The Efficacy of Various Food Preservation Methods Against the Growth of Escherichia Coli in a Controlled Environment
Abstract:
Bibliography/Citations:
Islam, Shirmin et al. “Probiotic potential of Lactobacillus plantarum DMR14 for preserving and extending shelf life of fruits and fruit juice.” Heliyon vol. 9,6 e17382. 19 Jun. 2023, doi:10.1016/j.heliyon.2023.e17382
Łojewska, Ewelina, and Tomasz Sakowicz. “An Alternative to Antibiotics: Selected Methods to Combat Zoonotic Foodborne Bacterial Infections.” Current microbiology vol. 78,12 (2021): 4037-4049. doi:10.1007/s00284-021-02665-9
Punia Bangar, Sneh et al. “Natural Antimicrobials as Additives for Edible Food Packaging Applications: A Review.” Foods (Basel, Switzerland) vol. 10,10 2282. 26 Sep. 2021, doi:10.3390/foods10102282
Teneva, Desislava, and Petko Denev. “Biologically Active Compounds from Probiotic Microorganisms and Plant Extracts Used as Biopreservatives.” Microorganisms vol. 11,8 1896. 27 Jul. 2023, doi:10.3390/microorganisms11081896
Udayakumar, S.; Rasika, D.M.D.; Priyashantha, H.; Vidanarachchi, J.K.; Ranadheera, C.S. Probiotics and Beneficial Microorganisms in Biopreservation of Plant-Based Foods and Beverages. Appl. Sci. 2022, 12, 11737. https://doi.org/10.3390/app122211737
Additional Project Information
Project files
Research Plan:
Rationale
Although industrial food preservation has achieved great wide-scale efficacy, many food products remain susceptible to harmful bacterial growth after time under refrigeration. A common culprit of this growth is the specimen, Escherichia Coli (E. coli). If consumed, coli-contaminated food can incite a variety of symptoms: including but not limited to, nausea, diarrhea, fever, congestion, cough, and rarely paralysis or seizure. This experiment aims to help consumers determine the best and most optimal form of at-home food preservation for the dangerous bacteria.
Hypotheses
The probiotic treated agar plates will experience the least growth of Escherichia Coli (as compared to the salt and turmeric-garlic treated plates) under a controlled environment since the probiotics utilize competitive exclusion, promoting the growth of specific bacterial strains which outcompete with the growth of E. Coli.
Methods, Materials and Procedures
Materials
1. 125 ml Tryptic Soy Agar [Carolina Biological]
2. 16 (20mm) petri dishes [Carolina Biological]
3. 1 head of lettuce
4. 50 grams iodized salt crystals
4. 25 grams ginger powder
5. 25 grams turmeric powder
6. 50 grams Lactobacillus Rhamnosus GG powder. [Culturelle Kids]
7. 1 Culture Escherichia Coli B [Ward's ScienceX]
8. 10 vials (125 ml) Escherichia Coli B broth [Ward's ScienceX]
9. 500 grams distilled water (ml)
Storage
1. Refrigerate Ward's ScienceX Escherichia coli B vial at 4º Celsius.
2. Refrigerate Ward's ScienceX Escherichia coli B culture broth at 4º Celsius
3. Store Culturelle Kids' Lactobacillus rhamnosus GG at 25º Celsius
4. Refrigerate Lettuce Leaf Dilution at 4º Celsius
5. Store Turmeric-Ginger Dilution at 25º Celsius
6. Store Salt Dilution at 25º Celsius
Procedure
24 hours before the experiment
1. Place 12 sterilized petri dishes on the prepared work surfaces
1a. Label 3 of the petri dishes "10% NaCl" (salt treatment)
1b. Label 3 of the petri dishes "10% GT" (ginger turmeric treatment)
1c. Label 3 of the petri dishes "10% LBR" (probiotic treatment)
1d. Label 3 of the petri dishes "– C" (refrigeration treatment)
2. Place the bottle of TS Agar into a microwave with the lid loosened, and microwave in undetermined increments until the medium is completely liquid.
