The Antibacterial Effects of Honey on Microbial Flora

Ryan NguyenGrade 10

Central Catholic High school

Antimicrobials

Artificial• Antibiotics• Disinfectants• Sulfa drugs• Anti-virals• Anti-fungals• Anti-protozoals• Iodine• Tryclosan• Bleach

Natural• Onions and garlic• Cruciferous vegetables

– Cabbage– Spinach– Kale

• Fermented foods– Loaded with microflora

• Herbs• Honey

Background Information• Known for its healing properties for

thousands of years• Ancient Greeks used it along with other

groups of people• Up to second World War honey was still

used• Modern antibiotics reduced honey in

medicinal use• Some honey can kill certain strains of

antibiotic-resistant bacteria• Mixed with water and oxygen, honey

creates glutonic acid and hydrogen peroxide which are antibacterial

Honey Tree’s Sugar Free Imitation Honey®

• Artificial honey created as a substitute • Sugar free• Said to be perfect for diabetics• Ingredients

– Malitol syrup– Natural and artificial flavors– Acesulfame K– Malic acid

Thrifty Bee™ Honey

• Dark & robust real honey• Low in fat & sodium• Extremely high in sugar• Ingredients

– Honey

Human Bacterial Flora

• Microorganisms that reside in or on the surface of the body – Respiratory, urinary, digestive tracts and skin

• Consists of mostly bacteria, protists, fungi and archea

• Most are harmless, but some can cause disease• Most studies based on ingested materials on

human cells, BUT humans are a collection of cells and symbionts

• Estimated 10% of body mass is symbionts

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

• One of most common forms of bacteria found in many environments

• Gram (-) bacilli• Part of human flora; found in colon and digestive

tract• Reproduction time of 30 minutes• Most are non-pathogenic• Aerobic

Staphylococcus epidermidis

• Common surface symbiont in mammals• Gram (+) cocci• Most are non-pathogenic• Part of human skin flora• Forms biofilms• Aerobic

Previous Studies

• Raw and processed honey prevented bacterial growth and found to be an effective bactericide

• Honey produces hydrogen peroxide, contributing to its antibacterial effects

Purpose

• To determine if two brands of honey, Honey Tree’s Sugar Free Imitation Honey® and Thrifty Bee™ Honey, will significantly affect the survivorship of E. coli or Staph

Hypothesis

• Null Hypothesis:Honey Tree’s Sugar Free Imitation Honey® and Thrifty Bee™ Honey will not significantly affect the survivorship of Staph or E. coli.

• Alternative Hypothesis:Honey Tree’s Sugar Free Imitation Honey® and Thrifty Bee™ Honey will significantly reduce the survivorship of Staph and E. coli.

Materials• LB Media (0.5% yeast extract, 1%

tryptone, 1% sodium chloride)• Luria Broth agar plates • E. coli DH5 Alpha• Staphylococcus epidermidis• Pipets• Sterile Dilution Fluid [SDF] (100

mM KH2PO4, 100 mM K2HPO4, 10 mM MgSO4, 1mM NaCl)

• Sterile test tubes

• Sterile spreader bars• Incubator• Ethanol• Matches• Turntable• Bunsen burner• Vortex• Thrifty Bee™ Honey• Sugar Free® Honey• Sidearm flask• Klett spectrophotometer

Procedures – Liquid Pulse1. E. Coli & Staph was grown overnight in sterile LB media.2. Sample of the overnight culture was added to fresh media

in a sterile sidearm flask.3. The culture was incubated at 37°C until a density of 50

Klett spectrophotometer units was reached (Cell density approximately 108 cells/mL).

4. The culture was diluted in a sterile dilution fluid to a concentration of approximately 105 cells/mL.

5. The selected honey variables were diluted with sterile dilution fluid to the chosen concentrations to a total of 9.9 mL.

Procedures – Liquid Pulse

6. 100 µL of cell culture was then added to the test tubes, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL.

