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ProducingSourdough To Illustrate I- the Use of Industrial

STEPHENC. WAGNER

E he use of beneficialmicroorganisms for the LS .This cultureis subsequentlyused to pro- productionof pharmaceuticals,food, food additives, duce various starter cultures (sizeable cultures of beverages,and chemicalsis a very importantaspect the LS strainto be used in the fermentationprocess) of microbiology.Despite its importance,students in for continuous propagation of the bacterium. the typicalmicrobiology laboratory course arerarely Studentsuse the startercultures to make their own exposed to these beyond growing and sourdoughbread and compareand contrastthem in observingthem in pure culture.In orderto familiar- terms of odor, texture,color, and appearance.These ize our students with a typicalindustrial microbiol- approaches, with appropriatemodifications, have ogy process from startingwith a microorganismto also been found to be suitable for biology lab cours- ending with a useful product,a simple,cost effective es for students not majoringin biology or microbi- laboratory exercise was developed that utilized ology. A number of these modifications are also Lactobacillussanfranciscensis (LS), the bacterium described. used to make San Franciscosourdough . This article describes an approach where stu- Background Information dents first develop and make a medium to grow an For centuries, ancient Egyptians,Romans, and Greeksused grains,such as barleyand oats, to make ratherunappetizing porridge or flat cakes. At some STEPHEN C. WAGNER is Associate Professorin the Department of Biologyat Stephen F. Austin State University,Nacogdoches, point the Egyptiansmade two significantadvances TX 75962; e-mail: [email protected]. that allowed them to produce something much

96 THEAMERICAN BIOLOGY TEACHER, VOLUME 67, NO.2, FEBRUARY2005 more enjoyableto eat. First, they started cultivating Table1. wheat (Triticum sp.) along Comparisonof"wild" with standard breadmaking ( cerevisiae). with other grains, such as barley. Secondly, the Criteria "Wild"Yeasts Saccharomycescerevisiae Egyptiansdiscovered, prob- SpeciesNumber Several One ablyby accident,that wheat did somethingnever OptimalpH Acidic(pH 2.5 - 5.5) Neutral/alkaline(pH 6.0 - 8.0) seen before after being set BreadTextures Determinedby yeasts in mixture Uniformtexture aside; it began to ferment and leaven. Because the RisingTime > 10 hr. < 2hr. wheat contained gluten, the protein in wheat that gives the dough its cohe- siveness and elasticity,some of the gases produced by tion. The yeasts were trappedfrom the environmentby leavening were trapped within the dough. When the simply exposing a mixture of wheat and or Egyptiansbaked this newfounddough, the productwas to the air. The resulting mixture of yeast species Downloaded from http://online.ucpress.edu/abt/article-pdf/67/2/96/52227/4451793.pdf by guest on 26 September 2021 completely different from anything they had experi- depended upon the type of flour used and the environ- enced. It was the first recognizableloaf of mental conditions that prevailedduring the fermenta- bread, puffy and crumbly inside with a dark, fragrant tion process. In contrast,today's mass-producedbread crust (Jacob,1944; Wood, 1996). is carriedout by the fermentationand leaveningaction of a single yeast, Saccharonycescerevisiae. A sourdough The Egyptianssoon became experts at bakingsour- starterculture begun with S. cerevisiaewill differin sev- dough bread, developing 50 differentvarieties. Bread eral ways comparedwith the starterbegun with yeasts became the major food as well as the currencyof the trapped from the environment,namely in the number day with wages paid in loaves of bread for centuries.In of yeast species found in the starterand the pH at which fact, a painting of the royalbakery of King Ramseswas these culturescan grow (Table 1; Wood, 1996). found in an Egyptiantomb UJacob,1944). The Egyptian art of bread making found its way LactobacillusCultures for Sourdough fromone generationto anotherand fromone cultureto another,eventually finding a place in Americanhistory. The other chief component of sourdoughis the bac- On January24, 1848, more than 4,000 years since the terium .This bacterium requires pyramidswere built,James Marshalldiscovered gold in (found in flour) to grow and is able to fermentit while Californiaat Sutter'sMill. San Franciscocaught "gold the yeast(s) cannot. The Lactobacillithrive in the acidic fever"so swiftly that within a few months most of the environment caused by the that they pro- men had abandoned the town to find their own for- duce. This process,together with the 'sability to tunes (Blumberg,1989); the CaliforniaGold Rush had producean antibioticthat controlsother organismsthat begun. Soon, gold would also be discovered in the might contaminatethe sourdoughstarter, keeps it from YukonTerritory. getting overtakenby other microorganisms.The bacte- ria also producealdehydes and esters that are unique to Once again sourdoughbread would play an impor- sourdoughbread and may contributeto its flavor(Seitz, tant role in history.Yukon prospectors became known Chung & Rengarajan,1998). as ""because they carriedhomemade yeast starter made of soured milk and flour (Morrell, Specific Organisms Found in San 1941).Thesourdoughs always carried a small amountof starterthat had been passed on to them from another Francisco Sourdough prospector.After mixing it with waterand flourto make Although continue to use dough, they baked sourdough bread, always saving dough made from starter cultures formulated during some starterfor the next camp. Some West Coast and the Gold Rush, the specific organisms responsible for Alaskan bakeries claim that their sourdough cultures this bread remained a mystery until the early 1970s. originated from some of these starters (Dworkin & The yeast responsible for the leavening action in San Dworkin, 1987). Francisco sourdough was identified as Saccharomyces exiguus(Sugihara, Kline & Miller,1971). This yeast has Yeast Cultures for Sourdough since been reclassified as Candida humilis (Wood, 2001). It is capableof growingin an acidic environment starter culture required the pres- The sourdough and unable to utilize maltose. Consequently the yeast ence of at least one species of yeast to initiatefermenta-

