Application note Received: 24 February 2014
Revised: 30 June 2014
Accepted: 2 July 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3430
Monitoring photooxidation-induced dynamic changes in the volatile composition of extended shelf life bovine milk by PTR-MS† Jonathan Beauchamp,a* Erika Zardin,a,b Patrick Silcockc and Phil J Bremerc Exposure of milk to light leads to photooxidation and the development of off-flavours. To follow these reactions, semi-skimmed (1.5% fat) and whole (3.8% fat) extended shelf life (ESL) bovine milk samples were exposed to fluorescent light for up to 20 h at room temperature, and the volatiles in the samples’ headspace were measured in real time using proton-transfer-reaction mass spectrometry (PTR-MS). Compounds tentatively identified as methanethiol, acetone/propanal, pentanal/octanal/nonanal/1octen-3-ol, hexanal, diacetyl, dimethyl disulphide, heptanal and benzaldehyde displayed dynamic release profiles relating to the changes occurring in milk upon exposure to light. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: PTR-MS; ESL milk; photooxidation; VOCs; off-flavour
Introduction
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Milk is susceptible to oxidation, particularly photooxidation. It has been known for almost a century that exposure to light can have a detrimental impact on the flavour of liquid milk,[1] with ultraviolet (λ = 180–380 nm) and visible (λ = 380–780 nm) light both playing a major role in the development of light-induced offflavours.[2] Unstable primary compounds are formed when absorbed radiation energy is transferred to unsaturated fatty acids, proteins and amino acids, resulting in the production of secondary volatile products such as carbonyl and sulphur compounds, e.g. aldehydes, ketones, alkenes or dimethyl disulphide (DMDS).[3] Continuous exposure of milk to natural or artificial light (particularly at λ = 420–520 nm) can induce chemical reactions that appear as flavour defects upon consumption.[2] Two distinctive offflavours in milk that are attributed to light irradiation are described as sunlight and cardboard-like or metallic flavours.[3,4] A sunlight offflavour imparts a burnt, oxidised odour in milk after 2- to 3-day light exposure and has been tentatively attributed to the formation of DMDS and methional that are derived from methionine.[5–7] The cardboard-like or metallic off-flavour occurs after prolonged intense light exposure due to the formation of secondary lipid oxidation products including carbonyl compounds (pentanal, hexanal, heptanal and ketones), alcohols and hydrocarbons.[8–10] The flavour quality of retail milk generally does not deteriorate significantly on exposure to the limited light conditions found in commercial display cabinets.[11] The analysis of light-induced flavour compounds in milk has commonly been carried out using gas chromatography–mass spectrometry (GC-MS) following diverse methods of headspace sampling or derivatisation.[12–14] Despite the strengths of this technique, GC-MS has a limited analytical time resolution due to the necessary sample workup and intrinsic time for analyte separation in the GC column, and this approach is therefore less suitable for characterising fast processes. Proton-transfer-reaction mass spectrometry (PTR-MS) is a chemical ionisation technique that enables real-time analysis of gas-phase volatile organic
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compounds (VOCs).[15] It has been used in diverse studies of food volatiles including investigating milk and milk-related products.[16,17] In the present investigation, PTR-MS was applied to monitor the release of volatile light-induced oxidation products of extended shelf life (ESL) milk. Dynamic headspace analyses were made in parallel on milk samples of different fat content and/or under different light-exposure conditions to demonstrate the capability of PTR-MS to continuously and sensitively detect volatile oxidation products in real time and provide indications on the dynamics of such processes.
Experimental Milk Semi-skimmed (1.5% fat) and whole (3.8% fat) extended shelf-life (ESL) bovine milks (500 ml, Molkerei Weihenstephan GmbH & Co. KG, Freising, Germany) were obtained from a local supermarket and stored at 4.7 ± 0.1 °C. The fat contents of the two milk types were taken as stated on the milk carton packaging and were not additionally determined experimentally. Samples were analysed at least 8 days before the best-before date to minimise interferences from autooxidation or microbial spoilage. The milk
* Correspondence to: Jonathan Beauchamp, Department of Sensory Analytics, Fraunhofer Institute for Process Engineering and Packaging IVV, Freising, Germany. E-mail: [email protected] †
This article is part of the Journal of Mass Spectrometry special issue entitled “3rd MS Food Day” edited by Gianluca Giorgi.
a Department of Sensory Analytics, Fraunhofer Institute for Process Engineering and Packaging IVV, Freising, Germany b Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich-AlexanderUniversität Erlangen-Nürnberg, Erlangen, Germany c Department of Food Science, University of Otago, Dunedin, New Zealand
Copyright © 2014 John Wiley & Sons, Ltd.
Photooxidation-induced volatiles in ESL milk was held in the unopened opaque carton at room temperature for at least 1 h before experimentation.
(~120–150 Td), and tentative identifications can be made based on these and on previously reported GC studies on milk volatiles.