7. The solution was then mixed by vortexing and allowed to sit at room temperature for 5 minutes.

8. After vortexing to evenly suspend cells, 100 µL aliquots were removed from the tubes and spread on LB agar plates.

9. The plates were incubated at 37°C for 24 hours.10. The resulting colonies were counted visually. Each colony is

assumed to have risen from one cell.

Chart of Concentrations (mL)

0% Concentration

1% Concentration

10% Concentration

50% Concentration

Microbe 0.1 mL 0.1 mL 0.1 mL 0.1 mL

SDF 9.9 mL 9.8 mL 8.9 mL 4.9 mL

Honey Brand 0 mL 0.1 mL 1 mL 5 mL

Total 10 mL 10 mL 10 mL 10 mL

Agar Infusion1. Honeys were directly infused into molten LB agar at

the following concentrations (0%, 1%, 10%)2. 100 mL aliquots from the liquid pulse control tube

(0% honey) were plated on to the infused plates3. The plates were incubated for 24 hours at 37° and

the resulting colonies counted.

Statistical Analyses

ANOVA: Single Factor• Analysis of Variation• Statistical test that

compares the means of multiple groups

• P-value from ANOVA testing identifies significance if P-value is < 0.05

Dunnett’s Test• Statistical test that

compares an experimental group directly to the control group

• Identifies that the experimental group is significantly different than the control if t-value > t-crit

P-value: 1.44E-17

P-value: 1.01E-16Sig: 0.01059

Sig: 0.00746

Sig: 0.00941

Variable Concentration T-value Interpretation

T-Crit: 3.10E. coli 1% Sugar Free 9.23 Significant

Thrifty Bee 11.20 Significant

10% Sugar Free 17.73 Significant

Thrifty Bee 18.04 Significant

50% Sugar Free 38.40 Significant

Thrifty Bee 35.15 Significant

Dunnett’s Test: Liquid Pulse

P-value: 2.59E-12

P-value: 7.92E-14Sig: 0.04997

Not Sig: 0.22589

Sig: 0.00884

Variable Concentration T-value Interpretation

T-Crit: 3.10

Staph 1% Sugar Free 10.85 Significant

Thrifty Bee 8.44 Significant

10% Sugar Free 28.53 Significant

Thrifty Bee 37.20 Significant

50% Sugar Free 42.48 Significant

Thrifty Bee 58.29 Significant

Dunnett’s Test: Liquid Pulse

P-value: 1.38E-22P-value: 4.66E-18Sig: 1.17E-06

Sig: 0.04044

Variable Concentration T-value Interpretation

T-Crit: 2.61E. coli 1% Sugar

Free10.65 Significant

Thrifty 9.51 Significant10% Sugar

Free24.21 Significant

Thrifty 22.03 Significant

Dunnett’s Test: Agar Infusion

P-value: 5.72E-21

P-value: 3.66E-21

Sig: 0.00079

Not Sig: 0.62660

Variable Concentration T-value Interpretation

T-Crit: 2.61

Staph 1% Sugar Free

9.59 Significant

Thrifty 15.50 Significant

10% SugarFree

21.97 Significant

Thrifty 29.15 Significant

Dunnett’s Test: Agar Infusion

Conclusions• Null Hypothesis: Rejected

• Sugar Free Honey (Liquid Pulse & Agar Infusion):– significantly reduced survivorship of E. coli at all

concentrations– significantly reduced survivorship of S. epidermidis at

all concentrations• Thrifty Bee Honey (Liquid Pulse & Agar Infusion):

– significantly reduced survivorship of E. coli at all concentrations

– significantly reduced survivorship of S. epidermidis at all concentrations

Limitations• Measuring honey was

difficult– Viscosity of honey impaired

pipetting

• Slight lag in synchronization of plating

• Impurities in the honey (spores)

