The information we identify in this report was designed to serve as a standard to compare and contrast experimentally microbe-produced compounds to existing chemically synthesized options. Gas chromatography-mass spectroscopy (GCMS) is the industry standard for identifying unknown substances, especially those produced by microbes. Since many teams including our own do not own a GCMS machine, it is essential to use other methods to identify physical properties associated with products of genetically modified microbes. Smell testing is one of these methods, and since scent is often considered a more variable or nuanced sensation, clarifying how to objectively analyze scent like we do in this report can be critical for many iGEM teams and industries. This deliverable outlines our process as relevant to chocolate flavoring and scents, but this smell testing format can be used by any team to analyze a number of products--assuming they are safe for inhalation. Our smell test was used for 4 different essential flavor compounds in chocolate: pyrazine, theobromine, phenylethylamine, and HDMF. This enabled participants to safely and effectively assess the distinct aromas of each compound.
To begin, deionized water is added to a beaker to serve as a solvent for the solution. Deionized water is used because it lacks impurities that could interfere with the compound. Next, the powdered compound is measured and added to the water and mixed thoroughly to evenly disperse the particles for a consistent smell profile across the mixture. To safely smell the solution, participants waft to prevent direct inhalation of the vapor, comparing it to the smell of Sweet Leaf brand chocolate extract as a standard. Our protocol was based off of the one used by the 2019 Eau That Smell team.
Standards are one of the most essential components of both smell tests and GCMS, as they provide a baseline for comparison. Since smell testing is often variable, standards are essential to ensure an accurate goal to compare your results to. As previously mentioned, we used Sweet Leaf brand chocolate extract for our standard, and in addition, we compared said standard to several chocolate products of varying qualities to confirm its aroma was accurate in turn.
We designed a confidential survey to be taken after our compounds are dissolved in water. First, we asked respondents if the smell was recognizable and to describe the smell in their own words. Then, we had participants rank the scents from 1-5 in several categories derived from what we saw in the literature and from general knowledge associated with one of more of the compounds which include :
Pyrazine is an aromatic organic compound known for its role in enhancing the flavor and aroma profiles of various foods, particularly in roasted and nutty products like chocolate, coffee, and roasted nuts. Even in small concentrations, pyrazine has a strong, noticeable aroma. Pyrazine is slightly hydrophobic, meaning that it requires a relatively large amount of water and more mixing to dissolve into the solvent. Pyrazine plays a central role in the sensory experience of chocolate, providing a foundation for its depth of flavor and aromatic richness.
Theobromine is a naturally occurring compound found primarily in cocoa beans, making it a key component in the flavor and sensory profile of chocolate. Chemically similar to caffeine, theobromine contributes to chocolate’s slightly bitter taste with mild stimulating effects. Theobromine is relatively stable and exhibits low water solubility. While it has a less direct influence on aroma compared to other flavor compounds, its presence is essential to the overall complexity of chocolate’s taste. Theobromine also has a smooth, lingering bitterness that balances the sweetness and richness of chocolate, enhancing the depth of its flavor.
Due to theobromine’s poor water solubility, we tried several different solvents, including ethanol at 99%, 50% and 25% concentrations, which all worked poorly, and DMSO, which worked well in low quantities. Due to the strong aromas of each solvent, we ultimately determined they were inappropriate for smell testing. Experimentally, we determined theobromine had a “fishy” odor, which is not associated with chocolate.
Phenylethylamine is a compound which is a naturally occurring monoamine alkaloid chemical. Phenylethylamine is found naturally in the human body, where it plays a role as a neurotransmitter and neuromodulator in the central nervous system. It plays a role in the release of dopamine, norepinephrine and serotonin, which are all important chemicals involved in mood regulation, motivation, and pleasure. Phenylethylamine is also found in certain foods such as chocolate, which is why chocolate is often thought of as a feel-good food. Much like theobromine, phenylethylamine has a distinctly fishy odor. This means it is easily detectable using smell testing. However, its mood enhancing effects are often short-lived in supplements because the body rapidly metabolizes it. Due to the acute toxicity of phenethylamine to aquatic life when dissolved in water we used proper biohazard waste disposal systems.
HDMF is an organic compound which has a distinctive sweet, caramel-like aroma. It is naturally found in many fruits such as strawberries, pineapples and tomatoes. HDMF plays a major role in food flavoring, both in its natural and synthetic form when added as an additive in the food industry, where it can be used to enhance or replicate aromas in products. Additionally, HDMF is often used in perfumes, and personal care products. In research, HDMF is often used in smell tests to study the sense of smell. Its strong yet familiar scent makes it ideal for such purposes. Overall, we found that while we received valuable insights into the individual values of our compounds, HDMF’s overpowering fragrance meant while working with it in a mixture, even when we increased all other compounds 10 fold and decreased HDMF 10 fold, it was impossible to detect any influences from other substances.
GCMS is essential for the identification of our compounds once we produce them. GCMS is useful because it provides numerous pieces of information about different compounds present in an analyte, such as their mass and structure. Smell testing is the only other method that can be used for many of the compounds we work with. However, because many compounds either have no odor or are dangerous, many iGEM teams have to use GCMS if they produce compounds.
First, the sample is injected into a high-temperature gas chromatography inlet, which evaporates the sample. The sample, now a gas, is transferred through a progressively heated tube that collects, identifies, and separates compounds traveling through it based on boiling point. Once this process is completed, the gas is transferred to the mass spectrometer. The compound is shot with electrons, which break apart and ionize it, and the combination of mass and charge is analyzed by the machine. This identifies the present compounds.
First, ensure you have a standard to compare your samples to. As mentioned above, standards are incredibly important, so they should be carefully prepared before measurement.
Sample prep can be complex. There are numerous ways to prepare your specific sample, and that will change depending on your compound. Here are some ways to figure out what technique is right for you:
Source: ThermoFisher
This information was also provided in our interview with Dr. Garrett Miller, a scientist who works on finding biosynthetic pathways for production and analysis of chemicals. He also shared with us the following information about how to select a solvent:
“Different solvents will have different properties which influence whether they are good at dissolving or not. Polarity is the most essential when determining solvents to use for your compound, according to the ‘like-dissolves-like’ rule of general chemistry. That is–polar solvents generally dissolve polar substances, and non-polar solvents generally dissolve non-polar substances.
“Hexane works well for something ‘really oily’, or non-polar, while ethyl acetate is better for something a bit non-polar or ‘oily’ but also has oxygens attached. Methanol is good for really polar substances, but it is not optimally safe in most scenarios. Something like ethanol is safer for human handling, but might be less effective.”
First, it’s important to understand that your data will be displayed in graph form, with the X-axis representing retention time, and the Y-axis representing sample concentration.
Your “goal” is essentially to see how well your sample lines up with the known and identified standard. Your standard is essential in this stage, as it is what is used to determine the identity and accuracy of your sample. Only then can you be sure that your samples contains the compounds you think it does.
The retention time (X-axis) in gas chromatography is significantly impacted by the type of column and GC parameters, like flow rate and temperature. To ensure accurate comparisons of retention times, it’s essential to use consistent parameters across different analyses or labs.
Source: InnovaTech
GCMS machines are relatively expensive, so not every lab has one. Here are some tips for locating a lab which you may be able to send samples to:
The results of our smell testing can be found on the results page of our wiki!