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Article

The Use of Proton-Transfer-Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) to Determine the Volatile Organic Compounds (VOCs) Produced by Different Lactic Acid Bacterial Strains Growing in Defined Media

by
Sarathadevi Rajendran
1,2,3,
Iuliia Khomenko
2,
Patrick Silcock
1,
Emanuela Betta
2,
Franco Biasioli
2 and
Phil Bremer
1,*
1
Department of Food Science, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand
2
Sensory Quality Unit, Research and Innovation Centre, Fondazione Edmund Mach, 38098 Trento, Italy
3
Department of Agricultural Chemistry, Faculty of Agriculture, University of Jaffna, Kilinochchi 44000, Sri Lanka
*
Author to whom correspondence should be addressed.
Appl. Microbiol. 2025, 5(1), 33; https://doi.org/10.3390/applmicrobiol5010033
Submission received: 21 February 2025 / Revised: 11 March 2025 / Accepted: 14 March 2025 / Published: 20 March 2025
Browse Figures
Versions Notes

Abstract

:
Lactic acid bacteria (LAB) fermentation has been claimed as an effective way of modifying the sensory properties of plant-based foods. However, not much has been published on the influence of different LAB strains on the flavour of the volatile organic compounds (VOCs) produced. Using a defined medium (DM) and proton-transfer-reaction time-of-flight mass spectrometry (PTR-ToF-MS), we assessed the VOCs produced by seven LAB strains, Levilactobacillus brevis WLP672 (LB672), Lactobacillus delbrueckii WLP677 (LD677), Pediococcus damnosus WLP661 (PD661), Lactiplantibacillus plantarum LP100 (LP100), Pediococcus pentosaceus PP100 (PP100), Pediococcus damnosus 5733 (PD5733), and Lentilactobacillus buchneri 5335 (LU5335), at three time points during fermentation (0, 7, and 14 days) at either 25 or 35 °C. Significant variations in VOC production were observed among LAB strains, growing in the same DM composition at either 25 °C or 35 °C. Specifically, the concentration of m/z 87.043 (t.i. diacetyl) was significantly (p < 0.05) higher at 7 days of fermentation at 35 °C by LP100, followed by PP100 at 35 °C and PD661 at 25 °C compared to the other strains at either 25 or 35 °C. The concentration of m/z 115.112 (t.i. 2-heptanone) was significantly (p < 0.05) higher at 7 days of fermentation at either 25 or 35 °C by LP100 compared to the other strains at all temperature and time points. The concentration of m/z 49.011 (t.i. methanethiol) was significantly (p < 0.05) higher after 7 days of fermentation at 35 °C by LB672 compared to the other strains at either 25 or 35 °C. The concentration of m/z 71.085 (t.i. 3-methyl butanol) was significantly (p < 0.05) higher after 7 days of fermentation at either 25 or 35 °C by PD661, LU5335, or PD5733 compared to the other strains studied. A notable increase in specific VOC concentrations was observed at 35 °C compared to 25 °C. This research demonstrates that LAB strains generate distinct VOC profiles in a DM based on strains and fermentation conditions. Therefore, this knowledge provides a basis for controlling and enhancing flavour in plant-based fermentations.