• Only three concentrations of honey were used

• Only two methods were used

Extensions• More trials• Different microbes• Smaller increments of

honey concentrations• Different types of honey

– Raw vs. artificial• Determine if the honey

affected the growth rate of the bacteria in addition to survivorship

• Use another method of experimentation

References• Peter B Olaitan, Olumfemi E Adeleke, and Iyabo O Ola

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2269714/• Burgget http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2269714/#R17• Water Activity http://www.wateractivity.org/• J.H Dustmann

http://www.apimondiafoundation.org/foundation/files/1979/J.H.%20DUSTMANN.pdf

• Sherlock O, Dolan A, Athman R, Power A, Gethin G, Cowman S, and Humphreys H http://www.ncbi.nlm.nih.gov/pubmed/20813024

• Bacteriostatic Definition http://www.wisegeek.com/what-is-a-bacteriostatic.htm

• D.P Mohapatra, V. Thakur, and S. K. Brarhttp://www.hindawi.com/journals/btri/2011/917505/

• Basson NJ and Grobler SRhttp://www.ncbi.nlm.nih.gov/pubmed/18627601

Honey Tree’s Sugar Free Imitation Honey®(Additional Info)

• Ingredients– Maltitol syrup

• Sugar free low carb sweetener– Natural & artificial flavors

• Artificial = non-natural products used to stimulate a desired flavor

• Natural = self-explanatory– Acesulfame K

• Sugar substitute– Malic Acid

• Acidulant & flavoring agent

Anova: E. Coli Liquid Pulse (Sugar Free)

SUMMARY

Groups Count Sum Average Variance

Column 1 7 1068 152.5714 34.28571

Column 2 5 640 128 18.5

Column 3 5 527 105.4 13.3

Column 4 5 245 49 14

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 32853.45 3 10951.15 506.8487 1.44E-17 3.159908

Within Groups 388.9143 18 21.60635

Total 33242.36 21

Anova: E. Coli Liquid Pulse (Thrifty)

SUMMARY

Groups Count Sum Average Variance

Column 1 7 1068 152.5714 34.28571

Column 2 5 585 117 36.5

Column 3 5 475 95 29.5

Column 4 5 198 39.6 24.3

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 38435.45 3 12811.82 406.7858 1.01E-16 3.159908

Within Groups 566.9143 18 31.49524

Total 39002.36 21

Anova: Staph Liquid Pulse (Sugar Free)

SUMMARY

Groups Count Sum Average Variance

Column 1 6 1143 190.5 19.5

Column 2 3 467 155.6667 56.33333

Column 3 3 296 98.66667 16.33333

Column 4 3 161 53.66667 20.33333

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 43450.9 3 14483.63 561.9752 2.59E-12 3.587434

Within Groups 283.5 11 25.77273

Total 43734.4 14

Anova: Staph Liquid Pulse (Thrifty)

SUMMARY

Groups Count Sum Average Variance

Column 1 6 1143 190.5 19.5

Column 2 3 508 169.3333 16.33333

Column 3 3 281 93.66667 20.33333

Column 4 3 116 38.66667 9.333333

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 54948.23 3 18316.08 1063.202 7.92E-14 3.587434

Within Groups 189.5 11 17.22727

Total 55137.73 14

Anova: E. Coli Sugar Free Infusion

SUMMARY

Groups Count Sum Average Variance

Column 1 20 3240 162 31.89474

Column 2 10 1396 139.6 41.82222

Column 3 10 1121 112.1 22.76667

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 16849.48 2 8424.738 262.5413 1.38E-22 3.251924

Within Groups 1187.3 37 32.08919

Total 18036.78 39

Anova: E. Coli Thrifty Infusion

SUMMARY

Groups Count Sum Average Variance

Column 1 10 2014 201.4 187.3778

Column 2 10 1603 160.3 42.01111

Column 3 10 1062 106.2 48.62222

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 45596.87 2 22798.43 246.0164 4.66E-18 3.354131

Within Groups 2502.1 27 92.67037

Total 48098.97 29

Anova: Staph Sugar Free Infusion

SUMMARY

Groups Count Sum Average Variance

Column 1 20 3813 190.65 40.87105

Column 2 10 1673 167.3 52.9

Column 3 10 1362 136.2 55.06667

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 19968.15 2 9984.075 211.3032 5.72E-21 3.251924