PRODUCINGSOURDOUGH BREADS 97 seems to be able to coexist with sourdoughbacteria that require or prefer maltose and produce organic acids Table2. upon its metabolism(Wood, 2001). Lactobacillussanfranciscensismedium (modified from Kline & The lacticacid bacteriumtypically found in the sour- Sugihara,1971). dough is Lactobacillussanfranciscensis. The bacteriaare Constituent Amount gram-positive,nonmotile, catalase-negativerods that number30 to 100 times more than the yeast cells found Maltose 20g in the dough.They produce both lacticand aceticacids as YeastExtract 3 g well as carbondioxide and requirelarge amounts of - ose in order to produce heavy growth.The acids make FreshlyPrepared 15mL sourdoughbread eight to ten times more acidic (pH 3.8 Tween80 to 4.3) than conventionalbread. The two organismsgrow 3g togetherin a mutualisticrelationship, each metabolizing a Trypticase 1.5g differentcarbohydrate that producesthe conditionsthat protectthe culturefrom contamination by other microor- DeionizedWater 1000mL ganisms. This allows the startercultures used to make Downloaded from http://online.ucpress.edu/abt/article-pdf/67/2/96/52227/4451793.pdf by guest on 26 September 2021 San Franciscosourdough bread for overa centuryto con- pHadjusted to4.0 with HCI tinue to be used (Sugihara,Kline & Miller,1971). (Tween80). Afterstudents preparethis medium, 30 mL Procedures aliquots are placed in 250 mL flasks; the flasks are capped with a foam or cotton plug and autoclavedfor Objectives 20 minutes (121?C, 15 psi). Afterthe media has cooled Studentswork as individualsor teams to: the students should add a small amount of a lypholized cultureof LS (AmericanType Culture Collection [ATCC] * Develop and preparea medium to grow a culture # 27651) and incubate these cultures on a reciprocal of LS. shaker(200 rpm) set at 32 C until the culturebecomes * Develop a starterculture of this . turbid(72 hours). * Use the starterculture to make sourdoughbread. Starter Culture Preparation. Each student or team is assigned the recipe for a particularstarter culture in * Compare and contrast their bread with those order to compare the effectivenessof several types of other students. produced by startercultures (Table 3). Alternativelythe students can be requiredto find their own starterculture recipes. The Week 1. Preparation & Inoculation of students preparetheir starterculture and place it in a Lactobacillus Medium & Starter Culture plastic or glass container (Ca 750 mL); the containers Students first preparea medium to grow pure cul- are loosely covered with a lid and incubated at room tures of LS.This medium requiresingredients that have temperature.Each culture is observedand stirredvigor- to be preparedfresh. The students learn how to prepare ously with a plastic spoon once a day until it is used to and properlysterilize the medium so it can be inoculat- make the bread.A successful starteris indicatedby the ed with a pure cultureof the bacterium. appearance of bubbles in about two days. If mold appearson the surfaceof the starterit is contaminated and should be discarded(Wood, 2001). Autilized Yeast Extract.The LS medium requires freshly-preparedyeast extract.This is preparedby auti- At one or two-dayintervals, aliquots of each starter lizing a suspension of compressedbaker's yeast. A cultureare removed in orderto determinethe pH of the 20% suspension of compressedbakers' yeast is auto- culture and thereby observe the culture'spH over the claved (121'C, 15 psi) for 30 minutes. Once the auto- incubation time. Twenty milliliters are removed and claved suspension settles overnightat 2-8? C, the stu- mixed with 40 mL deionized water.After the solution dents decant the suspension and furtherclarify the has equilibratedfor 10 minutes, the pH is measured supernatantby centrifugingfor 15 minutes at 6000 with a pH meter to reachor determinethe desired acid- rpm. The resultingsupernatant can be stored at 4 ?C ity. Alternatively,titratable or total acidity (acid content until the rest of the medium is prepared(Sugihara, determinedby titratingwith sodium hydroxide)of each Kline & Miller,1971). starterculture can be determinedby methods described by Drakeand McKillip Lactobacillussanfranciscensis Medium. In addition to (2000). freshly-preparedyeast extract,the LS medium (Table2) At Day 3, a 10 mL aliquot of the LS culture contains maltose, trypticase,and unsaturatedfatty acid describedabove is added to each starterand incubated

