Bacterial Growth and pH tests support antibacterial properties present in Manuka Honey while not found in other types

By:  “Agent Orange”
Lora Edwards, Bryan Jarvis, and Elizabeth Rutledge



    In this experiment, we investigated if there were chemical differences that made Manuka Honey a better healing agent than three other types of raw and unprocessed honeys (Buckwheat, Orange Blossom, and Pure Natural).  We define healing as the ability to inhibit bacterial growth which also results in promoting good health.  Manuka Honey has a special antibacterial agent which is only produced in the honey of bees that use the Manuka bush (Leptospermum Scoparium) of New Zealand as their pollen source (Molan, 2002).  The tests that were used in our investigation incorporated Sugar Tests:  Benedict’s test, Barfoed’s Test, Selivanoff’s Test, Bial’s Test, and the Iodine Test for Coiled Polysaccharides; Bacterial Growth tests; pH tests; and the Bradford Assay test.
    The sugar tests verified that all four honey samples contained sugars with a free ketose group, all were monosaccharides, and all were hexose furanoses, but none contained coiled polysaccharides.  Manuka Honey was the only honey that inhibited bacterial growth in the bacterial growth tests.  In the pH test, Manuka Honey was determined to have the lowest pH of the four honey samples as shown by the pH meter.  Finally, in the Bradford Assay, similar concentrations of protein were found in each of the honey samples.  Our test results suggest that because Manuka Honey inhibits the growth of bacteria and it has a low pH, both of which are important in the healing process, Manuka Honey has antibacterial properties that make it the best healing agent among those tested in our experimentation.