Graphical Abstract

1. Introduction

Consumers are seeking to decrease their consumption of animal-based foods due to concerns about the impact of its production on the environment, the reported adverse health effects of high meat diets, and animal welfare implications, which in turn is driving demand for plant-based meat and dairy analogues [1,2,3]. However, the overall flavour of these analogues still falls short of their traditional meat or dairy counterparts [4,5,6,7]. Flavour perception is complex, with volatile organic compounds (VOCs) present in the food either singularly or together playing a significant role in how consumers perceive the flavour associated with food [8,9,10,11].
Plant-based substrates may contain VOCs associated with meat or dairy flavours; however, directly obtaining these VOC is challenging as their concentration and recovery rate is low [12]. Fermenting plant-based substrates using lactic acid bacteria (LAB) has been proposed to be an efficient way to produce higher concentrations of the desired VOCs [13,14,15]. During growth on plant substrates, LAB produce VOCs as secondary metabolites [16]. However, due to the complexity of the compounds that are present within plant-based systems, it is challenging to relate the impact of substrate, fermentation conditions or LAB strain to the production of specific VOCs.
LAB require a relatively rich cultivation media for growth, as they are auxotrophic to many vitamins and amino acids [17,18]. However, the use of poorly defined cultivation medium means that it can be difficult to identify which VOCs are being produced in response to substrates being metabolised [19]. To overcome this challenge, a well-defined minimal medium (DM) can be used. While many researchers have investigated the growth of LAB in a DM [20,21,22,23,24], only a few studies have examined the VOCs produced by LAB in DM [25,26,27]. Further, to maximise the formation of desirable VOCs in a natural or DM, a better understanding of the metabolic pathways present in different LAB strains and the VOC they produce is required [28,29].
While gas chromatography–mass spectrometry (GC-MS) remains a gold standard for VOC analysis, it is limited by its inability to perform real-time VOC analysis. As an alternative, proton-transfer-reaction time-of-flight mass spectrometry (PTR-ToF-MS) offers highly sensitive, real-time VOC detection in a direct and non-invasive manner [30,31]. However, as PTR-ToF-MS can only detect exact protonated masses, GC-MS and fastGC-PTR-MS can be used in conjunction to support VOC identification [32,33,34].
Therefore, the current study determined the VOCs associated with seven LAB strains growing in a DM, using PTR-ToF-MS, HS-SPME-GC-MS, and fastGC-PTR-ToF-MS.

2. Materials and Methods

2.1. LAB Strains

Seven commercially available LAB strains, namely Levilactobacillus brevis WLP672 (thereafter referred to as LB672), Lactobacillus delbrueckii WLP677 (LD677), Pediococcus damnosus WLP661 (PD661), Lactiplantibacillus plantarum LP100 (LP100), Pediococcus pentosaceus PP100 (PP 100), Pediococcus damnosus 5733 (PD5733), and Lentilactobacillus buchneri 5335 (LU5335), were used (Table 1). These strains were deemed appropriate for this study and for the commercial production of VOCs as they are all readily available, of food-grade standard, grown in the temperature range of 20 to 35 °C [35], and relatively nutritionally robust. The inoculums used for the fermentation trials were prepared following the procedure described in our previous experiments [27,36].

2.2. Medium Composition

The DM was developed based on the findings of our previous research [27,37]. The DM contained D-glucose (20 g/L), peptone (enzymatic protein digest) (5 g/L), sodium acetate (12 g/L), mineral salts (MgSO4·7H2O (0.2 g/L), NaCl (0.01 g/L), FeSO4·7H2O (0.01 g/L), MnSO4·5H2O (0.04 g/L)), vitamins (calcium pantothenate (B5) (0.4 mg/L), nicotinic acid (B3) (0.2 mg/L), riboflavin (B2) (0.4 mg/L), thiamine HCl (B1) (0.2 mg/L)), and an amino acid mixture (0.4 g/L of each amino acid: L-leucine, L-isoleucine, L-phenylalanine, L-glutamic acid, L-aspartic acid, L-threonine, or L-methionine).

2.3. Fermentation

Before use, the DM was incubated at 25 °C for at least 3 days to ensure no bacterial growth (turbidity). Following the confirmation of the absence of contamination, 4 mL aliquots of DM were transferred into sterile headspace vials (20 mL) capped with PTFE/silicone septa (Agilent, Cernusco sul Naviglio, Italy). A 0.05 mL aliquot of each LAB cell suspension (1 × 109 CFU/mL) was subsequently inoculated into each headspace vial, which were flushed with N2 at a rate of 10 mL/min for 20 min to establish an anaerobic environment. The inoculated vials were placed in sample trays in a randomised order in an autosampler (MPS Multi-Purpose Sampler, Gerstel, Germany) and held at either 25 or 35 °C for 14 days. Eight analytical replicates were prepared from each sample, four of which were kept at either 25 °C or 35 °C. The controls included uninoculated DM. After 14 days of fermentation, growth was confirmed by measuring pH (inoLab Level 1/WTW, Weilheim, Germany) and optical density (BioPhotometer/Eppendorf, Hamburg, Germany) in a sub-sample of the fermented culture.