98 THEAMERICAN BIOLOGY TEACHER, VOLUME 67, NO. 2, FEBRUARY2005 Table3. Examplesof sourdoughstarters. pHafter incubation at32?C

Reference Flour Milk Water Inoculant Day0 Day1 Day3 Day6

Dworkin&Dworkin, 1987 250mL 250mL - - 6.0 4.6 3.6 3.7 Bilheuxetal.,1989 250mL - 250mL - 5.9 5.0 3.8 3.8 Nelson,2002 250mL - 250mL 7 g driedactive yeast 5.9 4.8 3.8 4.1 Wood,1996 250mL - 60mL 18g sourdoughstarter' 5.9 4.5 3.4 3.4

1Available from Sourdoughs International, P.O.Box 670, Cascade, ID83611 (http:/www.sourdo.com) forapproximately $15.00. Downloaded from http://online.ucpress.edu/abt/article-pdf/67/2/96/52227/4451793.pdf by guest on 26 September 2021 for four more days at 32?C. Afteranother two to three Students can compare the leavening action of the days, a layerof clearbrown fluid called "hooch"usually different starters by measuring the height that each develops on the surface of the starters,indicating that breadrises within a certainamount of time. This can be the culture is viable and is developing normally.The accomplishedby placing a small amount of the dough students mix this liquid into the culture and continue in a plastic drinkingstraw (by stabbing the dough with mixing every day.At Day 5, each starterculture is "fed" an empty straw). The end of the straw containing the by adding 150 g flour and 250 mL tap water. dough is pinched closed with a paperor binder clip and the level of the dough is marked.The filled straws are Week 2. Bread Making then incubated for 30 minutes and the level of the dough is measured. Preparing & the Bread Analyzing the Bread Manyrecipes for sourdoughbread allow the dough to fermentand rise for an extended period of time (up Studentscompare the sourdoughbreads by using a to 12 hours) in order for the bread to produce a suffi- sensory test with numericalscores to simulatea typical cient amount of leavening and lactic acid. This will food quality analysis. Criteria for evaluation include require the students to mix the dough the day before appearance,color, taste, odor, and texture (Table 4; the bread-makinglab. Alternativelya small amount of Casaburri& Gardner,1999). It should be noted that baker'syeast (Saccharomycescerevisiae) can be added to the Lactobacillussanfranciscensis is made availablefrom boost and speed up the leaveningaction. Sufficient leav- the ATTCfor researchpurposes and not for human con- ening will then occur within 1 to 1.5 hours. sumption. Consequently,although this procedureordi- narily includes tasting the food product, it is recom- Students should prepare their sourdough bread mended that instructors consider not requiring stu- accordingto the followingrecipe (modified from Wood, dents to taste the breadswhen a cultureof ATCCstrain 1996): is used to make them. Students should observe the fol- 1. Combine 250 mL of the starterwith: lowing criteriawhen analyzingthe breads: * 4 g yeast * Analyze the qualityof each product rather than personalpreference. * 9 g warm butter * A descriptiveterm should * 9 g accompanyeach cate- gory rating(Table 4). * 600 g flour * Each food should be given an overallrating that * 250 mL milk is not necessarilyan averageor total of the other * 26 g categories. 2. Knead the resulting dough and place it in a * Do not talk with other panelists during evalua- greasedbread pan, allowingit to rise for about 1 tions. hour. * Refrainfrom smoking, eating, or drinkingfor 60 3. Bake the bread at 132?C (350?F) until golden minutes prior to analyzing the food because brown (30-45 minutes). these activitiesmay influenceyour evaluation.

