Microbiology sourdough final paper PDF

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Rebecca Simmons Sourdough for Science Abstract In this project, a sourdough starter and its bread were successfully made from scratch, which helped the understanding of microbial influences on sourdough bread, like rise and flavor. Type I sourdough bread dates back thousands of years and is still made today, although our worlds staple bread is made with it relative, type II and III sourdough bread. Sourdough starters are affected by multiple factors like the ingredients used, the environment it is grown in, and the procedure in which it’s made. They involve complex microbes that perform many functions, and one is not likely to be exactly the same as the next. In conclusion, it takes time to make and take care of a type I sourdough starter and there is room to learn and get different results next time. Introduction For centuries, ancient Egyptians used grains like barley and oats to make unappetizing food staples, until stumbling upon an accident where wheat dough was set aside and they noticed it began to ferment and leaven, giving birth to the first recognizable loaf of type I sourdough bread (Wagner, 2005). Fast forward about 5,000 years and people still consume bread products as one of our necessary staples. However, with the recent discovery of producing yeast commercially, type I sourdoughs are not used to make bread products as often as they once were. Out of the 3 main types of sourdough breads, there are several differences and similarities between them. Type I is the most traditional type of sourdough bread and can have the most diverse outcomes and microbes involved. Type I is generally firm, has a pH of about 3.8 to 4.5, and is fermented at about 68-86 degrees Fahrenheit (Xu et al, 2019). With type I you can achieve a more diverse and complex outcome, like various microbiota, better flavor, aroma, taste, texture, and shelf life (Yan, 2019). Type II sourdough bread is used by industrial companies to easily produce mass amounts of bread products and is also utilized residentially for ease. Type II sourdoughs are not as firm, have a pH less than 3.5 and are fermented at about 86-122 degrees Fahrenheit (Xu et al, 2019). Type II applies commercial yeast (active dry yeast, baker, or instant yeast) which uses an inactive form of Saccharomyces cerevisiae to achieve dough leavening (Xu, et al, 2019). Studies have shown that S. cerevisiae can limit the diversity in aroma, end products, taste, and other factors (Aslankoohi et al, 2016). Type III

sourdough breads are made with a drying process, and mainly used at industrial levels as flavoring. Drying-resistant lactic acid bacteria like Pediococcus pentosaceus, Lactobacillus plantarum, and L. brevis are involved. As you can see, since type I sourdough bread ferments its own yeasts, it is the most unique and can vary the most out of the three breads, especially when talking about the microbes at work. While growing type I sourdough, the environment, flour type, other ingredients, and the procedures used, all effect and thus cause the selection of characteristic microflora that generally contain a diverse mix of lactic acid bacteria and yeasts. While studies have found vast amounts of compounds present, type I sourdough harbors Lactobacillus sanfranciscensis (a species of lactic acid bacteria) as its dominant microbiota in association with Kazachstania species (predominantly Kazachstania humilus) (Xu et al, 2019). A study done in Italy, making type I sourdough, revealed over 50 lactic acid bacteria species were isolated. About 30 species belonged in the genus Lactobacillus, while over 20 yeast species belonged to the genera Saccharomyces and Candida, and the presence of non-Lactobacillus species, such as Enterococcus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus and Weissella (Reale et al, 2011). During the fermentation process the noticed that lactobacilli were highly adaptive to the environmental conditions, like temperature, pH, acidity, antimicrobial products, etc. (Reale et al, 2011). Therefore, lactobacilli are the dominant microbial group in mature sourdoughs. But where do these microbes come from? Basically, your environment, the type of flour and water used dictates what microbes will be present in your sourdough. To initiate fermentation of type I sourdough starters the presence of at least one species of yeast must be present in your environment. By simply having the mixture of flour and water and exposing it to the air and environment you are in, yeasts from that environment are entrapped. The number of yeast species found in the sourdough starter and the pH at which these cultures can grow will be drastically different if the culture had S. cerevisiae (type II) in it to start, versus yeasts trapped from the environment. Another component is the bacterium Lactobacillus, to grow it requires maltose (found in flour) (Wagner et al, 2005). So, the mix of yeast species your starter has or obtains depends upon the type of flour used and the environmental conditions during the fermentation process. Now, lets find out what and how all these microbes live together to form sourdough bread. The bacterium Lactobacillus, will ferment the flour while the yeasts cannot (Wagner et al, 2005). In the acidic environment that they create, Lactobacilli thrive. The bacteria can produce an antibiotic that controls other organisms that might contaminate the sourdough starter, this

