Lactic Acid Fermentation Lab Report PDF

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Professor: Frank Mallory...

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Changes in pH and texture of milk mixtures fermented by lactic acid bacteria (Streptococcus thermophilus and Lactobacillus bulgaricus) to produce yogurt Megan Brousseau BIOL 1506E Tuesday 1:15pm November 15th, 2016

Introduction

Lactic acid fermentation is a form of cellular respiration conducted by anaerobic organisms (Brosseau-Demore and Mallory 2016). Like other respiration processes, anaerobic respiration produces pyruvate through the application of glycolysis (Brosseau-Demore and Mallory 2016). However, in animals and microorganisms, the oxidation of pyruvate is done by fermentation to lactic acid (Brosseau-Demore and Mallory 2016). The fermentation process is activated by lactic acid bacteria (Rajendran and Ohta 1998). Lactic acid bacteria form a diverse group of microorganisms that can be beneficial to their hosts (Laiño et al. 2012). This faction of bacteria are found naturally in foods as well as the gastrointestinal tract (Laiño et al. 2012). In addition, fermentation can be of economic value when associated with food (Chelule et al. 2010). Fermented food products improve nutritional quality by developing detoxifying effects and increasing the availability of vitamins and proteins (Chelule et al. 2010). Furthermore, fermented foods have preservation properties due to their low pH levels (Chelule et al. 2010). When abundantly used, consumers can expect an immune system boost as well as an increase in the body’s ability to fight pathogenic bacterial infections (Chelule et al. 2010). Moreover, animals commonly experience lactic acid fermentation in the cytosol of their muscle cells during demanding activity (Russel et al. 2011). Such activity requires ATP at a rate that cannot be supported and results in not enough O2 for aerobic respiration to continue (Russel et al. 2011). Once the oxygen levels of the muscle cells return, pyruvate is regenerated and processed normally by the citric acid cycle and electron transport chain (Russel et al. 2011). The purpose of the experiment was to demonstrate lactic acid fermentation by preparing yogurt through the incubation of milk mixtures and to identify the live cultures that aid in the yogurt production process (Brosseau-Demore and Mallory 2016). Also, to observe the influence fermentation has on texture and pH of different mixtures. One of the mixtures was designed to

remain the same before and after incubation in order to provide a control for the experiment (Brosseau-Demore and Mallory 2016). The other mixture was intended to produce the yogurt that would be further studied (Brosseau-Demore and Mallory 2016). Materials and Methods Materials and methods as outlined in BIOL 1506E Laboratory Manual, Diffusion and Osmosis lab, Exp. B (Brosseau-Demore and Mallory, 2016). Results Throughout the experiment, texture and pH levels were monitored and recorded. Table 1 shows the changes each test tube experienced before and after the incubation period. To begin, test tube 1 remained relatively constant during the experiment. Prior to the incubation, both the milk as well as the solution containing milk and water measured an approximate 6.5 pH level. Once the incubation was complete, the pH was recorded at roughly 7. Additionally, the texture of the milk alone was a creamy liquid that became slightly thinner after the water was combined. Finally, the texture of the milk and water mixture before and after the incubation was the same. Test tube 2 experienced significantly different results in comparison to test tube 1. Before the incubation period, the pH of the milk alone was recorded at roughly 6.5 and dropped to approximately 5.3 after the yogurt was added. After the incubation, the pH remained at 5.3. Similar to the first test tube, the milk in test tube 2 was a creamy liquid. Prior to the incubation, the yogurt was added and the texture became a thicker consistency. Finally, after the incubation the texture changed to a creamy solid. Table 1. Changes in pH and texture of solutions containing milk and milk mixed with water or yogurt. Observations recorded before and after a 6-8 hour incubation period at 45°C.

Tube 1

pH 6.5

Tube 2

6.5

Pre Incubation milk milk + water or yogurt texture pH texture Creamy liquid 6.5 Thin, creamy liquid Creamy liquid

Post Incubation mixture pH texture 7 Thin, creamy liquid

5.3

Thick, creamy 5.3 Creamy solid liquid A wet mount was prepared from the yogurt produced in test tube 2 and was observed

under a microscope. Figure 1. displays the two bacterial cultures found in the yogurt. The first bacteria, Streptococcus thermophilus, looks like a chain made of spherical shapes. In addition, Lactobacillus bulgaricus appears similar to previously mentioned bacteria except the chain is made of rod-like shapes. Streptococcus thermophilus