2.4. Determination of Volatile Organic Compounds (VOCs)

2.4.1. PTR-ToF-MS

The VOCs produced during fermentation were measured at three time points (0, 7, and 14 days of fermentation) using a PTR-ToF-MS 8000 (Ionicon Analytik GmbH, Innsbruck, Austria). PTR-ToF-MS analysis was performed as previously described by Di Pierro, Franceschi [38], with some modifications [39,40,41].

2.4.2. HS-SPME-GC-MS

HS-SPME-GC-MS measurements were included to support with the identification of compounds detected by PTR-ToF-MS. At the end of fermentation (after 14 days), the samples were removed from the PTR-ToF-MS autosampler sample tray and transferred to a GC-MS autosampler sample tray held at either 25 or 35 °C. HS-SPME-GC-MS analysis was performed using methods described in our previous publications [36,37].

2.4.3. FastGC-PTR-ToF-MS

To assist with attributing each m/z to the correct compound and determining the number of isomeric compounds contributing to each m/z, fastGC-PTR-ToF-MS was carried out on all samples at each time point after performing SHS-PTR-ToF-MS measurements using methods described in previous publications [36,37,42].

2.5. Statistical Analysis

ANOVA was used to determine which sample m/z values were significantly (p < 0.05) higher than those in blanks.
A three-way ANOVA using a general linear model was performed to identify m/z values that significantly discriminated between treatments. The main effects considered were LAB strain, fermentation temperature, and fermentation time, and all possible interactions were examined. Tukey’s HSD test (p < 0.05) was used for post hoc mean separation. All statistical analyses were conducted using SPSS (IBM SPSS Statistics, v. 29.0.0.0, Armonk NY, USA).
To encompass variations between LAB strains and fermentation VOCs, a non-targeted approach was employed, generating heatmaps of significant m/z values. These heatmaps were created using R (version 4.2.1, R Foundation for Statistical Computing, Vienna, Austria). Log-transformed average VOC concentrations (ppbV) were used to create the heatmaps.
Selected VOCs (m/z) were plotted using two-way ANOVA for the main factors of the LAB strains, their fermentation temperature, and their interaction at 7 days of fermentation using the “ggplot2”, “dplyr”, “ggpubr”, “reshape”, “ggthemes”, “multcompView”, “readr”, and “scales” packages in R.

3. Results

3.1. Physicochemical Properties After Fermentation

All LAB strains except LD677 exhibited good growth in the DM, as shown by decreased pH (due to acid production) [43] and increased OD600 (indicating cell growth) (Table 2). Significantly, the greatest pH reduction was observed in the DM inoculated with LP100, followed by LB672 and PP100 at either 25 or 35 °C. The highest OD600 values were observed in the DM inoculated with LP100, followed by LB672 and PP100 at 25 °C and LP100, followed by LB672 at 35 °C.
As LD677 did not grow sufficiently in the DM, the detected VOCs (all replicates) were excluded in the subsequent analysis.
It should be noted that there were no changes in pH and OD600 values in all uninoculated controls after fermentation, suggest that all uninoculated controls remained sterile throughout the experiment.