Light
Experiment
Using a CAS 140CT Array Spectrometer (Instrument Systems Optische Messtechnik GmbH, Munich, Germany), the integral irradiance intensity of two or four 15-W fluorescent tubes (TL-D 15W/840, Philips) positioned 20 cm from the samples (Fig. 1) was measured as 8.32 or 10.64 W m 2, respectively. The spectral emission of these fluorescent tubes encompassed wavelengths between 405 and 640 nm, with peaks of maximum emission at 430, 550 and 610 nm (data not shown). Both the integral irradiance intensity and the wavelengths of the emission maxima of this lighting system are sufficient to degrade riboflavin, chlorins and porphyrins and are thus likely to induce oxidised/ sunlight off-flavours, as described in the literature.[18]
The headspace (150 ml) above the milk (400 ml) held in 500-ml modified impinger (impinger stem removed) glass bottles (Schott AG, Mainz, Germany) was sampled via connections in the lid that enabled dynamic flushing through one and headspace analysis via the other. Until exposure to light, the bottles were wrapped in aluminium foil, and milk samples were poured into the bottles under darkened laboratory conditions. A polytetrafluoroethylenecoated magnetic stir bar (100 rpm) stirred the milk throughout the measurements. The experimental setup was held at 27 °C in a ventilated oven (UFE 500, Memmert GmbH+ Co. KG, Schwabach, Germany), which acted as an enclosed light-exposure chamber. A gas calibration unit (advanced model, GCU-a; IONICON Analytik GmbH) provided a constant flow (200 ml min 1) of dry VOC-free air to the line upstream of the sampling bottles (Fig. 1) which was subsequently equally split between the sampling bottles. An external solenoid valve (model M443W2DTS-LT, Teqcom Industries, Santa Ana, CA, USA) was switched simultaneously with the PTR-MS inlet-selection valve to configure the flows such that both bottles were flushed regardless of the sampling configuration (either inlet 1 or 2 active). The PTR-MS was supplied with a flow of ~50 ml min 1 from inlets (1/8″ outer diameter, 0.04″ inner ™ diameter) of Silcosteel (Restek GmbH, Bad Homburg, Germany) held at 80 °C and connected to the sample bottles via a short length (~20 cm) of ¼″ outer diameter perfluoroalkoxy tubing. In experiment 1, a direct comparison was made between semiskimmed milk exposed to four lamps for 19 h and foil-wrapped semi-skimmed milk (dark). Experiment 2 directly compared semiskimmed milk with whole milk exposed to light (two lamps) for 16 h. In experiment 3, semi-skimmed and whole milk samples were repeatedly exposed to light (four lamps) and then darkness at ~1-h intervals to investigate how rapidly the light-induced changes occurred.
PTR-MS The VOCs in the headspace of the milk samples were measured using a high sensitivity proton-transfer-reaction mass spectrometer (hs-PTR-MS; IONICON Analytik GmbH, Innsbruck, Austria). The reaction chamber was held at an electric field strength to buffer gas number density ratio (E/N) of 132 Td (1 Td (Townsend) = 10 17 V cm2). The drift tube operating conditions were 600 V, 2.2 mbar and 60 °C. The PTR-MS had a custom-made dual inlet system, which allowed two samples to be measured sequentially in a continuous manner via a solenoid valve housed within the PTR-MS that switched between inlets typically every 9 min. As the mass resolving power of the PTR-MS instrument is limited to one atomic mass unit (1 amu), VOCs that form product ions at the same nominal m/z cannot be resolved. However, the fragmentation patterns of many (common) VOCs are well characterised under standard PTR-MS operating conditions
Analysis
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Figure 1. Experimental setup of dynamic headspace analysis of milk volatiles by PTR-MS during light-induced oxidation. Flows were configured so that the headspace of both sample bottles was always flushed while one was being actively sampled. The figure shows active sampling via PTR-MS inlet 1 (solid arrows) and flushing sample 2 via the overflow (dashed arrows). Either two or four lamps were on during light exposure, depending on the experiment.
The PTR-MS instrument was operated in mass scan mode, scanning all ions in the range m/z 20–130 at an integration time of 500 ms per m/z (cycle time: 55 s). Based on 10 cycles measured per sample, switching between samples occurred approximately every 9 min. The signal intensities, in counts-per-second (cps), were processed by first correcting each signal for its m/z-specific transmission efficiency inherent to PTR-MS. The data were then normalised to the primary ion signal (m/z 21 and 37) and drift tube pressure to produce normalised count-rates (n-cps), which are directly proportional to concentration.[19] In the data post-processing, the first two cycles after switching from one sample to the other were discarded to eliminate potential carry-over effects. The remaining eight cycles were averaged (generation of mean values plus standard deviation), which were used to plot the figures and show the release profiles. Since only relative signal intensities (in n-cps) are described in the paper, a quantitative limit of detection (LOD) for the detected compounds was not calculated. Furthermore, the observed changes in the signal intensities in relation to light/dark conditions (or milk of different fat content) clearly indicate detection of the tentatively identified compounds, thus demonstrating that the PTR-MS instrument was operating above the LOD at that m/z.
J. Beauchamp et al. Table 1. Tentative identifications of m/z detected during light-induced oxidation m/z
Tentative identification
Elemental composition
1
MW [g mol ]
Compound class
Natural isotope measured (theoretical)d [%] 13
45 49 59 69a
83a 87 95 97a 107
Acetaldehyde Methanethiol Propanone (acetone)b Propanalb Pentanalb Octanalb 1-Octen-3-olb Nonanalb Hexanal 2,3-Butanedione (diacetyl) Dimethyl disulphide (DMDS) Heptanal Benzaldehyde
C2H4O CH4S C3H6O
44.05 48.11 58.08
C5H10O C8H16O
86.13 128.21
C9H18O C6H12O C4H6O2 C2H6S2 C7H14O C7H6O
142.24 100.16 86.09 94.20 114.19 106.12
Aldehyde Thiol Ketone Aldehyde Aldehyde Aldehyde Alcohol Aldehyde Aldehyde Ketone Disulphide Aldehyde Aldehyde
C
1.9 (2.3) 1.6 (2.0) 3.4 (3.4) 5.6 (5.7)
6.7 (6.8) 6.2 (4.6) 5.3 (3.9) 9.1 (8.0)c 6.9 (7.9)
34
Ref.
