Name: ptr-tof-ms coupled with an automated sampling system and tailored data analysis for food studies: bioprocess monitoring, screening and nose-space analysis
Uploaded: 2017-05-11T12:30:00
Description: Watch this Scientific Journal Video about PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis at

This work is supported by the European Commission's 7th Framework Programme under Grant Agreement Number 287382. SY is a beneficiary of a European Commission's 7th Framework Programme Grant Agreement Number 287382. IK is a beneficiary of a FIRST doctoral school grant from the Fondazione Edmund Mach. For his work at University of Foggia, VC is supported by the Apulian Region in the framework of 'Future In Research' program (practice code 9OJ4W81).

The protocol follows the guidelines of our institutional committee on human research ethics.
1. Sample Preparation and Autosampler Conditions
<ol>
	<li>On-line bioprocess monitoring: detecting VOCs released during lactic acid fermentation of yogurt 
	NOTE: This section of the protocol represents part of the procedure reported by Benozzi et al.32
	<ol>
		<li>Add 5 mL of pasteurized milk to each vial (20 mL glass vials equipped with PTFE/silicone septa). Note ...

Proton transfer reaction-mass spectrometry (PTR-MS) coupled to time of flight (ToF) mass analyzers represent a valid compromise between the need for identification and quantification of volatile organic compounds and the necessity for rapid analytical profiling. The high mass resolution that characterizes the ToF mass analyzer gives/provides relevant sensitivity and mass spectra with considerable informational content. Furthermore, the application of PTR-ToF-MS coupled with an auto-sampler and tailored data analysis tool...

