BTC 504 Yeast-Based Fermentation

Introduction Yeasts have long been associated with raising, fermentation, carbonation, and transformational abilities Bread, beer, and wine are the main food products associated with yeast fermentations Saccharyomyces cerevisiae is the most widely used yeast species for these purposes

Bread Ancient societies used grains to produce porridges, gruels, and unleavened bread As societies became more sophisticated, leavened breads became more popular Archaeological remains (estimated ages of 2700 B.C.) of Egyptian baking ovens have been found

Bread In early times, bakeries were often attached to breweries so that the yeast by- product of brewing (S. cerevisiae) could be used for bread-making S. cerevisiae is commonly called “baker’s yeast”

Bread-making Mix flour, sugar (as a fermentable carbohydrate), fat (for texture), salt, and other ingredients Baker’s yeast is added in the form of dried powder, block, or cream at 1 – 6% on a weight basis per weight of flour (or a bit of dough from the last batch is used) The main role of the yeast is to produce the CO 2 that makes the bread rise

Bread-making The yeast also produces amylases that break down starch to the more fermentable glucose Water is added, and the dough is kneaded so that the gluten protein in the flour stretches and forms a viscoelastic (both thick and elastic) mass

Bread-making The bread is fermented or “proofed” at 28 – 32 o C for several hours Remixed or “punched” to evenly distribute the gas cells, portioned into loaves, and fermented again (loaves will then double in volume) Baked at ~200 o C for ~30 min.

Bread-making During baking, CO 2 expansion causes a 40% increase in bread volume, which is captured as the protein “sets” by denaturation For sourdough breads, a stable mixture of heterofermentative LAB (mainly Lactobacillus sanfranciscensis) is also added to add a characteristic “bite”, improve texture, and prevent spoilage

Beer

Chemical analysis of 7,000 year-old jugs puts invention of beer around the same time period as wine Addition of hops to beer occurred about one thousand years ago but before the 14th century spruce, ginger, wormwood, sage, and sweet mary were more popular Bavarian purity law passed, oldest known food purity law still in effect, limited ingredients of beer to hops, barley, and water. 16th century lager beer type accidentally invented during cold cave fermentation. Beers in America were largely heavy beers, until prohibition ended most American breweries. Now the beer market is nearly monopolized by Anheuser-Busch, who made a fortune producing cheaper, watery, light beer for women workers during World War II. The American pallet now reflects this with preference for lighter beer. History

Egyptian Beer-making

Essential Ingredients malted barley to provide fermentable carbohydrate hops for flavor and antimicrobial activity yeast to produce alcohol and CO 2 water hops

Yeast Saccharomyces sp.

Yeast It is usual to select strains of yeast for brewing from yeasts already in commercial use. Some breweries isolate, select and maintain their yeast strains but others engage specialist laboratories to provide this service. Can make a stock of your yeast.

Yeast can be found naturally on the surface of most plants including barley seeds. “Wild” yeast will most likely produce flavors that are undesirable. During the fermentation process, undesirable microbes must be kept out of the beer. Saccharomyces cervisiae is the species most often used for ales, its optimum fermentation temperature is C Saccharomyces uvarium is largely used in lagers, and steam beers. The optimum temperature for this fermentation is 2-13 C Over a 15 square mile area near Brussels the resident wild yeast and bacterial populations are perfect for spontaneously fermenting, beer. This fruity sour beer is known as a “lambic”.

What qualities should my yeast have? Rapid initiation of fermentation High fermentation efficiency High ethanol tolerance Desired flavor characteristics High genetic stability Range of alcohol production

Metabolism Special flavors and aromas of beers arise from minor biochemical reactions Major Reaction: Glucose to Carbon Dioxide and Ethanol

Yeast In the average brewery, a large inoculum of cells is used (ca 5-15 million cells/ml of wort). In each fermentation the cell density increases three-to-four-fold. Therefore, one-third to one-fourth of the yeast crop of each fermentation is used for inoculation of the next batch.

Beer German purity laws forbid the use of any other ingredients Different hops give different beers their distinctive flavors Other countries (including U.S.) allow less expensive cereals, grains, or corn to be used to replace malt as the carbohydrate beer /

Beer-making Process consists of malting, mashing, wort boiling, fermentation, and post- fermentation treatments Yeast cannot ferment starch in the barley, so it is malted and mashed Sweetness in beer comes from the malted barley

Malt Made from barley that has been allowed to germinate. Germination converts starch in the seeds into simpler sugars. These sugars are extracted in the mashing process. This malt extract is then used by the yeast in the fermentation process. Before mashing the malt may be roasted to darken the color and harden a beer. barley

Malting Grain is steeped in water for 24 – 48 hrs to induce germination This breaks down the cell wall and protein matrix that contains the starch granules and liberates amylases

Wort What’s in it: –Brewers' wort (145) commonly has 8-14% total solids. –90-92% are carbohydrates: glucose, fructose, maltose, sucrose, maltotriose. –Nitrogenous compounds, such as, amino acids. –Vitamins: biotin, inositol, pantothenic acid, pyridoxine, and thiamine are present in wort and utilized by Brewers' yeast. –Phosphates, chlorides, sulfates and other anions are present with the cations Na, K, Ca, Mg, Fe, Cu, and Zn.