S
N/A 6.5 (4.5) N/A N/A
N/A N/A 9.1 (8.9)c N/A N/A
13, 16 12, 21 22, 23 24, 25 8, 13, 18, 23, 26 12 24, 27 28, 29 10, 26 28 13 8, 23, 26, 28 30
a
Predominant fragment ion in PTR-MS. Individual compounds not separable in PTR-MS. 34 c There is a contribution from the natural S isotope of DMDS (detected at m/z 95) to the signal for heptanal at m/z 97. This was corrected for here and in Fig. 2. d The measured natural isotope values listed here are taken from the data of Experiment 1 by way of example. The theoretical (expected) isotopic ratios were calculated using IsoPro 3.1 open source software. Note that the value listed for DMDS was corrected for the contribution from heptanal, and vice versa. Note, also, that the accuracy of the measured values diminishes for compounds present at low concentrations (i.e. low signal intensities). Although the measured natural isotopic ratio can help to confirm the elemental composition of a detected compound, it does not allow discrimination between isomeric species. b
The compound DMDS (cf. Table 1), predominantly detected at m/z 95, makes a minor contribution to the signal at m/z 97 from its naturally occurring 34S isotope. This signal interferes with that of heptanal, which is also detected at m/z 97. The contribution of this isotope to the signal at m/z 97 equates to ~8.9% of the m/z 95 signal intensity (natural isotopic ratio of 34S to 32S is 0.042:0.950, with two S-atoms present in DMDS). The signal at m/z 97 was therefore corrected for DMDS by subtracting its expected value, resulting in a net signal intensity for hexanal. The hexanal signal plotted in Fig. 2 has been corrected for this interference.
Results and discussion Tentative compound identification
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In the present experiments, VOCs which clearly exhibited changes as a result of light exposure in comparison to darkness are listed in Table 1. These VOCs were tentatively identified based on published reports from compounds previously observed in photooxidised milk, their typical m/z fragmentation patterns in PTR-MS[20] and the relative intensities of their natural 13C and 34S isotopologues in comparison to the expected (theoretical) values, as indicated in Table 1. All others m/z signals in the measured range did not show prominent features. Compared to lists reported in the GC literature, the presented experiments observed only a limited number of VOCs undergoing significant change in intensity as a consequence of exposure to light. This might be due to the relatively low-power fluorescent tubes used in the present experiments (15-W fluorescent tubes, with integral irradiance intensities of 8.32 and 10.64 W m 2 for two and four lamps, respectively; as previously described) similar to the light intensity in commercial refrigerator displays and to the
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comparably brief light exposure period (cf. typically several days in other studies). Furthermore, a combination of dynamic flushing of the sample headspace, which may have prevented sufficient enrichment of those VOCs present in only trace amounts in the milk, and the PTR-MS operating conditions mean, that protonated compounds at m/z higher than m/z 107 may have been below the detection threshold. Experiment 1: Light-exposed versus unexposed semi-skimmed milk The dynamic release profiles of selected VOCs from semi-skimmed milk either exposed to light (semi-skimmed, light) or kept in the dark (semi-skimmed, dark) are compared in Fig. 2. An initial period in the dark of ~1 h was followed by 19-h light exposure and then a further 1 h in the dark. The prominent feature of the VOC profiles is the clear response to light exposure and the differing rates of change and length of the lag phase for different compounds. Upon exposure to light the formation of methanethiol (m/z 49) was very rapid. It was the first detected compound to change, and it reached a maximum headspace concentration within 2 h of light exposure, followed by depletion over the remainder of the experiment and an abrupt drop after switching from light to dark. Methanethiol has been reported to be formed from methional, which is a light-induced product of methionine.[7] The rapid formation of methanethiol suggests that the degradation of methional is also very fast and may explain why methional was not detected explicitly in this or previous studies.[8,13,18,21,22] In this experiment, hexanal (m/z 83) was clearly produced as a result of light exposure with its intensity decreasing after switching from light to dark. Hexanal has been reported to be the main oxidation product from the fat constituents of milk[23]
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Photooxidation-induced volatiles in ESL milk
Figure 2. Release profiles of selected VOCs from semi-skimmed milk either held in the dark or exposed to light. Note that the plot of heptanal has 34 been corrected for the contribution from the S isotope of DMDS (down-corrected by 8.9% of the signal at m/z 95; see also Table 1). The brief spike in both samples in the plot of heptanal between 13 and 15 h was due to a temporary humidity effect and does not represent an actual change in heptanal concentration.
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diacetyl (m/z 87) appeared to be generated during light exposure but was also observed to increase to a lesser degree in the sample under darkness, indicating that the release of this compound is not driven solely by photooxidation, although the very low and noise-prone signal intensity meant that definitive conclusions could not be drawn. Experiment 2: Continuous light-exposure to semi-skimmed and whole milk In the second experiment semi-skimmed and whole milk samples after an initial period of ~1 h in the dark were continuously exposed to light for ~15 h followed by a further 4 h in the dark (Fig. 3). The onset of the VOC increase correlated directly to switching on the light, after which each of the volatiles showed a different evolution during the extended period of light exposure. Methanethiol and pentanal/octanal/nonanal/1-octen3-ol displayed a steep increase, followed by a plateau and then an almost equally steep decrease, the latter occurring during light exposure. Acetaldehyde showed a constant increase after a steep onset and a decrease abruptly after ~11-h light exposure. This decrease was not seen in experiment 1 where a similar sample was exposed to four lamps for 19 h. The reason for the abrupt decrease in experiment 2 but not in experiment 1 remains unclear, although batch-to-batch variations in milk samples might play a role.
