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Citrus Fruit Antimicrobial Effects
By John SeabrookeCentral Catholic High School
Grade 9
Antimicrobials
Natural• Tea tree oil• Onion • Lemon juice• Grapefruit seed extract• Cinnamon
Artificial• Antibiotics• Bleach• Hydrogen peroxide• Triclosan• Benzethonium Chloride
Lemon Juice
• cooking and medicine• pH level of 2 (acidic)• Home remedy for killing bacteria • Acid is corrosive
Lemon Juice Chemical Composition
• Water • Citric acid• Phosphoric acid• Melic acid• Citronellol• Citral
• Pectin• Hesperidin• D- Limonene• Sesquiterpen• Phellandrene
Grapefruit Seed Extract (GSE)
• All-natural antimicrobial• found to contain synthetic preservatives– benzethonium chloride
• pH level of 2
GSE
• Ground grapefruit seeds and pulp• Polyphenolics are added and chemically
converted into quaternary compounds• quaternary compounds used industrially– microbials but are toxic to animal life
• Natural form of these industrial compounds
Escherichia coli (E. coli)
• Gram negative bacteria• Lab prokaryotic cell model• Many different types• Many are not harmful• Ubiquitous • Mammalian symbiote
Purpose
To determine the effect of lemon juice and grapefruit seed extract on E. coli survivorship
Hypotheses
• Null Hypothesis- The lemon juice and grapefruit seed extract will not have a significant effect on E. coli survivorship.
• Alternate Hypothesis- The lemon juice and grapefruit seed extract will significantly reduce E. coli survivorship.
Materials• E. coli (DH5 Alpha)• Ethanol• Latex gloves• Sicilia Lemon Juice• Nutribiotic Grapefruit Seed
Extract• Micropipettes • Microtubes• Sterile Dilution Fluid (per 1
liter)(100mMKH2PO4,100mMK2HPO4,10mMMgSO4,1mMNaCl)
• LB Agar Plates(LB Media) (1% Tryptone, 0.5% Yeast extract, 1% NaCl)
• Matches• Incubator• Spread bar• Turntable• Bunsen Burner• Vortex
Liquid Pulse Procedure
1. E. coli was 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 degrees Celsius)
until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10^8 cells/mL.
4. The cultures were diluted in sterile dilution fluid to a concentration of approximately 10^5 cells/mL.
5. The experimental variables were mixed with the appropriate amounts of SDF to create concentrations of 0%, 0.1%, 1%, 10%.
Liquid Pulse Procedure (Continued)0% 0.1% 1% 10%
Sterile Dilution Fluid
9.9 mL 9. 89 mL 9.8 mL 8.9 mL
Microbe 0.1 mL 0.1 mL 0.1 mL 0.1 mL
Variable 0 mL 0.01 mL 0.1 mL 1 mL
Total Volume 10 mL 10 mL 10 mL 10 mL
PH effect (Lemon Juice)
7 7 7 5
PH effect (GSE) 7 7 7 6
Concentration Chart- Lemon Juice and GSE
Liquid Pulse Procedure (Continued)
6. 0.1 mL of cell culture was then added to the solutions, yielding a final volume of 10 mL and a cell density of approximately 10^3 cells/mL.7. The solutions were vortexed and allowed to sit at room temperature for 15 minutes.8. After vortexing to evenly suspend the cells, 100 microliters aliquots were removed from the tubes and spread on LB-agar plates.9. The plates were incubated at 37 degrees Celsius for 24 hours.10. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.
Agar Infusion Procedure
1. Sterilized lemon juice and grapefruit seed extract were infused into the LB agar media in three concentrations, 0.1%, 1%, and 10% (with volumes of 2, 20, and 200 microliters of variable) and used to create the LB agar plates.
2. 0.1 mL of cell culture was then added to the solutions, yielding a final volume of 10 mL and a cell density of approximately 10^3 cells/mL.3. After vortexing to evenly suspend the cells, 100 microliters aliquots were removed from the control tube and spread onto the infused agar plates.4. The plates were incubated at 37 degrees Celsius for 24 hours.5. The resulting colonies were counted visually. Each colony was assumed to have arisen from one cell.
