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SHAD 2018 Chemical Composition Project

Analyzing Carcinogen Levels in Burnt and Charred Food Cooked Using Emergency Survival Methods

www.usask.ca

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Acknowledgement

We acknowledge the University of Saskatchewan for providing the facilities without which we could not succeed. The university is situated on Treaty 6 Territory and the Homeland of the Métis. We pay our respects to the First Nations and Métis ancestors of this area and reaffirm our relationship with one another.

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Who we are

  • Group of 8 students:
    • Eugene
    • Achilles
    • Chloe
    • Harrison
    • Yue Yang
    • Shreeya
    • Brian
    • William
  • From across Canada (Ontario)
  • Researched carcinogens under guidance of USask/CLS staff
  • Part of a summer enrichment program called SHAD

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What is SHAD?

  • Over 900 students from across Canada attend the STEAM-based month-long program.

  • Students attend one of 16 Canadian host universities, including the University of Saskatchewan.

  • Consists of lectures, workshops, projects and activities that develop an innovative and entrepreneurial mindset.

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SHAD Theme

“How can we help Canadian communities be more resilient in a natural disaster?”

  • Our project fit within this theme by analyzing carcinogenic properties in emergency food cooked using fire techniques available in emergency situations.

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Literature Search

Previous Research

  • Previous research has been done by University of Birmingham
  • Polycyclic Aromatic Hydrocarbons (PAH) are carcinogens released from burning
  • Correlation between burnt portions of bread and Acrylamide (carcinogen) has been found

Our research ideas

  • Wanted to explore how carcinogenic properties increase with burning
  • In the news and popular media
  • Determine what carcinogens are present
  • Presence of polycyclic aromatic hydrocarbons, where and what kind

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Situation

  • As part of the SHAD USask program students research a project using the synchrotron.

  • Unfortunately, the beam was down so we were unable to use it for our research so we devised an alternative project.

  • So, we used the Chemistry Department and Saskatchewan Structural Science Centre’s (SSSC) equipment instead.

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Research Questions

How does the degree of burn affect the presence and amount of carcinogens? What kind(s) of carcinogens are present?

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Hypothesis

  • We predict that as the degree of charring/burning of different foods roasted in open flames increases, the level of carcinogens present will increase as well.

  • This effect should be seen more in starch-based foods (potatoes) compared to fish (sardines) as noted by previous research.

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Sample Preparation

  • Roasted samples of potatoes and sardines over open fire

  • Freeze-dried and ground samples (used liquid nitrogen to aid)

  • Used various analytical techniques at the Chemistry Dept. and the SSSC to learn more about samples

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Sample Preparation - continued

Naming System:

POT → Potato Sample (Solanum tuberosum)

SAR → Sardine Sample (Clupea harengus)

A → Cooked Sample (no burn/slight burn)

B → Burnt Sample (Visible moderate burn)

C → Charred Sample (Completely blackened/charred)

Z → Control Sample (Raw)

Number → Sample Iteration

I.E. SAR - A - 1 → Sardine - Cooked - First Iteration

For Potatoes Only:

I → Interior Sample (Potato Flesh)

S → Skin Sample (Potato Peel)

I.E. POT - A - 1 - S → Potato - Cooked - First Iteration - Skin

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NMR - Nuclear Magnetic Resonance

  • Nuclear Magnetic Resonance spectroscopy is a chemistry technique used in research to determine the content and purity of a sample as well as its molecular structure.

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How does NMR work?

  • Sample dissolved in solvent (Chloroform-D in this case)

  • Particulate matter filtered out

  • Purified sample placed in magnetic field

  • Nuclei are excited by radio waves, creating the NMR signal

  • Signal is detected and graphed in a series of peaks

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Figure 1

A comparison of potato samples using NMR analysis - visible Aldehydes and PAHs (likely Benzene or Pyrene)

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Colour code:

  • Purple: C Sample (Charred)
  • Green: B Sample (Burnt)
  • Red: A Sample (Cooked)
  • Blue: Z Sample (Control)

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Figure 2

A comparison of sardine samples using NMR analysis. There are no significant differences or changes found.