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and is produced from the oxidation of n-6 polyunsaturated fatty acids (e.g. linoleic acid).[24] Hexanal is typically used as a quality marker for fresh milk[8,24] as, along with other odour-active compounds including pentanal and DMDS, it is associated with the unpleasant cardboard-like or metallic-like flavour note of milk.[10] A similar release profile is seen for the other oxidation-generated aldehydes and alcohols such as acetaldehyde (m/z 45), pentanal/ octanal/nonanal/1-octen-3-ol (all measured at m/z 69), heptanal (m/z 97, with a minor contribution from the 34S isotope of DMDS that has been corrected for in Fig. 2) and benzaldehyde (m/z 107), though the lag phase for acetaldehyde and the aldehydes at m/z 69 was shorter than for hexanal, suggesting that the reaction kinetics are different.[14] DMDS (m/z 95) showed a similar behaviour to hexanal. The initially high concentration of DMDS, in the dark, and its reduction prior to light exposure is presumably a by-product of previous oxidation of the sample. Acetone/ propanal (m/z 59) displays divergent behaviour from the other compounds (Fig. 2). After initially increasing steadily in both light-exposed and non-exposed samples and reaching a maximum concentration at ~4 h, a steady decline in concentration was observed, indicating the presence of acetone/propanal in the milk liquid phase that is gradually stripped into and then from the headspace during flushing. Furthermore, acetone/ propanal reached a higher concentration in the sample held in the dark, suggesting that it is a precursor for light-induced products and it is depleted during light exposure. The compound
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Figure 3. Release profiles of selected VOCs from semi-skimmed and whole milk after ~1h in the dark followed by continuous expose to light for ~15 h followed by a further 4 h in the dark.
The fat content in the milk affected the pattern of VOC release. Acetaldehyde, hexanal and acetone/propanal, for instance, were released at higher concentrations from semi-skimmed milk than from whole milk, while the intensity of methanethiol in both samples was similar. In contrast, pentanal/octanal/nonanal/1octen-3-ol had marginally higher intensities in whole milk than in semi-skimmed milk during light exposure. This data showed again the effect of fat content on volatile products, as previously reported for headspace solid-phase micro-extraction analyses where the concentrations of pentanal increased as the fat content of the milk increased from skim to 1%, 2% or whole milk in samples exposed to fluorescent light for 8 h at 4 °C.[6] Diacetyl
was released at similar concentrations in both milk samples and decreased prior to the light being turned off, suggesting that this compound became depleted from the liquid phase (data not shown). DMDS was unique in that it appeared to be generated in semi-skimmed but not whole milk during light exposure Experiment 3: Alternating light/dark exposure to semi-skimmed and whole milk A further experiment simultaneously exposed semi-skimmed and whole milk to alternating periods of light and dark of ~1 h each, for 7 h in total (Fig. 4). VOCs in the headspace showed a pattern
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Figure 4. Release profiles of selected VOCs from semi-skimmed and whole milk under alternating light/dark exposure.
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Photooxidation-induced volatiles in ESL milk of increase and decrease consistent with the light/dark period. Methanethiol and to a lesser extent DMDS appeared most responsive to the light/dark cycle. In the dark, both compounds showed a consistent decrease in concentration followed by an immediate increase on exposure to light. For other compounds such as acetaldehyde and pentanal/octanal/nonanal/1-octen-3ol, there appeared to be a plateau or reduction in the rate of increase during the dark period. This result suggests that the formation of the aldehydes is to a degree self-catalytic whereas the sulphur compounds require light to catalyse formation. The VOCs also exhibited general differences in concentration in relation to fat content. With the exception of acetaldehyde, for which the overall signal intensity from semi-skimmed milk was similar to that of whole milk, the other VOCs were less intensely released from the semi-skimmed milk than from whole milk. There were similarities and differences with the VOC release in experiment 2, but a direct comparison could not be made because of the shorter (1 h) and more intense (four lamps) light exposure in experiment 3.
Conclusions
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This work was financially supported by the Royal Society of New Zealand and the German Federal Office for Agriculture and Food (BLE). The authors would like to thank Landry Dawak Domché for assistance with the experimental setup and preliminary tests.
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This technical note highlights the applicability of PTR-MS for online monitoring of changes in the volatile composition of milk as a result of photooxidation and demonstrates the suitability of this technique to characterise such rapid processes. The chemical identification of VOCs was limited, and nominally isobaric compounds could not be resolved due to the low mass resolving power of PTR-MS. Nevertheless, the advantage of this technique over GC-MS lies in its ability to measure the dynamics of release and to detect the occurrence of compounds that are transitory, which may provide new insights into the kinetics of production and release of light-induced products, particularly in relation to off-flavour formation. Exposure to light resulted in the generation of several compounds. These included methanethiol, which increased rapidly after the transition from dark to light conditions and similarly decreased rapidly once conditions again became dark. Other compounds detected included a series of aldehydes including hexanal, acetaldehyde, pentanal/octanal/nonanal and heptanal. These are common oxidation products derived from the fat constituents of milk, and some have previously been linked to the light-affected flavour of milk. DMDS was also detected and was presumably a by-product from the previous oxidation of the milk samples. The signal related to acetone/propanal did not increase on exposure to light but rather decreased over the course of the experiments, indicating the initial presence of acetone/propanal in the milk liquid phase that gradually depleted over the course of the measurements. Diacetyl appeared to be generated during light exposure but was also observed to increase to a lesser degree in the sample under darkness, indicating that the release of this compound is not driven solely by photooxidation. The milk fat content also influenced the signal intensity of compounds detected, but this influence was dependent on the compound with some compounds having a higher intensity in the low fat milk compared to the higher fat milk, whereas others showed the opposite trend. The knowledge produced in this study can be useful in developing new strategies for processing, handling and storage to ensure stability in the volatile (flavour) composition of milk and milk products.