    The question investigated in this experiment was:  Are there differences in chemical properties, that make Manuka Honey a better healing agent than other types of raw and unprocessed honeys, specifically: Buckwheat, Orange Blossom, and Pure Natural.  Again, we define healing as the ability to inhibit bacterial growth which also results in promoting good health.  Furthermore, we originally hypothesized that Manuka Honey, as well as the other three honey varieties, would all contain coiled polysaccharides, however we realized that all of the sugar tests needed to be run so we then revised our hypothesis to state that Manuka Honey as well as the three other types tested would all contain a free aldehyde or ketone group, monosaccharides, ketoses, and furanoses, and coiled polysaccharides.  We also hypothesized that Manuka Honey would be the only one of our honey samples that would inhibit bacterial growth in the Bacterial Growth tests.  In addition to the above hypotheses, we hypothesized that Manuka Honey would have the lowest pH of all four of our samples.  Finally, we hypothesized that all of our samples would contain similar protein concentrations.
Our hypotheses were based on the following:  More than 40 years ago, it was discovered that there are differences in the antibacterial activity of different honeys.  A method was created to determine the “inhibine” number of honeys as an index of antibacterial activity.  The inhibine number relates to the degree of dilution to which honey will retain and/or maintain its antibacterial activity (Molan, 2002).  The enzyme catalase is derived mostly from nectar as well as from the pollen of certain plants which accounts for the large differences between honeys from different flower (nectar) sources.  Manuka Honey from New Zealand has a high antibacterial activity, with half of it having exceptionally high levels of non-peroxide activity.  On wounds, some hydrogen peroxide may be broken down, so honey with only hydrogen peroxide activity may be less effective.
In 1991, a researcher named Allen and his colleagues took a survey of the various honeys of New Zealand and found that when a catalase was added to destroy the hydrogen peroxide levels in each source, the honey made from the Manuka source had the most antibacterial properties present (Molan, 2001).  Manuka Honey is produced by bees that visit the Manuka bush (Leptospermum scoparium) of New Zealand which grows uncultivated throughout the entire country (Molan, 2002).  Active Manuka Honey is the only honey on the market which has been tested for its antibacterial properties.  Manuka Honey must be stored at a low temperature and out of light so that none of the glucose oxidase activity is lost.  It has what is known as the Unique Manuka Factor (UMF) because it has a special antibacterial agent which is only produced in the honey of the bees that use Leptospermum as their pollen source (Molan, 2002).
     To get a better idea of the composition of the honeys, we performed a series of tests to determine different chemical components of each of our honey samples:  Manuka, Buckwheat, Orange Blossom, and Pure Natural.  The tests we used are as follows: Benedict’s, Barfoed’s, Selivanoff’s, Bial’s, and the Iodine Test for Coiled Polysaccharides (Krha et al., 2003).  They tested for the presence of free aldehyde or ketone groups, monosaccharides, ketoses, furanoses, and coiled polysaccharides, respectively.  Our results showed that all of the honeys contained:  free aldehyde or ketone groups, monosaccharides, ketose, and furanose; they all lacked coiled polysaccharides (figures 2-6).  These results were as predicted, except for the iodine test; we originally predicted that all of the honeys would contain coiled polysaccharides; however, the results show the opposite.  Since the results showed that the four honeys have similar carbohydrate properties, there are no comparable differences between them that could lend support to the hypothesis that one honey is better than another in the inhibition of bacterial growth.
    To investigate the actual effect of honey on bacteria, we cultured bacteria and observed how its growth was affected as a result of honey being added to the culture.  We used E. Coli as our test bacteria and we prepared a lawn of these bacteria on an LB agarose media.  Plates were prepared for each of the four experimental honeys (Manuka, Buckwheat, Orange Blossom, and Pure Natural); after the bacteria had been poured onto the media, a drop of the respective honeys was placed in the center of the culture dish.  After a day of bacterial growth, we observed that at the site of honey application, bacterial growth was prevented by only the Manuka Honey (Figure 7a, 7b, 7c, 7d and 7e and Table 2).    We believe that this happened as a result of the bent rod being too hot and consequently killed the bacteria immediately, giving it no chance to actually grow while incubated.  The results of this test supported our hypothesis, as only the Manuka Honey inhibited bacterial growth while the other three honey samples did not.  This also supports the hypothesis that, “the antimicrobial properties of honey prevent microbial growth” (Molan, 1998); however, we are unable to draw conclusions as to what antimicrobial properties present in Manuka Honey are involved in the inhibition of bacteria growth.  The qualitative nature of this test allows us only to observe that Manuka Honey inhibits bacterial growth while the other three samples and the controls did not. 
    Evidence supports that pH plays a role in antibacterial action; low acidic pH can inhibit the growth of animal pathogens (Molan, 2002); also, an acidic pH inside the vacuoles of cells, is involved in the killing of ingested bacteria (Molan, 1998.)  The pH test that we ran was the second test to show significant differences between our four honey samples.  The results of this test (Figure 8 and Table 3) showed that although all of the 16.67 % honey solutions containing approximately 25 grams of honey and 150 ml distilled water had lower pH values than the distilled water; Manuka Honey was by far the lowest, there-fore making it the most acidic.  The low pH value of Manuka Honey supports our hypothesis that Manuka Honey would have the lowest pH reading and also lends support to the belief that Manuka Honey is a better healing agent due to its acidic properties that help it to fight bacteria. The reason that the 16.67% solutions were used in the pH test was because the honey samples themselves were too thick for the pH meter and may have damaged the apparatus.  Also, the pH readings are higher than they would normally be because the honey is combined with distilled water, which is neutral, and has a higher pH than the honey samples.
    The final test we ran was the Protein (Bradford) Assay which determined the total protein concentration in each of our honey samples.  This test supported our hypothesis as all four of the 16.67% honey solutions had a similar percentage of protein (Table 4).   There was a very low percentage of proteins in all of our honey samples; we believe this is due to honey having a composition primarily composed of sugars.   
    After completing this entire experiment and reflecting back onto the work we did, we found that there were quite a few possible places where human error or errors caused by other outside factors could have thrown off our results.  For instance, Manuka Honey is supposed to be kept in a dark container away from light of any kind.  Throughout the many days of testing, our Manuka Honey jar was opened and closed dozens of times.  From the frequent opening and closing of the jar, light was repeatedly let in.  This small amount of light let in each time could, over a long enough period, affect the properties of the Manuka Honey, therefore throwing off the results of our tests that were run later in the 8-week study. 
    Another place where possible error could occur is in the measuring portions of our tests.  The four honeys have distinctly different viscosities as well as densities.  These differences could pose a significant problem as far as measurement goes.  For example, the Manuka Honey is quite dense in comparison to the other three honey types.  This could pose a problem because when we measured the honeys we may not have been able to get the entire amount of honey out of our pipette.  Even that small amount left in the pipette could have skewed our results.