keeps it from getting engulfed by other microorganisms (Wegner et al, 2005). The bacteria also produce aldehydes and esters that are unique to sourdough bread and may contribute to its flavor. For type II sourdough, Saccharomyces exiguous or Candida humilis is capable of growing in an acidic environment and unable to utilize maltose. The yeast seems to coexist with sourdough bacteria that require or prefer maltose and produce organic acids upon its metabolism (Wegner et al, 2005). Lactobacillus sanfranciscensis require large amounts of maltose to grow and produce lactic and acetic acids as well as carbon dioxide. These acids make sourdough bread up to ten times more acidic (pH 3.8 to 4.3) than other bread. There is a balancing relationship with the two organisms as they metabolize different carbohydrates which simultaneously forms protection for the dough from other microorganisms, making the sourdough usable for years (Wegner et al, 2005). There are many more components but that is the gist of some functions occurring in sourdough starters. Methods I.

Preparing the Starter: Day one

To prepare the starter on day one, at 5 PM, two tablespoons of oat flour and two tablespoons of filtered water were placed in a glass mason jar and thoroughly mixed to form a paste. The sides of the jar were scraped with a spoon to reduce the chances of mold to form on the sides of the jar and so it was easier to observe how much the starter was rising. The mouth of the jar was covered with a paper towel and secured with a rubber band. This kept out any large debris and insects but allowed microbes from the environment to filter. The starter was placed under a warm lamp for 24 hours. II.

Refreshing and Feeding the Starter: Days 2 - 15

Every day, for at least 14 days, the starter was refreshed and fed. At 5 PM the paper towel lid and rubber band were removed, and a spoon was used to mix the starter thoroughly. Each day the starter was smelled and noted to observe subtle shifts happening in the microbial community. Every 3 days the starters pH was measured. One tablespoon of the starter was removed from the mason jar and discarded into the trash (this was done to keep the starter from growing exponentially large thus needing a massive amount of flour). Then four teaspoons of oat flour and one tablespoon of filtered water was added to the starter in the mason jar and mixed thoroughly. A spoon was used to scrape down the sides of the jar to ensure the entire mixture was together in the bottom of the jar. The paper towel lid was placed back on the mason

jar and secured with the rubber band. The starter was placed back under the warm lamp for the next 24 hours. To Test the pH: After the starter was mixed and the tablespoon was being discarded, that discarded tablespoon was used to measure the pH. One side of a pH paper strip touched the starter. The paper pH strip could soak, while keeping the other side clean to make the reading easier. The color on the paper was then matched to the color key and noted down. III.

Characterize Rise Time and Height

The day after the 14th day at 5 PM the starter was fed it was mixed thoroughly. Two tablespoons of the starter were removed and instead of being discarded it was added to a new glass mason jar. Three tablespoons of oat flour and two tablespoons of filtered water was also added to the new mason jar and mixed thoroughly. The height was recorded at 6.35 mm. A paper towel lid was secured on both jars, and they were set aside for the night. The next day at 9 AM the new height was recorded at 30.48 mm and pictures were taken for the Rob Dunn lab website. The aroma was observed as smelling bodily, earthy, and moldy. The second mason jar was then discarded. The original mason jar with the majority of the starter had its pH measured one last time. IV.

Bread Recipe

3 2/3 cups of oat flour and 1 ¾ teaspoons of salt were mixed in a bowl. 1/3 cup mature sourdough starter and 1 ½ cups plus 4 teaspoons water was mixed in a separate bowl. The wet and dry ingredients were then mixed for about 10 minutes, till the dough was smooth looking. The dough was placed on the counter and formed into a ball. The bowl was coated with oil and the dough ball was placed into the bowl, to coat the entire surface. The bowl with the dough inside was covered and placed on the counter for 12 hours. Then the bowl was placed in the refrigerator overnight. The oven was set to 500 degrees Fahrenheit with a baking sheet inside to warm it up. The bowl of dough was taken out of the refrigerator and removed gently from the bowl. Rice flower was sprinkled on the top surface of the dough ball. The baking sheet was removed from the oven and the dough ball was placed, rice flour side down on the sheet. A knife was used to score the top of the loaf. The baking sheet with the dough on it was placed into the oven on the middle rack. The oven temperature was then re-set to 475 degrees Fahrenheit. The dough was baked for 20 minutes at 475 degrees Fahrenheit then the temperature was lowered to 350 degrees Fahrenheit and baked another 20 minutes.