Within Groups 1748.25 37 47.25

Total 21716.4 39

Anova: Staph Thrifty Infusion

SUMMARY

Groups Count Sum Average Variance

Column 1 10 2320 232 54.88889

Column 2 10 1818 181.8 76.84444

Column 3 10 1376 137.6 24.93333

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 44616.8 2 22308.4 427.1821 3.66E-21 3.354131

Within Groups 1410 27 52.22222

Total 46026.8 29

Anova: E. Coli Liquid Pulse (Sugar Free vs. Thirfty 1%)

SUMMARY

Groups Count Sum Average Variance

Column 1 5 640 128 18.5

Column 2 5 585 117 36.5

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 302.5 1 302.5 11 0.010593 5.317655

Within Groups 220 8 27.5

Total 522.5 9

Anova: E. Coli Liquid Pulse (Sugar Free Vs. Thrifty 10%)

SUMMARY

Groups Count Sum Average Variance

Column 1 5 527 105.4 13.3

Column 2 5 475 95 29.5

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 270.4 1 270.4 12.63551 0.007459 5.317655

Within Groups 171.2 8 21.4

Total 441.6 9

Anova: E. Coli Liquid Pulse (Sugar Free Vs. Thrifty 50%)

SUMMARY

Groups Count Sum Average Variance

Column 1 5 245 49 14

Column 2 5 198 39.6 24.3

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 220.9 1 220.9 11.53525 0.009411 5.317655

Within Groups 153.2 8 19.15

Total 374.1 9

Anova: Staph Liquid Pulse (Sugar Free Vs. Thrifty 1%)

SUMMARY

Groups Count Sum Average Variance

Column 1 3 467 155.6667 56.33333

Column 2 3 508 169.3333 16.33333

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 280.1667 1 280.1667 7.711009 0.049978 7.708647

Within Groups 145.3333 4 36.33333

Total 425.5 5

Anova: Staph Liquid Pulse (Sugar Free vs. Thrifty 10%)

SUMMARY

Groups Count Sum Average Variance

Column 1 3 296 98.66667 16.33333

Column 2 3 281 93.66667 20.33333

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 37.5 1 37.5 2.045455 0.225891 7.708647

Within Groups 73.33333 4 18.33333

Total 110.8333 5

Anova: Staph Liquid Pulse (Sugar Free Vs. Thrifty 50%)

SUMMARY

Groups Count Sum Average Variance

Column 1 3 161 53.66667 20.33333

Column 2 3 116 38.66667 9.333333

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 337.5 1 337.5 22.75281 0.00884 7.708647

Within Groups 59.33333 4 14.83333

Total 396.8333 5

Anova: Staph Infusion (Sugar Free Vs. Thrifty 1%)

SUMMARY

Groups Count Sum Average Variance

Column 1 10 1673 167.3 52.9

Column 2 10 1818 181.8 76.84444

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 1051.25 1 1051.25 16.20493 0.000793 4.413873

Within Groups 1167.7 18 64.87222

Total 2218.95 19

Anova: Staph Infusion (Sugar Free Vs. Thrifty 10%)

SUMMARY

Groups Count Sum Average Variance

Column 1 10 1362 136.2 55.06667

Column 2 10 1376 137.6 24.93333

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 9.8 1 9.8 0.245 0.626606 4.413873

Within Groups 720 18 40

Total 729.8 19

Anova: E. Coli Infusion (Sugar Free Vs. Thrifty 1%)

SUMMARY

Groups Count Sum Average Variance

Column 1 10 1396 139.6 41.82222

Column 2 10 1603 160.3 42.01111

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 2142.45 1 2142.45 51.11213 1.17E-06 4.413873

Within Groups 754.5 18 41.91667

Total 2896.95 19

Anova: E. Coli Infusion (Sugar Free Vs. Thrifty 10%)

SUMMARY

Groups Count Sum Average Variance

Column 1 10 1121 112.1 22.76667

Column 2 10 1062 106.2 48.62222

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 174.05 1 174.05 4.876109 0.040443 4.413873

Within Groups 642.5 18 35.69444

Total 816.55 19