This semester, we dove into the field of honey testing to determine more about the contents within various types of honeys. Although many methods of honey testing exist, we carried out refractometry and DNA metabarcoding tests. Refractometry measures the level of moisture found within the honey, which can help to properly time honey harvests or potentially hint at the presence of honey additives within a sample. DNA metabarcoding takes an in-depth look at the pollen protein DNA present in the honey and sequences the DNA to match it to the genomes of various plant species. This testing method can help determine the composition of pollen sources in the sample and give insight into geographical origins of the honey!
Both of these tests can help to quantify the exact contents found within the honey bottle, which is the main goal of our honey testing research. Ultimately, our team would like to perform these honey tests to compare the contents of the honey with the label claims on the outside of the honey bottle. With this research, we can determine if honey producers are truly meeting the standards they claim to uphold, which can contribute to current knowledge on FDA honey requirements and can help to ensure that fully authentic honeys are available to the consumer.
The first step we took in this project was going to the grocery store and selecting five samples of honey for testing. As you can tell from the photo on the left, honey can be highly variable in its consistency and color! We tried to select honeys that looked different and had varied labeling claims of origin and processing. Below is a table that describes our five samples:
After we gathered and purchased our honeys, we sent out five samples to Jonah Ventures, a private research company that performs DNA metabarcoding tests on honey. Jonah Ventures sent us five kits with vials to fill with honey, and we shipped them off to be sequenced!
While we waited for our pollen DNA results to come back, we also performed refractometry tests on our honeys. We used a honey-specific refractometry tool to quantify the moisture content of each honey sample. Performing this test was very simple: we just added a small drop of honey to the refractometer plate, then thinly and evenly spread the droplet to allow light to pass through the sample. The tool then measures the amount of light refracted by the suspended solids in the honey, and gives a moisture percentage value.
Our five honey vials before being sent to Jonah Ventures for metabarcoding testing.
Loading up the refractometer plate with a small drop of honey.
Our results for these honey tests were very intriguing. For moisture content, all of the moisture percentage values fit within the threshold of what is considered proper honey moisture (anything at or below 18%). This was a good sign! For the pollen DNA metabarcoding tests, however, we received some unexpected results. Samples 2, 4, and 5 all yielded diverse pollen origins, which is to be expected from normal wildflower honeys. Further research on each plant family showed that each plant is cultivated in regions where our honeys were supposed to be sourced, showing that the labels for these three honeys were correct and meet the True Source Certified honey stamp.
For the other two honeys (samples 1 and 3), Jonah Ventures informed our team that these samples did not have sufficient DNA content to conduct thorough investigations. This was very unexpected, because pollen grains are so miniscule that they rarely escape even extensive honey filtering. Both of these honey samples should have contained an abundance of pollen DNA in them, especially sample 3 whose label claimed that the honey was from wildflowers and "contains pollen."
Although there was very little presence of DNA in these samples, we still received results for sample 3. The origin chart showed a 100% presence of Glycine protein (the soybean family). Although it is true that honey bees can pollinate soybean crops, it was very odd that the result showed only this plant family.
Honey samples and their corresponding moisture content.
Honey sample 2 pollen testing results
Honey sample 3 pollen testing results
Honey sample 4 pollen testing results
Honey sample 5 pollen testing results
We have a few theories on why samples 1 and 3 did not have sufficient pollen DNA content to be tested. Firstly, these honeys might have been overheated in pre-packaging processes in an attempt to prevent future crystallization. This overheating could have broken down the pollen DNA present in the honey so little intact pollen was left in the sample.
These samples also might contain a majority of a honey additive, and sample 3 specifically might have a soy-based food additive. Some beekeepers will add cheaper sweeteners to their honey to maximize profits, but will not label their honey as a honey blend. These honey additives are not made by the bees and will not contain pollen grains. This is a plausible reason on why both of these sample had little to no pollen content. However, more specific chemical testing should be performed on these samples to verify exact honey contents.
We can continue our honey testing research in many avenues. It would be very interesting to perform further tests, especially on samples 1 and 3, to find out why these samples did not have much pollen DNA. It would also be interesting to discover the potential presence and identity of any pesticides or antibiotics in our samples. Each of these honey content identification tests can be performed through advanced chemical testing such as nuclear magnetic resonance (NMR) profiling or mass spectroscopy. By unveiling the contents of store-bought honey, we can help to solve the problem of honey mislabeling and inform other researchers and beekeepers, agencies, and honey consumers about what exactly is within this sweet substance that we all enjoy.