3.2. VOCs Produced During Fermentation

The fermentation of the DM at either 25 or 35 °C by the six (LD677 removed as no growth) LAB strains resulted in 104 m/z that were significantly (p < 0.05) higher than the baseline, after the removal of isotopologues (Table S1). The tentative identification (t.i.) of each m/z was based on its exact mass, supported by HS-SPME-GC-MS identification for 32 of the 104 (Table 3, Table 4 and Table S1), fastGC-PTR-ToF-MS identification, and/or literature data.
The concentrations of all the 104 m/z compounds’ peaks were higher after 7 days compared to 14 days of fermentation. The reduction in VOC production after 7 days is likely a result of substrate depletion, leading to reduced microbial growth and metabolic activity. Additionally, the gas flushing of the headspace would have removed any remaining VOCs from day 7. Therefore, to more accurately assess the effects of the different LAB strains, only VOC data obtained from day 0 and day 7 were compared.
Three-way ANOVA was used to determine that of the 104 sample-related m/z; a total number of 81, 87, 61, 64, 52, 42, and 56 m/z were significantly (p < 0.05) differentiated based upon LAB strains, time (at 0 and 7 days), temperature (25 and 35 °C), strain × time interactions, strain × temperature interactions, time × temperature interactions, and strain × time × temperature interactions, respectively (Table S1). Finally, 53 m/z were selected for which there was a significant (p < 0.05) increase (production) in their concentration during fermentation as opposed to a decrease (utilisation) and significant (p < 0.05) differences in either the main effects (LAB strains, time, or temperature) or their interaction effects (Table 4 and Table 5).
Hierarchical clustering analysis and the heatmap visualisation of the selected m/z showed the VOCs (m/z) produced by the six differing LAB strains growing at either 25 °C (Figure 1) or 35 °C (Figure 2). In heatmap 1 (Figure 1), the VOCs, after 7 days of fermentation at 25 °C, were primarily grouped (column-wise) into two clusters based on the LAB strains used; cluster 1—LP100, PP100, and LB672 (subcluster-LP100 and PP100)—and cluster 2—LU5335, PD5733, and PD661 (subcluster-LU5335, and PD5733). The ketones (m/z 59.049 (t.i. acetone), m/z 87.043 (t.i. diacetyl)), and aldehydes (m/z 85.066 (t.i. 2-methyl-2-butenal and 3-methyl-2-butenal)) were present in higher proportions in the subcluster of cluster 1 LP100 and PP100, and the sulphur compounds (m/z 49.011 (t.i. methanethiol) and m/z 95.004 (t.i. dimethyl disulphide)) were present in higher proportions in cluster 1 LP100, PP100, and LB672. However, alcohols (m/z 43.054 (t.i. propanol fragment), m/z 57.070 (t.i. butanol fragment), m/z 71.085 (t.i. 3-methyl butanol fragment), m/z 91.072 (t.i. 2,3-butanediol), and m/z 109.059 (t.i. benzyl alcohol)) were present in a lower proportion in cluster 1 LP100, PP100, and LB672. In contrast, the alcohols (m/z 47.049 (t.i. ethanol), butanol, propanol, 3-methyl butanol, 2,3-butanediol, m/z 107.066 (t.i. methionol), and benzyl alcohol) were present in a higher proportion in cluster 2 LU5335, PD5733, and PD661, and the ketones (acetone and m/z 115.112 (t.i. 2-heptanone)), aldehydes (2-methyl-2-butenal and 3-methyl-2-butenal), and sulphur compounds (methanethiol and dimethyl disulphide) were present in a lower proportion. Further, the VOCs were row-wise clustered, mainly into two clusters (Figure 1 and Table S2); cluster 1 (orange) was characterised by a higher proportion of alcohols, while cluster 2 (pink) was characterised by sulphur compounds and ketones.
At 35 °C, the VOCs were also mainly clustered into two groups (column-wise) after LAB fermentation, as shown in heatmap 2 (Figure 2); cluster 1—LP100 and PP100—and cluster 2—LU5335, PD5733, PD661, and LB672 (subclusters—LU5335 and PD661; LU5335, PD661, and PD5733). The ketones (acetone, diacetyl, and 2-heptanone), aldehydes (2 and 3-methyl-2-butenal), and sulphur compounds (m/z 105.046 (t.i. methional) and dimethyl disulphide) were present in a higher proportion in the cluster 1 (LP100, and PP100), where alcohols (propanol, ethanol, butanol, 3-methyl butanol, 2,3-butanediol, methionol, and benzyl alcohol) were present in a lower proportion. In contrast, the ketones (acetone, 2-heptanone, and diacetyl) and aldehydes (2 and 3-methyl-2-butenal) were present in a lower proportion in cluster 2 (LU5335, PD5733, PD661 and LB672), and sulphur compounds (methional, methanethiol, and dimethyl disulphide) were present in a lower proportion in subcluster 2 LU5335, PD661, and PD5733. However, alcohols (propanol, butanol, 3-methyl butanol, and 2,3 butanediol) were present in a higher proportion in subcluster 2 LU5335, PD661, and PD5733. Further, VOCs were row-wise clustered into two groups (Figure 2 and Table S3); cluster 1 (orange) was characterised by having a higher proportion of alcohols, while cluster 2 (Pink) was characterised by having a higher proportion of ketones and sulphur compounds. The specific VOCs produced by the six differing LAB strains are discussed separately in the following sections.

