Effects of Pineapple Juice on Microbial Flora Jamison Beiriger Grade 9 Central Catholic High School
Pineapple Juice Popular drink worldwide Pineapples grow in tropical climates 131% Daily Value of Vitamin C High in sugar and citric acid
The Effects of Sugar Three main sugars in pineapple juice 1. FRUCTOSE 2. Glucose 3. Sucrose NegaMve Impacts when taken in excess: Obesity Diabetes Other diseases
TYPES OF PINEAPPLE JUICE TESTED Fresh Squeezed Dole Canned 100%
INGREDIENTS Fresh Squeezed Fructose Glucose Bromelain Sucrose Citric Acid Vitamin C Thiamine Vitamin B6 Magnesium Potassium Dole Canned 100% Ascorbic acid (vitamin C) Potassium Fructose Glucose Bromelain Sucrose Citric Acid Vitamin C Thiamine Vitamin B6 Magnesium
Escherichia coli Escherichia coli is a large and diverse group of gram (- ) bacteria Found in the intesmnal tract of most animals, including humans EsMmated to cause infecmon in more than 70,000 paments a year in the United States
Saccharomyces cerevisiae (Yeast) Easy to grow and culture Unicellular Most studied cell in the world Aerobic and anaerobic cell respiramon Similar cell cycle, biochemistry and genemcs to other eukaryomc cells, like those in humans
Purpose: To determine if pineapple juice has a significant effect on microbial flora survivorship.
Null Hypothesis Alternate Hypothesis Pineapple juice will not Pineapple juice will significantly significantly affect the survivorship of E. coli or yeast affect the survivorship of E. coli or yeast
Materials: E. coli Yeast Latex Gloves Micropipebes Micro tubes Fresh squeezed pineapple juice Canned Dole pineapple juice Sterile DiluMon Fluid (SDF) (per 1 liter) (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl) LB agar plates LB media (Per Liter:1% Tryptone, 0.5% Yeast Extract, and 1% NaCl) YEPD media and agar plates (1% yeast extract, 2% peptone, 2% glucose (dextrose), 1.5% agar) 2 syringe Sterile Filters Ethanol Matches Bunsen Burners Vortex Spread Bar Kleb Spectrophotometer Incubator
Procedure 1. Saccharomyces cerevisiae and E. coli were grown overnight in sterile YEPD and LB media. 2. Samples of the overnight cultures were added to fresh media in a sterile sidearm flask. 3. The cultures were placed in incubators (30 C and 37 C) until a density of 50 Klett spectrophotometer units were reached. This represents cell densities of approximately 10 7 cells/ml and 10 8 cells/ml. 4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/ml. 5. Fresh squeezed and canned pineapple juices were added with the appropriate amount of SDF to create a concentration of 10%, 1%, and a control of 0%.
E. coli Chart of ConcentraIon Control 1x Canned 10x Canned 1% Fresh Squeezed 10% Fresh Squeezed E. coli 0.1 ml 0.1 ml 0.1 ml 0.1ml 0.1ml Sterile DiluMon Fluid (SDF) 9.9 ml 9.8 ml 8.9 ml 9.8 ml 8.9 ml Fresh squeezed juice 0 ml 0 ml 0 ml.1 ml 1 ml Canned juice 0 ml.1 ml 1 ml 0 ml 0 ml Total Volume 10 ml 10 ml 10 ml 10ml 10ml
Yeast Chart of ConcentraMon Control 1% Fresh Squeezed 10% Fresh Squeezed 1% Canned 10% Canned Yeast.1ml.1ml.1ml.1ml.1ml Sterile DiluMon Fluid (SDF) Fresh Squeezed Juice 9.9 ml 9.8 ml 8.9 ml 9.8 ml 8.9 ml 0 ml.1 ml 1 ml 0 ml 0 ml Canned Juice 0 ml 0 ml 0 ml.1 ml 1ml Total volume 10ml 10 ml 10 ml 10ml 10ml
Procedure (Liquid Exposure conmnued) 7. 100 µl of cell culture was then added to the pineapple juice solumons, yielding a final volume of 10 ml and a cell density of approximately 10 3 cells/ ml. 8. The solumons were vortexed and allowed to sit at room temperature for 15 minutes 9. Aier vortexing to evenly suspend the cells, 100 µl aliquots were removed from the tubes and spread on YEPD and LB agar plates. 10. The plates were incubated at 30 C and 37 C for 24 hours. 11. The resulmng colonies were counted visually. Each colony was assumed to have arisen from one cell.