Results On day 1, preparing the starter was easy and went well. On day two, the first pH measurement was taken, and it read about 5.8 pH and the refreshing and feeding started. On day four small bubbles were observed for the first time in the starter, observed in figure 1, which indicated yeast was active and was creating gases as it was feeding off the flour. Over the course of the rest of the fermentation duration everything went smoothly with refreshing and feeding the starter, making sure it stayed at the proper 75 degrees Fahrenheit and measuring the pH occasionally. The pH measured 5.8, 5.5, 5.5, 5.3, 4.9, 4.5, 4.2, 4.0, and finally 3.8 pH. Over the course of the fermentation I found the pH to decrease from about 5.8 pH to about 3.8 pH on the last day, indicating a production of acid, figure 2 was the last day of fermentation and the starter grew well. The rise time and height were recorded on the last day to the Rob Dunn lab website and it was successful in rising over night, figure 3 and 4. The bread recipe was then followed, and the sourdough was baked. The bread in figure 5 did not come out perfect on the inside, it was most likely due to inaccurate oven temperatures.

Figure 1: Bubbles forming on day 4

Figure 2: Day 14 before making into bread

Figures 3: Arial shot of Characterizing rise time and Height

Figure 4: Side shot of Characterizing rise time and Height

Figure 5: Bread, Finished Product Discussion After writing up and reviewing my results I am very pleased with how my project turned out. I was able to properly ferment oat flour and filtered water over a course of 15 days. I know fermentation occurred because there were bubbles forming which indicated that yeast or other microbes were converting the flour into gases and sugars. I got a pH level that was in normal range for type I sourdough breads, decreasing over time, which also indicated successful formation of acid. When I cooked the bread, it looked like it came straight from a bakery which made me happy, figure 5. However, when I cut into my loaf the middle was not the best consistency. The bread was a bit dense, almost like it was not cooked all the way through. I am not sure what happened, other than my oven is a bit old and I don’t think the temperature was reading accurately. Over winter break my brother and I are going to try making it again. My roommates also enjoyed watching me complete this project and started making one oof their own. It took a lot of patience because I am not used to taking care of something so precisely every single day. For my first time making this I think it came out pretty well! References 1. Reale, A., Reale, A., Di Renzo, T., Di Renzo, T., Succi, M., Succi, M., Tremonte, P., Tremonte, P., Coppola, R., Coppola, R., Sorrentino, E., and Sorrentino, E. (2011) Identification of lactobacilli isolated in traditional ripe wheat sourdoughs by using molecular methods. World J.Microbiol.Biotechnol. 27, 237-244 2. Yan, B., Sadiq, F., Cai, Y., Fan, D., Zhang, H., Zhao, J., and Chen, W. (2019) Identification of Key Aroma Compounds in Type I Sourdough-Based Chinese Steamed Bread: Application of

Untargeted Metabolomics Analysisp. International journal of molecular sciences; Int J Mol Sci. 20, 818 3. Xu, D., Zhang, Y., Tang, K., Hu, Y., Xu, X., and Gänzle, M.,G. (2019) Effect of Mixed Cultures of Yeast and Lactobacilli on the Quality of Wheat Sourdough Bread. Frontiers in microbiology. 10, 2113 4. Aslankoohi, E., Herrera-Malaver, B., Rezaei, M.N., Steensels, J., Courtin, C.M., and Verstrepen, K.J. (2016) Non-Conventional Yeast Strains Increase the Aroma Complexity of Bread. PloS one; PLoS One. 11, e0165126 5. Wagner, S.C. (2005) FROM STARTER TO FINISH: Producing Sourdough Breads To Illustrate the Use of Industrial Microorganisms. The American Biology Teacher. 67, 96-101...