PRODUCINGSOURDOUGH BREADS 99 for Non- Modifications Table4. Majors Classes Criteriafor sensorypanel evaluation(Casaburri & Gardner, 1999).

Modifications of these procedures Item' DescriptiveTerms have specificallybeen used in a biology course for preserviceteachers to demon- Taste/Odor bitter,sweet, sour, salty, oxidized, rancid, stale, tasteless, strate the importance of beneficial metallic,flat, musty, yeasty, floral microbes. The students followed the same proceduredescribed previously for Texture crisp,soft, hard, stringy, tough, chewy, firm, fine, grainy, preparing the starter culture but used gummy,lumpy, mushy, pasty, rubbery, sticky, stiff, tender, only the recipe for the starterwith dried greasy,juicy yeast (Table 3; Nelson, 2002) and not Color/Appearance dull,lustrous, sparkling, bright, light, dark, greasy, glossy, with a pure culture of LS. Additionally, cloudy,old, pale the students used this starter to make sourdough instead of bread.

The class developed an experiment to Eachof the five items rated according tothe following scoring system: Downloaded from http://online.ucpress.edu/abt/article-pdf/67/2/96/52227/4451793.pdf by guest on 26 September 2021 determine the effect of the amount of on the quality of the pancakes. HighScores MidScores LowScores Each team was assigned a recipe with a differentamount of sugar in the batter 9 - LikeExtremely 6 - LikeSlightly 3 - DislikeModerately and cooked its pancakes according to the followingrecipe (Wood, 1996): 8 - LikeVery Much 5 - Neutral 2 - DislikeVery Much 1. Mix 500 mL of the liquid starter 7 - LikeModerately 4 - DislikeSlightly 1 - DislikeExtremely with the followingingredients: * 250 mL white flour * 125 mL water within three days, indicatingthe production of organic acids such as lacticand aceticacid (Table3). It is impor- * 0 to 36 g sugar tant to bear in mind that the startercultures inoculated * 9goil with dried active yeast (Saccharomycescerevisiae) may eliminate the wild yeasts and destroy the culture. On * 1 egg the other hand, some students may be more comfort- * 3 g salt able making this type of starter rather than trapping unknown wild yeasts.This may especiallybe the case in * 3 g baking soda a course such as a non-majorscourse where they arenot 2. Add an extra amount of flour to the batterif it is very familiarwith the microbialworld. not thick enough. Some instructorsmay want to simplify the proce- 3. Pour the batter onto a hot griddle to dures described by not growing and using a pure cul- form 5 to 5.5 cm rounds. Cook the cakes for two ture of Lactobacillussanfranciscensis. Students can use to four minutes until a number of bubbles form commerciallyavailable sourdough starters to do this. on the surface. Turn the pancakes and cook Dr. Ed Wood (SourdoughsInternational, P.O. Box 670, them for an additionaltwo minutes. Cascade, ID 83611; http://www.sourdo.com)has col- 4. The students can then evaluatethe pancakesby lected several starter cultures from across the world, conducting a sensory test as describedprevious- including San Francisco, Russia, Finland, France, ly. This can include tastingthe pancakesbecause Austria,Egypt, and Saudi Arabia.Students could com- the ingredientsused throughoutthe experiment pare the effectivenessof these startersby using the pro- are intended for human consumption. cedures described. They could also taste their breads because these startersare sold for human consumption. Discussion Effortsshould be made to eliminate the possibility of contaminationby unwantedmicroorganisms, such as All of the starter culture recipes described in this molds. We conduct the activitiesin a laboratorywhere article generate an adequate culture for sourdough other microorganismsare not handled. Also the stu- bread.Although the startercultures varied in terms of dents are instructed to inspect the cultures daily for pH change over time, all the culturesreached a low pH signs of contaminationsuch as the appearanceof molds

