further metabolized in the mitochondria.   This is the preferred route since much more ATP is made.

• If oxygen is absent (anaerobic conditions), the pyruvic acid undergoes fermentation.

III.  Fermentation

• Occurs in cytoplasm of cell

• Only occurs under anaerobic, no oxygen, conditions

• The purpose of fermentation is to keep glycolysis going by recycling materials needed for glycolysis.

• Types of fermentations - based upon end product

1. Alcohol:    pyruvic acid → ethyl alcohol + CO₂.   This type occurs in plants and yeast (fungi)

2. Lactic acid:   pyruvic acid →  lactic acid (3 carbons).   Occurs in animals, bacteria

IV.  Alcohol Fermentations

 A.  Organisms
   Many microbes can ferment sugars to alcohol.  Yeasts are the most important group because they are:  (a) common, found of the surface of plant structures; and (b) produce palatable products; many others have offensive byproducts.  Yeasts are single-celled fungi.  The most important yeast is Saccharomyces.  It tolerates more alcohol than other fungi.

B.  Products of alcohol fermentations
    Two commercially important products are alcohol and carbon dioxide.  Alcohol is obviously the product of choice in the beer, wine and distilled spirits industries, whereas the carbon dioxide is used by the bread makers.

V.  Beer

A.  General

• one of the oldest industries, dates back at least 6000 years

• important beverage that serves as:

1. an inebrient;

2. to improve flavor of water;

3. to preserve materials - i.e., water is safer to drink (acidity, anti-microbial activity of hops, alcohol)

• largest of the microbial-based industries; about 20 billion gal/year are consumed

• beer production occurs in 4 major steps

B.  Step 1.  Malting 
    The function of this step is to induce hydrolytic (digestive) enzymes that breakdown the starches in the grains to fermentable (simple) sugars like glucose.

• Steps in the malting process are:  barley → cleaned → soak → germinate (rotating drums) → dry in kiln (raise temp to about 180 F) → grind → screen → malt

• Malt - gets its name from the maltose (one of products of enzyme breakdown of starch)

• Preparation of malt will determine flavor of beer.  For example, stouts use malt that is caramelized.

C.  Step 2.  Mashing.
    The purpose of this step is to convert starch to simple (fermentable) sugar

• Malt + water + adjuncts (other materials, i.e., rice & corn added to US beers) → enzymes continue to convert starch into fermentable sugar → spent grain removed feed to animals; liquid → wort

D.  Step 3.  Brewing.
    The function of this step is to convert sugars into alcohol (and other flavor components)

• wort + hops (related to marijuana; use the female flowers; provides flavor, disguises sweetness from sugars in the beer; stops enzyme action, precipitates proteins, antimicrobial action) →  boil (stops enzyme action, extracts flavor, sterilizes) → spent hops removed (used for fertilizer) → add yeast to liquid → brew 5-12 days

• Saccharomyces cerevisae - ales top fermented (yeast floats), higher alcohol content, higher hops, paler color; lagers bottom fermented (yeast sinks). 

• The purpose of the yeast is to convert sugars to carbon dioxide and alcohol and to impart flavor from other metabolic products. 

E.  Step 4.  Lagering
    This is the aging and maturing step.  

• Beer is aged from 14 days to 3+ months.  

• The yeast is removed → transferred to lagering tanks where the proteins settle out and flavor improves → carbonated → pasteurized → bottled

• Carbonation can be done by: (a) adding beechwood chips and green beer to stimulate a final yeast growth (krausening); (b) introduce CO2 under pressure.

F.  Some notes

• The alcohol content ranges from 3-12%, usually around 4-6%.  

• Light beer - allow yeast to ferment more of sugars.

• Non-alcoholic beers - remove alcohol by evaporation/distillation/dilution

VI.  Wine Making – enology

A.  General

• Enology - science of wine making

• Viticulture - grape growing

• Wine is fermented from grapes, though any berry can be used.

• Wine vs. beer. 
       In wines, the sugars are already in a fermentable stage and they are fermented from grapes or sweet fruits; in beers the sugar must be converted to a fermentable form and beers typically fermented from grains

B.  Grapes

• Vines  

• Viticulture - the grapevines are pruned and trained to a trellis

• Commercially important wine grapes are a comprised of shoots of Old World plants (Vitis vinifera, native to area around Caspian Sea) grafted onto the roots of a N. American species (Vitis riparia).   North American grapes are not suitable for wine making, although Vitis labrusca can be used.  Red or green grape varieties can be used for making wine 

C.  Grape Harvest
    Grapes must have proper sugar content; tested at regular intervals to pick at proper moment.  The enologist must monitor the crop carefully.

D.  Preparation of the must
    The grapes are crushed and de-stemmed to release the juice from the grape.  The crushed grape/stem/skin mix is called the must.  It can be (a) pressed immediately to express the juice (as is done for white wines) or the mixture can be used intact (in red wine the mixture is pressed after fermentation)

E.  Fermentation
    Naturally occurring microbes are killed with sulfur dioxide (also remove excess oxygen from the juice to keep it anaerobic). Yeast is added.  Specialized wine strains are used and it is allowed to ferment - white wine (10-15 C; 1.5-6 weeks); for reds (25-30; 3-14 days).  Conversion of the sugar to alcohol occurs during the fermentation.  The yeast also produces other flavorings.  The fermentation is complete when the sugar runs out (dry wine, no residual sweetness) or is stopped early (filtering, more sulfur dioxide, lowering temperature) which will result in a sweeter wine.

F.  Clarification
    Remove suspended sediments by: (a) racking (allowing them to settle out); (b) adding clarifiers (gelatin, clay, egg whites); (c) centrifuging.