Mashing and Wort Boiling Amylases break the starch into fermentable carbohydrates After mashing, the liquid malt is almost ready to ferment Herbs, usually hops, are added for flavor and antimicrobial activity The mixture of hops and liquid malt is boiled and then cooled to ~20 o C

Humulus lupulus (hops)

Hops Flavouring beer is the sole major commercial use of hops. The flower of the hop vine is used as a flavouring and preservative agent in nearly all beer made today. The flowers themselves are often called "hops". Hops contribute a bitterness that balances the sweetness of the malt; Hops contribute floral, citrus, and herbal aromas and flavours to beer.

Hops contd. Hops have an antibiotic effect that favours the activity of brewer's yeast over less desirable microorganisms. aids in "head retention" the length of time that a foamy head created by carbonation will last. The acidity of hops is a preservative. Hop oils are produced in the Lupulin glands of the flower. The oils are made of α and β-acids, but α-acids contributes more to the bittering of a beer. These oils are non-polar, and can only be extracted through a short boiling.

Fermentation S. cerevisiae grows on top of the fermentation mix and is used for ales S. carlsbergensis settles to the bottom and is used for lagers Inoculum level is high at ~10 7 /ml and increases 8-fold over the course of fermentation

Fermentation

Lager Pale lagers are the most commonly consumed beers in the world. Lager yeast is a cool bottom- fermenting yeast (Saccharomyces pastorianus) and typically undergoes primary fermentation at 7–12 °C (the fermentation phase), and then is given a long secondary fermentation at 0–4 °C (the lagering phase). The cooler conditions also inhibit the natural production of esters and other byproducts, resulting in a "cleaner"-tasting beer. With improved modern yeast strains, most lager breweries use only short periods of cold storage, typically 1–3 weeks.

Fermentation The yeast from one fermentation is used to inoculate the next batch for 10 – 15 fermentations and then, a new inoculum is reintroduced The yeast uses the various sugars in the wort sequentially, with simple sugars being fermented first and maltotriose relatively later

Fermentation Ethanol production continues after the yeast stops growing Flavor compounds, such as aldehydes and higher alcohols, are produced

Post-fermentation Includes “aging” to remove the “green” flavors caused by diacetyl and acetaldehyde These can impact the taste adversely as humans can taste them at very low concentrations Aging in casks, storage in the presence of yeast at 15 o C, and avoiding O2 exposure helps to remove flavor defects

Post-fermentation Most beers are filtered until clear, although cloudiness of “boutique” beers is part of their charm Cellulose fiber or pumice are used as filter aids to enhance removal of the yeast by filtration

Pasteurization Membrane filtration can be used as a means of “cold pasteurization” Draft beer is not heated and must be kept refrigerated during shipping and storage Heat pasteurization involves holding beer for 5 – 30 min at 60 o C to kill the yeast before bottling

Steps in Beer Fermentation Process

Process Malted barley and specialty grains are run through roller mill and cracked open. This grist is then carried by an auger to the mash tun. Malted Barley and Specialty Grains

Process In the mash tun the grist is mixed with hot water to form a mash. In the mash, enzymes that exist in the grain become active and convert the starches to fermentable sugar. The sugar rich liquid from the mash, called wort, is drained from the mash tun.

Process The wort is drained from the mash tun and moved to the brew kettle. In the brew kettle the wort is boiled and hops are added. From the hops we can extract bitterness, which will help balance the sweetness of the wort.

Process After boiling, the wort is transferred through a chiller. While passing through the chiller the wort is instantly chilled to the appropriate temperature for fermentation.

Process After fermentation the fermented wort, now called beer, is transferred through a filter. The filter removes various proteins, hop residue and yeast cells.

Process From the chiller, the wort moves into a temperature controlled fermenter. Yeast is added and fermentation begins. In fermentation the yeast will ferment sugars in the wort and produce alcohol, carbon dioxide, and other flavor compounds.

Process From the filter, the beer moves to the beer servers. These are carbonating tanks, holding tanks and serving tanks.

Fermentation Systems Cylindroconical systems: –Produce ales and lagers. –Conical base, and pressure systems. Open systems: –Used for the fermentation of ales –Utilize skiming for repitching –Carbon dioxide can diffuse out

Fermentation of Ales Top fermenting-rise to the surface and create a think yeasty head. Warmer temps F More rapid growth Create more esters Complex and Fruity Ales, porters, stouts, and wheat beers.

Fermentation of Ales Lag phase is when yeast is building cell walls and reserves. Then yeast begin to divide. First visible sign of fermentation is bubbles starting to form, which spread until the surface is covered. After 18 hours the bubbles thicken and change to a light brown color. pH and specific gravity fall, and temperature and yeast count rise. Max fermentation is reached after hrs. White yeast head on top, with CO 2 escaping. Activity slows and the head changes from white to pale cream, as yeast rises to surface and replaces the foam.