Direct-Injection Mass Spectrometric (DIMS) technologies represent a class of analytical instrumental approaches that offer considerable mass and time resolution with high sensitivity and robustness, allowing the quick detection and quantification of volatile organic compounds (VOCs)1. These instrumental approaches include, among others, MS-e-noses, Atmospheric-Pressure Chemical Ionization Mass Spectrometry (APCI-MS), Proton-Transfer-Reaction Mass Spectrometry (PTR-MS), and Selected Ion-Flow-Tube Mass Spectrometry (SIFT-MS)1. The pros and the cons of each approach depend on: the kind of sample injection, the source and control of precursor ions, the control of the ionization process, and the mass analyzer1,2.
Proton-transfer-reaction mass-spectrometry (PTR-MS) was developed more than twenty years ago to monitor in real-time and with low detection limits (usually a few ppbv, part per billion by volume) most volatile organic compounds (VOCs) in air3,4. Current uses of PTR-MS range from medical applications, to food control, to environmental research5,6. The main features of this technique are: the possibility of rapid and continuous measurement, the intense and pure source of precursor ions, and the possibility to control ionization conditions (pressure, temperature and drift voltage). These features permit combining versatile uses with a high degree of standardization1,4. In fact, the method is based on reactions of hydronium ions (H3O+), which induce non-dissociative proton transfer in most volatile compounds (particularly in those characterized by a proton affinity higher than water), protonating neutral compounds (M) according to the reaction: H3O+ + M &#8594; H2O + MH+. In contrast to other techniques, e.g., APCI-MS, precursor ion generation and sample ionization are divided in two different instrumental compartments (a schematic representation of the PTR-MS instrument is given in Figure 1). An electrical discharge by water vapor in the hollow cathode ion source generates a beam of hydronium ions. After this phase, ions cross the drift tube, where the ionization of VOCs takes place7. Ions then enter a pulse extraction section and are accelerated into the TOF section. Through flight times, it is possible to determine the mass-to-charge ratios of the ions8. Each extraction pulse leads to a complete mass spectrum8 of the selected m/z range. Ion spectra are recorded by a fast data acquisition system7. A complete spectrum is typically acquired in one second although higher time resolution can be achieved according to the signal to noise level and a quantitative estimation of the VOC headspace concentration can be provided even without calibration9,10.
<img alt="Figure 1" src="/files/ftp_upload/54075/54075fig1.jpg" /> 
Figure 1: Schematic illustration of a PTR-MS. Schematic representation of the PTR-MS instrument. HC: external ion source with hollow cathode; SD: source drift; VI, venturi-type inlet; EM, electron multiplier; FC1-2, flow controllers. Reprinted with permission from Boschetti et al.7. <a href="http://cloudflare.jove.com/files/ftp_upload/54075/54075fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In general, the PTR technique assures fast analysis time, high detection sensitivity and a relatively compact instrument size, requires neither sample preparation nor sample destruction and thus allows real time investigations11. PTR is of considerable interest in the environmental, atmospheric, food, technological, medical and biological sciences12.
VOCs associated with food matrices are of outstanding interest in food science and technology because of their important role in the molecular basis of biological phenomena linked to odor and flavor perception and, thus, in food acceptance. Hence, our interest in real time and non-invasive detection of VOCs mainly deals with sensory qualities of food. In addition, if we consider the possibility to detect spoilage and pathogenic microorganisms by means of released VOCs13 and/or to monitor volatile organic compounds as markers following technological processes (e.g. Maillard by-products during thermal treatments)14, it becomes clear how VOC identification and quantification are fields of interest in food quality management6. Several recent uses of PTR-MS technologies for the rapid monitoring and quantification of VOCs in food matrices testify to the wide range of application of these analytical approaches (Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Food matrix</td>
			<td>Kind of application</td>
			<td>Brief description</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>Butter</td>
			<td>Screening/characterization</td>
			<td>Geographical origin of European butters</td>
			<td>15</td>
		</tr>
		<tr>
			<td>Yogurt</td>
			<td>Bioprocess monitoring</td>
			<td>Evolution during lactic acid fermentation</td>
			<td>16</td>
		</tr>
		<tr>
			<td>Cereal bars</td>
			<td>In vivo measurement</td>
			<td>Nosespace during consumption of cereal bars with varying sugar composition</td>
			<td>17</td>
		</tr>
		<tr>
			<td>Liquid model systems</td>
			<td>Simulated oral conditions</td>
			<td>Evaluation of tongue pressure and oral conditions in a model mouth</td>
			<td>18</td>
		</tr>
		<tr>
			<td>Apple</td>
			<td>In vivo measurement</td>
