3.1. Moisture content testing
The initial MC of brown and yellow flaxseed was 6.5 ± 0.1 % and 7.0 ± 0.2 % (w/w), respectively. The above-mentioned MCs are considered low and are consistent with the hot dry 2021 Manitoba growing season. Conditioning took two weeks to increase the MC of the flaxseed to a level of 9.2 ± 0.2 % (w/w).
In the Clēan Flow unit, the moistening action of the H2O2 spray seemed to counteract the drying effect of the fanned ozone for the brown flax. As a net result, the mean of the MC of the brown control flaxseed was not significantly different from that of the brown decontaminated flaxseed (Table 1). However, for the yellow flaxseed, there was a loss of MC to a value of 8.6 ± 0.2 % after decontamination.
The movement of gas, in the form of ambient air, through the grain, i.e. aeration, is commonly used in modern agriculture to lower MC. However, excessively fanning/blowing overly dry gasses of any kind, including ozone, over grain would have the risk of reducing the MC to a point where the seed becomes brittle and susceptible to mechanical breakage. Considering researchers have recently reported that MCs at or below 6% for flaxseed can make the seeds susceptible to mechanical damage (Nadimi 2022b), the MC of 8.6 % should not have any of such concerns. Considering very high MCs can also lead to spoilage, keeping the seeds at a safe MC is critical.
Based on the above discussions, drying grain during aeration with ozone or during advanced oxidative decontamination could be advantageous to storage, as lower MC is associated with a slower spoilage rate. Conversely, spraying excessive amounts of liquid H2O2 on the hygroscopic grain would have the risk of increasing grain MC, thereby accelerating spoilage.
During the subsequent 12 weeks of storage, the average weekly MCs of the control and decontaminated brown flaxseed were 9.1 ± 0.2 and 9.1 ± 0.3 %, respectively, which were not significantly different. The average weekly MCs of the stored yellow control (8.8 ± 0.3%) and decontaminated flaxseed (8.6 ± 0.2 %) were also not significantly different from each other. The recorded MCs indicate that it is likely that the equilibrium MC of the yellow variety is lower than that of the brown at our chosen temperature and RH.
Table 1
Moisture content of brown (CDC Glas and Bethune) and yellow (VT-50) flaxseed.
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Moisture content (%)
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Brown
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Yellow
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Mean
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SD
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Mean
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SD
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flaxseed from supplier
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6.5
|
0.1
|
|
7.0
|
0.2
|
control samples
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9.2
|
0.2
|
|
9.2
|
0.2
|
decontaminated samples
|
8.7
|
0.6
|
|
8.6
|
0.2
|
Stored control samples
|
9.1
|
0.2
|
|
8.8
|
0.3
|
Stored decontaminated samples
|
9.1
|
0.3
|
|
8.6
|
0.2
|
3.2. Germination testing
The germination rate of the brown and yellow flaxseed was initially 99 ± 2 and 97 ± 5 %, indicating good seed quality. This result is consistent with the short lag time between harvesting and testing, and good handling practices by the supplier. The advanced oxidative decontamination performed on the flaxseed with the Clēan Flow unit did not significantly lower the germination rate immediately. After decontamination, the germination rate was measured to be 96 ± 4 and 88 ± 4 % for brown and yellow flaxseed, respectively.
During storage, the germination rate decreased from its initial high value for both seed colours, including control and decontaminated flaxseed (Figure 4). At the 12-week timepoint at the end of the storage study, the brown control flaxseed had a germination rate of 80 ± 3 %, whereas the brown decontaminated flaxseed had a germination rate of 65 ± 6 %. This significant difference indicates some damage to the viability of brown flaxseeds that only becomes apparent after the passage of time and storage. In contrast, the 12-week stored yellow flaxseed did not have a significant difference in the germination rate between the control (61 ± 15 %) and decontaminated flaxseed (63 ± 10 %).