3. Hold the bottle of TS Agar at a 45º angle to prevent contamination
4. Hold the lid of one petri dish open at a 30º angle and carefully pour TS Agar until it covers the bottom of the plate (roughly 7.8 ml of agar)
4a. Repeat step 4 for every petri dish.
5. Set the dishes aside on a sanitized work surface and let them rest until they have cooled to a solid.
6. Place a sterilized test tube rack on a prepared work surface
7. Place 1 vial of the Ward's ScienceX Escherichia Coli B Broth in the test tube rack.
8. Using an inoculating loop, collect the E. coli culture and stir it into the vial of broth.
9. Place the vial, and the rack, into an incubator and incubate at 37º C until the experiment is ready to begin.
30 minutes before the experiment
1. Set head of lettuce and distilled water aside on a prepared work surface
2. Prepare a food processor
3. Separate the lettuce leaf blades and nodes
4. Using a scale, measure 100 grams of lettuce leaf blades and 100 grams of distilled water (ml).
5. In 10 second intervals, process the lettuce leaf blades and distilled water into a slurry
6. Place a sterilized beaker on a prepared work surface
7. Place a sterilized funnel on the beaker
8. Place a cheesecloth on the funnel
9. With a gloved hand, pour the lettuce leaf slurry through the cheesecloth
9a. Repeat step 9 until the liquid in the beaker turns a light green.
10. Seal the top of the beaker using a Parafilm strip.
11. Refrigerate the beaker containing the lettuce leaf dilution at 4º C until the experiment is ready to begin
Beginning the Experiment
12. Set a test tube rack on a sterilized work surface
13. With a gloved hand, place 3 sterilized test tubes into the rack and label them "10% NaCl," "10% GT" & "10% LBR"
14. Using a scale, measure 270 grams of distilled water (270 ml)
14a. measure 10 grams of salt and set aside in a sterilized dish
14b. measure 5 grams of ginger powder and set aside in a sterilized dish
14c. measure 5 grams of turmeric powder and set aside in a sterilized dish
14d. measure 10 grams of Lactobacillus Rhamnosus GG powder and set aside in a sterilized dish
15. Pipette 90 grams of distilled water into each of the test tubes
15a. Use a sterilized scoopula and place 10 grams of salt into the first test tube labeled "10% NaCl" and stir before sealing.
15b. place 5 grams of ginger powder into the second test tube labeled "10% GT" and stir before sealing.
15c. place 5 grams of turmeric powder into the second test tube labeled "10% GT" and stir before sealing.
15d. place 5 grams of Lactobacillus Rhamnosus GG powder into the third test tube labeled "10% LBR" and stir before sealing.
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16. Take the lettuce leaf dilution out of the refrigerator and let it sit at 25º C until it is required for the experiment.
17. Remove the test tube with the vial of Escherichia Coli B Broth and place it onto a prepared work surface.
18. Stir the liquid gently with a sterilized inoculating loop.
19. Extract the liquid with a pipette until there is 90 ml of the Escherichia Coli B Broth in the tube.
20. pipette 10 ml of the lettuce leaf dilution into the tube with the Escherichia Coli B Broth and stir.
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21. Set both of the test tube racks on the same work surface as the petri dishes.
22. Carefully pipette 1 ml of the "10% NaCl" solution, and 1ml of the Escherichia Coli B Broth x Lettuce Leaf Dilution, onto each of the dishes labeled "10% NaCl"
22a. Carefully pipette 1 ml of the "10% GT" solution, and 1ml of the Escherichia Coli B Broth x Lettuce Leaf Dilution, onto each of the dishes labeled "10% GT"
22b. Carefully pipette 1 ml of the "10% LBR" solution, and 1ml of the Escherichia Coli B Broth x Lettuce Leaf Dilution, onto each of the dishes labeled "10% LBR"
22c. Use masking tape to secure the lids of the dishes labeled "10% NaCl," "10% GT" or "10% LBR" and incubate at 37ºC for 48 hours.
26. Carefully pipette 1ml of the Escherichia Coli B Broth x Lettuce Leaf Dilution onto each of the dishes labeled "– C".
26a. Secure the petri dishes lids using masking tape.
26b. Refrigerate the plates labeled "– C" at 0º C for 48 hours.