			<td>Nosespace during consumption apple with different genetic, textural, and physicochemical parameters</td>
			<td>19</td>
		</tr>
		<tr>
			<td>Coffee</td>
			<td>Screening/characterization</td>
			<td>Differentiation of specialty coffees</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Grape must</td>
			<td>Screening/characterization</td>
			<td>Effect of cooking process</td>
			<td>21</td>
		</tr>
		<tr>
			<td>Flavored candies&#160;</td>
			<td>In vivo measurement</td>
			<td>Determination on panelists using different direct mass spectrometry methods</td>
			<td>22</td>
		</tr>
		<tr>
			<td>Ham&#160;</td>
			<td>Screening/characterization</td>
			<td>Effect of the pig rearing system</td>
			<td>23</td>
		</tr>
		<tr>
			<td>Bread</td>
			<td>Simulated oral conditions</td>
			<td>Simulating bread aroma during mastication</td>
			<td>24</td>
		</tr>
		<tr>
			<td>Milk</td>
			<td>Screening/characterization</td>
			<td>Monitoring photooxidation-induced dynamic changes in milk</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Coffee</td>
			<td>Screening/characterization</td>
			<td>Diversity in roasted coffees from different geographic origins</td>
			<td>26</td>
		</tr>
		<tr>
			<td>Bread</td>
			<td>Bioprocess monitoring</td>
			<td>Effect of different yeast starters during alcoholic fermentation</td>
			<td>27</td>
		</tr>
		<tr>
			<td>Coffee</td>
			<td>In vivo measurement</td>
			<td>Nosespace during consumption of different roasted coffee preparations</td>
			<td>28</td>
		</tr>
		<tr>
			<td>Tomatoes</td>
			<td>Screening/characterization</td>
			<td>Impact of Production Location, Production System, and Variety&#160;</td>
			<td>29</td>
		</tr>
		<tr>
			<td>Bread</td>
			<td>Bioprocess monitoring</td>
			<td>Effect of flour, yeast and their interaction during alcoholic fermentation</td>
			<td>30</td>
		</tr>
		<tr>
			<td>Mushrooms&#160;</td>
			<td>Screening/characterization</td>
			<td>Shelf life of dried porcini mushrooms&#160;</td>
			<td>31</td>
		</tr>
		<tr>
			<td>Yogurt</td>
			<td>Bioprocess monitoring</td>
			<td>Effect of different starter cultures during lactic fermentation</td>
			<td>32</td>
		</tr>
		<tr>
			<td>Apple</td>
			<td>Screening/characterization</td>
			<td>Diversity in an apple germplasm collection</td>
			<td>33</td>
		</tr>
		<tr>
			<td>Coffee</td>
			<td>Screening/characterization</td>
			<td>Tracing coffee origin&#160;</td>
			<td>34</td>
		</tr>
		<tr>
			<td>Coffee</td>
			<td>In vivo measurement</td>
			<td>Combination of a dynamic sensory method and in-vivo nosespace analysis to understand coffee perception</td>
			<td>35</td>
		</tr>
	</tbody>
</table>
Table 1: List of scientific studies using PTR-ToF-MS in the food sector. Non-exhaustive list of scientific studies using PTR-based approaches to monitor VOC content in food-related experiments.
In recent studies, we reported on the application of PTR-ToF-MS coupled with an automated sampling system and tailored data analysis tools to increase sampling automation and reliability and, consequently, to enhance the potential of this technique7,10,13. This allowed us to screen, in terms of VOC content, large sample sets (e.g. food of different origins with many replicates, entire germoplasms), to analyze the influence of several experimental modes on VOC release (e.g. different concentrations of a given ingredient, diverse intensities of a specific technological parameter), and to monitor VOCs associated with a given bioprocess (e.g. enzymatic oxidation, alcoholic fermentation). Here, in order to exemplify the potential of PTR-ToF-MS in the agri-food sector, we present three paradigmatic applications: the detection of VOCs released during lactic acid fermentation of yogurt induced by different microbial starter cultures (on-line bioprocess monitoring), the monitoring of VOCs associated with different apple cultivars (large-scale screening) and the in vivo study of retronasal VOC release while drinking coffee (nosespace analysis).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PTR-TOF 8000 High-Resolution PTR-TOF-MS</td>
			<td>Ionicon Analytik Ges.m.b.H.</td>
			<td>PTR-TOF 8000</td>
			<td>An detector for volatile organic compounds (VOCs) that allows for continuous VOC quantification with a very high mass resolution</td>
		</tr>
		<tr>
			<td>GERSTEL&#160;MPS 2XL</td>
			<td>Gerstel</td>
			<td></td>
			<td>A multifunctional autosampler&#160;</td>
		</tr>
		<tr>
			<td>Gas Calibration Unit</td>
			<td>Ionicon Analytik Ges.m.b.H.</td>
			<td>GCU-s / GCU-a</td>
			<td>A dynamic gas dilution system that provides variable but known quantities of different standard compounds in a carrier gas stream</td>
		</tr>
		<tr>
			<td>TofDaq</td>
			<td>Tofwerk AG</td>
			<td>free available at http://soft.tofwerk.com/&#160;&#160;&#160;</td>
			<td>A data acquisition software&#160;(for spectra&#160; acquisition)</td>
		</tr>
		<tr>
			<td>MATLAB&#160;</td>
			<td>MathWorks</td>
			<td>http://it.mathworks.com/products/matlab/</td>
			<td>A technical computing language and interactive environment for algorithm development, data visualization, and data analysis</td>
		</tr>
		<tr>
			<td>R</td>
			<td>The R Foundation</td>
			<td>free available at https://cran.r-project.org/mirrors.html&#160;&#160;</td>
			<td>A language and environment for statistical computing and graphics</td>
		</tr>
	</tbody>
</table>