Fermentation of Lager Bottom fermenting-yeast settle to the bottom of the fermenter as fermentation reaches completion. Colder tempeatures: 47-58F Slower growth Crisp and hoppy like a pilsner or sweet and malty like a Dopplebock. Examples: Pilsners, Bocks, and American malt liquors.

Fermentation of Lager Time-temperature profiles vary widely. Pitch the yeast at C and raise to C. This makes better beer because the low temps retard the development of by-products which are inappropriate in lager (esters, fusel alcohols, diacetyl). Lag period is longer at lower temperatures though. After primary fermentation the temperature is dropped by 1-1.5C each day and then is transferred to a lager cellar at 45-50C. The starting temp and the rate of temp increase can vary.

Compounds produced in primary fermentation and not reduced during lagering Fusel or higher alcohols:  By-product of amino acid metabolism.  Levels are affected by yeast strain.  Spicy, wine-like, and alcoholic taste. Organic acids:  Formed from carbohydrate metabolism  Contribute to the sourness or acid taste (also pH) of beer.

Esters:  By-product of lipid metabolism in a reaction between an alcohol and intermediates of lipid synthesis.  Usually impart a fruity character to the beer. There are two types:  Acetate esters  ethyl acetate (solventy, fruity, sweet)  isoamyl acetate (banana, fruity, sweet)  phenethyl acetate (roses, honey, apple, sweet).  Fatty acid ester  ethyl caproate (apple, aniseed, sweet)  ethyl caprylate (apple, fruity, sweet) Ale yeast strains produce more esters

Compounds produced in primary fermentation and reduced during lagering Diacetyl  Byproduct of amino acid metabolism.  Tastes like butter, butterscotch, and feels slick on the palate. Pentadione  Similar to diacetyl.  Milder flavor similar to honey or butter.

Acetaldehyde  Intermediate of ethanol production.  It can form in autolysis of yeast during lagering if yeast is in poor condition.  It can also form if post-fermentation beer is exposed to air (oxygen).  Ethanol can be oxidized to acetaldehyde. Acetoin  Formed from a secondary reaction of alcoholic fermentation.  Pyruvate decarboxylase converts pyruvate to acetaldehyde, which is then converted to ethanol and CO 2.  Pyruvate decarboxylase sometimes joins two acetaldehyde molecules to form acetoin.

Wine Wine production started in ancient times, probably when the natural yeasts accidentally contaminated grape juice In the late 19 th century, the French microbiologist Louis Pasteur discovered the role of microbes in this fermentation Pasteur also discovered the microbes responsible for wine spoilage

Wine Pasteurization was invented to kill these spoilage microbes in wine and was later used in milk France remains the biggest wine producer in the world, followed by Italy, Spain, and the U.S. Together, these four countries produce more than 15 million tons of wine per year

Wine Riesling, chardonnay, and sauvignon blanc are among the varieties used for white wines Merlot, pinot noir, and shiraz can be used for red wines Grapes can be fermented in barrels, but large stainless steel tanks are more commonly used for mass-produced wines

Grape/Wine Flavor Depends On: sugar-to-acid ratios climate soil vine age

White Wines The juice is drained away from the skins immediately after crushing The juice is then clarified and sent to the fermentation tank White wine fermentation is usually complete in 1 – 2 weeks at 10 – 18 o C The low temperature helps retain important volatile flavors

Red Wines The juice and skins go directly to the fermentor where the skins float and the juice is pumped over them This extracts the purple and red anthocyanins and phenolics that give red wines their color and possibly their health benefits

Red Wines After fermentation has started, the liquid is pressed through the skins into a fermentation tank Fermentation usually lasts about 7 days at 20 – 30 o C to extract the red color Fermentation ends when glucose and fructose are completely utilized

Fermentative Organisms Starters are not always added but sometimes S. cerevisiae or S. bayanus are to give a more consistent product Native grape yeasts are mostly Kloeckera and Hansenia spp. (Candida, Pichia, and Hansenula spp. are also found) All of these yeasts are present in low numbers on immature fruit, but increase exponentially as the grapes ripen

Products of Fermentation Ethanol and CO2 are the main products of fermentation Glycerol is sometimes produced and it can smoothen the taste and impart viscosity Higher-alcohol esters and aldehydes can be produced as flavor compounds

Unpleasant By-products Succinic and acetic acid production causes wine defects Malic acid occurs naturally in some grapes and can give wine a very sour taste (it is used to produce SweetTarts candy) Malolactic fermentation using Oenococcus oeni can decrease malic acid content thus lowering acidity of the wine

Post-fermentation Red wines are stored in oak barrels for 1 – 2 years for flavor development Barrels may be made with smoked woods, resins, or other flavor-induced mechanisms White wines are usually not aged

Post-fermentation Potassium sorbate can be added to red or white wine to control yeast spoilage Wine is then filtered to clarity and bottled