Acknowledgements
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Copyright © 2014 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2014, 49, 952–958
Revised: 30 June 2014
Accepted: 2 July 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3430
Monitoring photooxidation-induced dynamic changes in the volatile composition of extended shelf life bovine milk by PTR-MS† Jonathan Beauchamp,a* Erika Zardin,a,b Patrick Silcockc and Phil J Bremerc Exposure of milk to light leads to photooxidation and the development of off-flavours. To follow these reactions, semi-skimmed (1.5% fat) and whole (3.8% fat) extended shelf life (ESL) bovine milk samples were exposed to fluorescent light for up to 20 h at room temperature, and the volatiles in the samples’ headspace were measured in real time using proton-transfer-reaction mass spectrometry (PTR-MS). Compounds tentatively identified as methanethiol, acetone/propanal, pentanal/octanal/nonanal/1octen-3-ol, hexanal, diacetyl, dimethyl disulphide, heptanal and benzaldehyde displayed dynamic release profiles relating to the changes occurring in milk upon exposure to light. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: PTR-MS; ESL milk; photooxidation; VOCs; off-flavour
Introduction
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Milk is susceptible to oxidation, particularly photooxidation. It has been known for almost a century that exposure to light can have a detrimental impact on the flavour of liquid milk,[1] with ultraviolet (λ = 180–380 nm) and visible (λ = 380–780 nm) light both playing a major role in the development of light-induced offflavours.[2] Unstable primary compounds are formed when absorbed radiation energy is transferred to unsaturated fatty acids, proteins and amino acids, resulting in the production of secondary volatile products such as carbonyl and sulphur compounds, e.g. aldehydes, ketones, alkenes or dimethyl disulphide (DMDS).[3] Continuous exposure of milk to natural or artificial light (particularly at λ = 420–520 nm) can induce chemical reactions that appear as flavour defects upon consumption.[2] Two distinctive offflavours in milk that are attributed to light irradiation are described as sunlight and cardboard-like or metallic flavours.[3,4] A sunlight offflavour imparts a burnt, oxidised odour in milk after 2- to 3-day light exposure and has been tentatively attributed to the formation of DMDS and methional that are derived from methionine.[5–7] The cardboard-like or metallic off-flavour occurs after prolonged intense light exposure due to the formation of secondary lipid oxidation products including carbonyl compounds (pentanal, hexanal, heptanal and ketones), alcohols and hydrocarbons.[8–10] The flavour quality of retail milk generally does not deteriorate significantly on exposure to the limited light conditions found in commercial display cabinets.[11] The analysis of light-induced flavour compounds in milk has commonly been carried out using gas chromatography–mass spectrometry (GC-MS) following diverse methods of headspace sampling or derivatisation.[12–14] Despite the strengths of this technique, GC-MS has a limited analytical time resolution due to the necessary sample workup and intrinsic time for analyte separation in the GC column, and this approach is therefore less suitable for characterising fast processes. Proton-transfer-reaction mass spectrometry (PTR-MS) is a chemical ionisation technique that enables real-time analysis of gas-phase volatile organic
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compounds (VOCs).[15] It has been used in diverse studies of food volatiles including investigating milk and milk-related products.[16,17] In the present investigation, PTR-MS was applied to monitor the release of volatile light-induced oxidation products of extended shelf life (ESL) milk. Dynamic headspace analyses were made in parallel on milk samples of different fat content and/or under different light-exposure conditions to demonstrate the capability of PTR-MS to continuously and sensitively detect volatile oxidation products in real time and provide indications on the dynamics of such processes.
Experimental Milk Semi-skimmed (1.5% fat) and whole (3.8% fat) extended shelf-life (ESL) bovine milks (500 ml, Molkerei Weihenstephan GmbH & Co. KG, Freising, Germany) were obtained from a local supermarket and stored at 4.7 ± 0.1 °C. The fat contents of the two milk types were taken as stated on the milk carton packaging and were not additionally determined experimentally. Samples were analysed at least 8 days before the best-before date to minimise interferences from autooxidation or microbial spoilage. The milk
* Correspondence to: Jonathan Beauchamp, Department of Sensory Analytics, Fraunhofer Institute for Process Engineering and Packaging IVV, Freising, Germany. E-mail: [email protected] †
This article is part of the Journal of Mass Spectrometry special issue entitled “3rd MS Food Day” edited by Gianluca Giorgi.
a Department of Sensory Analytics, Fraunhofer Institute for Process Engineering and Packaging IVV, Freising, Germany b Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich-AlexanderUniversität Erlangen-Nürnberg, Erlangen, Germany c Department of Food Science, University of Otago, Dunedin, New Zealand
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Photooxidation-induced volatiles in ESL milk was held in the unopened opaque carton at room temperature for at least 1 h before experimentation.
(~120–150 Td), and tentative identifications can be made based on these and on previously reported GC studies on milk volatiles.