ptr-tof-ms coupled with an automated sampling system and tailored data analysis for food studies: bioprocess monitoring, screening and nose-space analysis

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that belongs to the Direct-Injection Mass Spectrometric (DIMS) technologies. These techniques allow the rapid determination of volatile organic compounds (VOCs), assuring high sensitivity and accuracy. In general, PTR-MS requires neither sample preparation nor sample destruction, allowing real time and non-invasive analysis of samples. PTR-MS are exploited in many fields, from environmental and atmospheric chemistry to medical and biological sciences. More recently, we developed a methodology based on coupling PTR-ToF-MS with an automated sampler and tailored data analysis tools, to increase the degree of automation and, consequently, to enhance the potential of the technique. This approach allowed us to monitor bioprocesses (e.g. enzymatic oxidation, alcoholic fermentation), to screen large sample sets (e.g. different origins, entire germoplasms) and to analyze several experimental modes (e.g. different concentrations of a given ingredient, different intensities of a specific technological parameter) in terms of VOC content. Here, we report the experimental protocols exemplifying different possible applications of our methodology: i.e. the detection of VOCs released during lactic acid fermentation of yogurt (on-line bioprocess monitoring), the monitoring of VOCs associated with different apple cultivars (large-scale screening), and the in vivo study of retronasal VOC release during coffee drinking (nosespace analysis).

The volatile profile of samples resulted in a complete mass spectrum for the desired mass range acquired each second. In Figure 2, an example of the acquired average spectra during the yogurt on-line bioprocess is given32. In every spectrum, more than 300 mass peaks in the m/z range up to 250 Th can be identified32.
<img alt="Figure 2" src="/files/ftp_upload/54075/...

Watch this Scientific Journal Video about PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis at 

PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis

Proton Transfer Reaction Time of Flight Mass Spectrometry allows high-sensitivity, rapid and non-invasive analysis of volatile organic compounds. To demonstrate its potential, we give three examples: lactic acid fermentation of yogurt (on-line bioprocess monitoring), different apple genotypes (large-scale screening), and retronasal space after drinking coffee (nosespace analysis).

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that ...