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Colour code:

  • Purple: C Sample (Charred)
  • Green: B Sample (Burnt)
  • Red: A Sample (Cooked)
  • Blue: Z Sample (Control)

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Data Analysis

  • Aldehydes are correlated with the formation of Acrylamide within the Maillard Reaction (Mottram, D. S., Wedzicha, B. L., & Dodson, A. T. (2002, October 03). Food chemistry: Acrylamide is formed in the Maillard reaction. Retrieved from https://www.nature.com/articles/419448a)

  • Polycyclic Aromatic Hydrocarbons like Benzene or Pyrene are also carcinogens

  • NMR data does not guarantee the exact listed compounds, though our graphs consistently show this data

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FTIR - Fourier-Transform Infrared Spectroscopy

  • Sample analysis technique which collects absorption and emission spectral patterns

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FTIR - How does it work?

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  • Interferometry with multi-frequency wavelengths
  • Fourier Transform

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FTIR - Data Collection Procedure

  • IR Machine prepared using liquid nitrogen
  • Clean slide set up with ground up sample
  • Laser chamber secured with sample in it
  • IR Machine activated to direct multi-frequency rays onto sample
  • Absorption spectra is recorded by IR module and mapped onto 2D graph using Fourier Transform

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What the y-axis/intensity means

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Y-axis meaning - continued

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Acrylamide chemical structure and its relevant functional groups

C = O C - N = C - H C = C

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Blue: 1% intensity

Green: 5% intensity

Red: 100% intensity

Figure 3

Raman analysis of sardine samples - Raman spectroscopy is one of the vibrational spectroscopic techniques used to provide information on molecular vibrations and crystal structures.

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Z1 - control

B1 - burned

C1 - charred

Figure 4

FTIR analysis of potato flesh - showed almost no difference when comparing the results between the samples

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Z1 - control

A1 - cooked

B1 - burned

C1 - charred

Figure 5

FTIR analysis of potato skins - showed to have several differences when comparing all of the samples to the most charred C sample

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Aldehyde

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Z1 - control

A1 - cooked

B1 - burned

C1 - charred

Figure 6

Potato Skin FTIR first discrepancy - 2850 peak indicates aldehyde

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Z1 - control

A1 - cooked

B1 - burned

C1 - charred

Figure 7

FTIR analysis of potato skins - showed to have several differences when comparing all of the samples to the most charred C sample

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Carboxylic acids

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Z1 - control

A1 - cooked

B1 - burned

C1 - charred

Figure 8

FTIR analysis of potato skins - showed to have several differences when comparing all of the samples to the most charred C sample

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Amine

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Z1 - control

A1 - cooked

B1 - burned

C1 - charred

Figure 9

Potato Skin FTIR second discrepancy - 1320 peak indicates amine

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Z1 - control

A1 - cooked

B1 - burned

C1 - charred

Figure 10

FTIR analysis of potato skins - showed to have several differences when comparing all of the samples to the most charred C sample

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Alkene

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Z1 - control

A1 - cooked

B1 - burned

C1 - charred

Figure 11

Potato Skin FTIR third discrepancy - 885 peak indicates alkenes

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What does this all mean?

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  • The increased levels of alkenes, in combination with amines and carboxylic acids in the charred sample support that as the potato peel burns, the levels of acrylamide rise with it
  • Also, the increase of aldehydes support the same former statement as well

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XPS - Axis Sapra

What is it?

  • X-ray photoelectron spectroscopy (XPS) is a surface analysis technique that measures the elemental composition and chemical state of a material using the feedback from an induced photoelectron effect

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Why was it important in this experiment?

