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Identifying the presence of mould on bread by ASAP-MS
Sam Ray1, Scott J. Campbell2, John H. Moncur2, David Douce3
1The Fallibroome Academy, Macclesfield, UK, 2SpectralWorks Ltd, Runcorn, UK, 3Waters Corporation, Wilmslow, UK.
First presented: BMSS Ambient Ionisation SIG Meeting February 2025
Introduction
According to healthline1, any type of bread mould should be assumed to be harmful and not be consumed. Mould is a fungus, which survives by absorbing and digesting the nutrients from the material on which they grow. It is advised not to eat mouldy bread, even parts with no visible mould, due to the mould quickly spreading throughout the bread. The ability to recognise the mould contents of mouldy bread can help us to assess whether bread spoilage is an issue. Here we show the use of an Atmospheric Solids Analysis Probe (ASAP) Mass Spectrometer (MS) to identify whether there is mould on bread even where it is not visible to the human eye.
Experiment
A glass rod was used to spot pieces of mouldy bread (3 weeks growth) to use as a mould standard. A slice of bread that had been left for 2 weeks was used to test for regions of mould. This was done by marking the area to sample different pieces of bread where the glass rod was used to analyse for the presence of mould. This was done at approximately the same distance apart at 30 locations, as shown in Figure 1.
This gave us 5 rows and 6 columns. The piece of bread was spotted at around 20 mm apart to cover the entire surface of the bread. The results were acquired on the Waters’ RADIAN ASAP-MS in full scan mode (m/z 50-1000) with a cone voltage of 15 V. Acquisition time was 30 seconds with the sample being inserted into the RADIAN for 10 seconds. Sample submission and data acquisition were performed using RemoteAnalyzer® and AnalyzerPro® XD from SpectralWorks. Between each sample, the rods were replaced and then baked out at 450 °C for 60 seconds to remove any contaminants or unwanted substances. Furthermore, a blank between each sample was used as a comparison to the sample and to account for any interference by other species present. Overall, 30 different samples were acquired covering the area of the bread.
Results
Initial analysis between the blanks and the mould standards were well separated by Principal Component Analysis (PCA), as shown in Figure 2.
Samples taken from 6 unknown samples, of which 3 were from areas of no visible mould and 3 which were taken directly from the mould show 2 different regions on the PCA analysis, as shown in figure 3.
The unknowns, taken directly from visible mould areas, show some of the ions observed in the mould standard. The difference in the PCA plot is likely to be due to different stages of metabolism, because of the mould fungus breaking down the carbohydrates. In the mould standard, the ions between m/z 800-900 are only present at low levels. The unknowns taken from the mould were at a higher level because the digestion of the bread was incomplete.
Figure 4 shows a heatmap of m/z 175 overlayed on a slice of bread, as this m/z was more
prevalent in areas where mould was present.
Conclusions
Using ASAP-MS and PCA it was possible to differentiate the presence or absence of mould. Separation was shown in PCA analysis of moulds of different ages, potentially caused by the incomplete digestion of carbohydrates in younger moulds.
Future research would include spotting pieces of the bread at more locations and the use of more precise methods of measurements. This would allow the determination of where the mould was starting to form.
1. https://www.healthline.com/nutrition/can-you-eat-bread-mold.