Observation and Analysis
27. Sterilize a light tray and place it onto a prepared work surface.
28. Take each of the petri dishes out of the incubator.
29. Place the petri dishes on a prepared work surface.
30. Sanitize a tripod and position the photography device above the light tray.
31. Place any of the 12 petri dishes on the light tray and take a picture
31b. repeat step 31 for the remaining plates.
32. Crop each image so that each border is touching the petri dishes' rim.
32a. Use a graphic measuring tool (iOS image markup) and confirm that the surface area of the petri dishes are consistent throughout each picture.
32b. If the petri dish surface areas are not consistent, retake the pictures after stabilizing the tripod.
33. Measure the surface area of each individual bacterial colony on a single petri dish and add them together
33a. Divide the surface area of the bacterial colonies by the surface area of the petri dish.
33b. Denote the bacterial surface area percentage in a table.
33c. Compare the percentages and form a conclusion (a lower percentage of bacterial surface colonies indicates a more effective form of preservation).
Bibliography
Islam, Shirmin et al. “Probiotic potential of Lactobacillus plantarum DMR14 for preserving and extending shelf life of fruits and fruit juice.” Heliyon vol. 9,6 e17382. 19 Jun. 2023, doi:10.1016/j.heliyon.2023.e17382
Łojewska, Ewelina, and Tomasz Sakowicz. “An Alternative to Antibiotics: Selected Methods to Combat Zoonotic Foodborne Bacterial Infections.” Current microbiology vol. 78,12 (2021): 4037-4049. doi:10.1007/s00284-021-02665-9
Punia Bangar, Sneh et al. “Natural Antimicrobials as Additives for Edible Food Packaging Applications: A Review.” Foods (Basel, Switzerland) vol. 10,10 2282. 26 Sep. 2021, doi:10.3390/foods10102282
Teneva, Desislava, and Petko Denev. “Biologically Active Compounds from Probiotic Microorganisms and Plant Extracts Used as Biopreservatives.” Microorganisms vol. 11,8 1896. 27 Jul. 2023, doi:10.3390/microorganisms11081896
Udayakumar, S.; Rasika, D.M.D.; Priyashantha, H.; Vidanarachchi, J.K.; Ranadheera, C.S. Probiotics and Beneficial Microorganisms in Biopreservation of Plant-Based Foods and Beverages. Appl. Sci. 2022, 12, 11737. https://doi.org/10.3390/app122211737
Questions and Answers
Iniitial project questions
1. What was the major objective of your project and what was your plan to achieve it? I wanted to determine which method of preservative food method, such as brining or marination, would be most effective at inhibiting bacterial growth (amongst salt, probiotics, and ginger-turmeric).
a. Was that goal the result of any specific situation, experience, or problem you encountered? [n/a]
b. Were you trying to solve a problem, answer a question, or test a hypothesis? I was trying to answer a question but
2. What were the major tasks you had to perform in order to complete your project? Pipetting, Agar Pouring, Sterile Technique, Disposal
a. For teams, describe what each member worked on. [n/a]
3. What is new or novel about your project?
a. Is there some aspect of your project's objective, or how you achieved it that you haven't done before? This is the first time I have utilized probiotics for competetive inhibition when researching the restriction of bacterial growth.
b. Is your project's objective, or the way you implemented it, different from anything you have seen? Not particularly.
c. If you believe your work to be unique in some way, what research have you done to confirm that it is? [n/a]
4. What was the most challenging part of completing your project? Conducting an experiment in school restricts the window of time in which procedure can take place.
a. What problems did you encounter, and how did you overcome them? In the initial round of testing, the incubator shut off due to weather resulting in school closure.
b. What did you learn from overcoming these problems? Research procedure must be completed as swiftly as possible and must anticipate any possible obstructions.
5. If you were going to do this project again, are there any things you would you do differently the next time? I would include an ecoliphage treatment as well as phage-based preservative technology has proven effective. This would diversify the comparability of the experiment. I would also increase the concentrations of each treatment and compare the effects on bacterial growth inhibition.
6. Did working on this project give you any ideas for other projects? I was inspired to look further into the applications of probiotics as promoters of competetive inhibition.
7. How did COVID-19 affect the completion of your project? It did not.