Light
Experiment
Using a CAS 140CT Array Spectrometer (Instrument Systems Optische Messtechnik GmbH, Munich, Germany), the integral irradiance intensity of two or four 15-W fluorescent tubes (TL-D 15W/840, Philips) positioned 20 cm from the samples (Fig. 1) was measured as 8.32 or 10.64 W m 2, respectively. The spectral emission of these fluorescent tubes encompassed wavelengths between 405 and 640 nm, with peaks of maximum emission at 430, 550 and 610 nm (data not shown). Both the integral irradiance intensity and the wavelengths of the emission maxima of this lighting system are sufficient to degrade riboflavin, chlorins and porphyrins and are thus likely to induce oxidised/ sunlight off-flavours, as described in the literature.[18]
The headspace (150 ml) above the milk (400 ml) held in 500-ml modified impinger (impinger stem removed) glass bottles (Schott AG, Mainz, Germany) was sampled via connections in the lid that enabled dynamic flushing through one and headspace analysis via the other. Until exposure to light, the bottles were wrapped in aluminium foil, and milk samples were poured into the bottles under darkened laboratory conditions. A polytetrafluoroethylenecoated magnetic stir bar (100 rpm) stirred the milk throughout the measurements. The experimental setup was held at 27 °C in a ventilated oven (UFE 500, Memmert GmbH+ Co. KG, Schwabach, Germany), which acted as an enclosed light-exposure chamber. A gas calibration unit (advanced model, GCU-a; IONICON Analytik GmbH) provided a constant flow (200 ml min 1) of dry VOC-free air to the line upstream of the sampling bottles (Fig. 1) which was subsequently equally split between the sampling bottles. An external solenoid valve (model M443W2DTS-LT, Teqcom Industries, Santa Ana, CA, USA) was switched simultaneously with the PTR-MS inlet-selection valve to configure the flows such that both bottles were flushed regardless of the sampling configuration (either inlet 1 or 2 active). The PTR-MS was supplied with a flow of ~50 ml min 1 from inlets (1/8″ outer diameter, 0.04″ inner ™ diameter) of Silcosteel (Restek GmbH, Bad Homburg, Germany) held at 80 °C and connected to the sample bottles via a short length (~20 cm) of ¼″ outer diameter perfluoroalkoxy tubing. In experiment 1, a direct comparison was made between semiskimmed milk exposed to four lamps for 19 h and foil-wrapped semi-skimmed milk (dark). Experiment 2 directly compared semiskimmed milk with whole milk exposed to light (two lamps) for 16 h. In experiment 3, semi-skimmed and whole milk samples were repeatedly exposed to light (four lamps) and then darkness at ~1-h intervals to investigate how rapidly the light-induced changes occurred.
PTR-MS The VOCs in the headspace of the milk samples were measured using a high sensitivity proton-transfer-reaction mass spectrometer (hs-PTR-MS; IONICON Analytik GmbH, Innsbruck, Austria). The reaction chamber was held at an electric field strength to buffer gas number density ratio (E/N) of 132 Td (1 Td (Townsend) = 10 17 V cm2). The drift tube operating conditions were 600 V, 2.2 mbar and 60 °C. The PTR-MS had a custom-made dual inlet system, which allowed two samples to be measured sequentially in a continuous manner via a solenoid valve housed within the PTR-MS that switched between inlets typically every 9 min. As the mass resolving power of the PTR-MS instrument is limited to one atomic mass unit (1 amu), VOCs that form product ions at the same nominal m/z cannot be resolved. However, the fragmentation patterns of many (common) VOCs are well characterised under standard PTR-MS operating conditions
Analysis
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Figure 1. Experimental setup of dynamic headspace analysis of milk volatiles by PTR-MS during light-induced oxidation. Flows were configured so that the headspace of both sample bottles was always flushed while one was being actively sampled. The figure shows active sampling via PTR-MS inlet 1 (solid arrows) and flushing sample 2 via the overflow (dashed arrows). Either two or four lamps were on during light exposure, depending on the experiment.
The PTR-MS instrument was operated in mass scan mode, scanning all ions in the range m/z 20–130 at an integration time of 500 ms per m/z (cycle time: 55 s). Based on 10 cycles measured per sample, switching between samples occurred approximately every 9 min. The signal intensities, in counts-per-second (cps), were processed by first correcting each signal for its m/z-specific transmission efficiency inherent to PTR-MS. The data were then normalised to the primary ion signal (m/z 21 and 37) and drift tube pressure to produce normalised count-rates (n-cps), which are directly proportional to concentration.[19] In the data post-processing, the first two cycles after switching from one sample to the other were discarded to eliminate potential carry-over effects. The remaining eight cycles were averaged (generation of mean values plus standard deviation), which were used to plot the figures and show the release profiles. Since only relative signal intensities (in n-cps) are described in the paper, a quantitative limit of detection (LOD) for the detected compounds was not calculated. Furthermore, the observed changes in the signal intensities in relation to light/dark conditions (or milk of different fat content) clearly indicate detection of the tentatively identified compounds, thus demonstrating that the PTR-MS instrument was operating above the LOD at that m/z.
J. Beauchamp et al. Table 1. Tentative identifications of m/z detected during light-induced oxidation m/z
Tentative identification
Elemental composition
1
MW [g mol ]
Compound class
Natural isotope measured (theoretical)d [%] 13
45 49 59 69a
83a 87 95 97a 107
Acetaldehyde Methanethiol Propanone (acetone)b Propanalb Pentanalb Octanalb 1-Octen-3-olb Nonanalb Hexanal 2,3-Butanedione (diacetyl) Dimethyl disulphide (DMDS) Heptanal Benzaldehyde
C2H4O CH4S C3H6O
44.05 48.11 58.08
C5H10O C8H16O
86.13 128.21
C9H18O C6H12O C4H6O2 C2H6S2 C7H14O C7H6O
142.24 100.16 86.09 94.20 114.19 106.12
Aldehyde Thiol Ketone Aldehyde Aldehyde Aldehyde Alcohol Aldehyde Aldehyde Ketone Disulphide Aldehyde Aldehyde
C
1.9 (2.3) 1.6 (2.0) 3.4 (3.4) 5.6 (5.7)
6.7 (6.8) 6.2 (4.6) 5.3 (3.9) 9.1 (8.0)c 6.9 (7.9)
34
Ref.
