Antimicrobial Effects of Orange/Mango Juice Jack O'Toole Grade 9 Pittsburgh Central Catholic High School

Problem What are the effects of orange/mango juice on two different types of bacteria?

Rationale Almost all consumables are tested for their effects on human cells. The micro flora, however, should also be considered. Do consumables such as orange/mango juice cause a disruption in the flora of the body?

Micro Flora Present in the respiratory, urinary, and digestive tracts as well as the skin. Consists of mostly bacteria, few protists and fungi. Most are harmless, but some can cause diseases. Influences human anatomy, physiology, and overall health. Over 1000 different types of bacteria are harbored within and on humans. Ingested substances may have an effect on the population size of the flora

Gram(-) vs. Gram(+) Bacteria Cell wall contains an extra layer of lipopolysaccharides for extra protection. Outer membrane protects bacteria from several antibiotics. Most pathogenic bacteria in humans are Gram (+) organisms. Simple cell wall. Some antibiotics work against the formation of the cell wall.

Escherichia Coli (E.coli) Large and diverse group of gram (-) bacteria. Free living, symbionts, or pathogens. Live in the intestinal tract of many mammals. Most strains are not pathogenic. Serve as a common prokaryotic cell model.

Staphylococcus Epidermidis (Staph) Common surface symbiont in many mammals (Human). Gram (+) bacteria Most strains considered non-pathogenic. Pathogenic strains can be life-threatening.

Health Benefits of Orange Juice one of the healthiest beverage boost immune system function reduce signs of aging protect against cancer boost cellular repair and metabolism detoxify the body improve circulation improves blood pressure reduces inflammation lowers cholesterol levels

Health Benefits of Mango Juice very low in saturated fat, cholesterol and sodium excellent source of dietary fiber, vitamin B6, vitamin A and vitamin C. rich in minerals like potassium, magnesium and copper one of the best sources of quercetin, betacarotene, and astragalin. These powerful antioxidants neutralize free radicals which cause Ailments like heart disease, premature aging, cancer and degenerative diseases throughout the body

Previous Studies E.coli and Staph are common cell models used in many Biomedical investigations. Other fruit juices have been tested, revealing interesting anti-microbial properties.

Purpose To assess the effects of Orange/Mango Juice on the survivorship of Staphylococcus epidermidis and Escherichia coli .

Hypotheses Null Hypothesis: Orange/Mango Juice will have no significant effect on the survivorship of E.coli and Staph. Alternative Hypothesis: Orange/Mango will have significantly reduce the survivorship of E.coli and Staph.

Materials Incubator Gloves/goggles Klett Spectrophotometer Micro and Macro pipettes + Sterile tips Spreader bars Escherichia coli Staphylococcus epidermidis (Staph) Sterile Dilution Fluid (100mM KH2PO4, 100mM K2HPO4, 10mMMgSO4, 1mM NaCl) Bunsen Burner Turn-table Vortex Incubator Gloves/goggles Klett Spectrophotometer 20 Sterile Test Tubes Ethanol Luria Broth Agar Plates (0.5% yeast extract, 1% tryptone, 1% NaCl) Simply Orange with Mango

Procedure (Liquid Pulse) 1. Bacteria (E.coli and Staph) were grown overnight in sterile LB Media. 2. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 3. The cultures were placed in an incubator (37°C) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10⁸ cells/mL. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10⁵ cells/mL. 5. The Orange/Mango Juice was sterile filtered 6. The experimental variables were mixed with the appropriate amounts of SDF to create concentrations of 0%, 1%, 5% and 10%.

Chart of Liquid Concentration 0% 1% 5% 10% Microbe 0.1mL SDF 9.9mL 9.8mL 9.4mL 8.9mL Orange/Mango Juice 0mL 0.5mL 1mL Total 10mL

Procedure (Liquid Pulse) 7. 100 µL of cell culture was then added to the Orange/Mango juice solutions, yielding a final volume of 10 mL and a cell density of approximately 10^3 cells/mL. 8. The solutions were vortexed. 9. After vortexing to evenly suspend the cells, 100 µL aliquots were removed from the tubes and spread on LB- agar plates. 10. The plates were incubated at 37 C for 24 hours. 11. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.

Procedure (Agar Infusion) Plates were infused as follows: 2. The plates were incubated for 1 hour (37°C) to promote infusion. 3. 100µL from the control tube (0%) was spread onto the plates. 4. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell. Concentration SDF Variable Low 180µL 20µL High 0µL 200µL

Orange/Mango Juice Effects on E.Coli Survivorship (Liquid Pulse) P-value < 0.1^-6

Orange/Mango Juice Effects on E.Coli Survivorship (Agar Infusion) P-value < 0.1^-6

Orange/Mango Juice Effects on Staph Survivorship (Liquid Pulse) P-value < 0.1^-6

Orange/Mango Juice Effects on Staph Survivorship (Agar Infusion) P-value < 0.1^-6

Anova Abbreviation for analysis of variance Statistical test comparing variation within and between experimental groups If the P- value is lower than the alpha value (.05), then the result is significant (a result of the variable influence)

Dunnett's Test Follow up test to the Anova Localizes source of variation compared to the control. T Value is greater than your T Critical value than the difference is significant.

Dunnett's Test Staph (Liquid Pulse) Concentration Groups T-Value T-Crit Value Results 1% 5.634 2.94668 Significant 5% 18.008 10% 38.361

Dunnett's Test Staph (Agar Infusion) Concentration Groups T-Value T-Crit Value Results Low 68.748 4.9646 Significant High 83.334

Dunnett's Test E.Coli (Liquid Pulse) Concentration Groups T-Value T-Crit Value Results 1% 5.129 2.94668 Significant 5% 12.46 10% 21.002

Dunnett's Test E.Coli (Agar Infusion) Concentration Groups T-Value T-Crit Value Results Low 2.112 4.9646 Insignificant High 15.682 Significant

Conclusion Reject the null hypothesis that there will be no significant effect on the survivorship of Staph and E. coli. Each tested concentration of Orange/Mango significantly reduced the survivorship of both E. Coli and Staph except for the Low concentration of the E.coli agar infusion.

Limitations Only two Bacteria tested. Only one exposure time. Only one brand of orange/mango juice tested Study doesn't account for other factors affecting the bacteria

Extensions Use other brands Use other bacteria Use multiple exposure times Do more trials per group More control over human interference

Works Cited https://www.organicfacts.net/health- benefits/beverage/orange-juice.html https://www.organicfacts.net/health- benefits/fruit/mango.html

E.Coli Anova (Liquid Pulse) P-value < 0.1^-6

E.Coli Anova (Agar Infusion) P-value < 0.1^-6

Staph Anova (Liquid Pulse) P-value < 0.1^-6

Staph Anova (Agar Infusion) P-value < 0.1^-6