250 200 Yeast Results P Value= 3.5E- 04 P Value= 8.89E- 06 P Value= 1.55E- 07 Colonies 150 100 P value= 1.77E- 5 FS Canned 50 0 0% 1% 10% % ConcentraIon of Pineapple Juice
Key QuesMons Yeast: Alpha =.05 1. Did fresh squeezed pineapple juice have a significant effect on Yeast? P Value= 8.89E- 06, this is less then.05, so it had a significant effect. 2. Did canned pineapple juice have a significant effect on Yeast? P Value= 1.55E- 07, this is less then.05, so it had a significant effect. 3. Was there significant variamon between the groups at 1% variable? P Value= 3.5E- 04, this is less then.05, so the variamon is significant. 4. Was there significant variamon between the groups at 10% variable? P value= 1.77E- 5, this is less then.05, so the variamon is significant.
Colonies 300 250 200 150 100 E. coli results P value=.586 P value=.783 P- value= 5.1E- 13 P- value= 2.61E- 13 FS Canned 50 0 0% 1% 10% % ConcentraIon of Pineapple Juice
Key QuesMons E. coli: Alpha =.05 1. Did fresh squeezed pineapple juice have a significant effect on E. coli? P- value= 5.1E- 13, this is less then.05, so it had a significant effect. 2. Did canned pineapple juice have a significant effect on E. Coli? P- value= 2.61E- 13, this is less then.05, so it had a significant effect. 3. Was there significant variamon between the groups at 1% variable? P value=.586, this is greater then.05, so the variamon is insignificant. 4. Was there significant variamon between the groups at 10% variable? P value=.783, this is greater then.05, so the variamon is insignificant.
DunneM s Test Analysis: Yeast T CriMcal= 3.49 (Significant) Variable ConcentraIon T Value InterpretaIon 1% Fresh Squeezed 1.74201 Insignificant 10% Fresh Squeezed 6.47758 Significant 1% Canned 4.44805 Significant 10% Canned 8.49020 Significant
DunneM s Test Analysis: E. coli T CriMcal= 3.49 (Significant) Variable ConcentraIon T Value InterpretaIon 1% Fresh Squeezed 2.421409 Insignificant 10% Fresh Squeezed 11.13247 Significant 1% Canned 1.296106 Insignificant 10% Canned 10.93150 Significant
CONCLUSIONS The null hypothesis can be rejected at the 10% concentramon of each juice for both yeast and E. Coli. It can also be rejected at the 1% concentramon of canned juice on yeast. This is the only 1% variable that was significant.
LIMITATIONS AND FUTURE STUDIES Only two types of Pineapple juices were tested Only two microorganisms were tested Slight variamons in plamng Mme Only one type of exposure(liquid pulse) Only one exposure Mme Test different types of exposure (agar infusion) SynergisMc effects Test different exposure Mmes Test different juices
REFERENCES hbp://www.exploreyeast.com/ hbp://textbookonacteriology.net/e.coli.html hbp://people.ku.edu/~jbrown/ecoli.html hbps://www.addgene.org/plasmid_protocols/bacterial_plates/ hbp://www.nymmes.com/1991/06/11/news/fruit- juice- and- infecmon- prevenmve- effect- confirmed.html hbp://www.livestrong.com/armcle/159381- health- benefits- of- pineapple- juice/
ANOVA AbbreviaMon for analysis of variance StaMsMcal test comparing variamon 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) Anova: Single Factor SUMMARY Groups Count Sum Average Variance 0% no UV 6 464 77.33333 140.6667 0% UV 6 189 31.5 91.5 ANOVA Source of VariaMon SS df MS F P- value F crit Between Groups 6302.083 1 6302.083 54.2893 2.4E- 05 4.964603 Within Groups 1160.833 10 116.0833 Total 7462.917 11 Sample ANOVA (not used in experiment)
Gram (- ) vs. Gram (+) Bacteria } Cell was contains an extra layer of lipopolysaccharides for extra protecmon. } Outer membrane protects bacteria from several anmbiomcs. Most pathogenic bacteria in humans are Gram (+) organisms. Simple cell wall. Some anmbiomcs work against the formamon of the cell wall.
Data Results Yeast Control 221 222 218 213 1% Fresh Squeezed 183 199 172 217 10% Fresh Squeezed 114 123 139 115 1% Canned 146 173 158 134 10% Canned 95 98 95 84
Data Results E. coli Control 270 263 281 257 302 278 1% Fresh Squeezed 232 247 218 221 252 240 10% Fresh Squeezed 92 97 80 83 86 105 1% Canned 250 273 264 241 236 258 10% Canned 89 96 103 101 90 84