S
N/A 6.5 (4.5) N/A N/A
N/A N/A 9.1 (8.9)c N/A N/A
13, 16 12, 21 22, 23 24, 25 8, 13, 18, 23, 26 12 24, 27 28, 29 10, 26 28 13 8, 23, 26, 28 30
a
Predominant fragment ion in PTR-MS. Individual compounds not separable in PTR-MS. 34 c There is a contribution from the natural S isotope of DMDS (detected at m/z 95) to the signal for heptanal at m/z 97. This was corrected for here and in Fig. 2. d The measured natural isotope values listed here are taken from the data of Experiment 1 by way of example. The theoretical (expected) isotopic ratios were calculated using IsoPro 3.1 open source software. Note that the value listed for DMDS was corrected for the contribution from heptanal, and vice versa. Note, also, that the accuracy of the measured values diminishes for compounds present at low concentrations (i.e. low signal intensities). Although the measured natural isotopic ratio can help to confirm the elemental composition of a detected compound, it does not allow discrimination between isomeric species. b
The compound DMDS (cf. Table 1), predominantly detected at m/z 95, makes a minor contribution to the signal at m/z 97 from its naturally occurring 34S isotope. This signal interferes with that of heptanal, which is also detected at m/z 97. The contribution of this isotope to the signal at m/z 97 equates to ~8.9% of the m/z 95 signal intensity (natural isotopic ratio of 34S to 32S is 0.042:0.950, with two S-atoms present in DMDS). The signal at m/z 97 was therefore corrected for DMDS by subtracting its expected value, resulting in a net signal intensity for hexanal. The hexanal signal plotted in Fig. 2 has been corrected for this interference.
Results and discussion Tentative compound identification
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In the present experiments, VOCs which clearly exhibited changes as a result of light exposure in comparison to darkness are listed in Table 1. These VOCs were tentatively identified based on published reports from compounds previously observed in photooxidised milk, their typical m/z fragmentation patterns in PTR-MS[20] and the relative intensities of their natural 13C and 34S isotopologues in comparison to the expected (theoretical) values, as indicated in Table 1. All others m/z signals in the measured range did not show prominent features. Compared to lists reported in the GC literature, the presented experiments observed only a limited number of VOCs undergoing significant change in intensity as a consequence of exposure to light. This might be due to the relatively low-power fluorescent tubes used in the present experiments (15-W fluorescent tubes, with integral irradiance intensities of 8.32 and 10.64 W m 2 for two and four lamps, respectively; as previously described) similar to the light intensity in commercial refrigerator displays and to the
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comparably brief light exposure period (cf. typically several days in other studies). Furthermore, a combination of dynamic flushing of the sample headspace, which may have prevented sufficient enrichment of those VOCs present in only trace amounts in the milk, and the PTR-MS operating conditions mean, that protonated compounds at m/z higher than m/z 107 may have been below the detection threshold. Experiment 1: Light-exposed versus unexposed semi-skimmed milk The dynamic release profiles of selected VOCs from semi-skimmed milk either exposed to light (semi-skimmed, light) or kept in the dark (semi-skimmed, dark) are compared in Fig. 2. An initial period in the dark of ~1 h was followed by 19-h light exposure and then a further 1 h in the dark. The prominent feature of the VOC profiles is the clear response to light exposure and the differing rates of change and length of the lag phase for different compounds. Upon exposure to light the formation of methanethiol (m/z 49) was very rapid. It was the first detected compound to change, and it reached a maximum headspace concentration within 2 h of light exposure, followed by depletion over the remainder of the experiment and an abrupt drop after switching from light to dark. Methanethiol has been reported to be formed from methional, which is a light-induced product of methionine.[7] The rapid formation of methanethiol suggests that the degradation of methional is also very fast and may explain why methional was not detected explicitly in this or previous studies.[8,13,18,21,22] In this experiment, hexanal (m/z 83) was clearly produced as a result of light exposure with its intensity decreasing after switching from light to dark. Hexanal has been reported to be the main oxidation product from the fat constituents of milk[23]
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Photooxidation-induced volatiles in ESL milk
Figure 2. Release profiles of selected VOCs from semi-skimmed milk either held in the dark or exposed to light. Note that the plot of heptanal has 34 been corrected for the contribution from the S isotope of DMDS (down-corrected by 8.9% of the signal at m/z 95; see also Table 1). The brief spike in both samples in the plot of heptanal between 13 and 15 h was due to a temporary humidity effect and does not represent an actual change in heptanal concentration.
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diacetyl (m/z 87) appeared to be generated during light exposure but was also observed to increase to a lesser degree in the sample under darkness, indicating that the release of this compound is not driven solely by photooxidation, although the very low and noise-prone signal intensity meant that definitive conclusions could not be drawn. Experiment 2: Continuous light-exposure to semi-skimmed and whole milk In the second experiment semi-skimmed and whole milk samples after an initial period of ~1 h in the dark were continuously exposed to light for ~15 h followed by a further 4 h in the dark (Fig. 3). The onset of the VOC increase correlated directly to switching on the light, after which each of the volatiles showed a different evolution during the extended period of light exposure. Methanethiol and pentanal/octanal/nonanal/1-octen3-ol displayed a steep increase, followed by a plateau and then an almost equally steep decrease, the latter occurring during light exposure. Acetaldehyde showed a constant increase after a steep onset and a decrease abruptly after ~11-h light exposure. This decrease was not seen in experiment 1 where a similar sample was exposed to four lamps for 19 h. The reason for the abrupt decrease in experiment 2 but not in experiment 1 remains unclear, although batch-to-batch variations in milk samples might play a role.