Lactobacillus bulgaricus

Figure 1. Live cultures found in yogurt. Discussion During the experiment, yogurt was produced through the incubation of milk mixtures and the live cultures present were observed and studied. The production of yogurt relies on the two thermophilic lactic acid bacteria, Streptococcus thermophilus and Lactobacillus bulgaricus (Brosseau-Demore and Mallory 2016). The symbiosis between both cultures carried out fermentation to digest the milk sugars and produce lactic acid (Angelov et al. 2009). The lactose from the milk was metabolized by the lactic acid bacteria and convert into glucose (Angelov et

al. 2009). Glycolysis was conducted and the pyruvate was oxidized to complete the reaction and produce lactic acid (Brosseau-Demore and Mallory 2016). Since S. thermophilus and L. bulgaricus thrive at relatively high temperatures, the 45°C incubation period was necessary in order for the bacteria to be efficient in the fermentation process (Marteinsson et al. 1997). Moreover, the milk mixtures were cooled after incubation to stop further acidification and to preserve and maintain the live cultures (Greaves 1956). When lactic acid bacteria are cooled, their rate of activity is decreased (Greaves 1956). However, it does not stop completely (Greaves 1956). Throughout the duration of the experiment, each test tube reacted differently. To begin, test tube 1, containing milk and water, was designed to act as the control for the experiment and was intended to provide results from a scenario where lactic acid fermentation did not occur. Therefore, the results obtained from test tube 1 were expected. The slight pH change and miniscule texture difference was due to the addition of water to the milk. However, test tube 2, containing milk and yogurt, experienced distinct changes in both texture and pH. To start, an initial change in pH occurred when the yogurt was added to the milk. The pH level dropped from 6.5 to 5.3, making the solution acidic (Tro et al. 2014). Since yogurt has an average pH value of 4.4, the pH decrease was expected (Campbell 2016). After the incubation was complete, the pH value remained at 5.3. The pH results obtained were slightly less acidic than anticipated and possible sources of error were taken into consideration. For example, the pH indicator strips used in the experiment did not give an exact pH value, but provided a range of possible values. Therefore, the actual pH of the mixtures were unknown and an approximation was recorded. This factor could have caused the recorded results to be inaccurate compared to the actual results.

In addition, the texture of the milk in test tube 2 was originally observed as a creamy liquid. The texture changed to a much thicker liquid after the yogurt was combined. This result was expected due to yogurt’s thick consistency. Once the incubation was complete, the texture drastically changed from a liquid to a creamy solid. The thick, creamy milk mixture was fermented by lactic acid bacteria to produce yogurt. Due to the contents of the mixture and the methods used in the experiment, the production of yogurt was expected. To conclude, the process of lactic acid fermentation was distinctly demonstrated through the fermentation of milk into yogurt. Lactic acid bacertia, Streptococcus thermophilus and Lactobacillus bulgaricus, proved to be essential in the production of the yogurt. Working in symbiosis the two live cultures thrived in a hot environment to ferment lactose and produce lactic acid. The fermentation resulted in an acidic pH of approximately 5.3 and solid texture. References Angelov, M., Kostov, G., Simova, E., Beshkova, D., and Koprinkova-Hristova, P. 2009. Proto cooperation factors in yogurt starter cultures. E-Rev. Génie Ind. 3: 1-7. Brosseau-Demore, L. and Mallory, F., 2016, BIOL 1506E Laboratory Manual, Laurentian University, Sudbury, Ont. Campbell, M., 2016, Is Yogurt Alkaline or Acidic?, (Nov. 12, 2016), retrieved from: http://www.livestrong.com/article/483061-is-yogurt-alkaline-or-acidic/ Chelule, P.K., Mokoene, M.P., and Gqaleni, N. 2010. Advantages of traditional lactic acid bacteria fermentation of food in Africa. Tech. and Ed. Topics in Applied Microbiol. and Microbial Biotechnol. 2: 1160-1167.

Greaves, R.I.N., 1956. The preservation of bacteria. Can. J. of Microbiol. 2(3): 365-371 Laiño, J.E., Leblanc, J.G., and Savoy de Giori, G. 2012. Production of natural folates by lactic acid bacteria starter cultures isolated from artisanal Argentinean yogurts. Can. J. of Microbiol. 58(5): 581-588. Marteinsson, V.T., Birrien, J., and Prieur, D. 1997. In situ enrichment and isolation of thermophilic microorganisms from deep-sea vent environments. Can. J. of Microbiol. 43(7): 694-697. Rajendran, R. and Ohta, Y. 1998. Binding of heterocyclic amines by lactic acid bacteria from miso, a fermented Japanese food. Can. J. of Microbiol. 44(2): 109-115. Russel, P.J., Hertz, P.E., Mcmillian, B., Fenton, B.M., Addy, H., Maxwell, D., Haffie, T., and Milsom, B. 2011. Biology: Exploring the Diversity of Life. 2nd edition. Nelson Education Ltd, Toronto, Ont. Tro, N.J., Fridgen, T.D., and Shaw, L.E. 2014. Chemistry: A Molecular Approach. 2nd Canadian edition. Pearson Education Inc., Upper Saddle River, New Jersey....