G.  Sterilization
   Kill yeast and any other microbes by (a) cold sterilizing; (b) pasteurization, (c) adding preservatives

H.  Aging/bottling
   Flavor development, especially important in red wines.  Age in oak barrels. In some wine, a secondary fermentation removes excess acids using Lactobacillus bacteria).  Then, wine is bottled and corked. 

I.  Diseases
   Phylloxera is a root aphid that does serious damage to V. vinifera grapes.  When American grapes were transplanted to France, they also brought the root parasite with them.  It nearly wiped out the French wine industry.  To solve problem, they grafted V. vinifera stocks on rootstocks of less susceptible V. riparia. 

    Another problem is powdery mildew, a fungus also introduced from N. America.  These fungi grow on leaves and form a whitish powder on the surface.  You've probably seen them in the fall and didn't realize it - for example, lilac leaves often have lots of powdery mildew by the end of the summer.  Again, this caused great problems for the French until a French botanist noticed that plants growing along a road were doing well.  These had been sprayed with a copper mixture by a farmer to prevent people from eating the grapes.  Thus, copper sulfate became a big component of the standard treatment, called the Bordeaux mix.

J.  Alcohol Content
    Naturally fermented wine has maximum alcohol content of ca 14%.   Why no higher?  cuz the yeast pickle themselves.  Why is the alcohol content of wine higher than beer?  (a) more tolerant yeast; and (b) beer yeast has less sugar to start with therefore makes less alcohol.

    What about fortified wines like sherry and port that have higher alcohol contents (15 - 21%)?  These have brandy added to them.  This practice presumably arose from the need to insure wine preservation by increasing the alcohol content.

VII.  Distilled Spirits.

• grains or juices are fermented into beer/wine.  These are then distilled and the alcohol is concentrated. 
• Pot still - all at once.
• Patent still - continuous feed by adding beer or wine into top of column with plates to affect a separation.
• In both types of stills, attempts are made to get rid of the "heads" that volatilize first at lower temperatures and the "tails" that volatilize latter in the distillation at higher temperatures.  Both produce a harsher tasting beverage.  Continuous feed stills are easier to control and avoid the heads and tails.
• Proof = twice alcohol content.  Grain spirit or alcohol high alcohol content, over 190 proof. 

 VIII.  Interesting alcoholic beverages.

1. Champagne - Dom Perignon, 17th Century Benedictine monk, one of first to produce.  A wine undergoes a secondary fermentation in the bottle (although cheaper products may artificially introduce carbon dioxide).  The final pressure of carbon dioxide in the bottle is 75-90 lbs/in².  Riddling is the process of removing the sediments. 
2. Mead - honey
3. Pulque - fermented agave
4. Mescal - distilled pulque.  Tequila is mescal from a particular region in Mexico.
5. Sake - fermented steamed rice.  Starch is hydrolyzed by black bread mold (Rhizopus) prior to fermentation
6. Chicha - salivary amylase is to hydrolyze the starch in the grains.  Can be made from corn, peanuts, potatoes.
7. Gin - grains or potatoes; flavored with juniper berries
8. Vodka - potatoes
9. Rum - molasses
10. Whiskeys:  scotch - barley malt, heated over a peat fire; bourbon - corn, aged in charred barrels; rye - rye
11. Brandy - distilled grape wine; aged in oak.  VSOP - long aging, sign of quality

 IX.  Bread

• Takes advantage of the carbon dioxide released during fermentation.
• Known very early in Egypt.
• Unleavened bread first ones eaten, leavened breads came later.  They probably arose as a result of spontaneous growth of yeast in mix.
• The leavening (1)  increases the volume of the dough - CO₂ trapped in gluten fibers causing it to rise; (2) Changes the structure and texture; (3) Flavors; (4) increases the nutritive value.
• Sourdough bread - yeast mix contains additional microbes (bacteria) that flavor the bread.  Different types of microbes in different areas.
• Why don't you get drunk from a loaf of bread?  Alcohol evaporates during cooking.

X.  Lactic Acid Fermentations.
   Commercially important in products like: (a) cultured dairy products (cheese, yogurt, buttermilk - i.e., preserving milk); (b) silage (fermentation lowers pH so no other organisms can grow) and (c) preservation of olives, pickles, sauerkraut, cucumbers.

A.  Cheese Production.  
    The type of cheese is determined by:  (1) bacterial flora that is present; (b) temperature of manufacture; and (c) presence/absence of secondary microbes on the cheese.

B.  Basic process.
    Milk → inoculate with bacteria → incubate, souring begins → add proteolytic enzymes (rennet) → coagulates proteins → liquid (whey) removed and discarded or used to make wine or feed pigs; curd → dried → processing further.

C.  Cheese and water content.  
    Cheese is drained, heat, pressed.  Soft cheese has a hi water content (50-80%; Camembert, Brie); semi-hard cheese - cooked briefly, reduce water content to ca 45% (blue, Muenster, Monterrey); hard 40% or less water (Colby, Cheddar, Gouda, Edam, Swiss).

D.  Processing. 
    Unripened cheese - finished product undergoes additional processing.  ripened cheese -allow for secondary growth of bacteria or fungi for flavor (blue, camembert).

E.  Some assorted products

• Yogurt - fermented from milk.  Flavor due to lactic acid and acetaldehyde which gives it tartness.  
• Kefir - Middle East, includes yeast
• Sour cream - pasteurized cream fermented with lactic acid bacteria
• Buttermilk - fermented skin milk (Lactobacillus)

Last updated:  10/24/2005     � Copyright  by SG Saupe