  • Used to see the chemical composition of the outer crust of the samples

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XPS - Axis Sapra

Procedure

    • XPS holder had 8 pieces of double sided carbon tape on it
    • One sample per piece
    • Nitrogen gas went into the system to bring the anti chamber to atmosphere
    • Screw latch into place and press vent to move arm

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XPS - Axis Sapra

Issues/Holdbacks

    • Could not test 2 samples due to dampness
    • No oily samples were allowed to be used in vacuum machines, therefore no sardines

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Amide

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XPS - Axis Sapra

Results:

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Figure 12:

XPS analysis of potato skins - showed to have several differences when comparing all of the samples to the most charred C sample

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XPS - Axis Sapra

Results:

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Figure 13:

XPS analysis of potato flesh - showed significant differences when comparing the results between the samples

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XPS - Axis Sapra

Analysis

    • Acrylamide is an organic compound belonging to the amide functional group.
    • The National Toxicology Program's Report on Carcinogens considers acrylamide to be reasonably anticipated as a human carcinogen.
    • These group of organic compounds have the general formula of :

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XPS - Axis Sapra

Analysis

    • These compounds are characterized by carbon atoms double bonded with oxygen atoms and single bonded to nitrogen atoms.
    • As the potato skin samples were burnt further, the concentration of C=O bonds increased significantly
    • This was indicative of an increase in the concentration of acrylamide in the skins.

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Gas Chromatography Mass Spectrometry (GC-MS)

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Steps we took:

  1. Sample preparation - Extraction process
  2. GC-MS data collection
  3. Data analysis/Results

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Step 1: Sample Preparation

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  • Took roughly half of each unprocessed sample after the campfire (wet burnt samples)
  • Manually removed the surface from the wet samples
  • Created 6 samples POT-A, B, C and SAR-A, B, C
  • Mass of samples
    • POT: 2.0g - 2.9g
    • SAR: 0.5g - 1.0g

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Step 1: Sample Preparation - continued

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Extraction Process:

  • Used methylene chloride (CH2Cl2) as solvent
  • Homogenized solution using an ultrasonic homogenizer

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Step 2: GC-MS Data Collection

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How it works:

  • GC
    • Injects minute quantity of solution into capillary column
    • Helium carrier gas is pumped through column
    • GC is gradually heated up to vaporize molecules

  • MS
    • Tungsten filament is heated up and electrons hit sample
    • Molecules fragment
    • Positive repeller pushes positive ions into analyzer

  • About 34 minutes for each sample

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Step 3: Data Analysis

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Overview

  • Potato - notable compounds:
    • Benzene - carcinogenic
    • Benzamide
    • Carbamic Acid
    • Docosenamide
  • Sardine - notable compounds:
    • Pristane - toxic
    • Cholesterol
    • Pyrene - carcinogenic
    • Oleamide
  • Sugars, starch, cholesterol, MeCl2 caused noise in data

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Figure 14

Chromatogram view of POT-C sample.

Boxes show peak intensities of certain compounds:

  • benzene,
  • benzamide,
  • carbamic acid,
  • docosenamide.

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Figure 15

Chromatogram view of all POT samples at around time 9.50.

Each peak is an increase in intensity of benzene measured.

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Peak ~9.50

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Figure 16

Spectrum view and library search of POT-C sample at time 9.50.

Red spectrum is experimental, blue spectrum is library theoretical

Many isomers of benzene matched - above had a match score of 924/1000.

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Figure 17

Chromatogram view of all POT samples with atomic mass within spectrum of 202.2 amu - 202.3 amu (mass of pyrene = 202.25 amu).

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Figure 18

Chromatogram view of SAR-C sample.

Boxes show peak intensities of certain compounds:

  • pristane,
  • pyrene,
  • oleamide,
  • cholesterol.

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Figure 19

Chromatogram view of all SAR samples at around time 19.44.

The middle red peak indicates pyrene.

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Figure 20

Spectrum view and library search of SAR-C sample at time 19.44.

