1. Soy Product Effects on Microbial Flora Soy Product Effects on Microbial Flora Cameron Herbst Pittsburgh Central Catholic High School

2. The Problem  Humans consume many different things, but how can we know to what extent these things affect our physiology?  A viable question which can be asked is: Do ingested consumables have adverse effects on symbiotic organisms in particular, internal flora?

3. The Problem  Countless studies have evaluated the affect of ingested material on human cells and physiology.  Another key component of the body is the natural flora especially those within the alimentary canal. These belong to the families: Clostridium, Fusobacterium, Escherichia, Bacteroides, Eubacterium, and Lactobacillus, among others.

4. Soy Protein Extract  Isolated Soy Protein Mix: A highly refined form of soy protein which contains at least 90% protein on a moisture free basis  Ingredients: Supro ® Brand Soy Protein Isolate, Fructose, Natural and Artificial Flavor, FD&C Red 40, Salt, Sucralose

5. Production and Uses of Soy Protein Isolates  Made from defatted soy flour where most of the fats, carbohydrates, and other non-protein components have been removed  Used to improve texture of meat products, increase dietary protein content, and as an emulsifier

6. The Dangers of Soy  High levels of estrogen – in baby formula, equivalent of 5 birth control pills per day  Causes weak thyroid gland which leads to excess fat and heart problems  Possibly linked to an increase of Leukemia cases in children  Suppresses testosterone levels; can be fatal in extreme cases

7. E. coli E. coli  One of the most common forms of bacteria; Free living, symbiotic or pathogenic  Has been utilized as the most studied prokaryote  There are many of different strains of E. coli, most of which are non- pathogenic. However, there are strains which can produce fatal disease

8. Purpose  To determine whether soy protein isolate in various concentrations will affect the survivorship of intestinal flora (E. coli)

9. Hypothesis  Null – Survivorship of E. coli in varying concentrations of soy protein isolate would not vary significantly from the control.  Alternative – Survivorship of E. coli in varying concentrations of soy protein isolate would vary significantly from the control.

10. Materials  Ethanol (sterilization of instruments)  Latex gloves  E. coli DH5 alpha  Micropipette  Tube rack  4 Tubes  SDF (per 1 liter) (100mM KH 2 PO 4, 100mM K 2 HPO 4, 10mM MgSO 4, 1mM NaCl)  Turn table  LB agar plates  LB media (0.5% yeast extract, 1% tryptone, 1% sodium chloride)  Bunsen burner  Spreader bar  Matches  Sterile pipette tips  Incubator  Vortex  Klett spectrophotometer  Scale  Weigh Boat  Supro ® Soy protein isolate

11. Procedure 1)E. coli was grown overnight in sterile LB media. 2)A sample of the overnight culture was added to fresh media in a sterile sidearm flask. 3)The culture was placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 8 cells/mL. 4)The culture was diluted in sterile dilution fluid to a concentration of approximately 10 3 cells/mL. 5)Soy protein isolate was weighed into appropriate amounts using the scale and sterile weigh boats. 6)The tubes were prepared as follows:

12. Procedure Tube # Soy Protein (g) E. coli (mL) SDF (mL) Final Volume (mL) Final % Soy Protein 100.19.9010.00 20.010.19.8910.00.1 3 9.8010.01.0 4 0.18.9010.0

13. Procedure 7) The E. coli was allowed to remain exposed to the soy protein extract for 15 minutes. 8) 100 µL aliquots were removed from the tubes and spread on LB plates. (8 replicates) 9) The plates were incubated at 37 degrees Celsius for 24 hours. 9) The resulting colonies were quantified. Each colony is assumed to have arisen from one cell.

14. E. coli Soy Survivorship Graph 0.110

15. Dunnett’s Test Variable Comparisont-valueInterpretation 0.1% versus 0% (control)3.20Not Significant 1.0% versus 0% (control)0.359Significant 10.0% versus 0% (control) 9.13Significant α = 0.01 T-critical = 3.15

16. Interpretation  There appeared to be a correlation between the percent concentration of soy protein isolate and cell survivorship. Higher concentrations of soy protein isolate resulted in fewer surviving colonies.

17. Conclusion  The null hypothesis was rejected for the 1% and 10% concentrations of E. coli.  A 10% concentration of Soy protein isolate appeared to greatly reduce E. coli survivorship.

18. Limitations  This experiment was limited in that pure soy protein was not able to be used.  Plating was not exactly synchronized, which could have resulted in extra time for bacterial replication.

19. Extensions  In future studies, synergy of soy effects on other foods could be tested. (2 factor ANOVA)  Varying exposure time of E. coli in the tubes before plating could be tested.  To allow for longer exposure, soy protein could be infused directly into the LB agar.

20. Cited Sources  http://e-colibasics.com/  http://www.soyfoods.org/products/soy- fact-sheets/soy-protein-isolate-fact-sheet  http://www.mercola.com/article/soy/avoid_ soy.htm  http://www.soya.be/soy-protein-isolate.php  http://www.karinya.com/soydangers.htm