The living zygote within a seed is vulnerable and is known to be damaged by overly high concentrations of ozone (Pandiselvam et al., 2020). Oxidative stress is implicated in many human diseases but is also relevant to seed germination. Germination involves rapidly dividing cells with an intense metabolic activity that produces ROS. Omega-3 fatty acid antioxidant properties neutralize oxidative stress, the imbalance between the production and detoxification of ROS. Low levels of ALA in flaxseed of certain genetic backgrounds have been related to a decline in seed vigor in stressed conditions (Saeidi et al. 1999). Oxidative decontamination could be considered a stressful condition for the flaxseed, and the different seed vigor/germination rate responses observed in the flaxseed varieties/colours may be related to their ALA level.
3.3. Saltwater Filter Paper testing
SFP testing showed that the percentage of seeds without visible mould was at or near 100% at the beginning of the storage study (Figure 5). The percentages decreased over the course of the storage study. Twelve weeks of storage was sufficient time for the visible mould percentage of the control yellow flaxseed to drop completely to a value that is not significantly different from 0% (i.e. 4 ± 4%). Differences between the control and decontaminated flaxseed became apparent only after 8 weeks of storage. The decontaminated flaxseed showed higher percentages of seeds without visible mould growth for both colours.
Our observation at the 12th-week timepoint at the end of the storage indicates a significantly higher percentage of seeds without visible mould for decontaminated yellow flaxseed compared to the control. The brown samples also decreased the percentage of seeds with visible mould growth after 12 weeks of storage, but the difference was insignificant at p < 0.05.
3.4. FAV testing
The FAV was not significantly affected by decontamination at 0 weeks of storage for both colours (Figure 6). After 12 weeks of storage, there was an increase in the FAV of the brown decontaminated flaxseed compared to the control. This observation indicates that the fatty acid contents of the seed were degraded faster in the brown seed after the advanced oxidative decontamination. On the other hand, no significant changes were observed in the yellow variety after 12 weeks.
Interestingly, our results are the opposite of what was reported in a study of ozone fumigation of flaxseed by Bechlin et. al (2019), where ozone fumigation caused an increase in the FFA increased in a yellow, but not in a brown variety. This inconsistency could be due to several differences between the experimental design of the two studies. For example, herein, we used unpackaged flaxseeds under advanced oxidative decontamination with ozone concentrations of 3-4 ppm for 30 seconds. In the other study, packaged flaxseeds were utilized with ozone fumigation at a concentration of 10 ppm for 120 minutes. The increase in FFA in response to ozone exposure is problematic as it indicates that the lipid contents of the seeds were oxidized and that rancidity was accelerated. Concerningly, the increase in rancidity was not apparent immediately after the oxidative treatment, but rather as a delayed effect that only became measurable with the passage of time. Our literature review has indicated that storage studies are necessary for assessing the full/long-term consequences of oxidative decontamination on lipid-rich commodities.
3.4.1. Percent mass of lipid and mass of flaxseeds
The control brown flaxseed studied was 36 ± 3% (w/w) oil. The yellow flaxseed (VT-50) was 44 ± 1% w/w oil. It is reported that the VT-50 variety has a 67.8% ALA (alpha-linolenic acid) content as a percent of total fatty acid composition and that the CDC Bethune and Glas (2012) varieties had an ALA percentage of 54.2 and 58.4%, respectively (You et al., 2016). It follows that the yellow flaxseed studied here should have a higher percentage of ALA per mass dry weight of seed than the brown.
3.5. FTIR spectroscopy
3.5.1. Transmission FTIR imaging
The internal anatomical features of the flaxseed can be seen when the seed is cut in either the longitudinal (Figure 7) or transverse (Figure 8A) direction. The flaxseed was cryo-sectioned in the transverse direction after soaking and embedding (Figure 8B), but the tissue was prone to tearing/separating at the mechanically weak region between the endospermic and the cotyledonous tissue.