Red spectrum is experimental, blue spectrum is library theoretical

Many variations of pyrene matched - above had a match score of 700/1000.

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Intensity of Carcinogens Across Samples

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Sample

Ion Intensity at Time 9.50 (Benzene)

POT - A

200 000

POT - B

600 000

POT - C

510 000

Sample

Ion Intensity at Time 19.44 (Pyrene)

SAR - A

170

SAR - B

160

SAR - C

360

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Conclusion

  • Through the analysis of results taken from NMR, XPS, FTIR, and GC-MS, there was a direct relation between the increased degree of burn and the level of carcinogens in potato peel, with a sharp linear correlation

  • The flesh of the potatoes was found to increase in carcinogenic level almost negligibly as the level of burn increased

  • The sardines were found to have some minor increases in carginocens, but not as much as the potato peels.

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If we were to replicate this experiment, we would...

  • Include more consistent timing and use a more consistent heating method (ex: gas stove)

  • Prepare our samples in a more precise manner (measurements, volume, mass)

  • Investigate into different extraction methods

  • Operate on a longer timeline

  • Run multiple replicate samples

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Next Steps

With the CLS:

  • Possible analysis of samples using the soft x-ray spectromicroscopy (SM) beamline
    • Allows for biological analysis
    • Scanning Transmission X-ray Microscopy (STXM) would allow for organic chemical imaging
      • Can pierce through water; wet samples can be used
      • Can map chemical compounds with structure

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Thank You

  • We acknowledge the Saskatchewan Structural Sciences Centre (SSSC) for providing facilities to conduct this research. Funding from the CFI, NSERC and the U of S for support research at the SSSC.

  • Thanks to the Chemistry Dept. at the University of Saskatchewan for allowing use of their equipment and labs

  • Thanks to the CLS Staff for their supervision and guidance

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Thank You

  • Supervisors:

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  • Jason Maley
  • Joyce Mcbeth
  • Danielle Covelli
  • Tylor Sove
  • Marla Cheston
  • Rob Blythe
  • Stephen Urquhart
  • Peter Zhu
  • Ken Thoms
  • Anna-Maria Boechler
  • Cooper Skjeie
  • Ramaswami Sammynaiken
  • Thomas Ellis

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Questions?

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References

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Carcinogenic factors in food with relevance to colon cancer development. (2003, January 10). Retrieved from https://www.sciencedirect.com/science/article/pii/002751079390031A

  • Effects of Asparagine, Fructose, and Baking Conditions on Acrylamide Content in Yeast-Leavened Wheat Bread. (n.d.). Retrieved from https://pubs.acs.org/doi/pdf/10.1021/jf034999w

  • Mottram, D. S., Wedzicha, B. L., & Dodson, A. T. (2002, October 03). Food chemistry: Acrylamide is formed in the Maillard reaction. Retrieved from https://www.nature.com/articles/419448a

  • The National Institute for Occupational Safety and Health (NIOSH). (2014, December 04). Retrieved from https://www.cdc.gov/niosh/idlh/1333864.html

  • Olesen, P. T., Olsen, A., Frandsen, H., Frederiksen, K., Overvad, K., & Tjønneland, A. (2008, January 08). Acrylamide exposure and incidence of breast cancer among postmenopausal women in the Danish Diet, Cancer and Health Study. Retrieved from https://onlinelibrary.wiley.com/doi/abs/10.1002/ijc.23359

  • Stocks, B. J., Fosberg, M. A., Lynham, T. J., Mearns, L., Wotton, B. M., J., . . . McKENNEY, D. W. (n.d.). Climate Change and Forest Fire Potential in Russian and Canadian Boreal Forests. Retrieved from https://link.springer.com/article/10.1023/A:1005306001055

  • S. (1978, January 01). Carcinogenic effects of metals. Retrieved from https://www.osti.gov/biblio/5204616