The overall goal of this paper is to demonstrate the potential of PTR-ToF-MS for direct volatile organic compound analysis in food science and technology and in particular the importance of automated sampling and tailored data analysis. Volatile organic compound play a key role in food science and technology both because they control flavor and odor perception and because they provide a tool for the monitoring of food quality. The high sensitivity and the rapid and direct injection make PTRMS one of the best choices for the monitoring of volatile organic compound release for bio process monitoring, for the screening of large data sets and for nose space analysis.We also develop proper data and the analysis tools which are necessary for proper exploitation of large and complex data sets such as there was used by PTRMS in a very short time. Future demonstration of our methodology can inspire searches from different statistical and scientific fields such as SENSOMICS, metabolomics, path analysis, and phonemics. To begin the first experiment, add five milliliters of pasteurized milk to 20 milliliter glass vials equipped with PTFE silicone septa.Then transfer the samples to a multi functional GC auto sampler equipped with a temperature control tray. Use the robotic arm of the auto sampler to inoculate the vials with the microbial starter cultures, set the incubation time according to the typology of the desired process and the specifications reported by the starter culture manufacturer. Then set the auto sampler to analyzer one sample after the other obtaining an online volatile organic compound monitoring of lactic acid fermentation during yogurt preparation.The second experiment select at least five apples at the desired phase of ripening without any visible damage for each variety. Store the apples for the desired period at four degrees celsius. Following this, collect five cylindrical disks from each apple with a flesh blade sampler including part of the cortex tissue and avoiding the core portion with seeds.After freezing the samples and liquid nitrogen homogenize them. Place three replicates of 2.5 gram apple samples from each variety into 20 milliliter glass vials. Mix each sample with an antioxidant solution obtained by dissolving one gram of sodium chloride, 12.5 milligrams of ascorbic acid and 12.5 milligrams of citric acid in 2.5 milliliters of deionized water.Incubate the samples at 40 degree Celsius. Then set the auto sampler to automatically analyze the volatile organic compounds. For the last experiment, prepare brewed coffee from ground coffee samples with a six cup stove top coffee maker using 450 milliliters of water and 30 grams of coffee powder.Transfer the brewed coffee to a bottle and place the bottle in a 60 degree celsius thermostatic water bath. Report the water powder ratio, the type of mineral water used the type of coffee maker and the procedure adopted to obtain the coffee beverage. For each coffee brew, transfer 7.5 milliliter aliquots to a 40 milliliter polystyrene cup with a plastic cap.Have each panelist taste the beverage according to the protocol. While PTR-ToF-MS continuously samples the Nose space concentration via a single use ergonomic silicon rubber nose piece at the nose of each panelist. This nose piece is connected to the PTR-ToF-MS by a PIC tube with the part in contact with panelist at room temperature and the remaining tube heated at 110 degree Celsius and an inlet hose that connects the sampling interface with the instrument.To perform head space measurements of the samples directly inject air into the PTRMS drift tube head space by inserting the end of the PTRMS inlet into the sample head space. Before and during measurements, set and constantly verify the following ionization conditions in the drift tube to lead to an E/N ratio of 140 Townsend. If an auto sampler is used, set the minimum inlet flow to 40 standard cubic centimeters.Set the sampling time per channel of ToF acquisition to a master charge ratio range. Use the self developed software to correct the count losses due to the ion detector dead time and perform internal calibration, compound annotation noise reduction, and peak extraction. Mine the data by performing principle compound analysis analysis of variance Tukey's post-hoc test and other statistical analysis adapting existing packages developed using Matlab.The three experiments provide examples of applying our methodology bio process monitoring, screening of sample sets, and in vivo nose space analysis. The online volatile organic compound profile of samples gives a complete mass spectrum every second and more than 300 mass peaks can be identified in the acquired average spectra during the yogurt fermentation. Fermentation kinetics of nine selected mass peaks during yogurt fermentation using four different starter cultures are shown here.Most of these volatiles displayed classical microbial like kinetics with an initial lag phase followed by a growth phase and a post-log phase. Interestingly the online analysis highlighted particular depletion kinetics for four sulfur containing compounds which can have a sensory effect even at a very low concentration. The dendrogram based on volatile organic compounds associated with a large apple collection represented by a 198 sessions highlights the presence of six main clusters mainly determined by esters and alcohols.The cumulative profiles for five coffee testers represented by means of radial plots are shown here. The results demonstrate the presence of reproducible and relevant differences between panelists as evident for panelist P one and P two. The difference of samples, the most tried out methodology can provide reliable data on volatile organic compound release, which when compared with our methods can be obtained in a shorter time.The possibility of measuring many samples the key feature of our approach. Because it allows controlling and tracing viability of biological samples. After this development, this methodology paid well for the researchers in different fields related to volatile organic compound monitoring to explore the link between their release and genetics and the screening of populations.After watching this video, you should have a good idea of how to explore the potential of PTR-ToF-MS in food and technology analysis to analyze volatility by fruits. Please remember that samples and measuring line are exposed to air during your experiment. So avoid to introduce some sources of volatile organic compounds.Another advantage of PTRMS is that it doesn't involve the use of solvents or hazardous dyes and it is a green chemistry technique. However don't forget that your samples can be extremely hazardous.

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