Swaths of tissues were intact enough to acquire transmission-mode FTIR hyperspectral images, as seen in Figure 9. The blacked-out area of the image in Figure 9B had low or no amide I and II bands in the FTIR spectra, indicating no protein and a lack of tissue where a rip occurred. Examining a ratio, rather than the peak alone, was used as a strategy to deemphasize changes to spectral peaks that are a consequence of a longer/shorter path length caused by uneven, thicker tissue sectioning. The FTIR images of the brown flaxseed had pixel spectra with moderate calculated ratios (green and yellow pixels Figure 9B) of two spectral features: 1) the area under the peak at 3012 cm−1 and 2) the area under the total CH2 CH3 stretching region between 2844 and 2857 cm−1. For the yellow flaxseed (Figure 9C) the FTIR images have more pixel spectra with a higher calculated ratio (red pixels, Figure 9D). An increase in the ratio of the area under the olefinic peak at 3012 cm−1 to the CH2 CH3 stretching region area was only subtly apparent in the unintegrated FTIR spectra and was not present in all pixel spectra (Figure 9E).
The spectra of the flaxseed have a relatively larger absorbance between 1200 and 900 cm-1 compared to the mammalian tissue that is rich in PUFA reported by Stitt et al. (2012). Absorbance in this spectral region could be due to the presence of C-O-H and C-O-C bending, and the C-O and C-C stretching of carbohydrates (Wiercigroch et al., 2017). Absorption in the range of 1690–1727 cm−1 is also substantial, and this is consistent with the presence of carbonyl (C=O) associated with FFA, which has been studied in flaxseed oils (Schönemann 2011).
Overall, the transmission FTIR micro-spectroscopy of the flaxseed was challenging, and due to the poor quality of the tissue, only the control flaxseed could be analyzed. In thin layers, flaxseed oil can oxidize in as little as five hours in ambient conditions (Van der Weerd et al., 2005). The thinness of the cryosections makes them vulnerable to oxidation due to ambient atmospheric oxygen during sectioning and imaging. One possible solution to the problem of tissue quality would be to dissect and remove the seed coat prior to sectioning. The seed coat readily separates after the seed is transversely bisected. However, we did not attempt imaging without the seed coat, due to the relevance of the seed coat to surface decontamination.
3.5.2. Handheld ATR-FTIR
During the handheld ATR FTIR spectra of ground flaxseed, it was observed that the yellow and brown flaxseed had a consistent difference in the areas of the peak 3012 cm-1 relative to the area of the CH2 CH3 stretching region (Figure 10). After the handheld ATR FTIR spectra were processed, it became apparent that all the stored yellow flaxseed spectra had a higher peak ratio than the stored brown flaxseed spectra (Figure 11). The intensity of the olefinic 3012 cm−1 peak relative to the CH2 CH3 stretching region did not decrease significantly over time during the storage study or due to decontamination.
Van der Weerd et al. (2005) observed a rapid (5-hour) decrease in the olefinic peaks in thin films of flaxseed oils. Our results on the stability of the olefinic peaks over the longer term (3 months) may be explained by the fact that the studied flaxseed tissue structure is intact during storage. Thin layers, such as cryosections or films, have a higher surface area exposed. The intact tissue and the outer hull would also offer some protection from oxygen.
The sample preparation and analysis involved with acquiring handheld ATR-FTIR spectra of ground flaxseed was simple and effectively showed spectral contrast between yellow and brown flaxseed. Hence, our experiment suggests that ATR-FTIR is a faster tool than transmission FTIR for studying the seed as a whole, when tissue-specific changes are not required, and when exposure to ambient oxygen is an issue and data must be acquired quickly.
The higher percentage of ALA and other unsaturated compounds in the yellow flaxseed relative to the amount of long-chain fatty acids explains the higher ratio of areas (3012 cm-1 / CH2 CH3 stretching region) observed in the FTIR spectra. These unsaturated compounds may be relevant to reducing damage to quality. A study by Mohanan et al. (2018) found that some natural antioxidants in flaxseed oil have an ability to prevent lipid oxidation that is on par with synthetic antioxidants. It may be possible that the increased presence of antioxidants in the yellow flaxseed counteracts lipid oxidation and oxidative damage due to oxidative decontamination, preventing the promotion of oxidative rancidity during storage. But there could be a more complex relationship here, as biological systems that are rich in olefins often have other antioxidant systems that protect them from lipid peroxidation. So it may not be the unsaturated lipids acting directly to detoxify oxygen, but rather related systems at work.