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and is produced from the oxidation of n-6 polyunsaturated fatty acids (e.g. linoleic acid).[24] Hexanal is typically used as a quality marker for fresh milk[8,24] as, along with other odour-active compounds including pentanal and DMDS, it is associated with the unpleasant cardboard-like or metallic-like flavour note of milk.[10] A similar release profile is seen for the other oxidation-generated aldehydes and alcohols such as acetaldehyde (m/z 45), pentanal/ octanal/nonanal/1-octen-3-ol (all measured at m/z 69), heptanal (m/z 97, with a minor contribution from the 34S isotope of DMDS that has been corrected for in Fig. 2) and benzaldehyde (m/z 107), though the lag phase for acetaldehyde and the aldehydes at m/z 69 was shorter than for hexanal, suggesting that the reaction kinetics are different.[14] DMDS (m/z 95) showed a similar behaviour to hexanal. The initially high concentration of DMDS, in the dark, and its reduction prior to light exposure is presumably a by-product of previous oxidation of the sample. Acetone/ propanal (m/z 59) displays divergent behaviour from the other compounds (Fig. 2). After initially increasing steadily in both light-exposed and non-exposed samples and reaching a maximum concentration at ~4 h, a steady decline in concentration was observed, indicating the presence of acetone/propanal in the milk liquid phase that is gradually stripped into and then from the headspace during flushing. Furthermore, acetone/ propanal reached a higher concentration in the sample held in the dark, suggesting that it is a precursor for light-induced products and it is depleted during light exposure. The compound
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Figure 3. Release profiles of selected VOCs from semi-skimmed and whole milk after ~1h in the dark followed by continuous expose to light for ~15 h followed by a further 4 h in the dark.
The fat content in the milk affected the pattern of VOC release. Acetaldehyde, hexanal and acetone/propanal, for instance, were released at higher concentrations from semi-skimmed milk than from whole milk, while the intensity of methanethiol in both samples was similar. In contrast, pentanal/octanal/nonanal/1octen-3-ol had marginally higher intensities in whole milk than in semi-skimmed milk during light exposure. This data showed again the effect of fat content on volatile products, as previously reported for headspace solid-phase micro-extraction analyses where the concentrations of pentanal increased as the fat content of the milk increased from skim to 1%, 2% or whole milk in samples exposed to fluorescent light for 8 h at 4 °C.[6] Diacetyl
was released at similar concentrations in both milk samples and decreased prior to the light being turned off, suggesting that this compound became depleted from the liquid phase (data not shown). DMDS was unique in that it appeared to be generated in semi-skimmed but not whole milk during light exposure Experiment 3: Alternating light/dark exposure to semi-skimmed and whole milk A further experiment simultaneously exposed semi-skimmed and whole milk to alternating periods of light and dark of ~1 h each, for 7 h in total (Fig. 4). VOCs in the headspace showed a pattern
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Figure 4. Release profiles of selected VOCs from semi-skimmed and whole milk under alternating light/dark exposure.
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Photooxidation-induced volatiles in ESL milk of increase and decrease consistent with the light/dark period. Methanethiol and to a lesser extent DMDS appeared most responsive to the light/dark cycle. In the dark, both compounds showed a consistent decrease in concentration followed by an immediate increase on exposure to light. For other compounds such as acetaldehyde and pentanal/octanal/nonanal/1-octen-3ol, there appeared to be a plateau or reduction in the rate of increase during the dark period. This result suggests that the formation of the aldehydes is to a degree self-catalytic whereas the sulphur compounds require light to catalyse formation. The VOCs also exhibited general differences in concentration in relation to fat content. With the exception of acetaldehyde, for which the overall signal intensity from semi-skimmed milk was similar to that of whole milk, the other VOCs were less intensely released from the semi-skimmed milk than from whole milk. There were similarities and differences with the VOC release in experiment 2, but a direct comparison could not be made because of the shorter (1 h) and more intense (four lamps) light exposure in experiment 3.
Conclusions
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This work was financially supported by the Royal Society of New Zealand and the German Federal Office for Agriculture and Food (BLE). The authors would like to thank Landry Dawak Domché for assistance with the experimental setup and preliminary tests.
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This technical note highlights the applicability of PTR-MS for online monitoring of changes in the volatile composition of milk as a result of photooxidation and demonstrates the suitability of this technique to characterise such rapid processes. The chemical identification of VOCs was limited, and nominally isobaric compounds could not be resolved due to the low mass resolving power of PTR-MS. Nevertheless, the advantage of this technique over GC-MS lies in its ability to measure the dynamics of release and to detect the occurrence of compounds that are transitory, which may provide new insights into the kinetics of production and release of light-induced products, particularly in relation to off-flavour formation. Exposure to light resulted in the generation of several compounds. These included methanethiol, which increased rapidly after the transition from dark to light conditions and similarly decreased rapidly once conditions again became dark. Other compounds detected included a series of aldehydes including hexanal, acetaldehyde, pentanal/octanal/nonanal and heptanal. These are common oxidation products derived from the fat constituents of milk, and some have previously been linked to the light-affected flavour of milk. DMDS was also detected and was presumably a by-product from the previous oxidation of the milk samples. The signal related to acetone/propanal did not increase on exposure to light but rather decreased over the course of the experiments, indicating the initial presence of acetone/propanal in the milk liquid phase that gradually depleted over the course of the measurements. Diacetyl appeared to be generated during light exposure but was also observed to increase to a lesser degree in the sample under darkness, indicating that the release of this compound is not driven solely by photooxidation. The milk fat content also influenced the signal intensity of compounds detected, but this influence was dependent on the compound with some compounds having a higher intensity in the low fat milk compared to the higher fat milk, whereas others showed the opposite trend. The knowledge produced in this study can be useful in developing new strategies for processing, handling and storage to ensure stability in the volatile (flavour) composition of milk and milk products.
Acknowledgements
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