100 THEAMERICAN BIOLOGY TEACHER, VOLUME 67, NO.2, FEBRUARY2005 on the surface of the cultures and atypical odors. as in general biology lab courses. It offers an effective Alternatively, the cultures could be prepared under a and doable strategy to teaching about industrial micro- sterilized laminar flow hood in order to eliminate possi- biology processes and the types of organisms used in ble exposure to contaminating microorganisms. these endeavors. Students at some institutions may not be permitted to ingest something that they prepared in a laboratory. Acknowledgments However, as described above, students can collect an adequate amount of data without tasting the product. The development of this laboratory activity was par- The instructor(s) should consult the rules and regula- tially funded by a grant from the National Aeronautics tions of their particular institution before allowing or and Space Administration Opportunities for Visionary requiring the students to taste their foods. For colleges Academics (NOVA) program. and universities, instructors should consult their cam- pus Health and Safety Office or Office of Environmental Health and Safety to determine rules and regulations References regarding such issues. Bilheux, R., Excoffier, A., Herve, D. & Pouradier, J.M. (1989).

Because an is required to bake the bread, the Special and Decorative Breads. New York, NY: Van Downloaded from http://online.ucpress.edu/abt/article-pdf/67/2/96/52227/4451793.pdf by guest on 26 September 2021 instructor may have to make arrangements to use a lab- Nostrand Reinhold. oratory in an allied program such as a Human Nutrition Blumberg, R. (1989). The Great Amer-icanGold Rush. New or Food Science department. Alternatively the sour- York, NY: Bradbury Press. dough starter could be used to make the sourdough pancakes as described above. Relatively inexpensive Casaburri, A. & Gardner, C.A. (1999). Space Food and Nutr-ition.An Educator'sGuide with Activities in Science and tabletop pancake griddles can be found at department Mathenmatics. National Aeronautics and Space will stores and are easy to use. Students still be able to Administration (EG-1999-02-1 15-HQ-1): Washington, follow the experiment from starting with a specific DC. microbial culture to making a final, edible product. Drake, M. & Mckillip, J. (2000). microbiology The course for preservice teachers is an inquiry- making cheese, yogurt & buttermilk as a lab exercise. The based course where lecture and laboratory activities are AmiericanBiology Teacher;62(1), 66-67. combined. This activity provides a good approach to introducing the students to the microbial world (specif- Dworkin, F. & Dworkin, S. (1987). Bake YourOwn Bread. New York, NY: Nal Penguin, Inc. ically beneficial microbes) as well as teaching them about biological concepts. For example, the use of yeast Jacob, H.E. (1944). Six ThousandYears of Bread. New York, NY: and bacterial cultures helps students grasp the concept The Lyons Press. of mutualistic between relationships microorganisms Kline, L. & Sugihara, T.F. (1971). Microorganisms of the San and how these microbes metabolize carbohydrates. It Francisco sour dough bread process. II. Isolation and also gives these future teachers a simple exercise that characterization of undescribed bacterial species respon- they can use to teach students about the scientific sible for the activity. Applied Micr-obiology,21(3), method and integrate these activities with other sub- 459-465. jects such as math, technology, and history. In addition, Morrell, W.P. (1941). The Gold Rushes. New York, NY: The the of bread and the history sourdough making people Macmillan Company. involved in this process could be incorporated as part of an introduction to the laboratory exercise. Nelson, P. (2002). How Sourdough Bread Works. Available online at: httpl /www.howstuffworks.com sourdough. Because microbiology students were exposed to htm ppintable. media development, continuous propagation, and pro- duction of final product for the yeast and Lactobacillus Seitz, L.M., Chung, O.K. & Rengarajan, R. (1998). Volatiles in cultures, this exercise gave them a more complete picture selected commercial breads. Cer-ealChemiiistt-y 75(6), 847- 853. of how industrial microorganisms are used. This exercise offered an ideal approach to illustrate metabolism, fer- Sugihara, T.F., Kline, L. & Miller, M.W. (1971). mentation, microbial ecology, symbiotic relationships Microorganisms of the San Francisco sour dough bread between microorganisms, use of beneficial organisms, process. I. Yeasts responsible for the leavening action. and microbiological media development and use. Applied Microbiology,21(3), 456-458. In summary, this paper describes an enjoyable, Wood, E. (1996). Wo-ld Sour-doughsfromi Antiquity. Berkeley, practical, hands-on experience that demonstrates the CA: Ten Speed Press. applications of beneficial microorganisms under "real Wood, E. (2001). Classic Sourdoughis.Berkeley, CA: Ten Speed world" conditions for students in microbiology as well Press.

PRODUCINGSOURDOUGH BREADS 101