Controlling Mastitis with Honey

Kery Griffiths and Amanda Weisbrod

The following science project was conducted and completed by Kery Griffiths and Amanda Weisbrod, sudents at the Lumsden High School. Their project, and their findings were submitted to a National Science competition where they were awarded second place. Their project involved Saskatchewan honey and strongly supported the healing properties of honey.

Sweet and Sour Milk

Streptococci, Staphylococci, Coliforms, Bacillus, Nocardia, Mycobacteria, and Mycroplasma bacteria cause Mastitis. (Harmon 1989, Schoemian 1999). Mastitis is normally treated with antibiotics (penicillin, dehydrostreptomycin, dexamthasone) and an antihistamine, which helps heal tissue and reduce inflammation (Swartz 1999). Some bacteria are not controlled with these antibiotics; other treatments are needed. Treatment with antibiotics could cause a build up of antibiotics in the treated animals, a condition implicated in the development of antibiotic resistant strains of bacteria, a current concern with humans.

Manuka honey, produced in New Zealand, proved to inhibit the growth of the Staphylococcus aureus and Streptococci bacteria mixed in a 1.8% solution with water (Molan, 1992). Low concentrations of Manuka honey also inhibited the growth of other common bacteria. Other honeys tested completely inhibited the bacteria in concentrations of 5% to 10%. Mastitis was completely inhibited with a 10% concentration of Manuka honey (Allen, Molan, 1994).

Canadian honey could prove to be an effective antibacterial agent if it has appropriate pH, hydrogen peroxide and moisture content. Hydrogen peroxide and non-peroxide are the main two agents for fighting bacteria. Sugar has osmotic effects. Lysosomes in honey and low pH also contribute antibacterial effects (Bogdanov, 1997).

The purpose of this project was to determine effectiveness of Canadian honey for controlling Mastitis bacteria. The researchers hypothesized borage honey would be the most effective Canadian honey because it is from a single source like Manuka honey, which has proven antibacterial properties.

The following procedures were followed to complete this project.

1. Honey was collected from beekeepers.
2. Pollen samples from golden rod, alfalfa, clover, canola, mustard and borage were collected and examined using a microscope at 400X magnification. The pollen was drawn so that it could be compared to the pollen in the honey samples.
3. Honey samples were examined using a microscope and the pollen grains in each honey sample were compared to those collected in the field to determine the nectar source for the honey.
4. Honey was tested for pH using pH paper, for hydrogen peroxide using test strips, and for moisture content using a refractometer.
5. Fresh cow milk was obtained from a local dairy and used to inoculate 12 petri plates of nutrient agar, with Mastitis bacteria.
6. 24 - 8mm filter paper discs were soaked in 10%, 25% and 50% solutions of the four different honey samples being tested and placed on the inoculated section of the petri plates. Three plates were set up for each type of honey; discs of the same concentration solution were placed in the each plate.
7. Plates were incubated for three days at 37^o C, after which time they were examined. The diameter of the area free of bacterial growth was measured. Bacteria growing in each dish were identified.
8. Results of pH, hydrogen peroxide content, moisture content and bacterial growth inhibition were compared to the results from the Manuka honey.

Results and Conclusions

Based on pollen comparisons, the origin of the nectar collected by the bees was identified and is summarized in Data Table 1: Honey Type by Pollen Identification.

Data Table 1: Honey Type by Pollen Identification

 

Honey	Plant Origin
1	Manuka
2	Borage
3	Alfalfa/Clover/Mustard
4	Alfalfa/Golden Rod

 

 

 

 

 

Results of pH were compared to standards published by the U.S. National Honey Board and to the Manuka honey, the proven antibacterial from New Zealand. The pH of the Manuka honey was slightly lower than the Canadian honey. The Manuka honey had a pH level of 5.67. All the Canadian honey had an average pH of 6 as can be seen in Data Table 2: pH of Honey. These results were then compared to pH levels required by Mastitis bacteria as summarized in Data Table 3: pH Requirements of Bacteria. The Canadian honey samples (2 - 4) would inhibit the growth of Staphylococcus, Diplococci, Bacillus and E. coli bacteria, but not Streptococci.

Data Table 2: pH of Honey

Substance	Average pH	Standards
Water	7	7
Honey #1	5.67	5 to 7
Honey #2	6	5 to 7
Honey #	6	5 to 7
Honey #4	6	5 to 7

Data Table 3: pH Requirements of Bacteria

Bacteria	pH Required	Honey #1	Honey #2-#4
Staphylococcus	6.5	5.67	6
Streptococci	5.3	5.67	6
Diplococci	7	5.67	6
Bacillus	7	5.67	6
E. coli	7	5.67	6

The hydrogen peroxide levels were tested using hydrogen peroxide test strips. All produced 15 mg of H₂O₂ per gram of honey, in one hour. Water and 3% hydrogen peroxide were used as controls. The water had no hydrogen peroxide in it while the 3% hydrogen peroxide had 70 mg.

Data Table 4: Hydrogen Peroxide Activity

Substance	Average H2O2 (mg)
Water	0
3 % H2O2	70
Honey #1	15
Honey #2	15
Honey #3	15
Honey #4	15

The percentage of moisture was tested to determine available water for each honey sample (Data Table 5: Available Water). The lower the level of available water, the greater the antibacterial effectiveness should be. The U.S. National Honey Board has published a chart that states the available water level of honey with respect to the moisture content. Most honey has a range of 0.5 to 0.7 available water. None of the honey tested had enough available water to support the growth of Mastitis bacteria

.

Data Table 5: Available Water

Bacteria	Av req. to grow	Canadian Honey	Manuka Honey
Staphylococcus	0.85	0.6	0.5
Streptococcus	0.95	0.6	0.5
Diplococci	0.9	0.6	0.5
Bacillus	0.9	0.6	0.5
E. Coli	0.95	0.6	0.5

 

Three solutions of honey were made for each sample of honey (10%, 25%, 50%). These solutions were tested on Mastitis bacteria using what is called the “diffusion method.” Nutrient agar plates were inoculated with Mastitis bacteria from cow milk. Eight millimeter discs soaked in 10%, 25%, and 50% solutions of the honeys being tested were placed on the milk. The diameter of the zone free of bacterial growth was recorded. The larger the zone, the more effective the honey solution was in inhibiting the growth of the bacteria.

The results of the impact of each honey on Mastitis bacterial growth in a petri plate is summarized in Data Table 6: Determining the Zone of Inhibition. The results for Canadian honey are compared to the results of Manuka honey.

Data Table 6: Determining the Zone of Inhibition

Zone Free of Bacterial Growth in mm.

Honey	10% Solution	25% Solution	50% Solution
1	12	14	26
2	10	12	20
3	12	14	16
4	8	8	18

 

In conclusion, Canadian Honey #3, derived from Alfalfa/Clover/Mustard was the most effective

antibacterial agent. It had a pH of 6 and hydrogen peroxide level of 15mg/g of honey. These values were equal to the Manuka honey’s values. It also had an available water of 0.6, which was close to the Manuka honey’s available water level. The zone of inhibition was 12mm at 10%, which was also equal to the Manuka honey’s value. Honey #2 (Borage) had a 10mm inhibition zone at 10%. Because there was no inhibition around the disc, honey #4 (Alfalfa/Golden Rod) would not be expected to inhibit Mastitis bacterial growth.

Acknowledgements

Peter Molan

Emily Brown

Wink Howland

Ron Kondra

Celeste Howland

Monica Foley

John Gruszka

Muriel Griffiths

Dawnlyn Ward

Alvey Halbgewachs

Rhonda Phillips

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