Lemon Juice (Liquid Pulse) Effect on E. coli survivorship
0.00% 0.10% 1.00% 10.00%0
100
200
300
400
500
600
700P value= 2.94055E-07
Colo
nies
Dunnett’s Test (Liquid Pulse)Lemon Juice Concentration T Value Interpretation
0.1% 2.613 Insignificant
1% 6.941 Significant
10% 9.284 Significant
Lemon Juice (Infusion) Effect on E. coli survivorship
0.00% 0.10% 1.00% 10.00%0
100
200
300
400
500
600
700P Value=0.000505063
Colo
nies
Dunnett’s Test (Infusion)Lemon Juice Concentration T Value Interpretation
0.1% 0.198 Insignificant
1% 3.431 Significant
10% 8.900 Significant
GSE (Liquid Pulse) Effect on E. coli survivorship
0.00% 0.10% 1.00% 10.00%0
100
200
300
400
500
600
Colo
nies
P Value=2.21499E-09
Dunnett’s Test (Liquid Pulse)GSE Concentration T Value Interpretation
0.1% 3.121 Insignificant
1% 5.114 Significant
10% 10.207 Significant
GSE (Infusion) Effect on E. coli survivorship
0.00% 0.10% 1.00% 10.00%0
100
200
300
400
500
600
Colo
nies
P Value= 0.0050967
Dunnett’s Test (Infusion)GSE Concentration T Value Interpretation
0.1% 1.935 Insignificant
1% 8.319 Significant
10% 10.529 Significant
Key Questions
• Did lemon juice have a significant effect on E. coli survivorship?
Yes, the p values were 2.94E-07 and 0.000505.
• Did grapefruit seed extract have a significant effect on E. coli survivorship?
Yes, the p values were 2.21E-09 and 0.005096.
Key Questions
• At which concentrations did lemon juice significantly reduce E. coli survivorship?
For both the liquid pulse and infusion tests, the 1% and 10% concentrations significantly reduced E. coli survivorship.• At which concentrations of GSE significantly reduce
E. coli survivorship?For both the liquid pulse and infusion tests, the 1% and 10% concentrations significantly reduced E. coli survivorship.
Conclusion
• For both experiments, the null hypothesis can be rejected and the alternate hypothesis can be accepted.
• Lemon juice had a significant negative impact on E. coli survivorship.
• Grapefruit Seed Extract had a significant negative impact on E. coli survivorship.
Limitations and Future Studies
Limitations• Only one
microorganism was tested
• Slight variations in plating time
• Only one exposure time• Unknown active
ingredient
Future Studies• More concentrations• Different times of
exposure• Synergistic effects• Narrow concentration
range• Attempt to find LD50• Try to identify the
antimicrobial agent
Works Cited
• http://www.biology-online.org/• http://www.nutribiotic.com/• http://www.biologycorner.com/• http://www.cdc.gov/ecoli/
Data0% 0.1% 1% 10%621 419 306 293592 518 344 243597 562 444 335578 522 372 325497 468 367 282
Lemon Juice (Liquid Pulse)
0% 0.1% 1% 10%
702 886 542 452
669 620 546 302
587 568 555 384
641 502 651 418
693 686 478 387
GSE (Liquid Pulse)
GSE (Infusion)Lemon Juice (Infusion)
0% 0.1% 1% 10%
576 486 380 227
516 479 458 272
627 427 403 279
549 506 399 302
568 465 421 209
0% 0.1% 1% 10%
702 487 284 365
669 465 402 284
587 629 387 218
641 762 345 274
693 597 361 236
Anova (Lemon Juice Liquid Pulse)SUMMARY
Groups Count Sum Average Variance
Column 1 5 2885 577 2240.5
Column 2 5 2489 497.8 3053.2
Column 3 5 1833 366.6 2550.8
Column 4 5 1478 295.6 1343.8
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 241082.6 3 80360.85 34.98399 2.94E-07 3.238872
Within Groups 36753.2 16 2297.075
Total 277835.8 19
Anova (Lemon Juice Infusion)SUMMARY
Groups Count Sum Average Variance
Column 1 5 2836 567.2 1651.7
Column 2 5 2363 472.6 868.3
Column 3 5 2061 412.2 867.7
Column 4 5 1289 257.8 1483.7
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 252911.4 3 84303.78 69.22345 2.21E-09 3.238872
Within Groups 19485.6 16 1217.85
Total 272397 19
Anova (GSE Liquid Pulse)SUMMARY
Groups Count Sum Average Variance
Column 1 5 3292 658.4 2152.8
Column 2 5 3262 652.4 21622.8
Column 3 5 2772 554.4 3848.3
Column 4 5 1943 388.6 3101.8
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 237910.2 3 79303.38 10.32405 0.000505 3.238872
Within Groups 122902.8 16 7681.425
Total 360813 19
Anova (GSE Infusion)SUMMARY
Groups Count Sum Average Variance
Column 1 6 3292 548.6667 73970.67
Column 2 6 2940.001 490.0002 69097.4
Column 3 6 1779.01 296.5017 22779.11
Column 4 6 1377.1 229.5167 15221.92
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 417999 3 139333 3.078007 0.0050967 3.098391
Within Groups 905345.5 20 45267.28
Total 1323345 23