3.2.1. Main Alcohols

Ethanol, which can be a key marker compound in fermentation studies, is produced by some LAB during sugar fermentation. In the present study, the concentration of m/z 47.049 (t.i. ethanol) was significantly (p < 0.05) higher after 7 days of fermentation at either 25 or 35 °C by PD661, LU5335, PD5733, and LB672 compared to the other two strains studied (Figure 3a). LU5335 and LB672 are heterofermentative LAB and produce ethanol via the phosphoketolase (PK) pathway. However, it seems that even though PD661 and PD5733 are classified as homofermentative LAB, the detection of ethanol suggests that under the fermentation conditions used in this study, glucose was fermented heterofermentatively [44]. Further, ethanol was not detected or was detected in very low concentrations after 7 days of fermentation by either LP100 or PP100, which are classified as being facultative heterofermentative or homofermentative LAB, respectively. Since acetaldehyde, an intermediate in the ethanol production pathway, was detected in DM after fermentation by LP100 and PP100 strains, these data suggest that these strains did not contain the alcohol dehydrogenase (AlcDH) enzyme. Besides sugar fermentation, ethanol can be produced from threonine amino acid [45,46,47]. Further, given the differences in the concentration of sugar (glucose) and threonine in the DM, it is speculated that the ethanol produced by PD661, LU5335, PD5733, and LB672 strains in this study was from mainly sugar metabolism.
The concentration of m/z 57.070 (t.i. butanol fragment), which is produced from sugars through the fatty acid biosynthesis pathway [48], was significantly (p < 0.05) higher after 7 days of fermentation at 35 °C by PD661, followed by LU5335 at 35 °C and PD661 at 25 °C compared to the other four strains studied, with PD5733 being significantly (p < 0.05) higher than the other three strains (Figure 3b).

3.2.2. VOCs Produced from Glucose and/or Aspartic Acid

Diacetyl, also known as 2,3-butanedione, is an important flavour compound across a range of dairy products, including in yoghurt [49]. It is generated from the metabolism of either sugar, citrate or aspartic acid, forming pyruvate [50]. Pyruvate is subsequently converted to acetaldehyde-TPP through a decarboxylation process, and then to α-acetolactate, facilitated by α-acetolactate synthase (ALS). ALS has a low affinity to pyruvate; therefore, α-acetolactate synthesis is favoured in the presence of excess pyruvate. α-acetolactate is an unstable intermediate, which, in the presence of molecular oxygen, can be converted to diacetyl through nonenzymatic oxidative carboxylation [51,52,53,54]. In the present study, the concentration of m/z 87.043 (t.i. diacetyl) was significantly (p < 0.05) higher after 7 days of fermentation at 35 °C for LP100, followed by PD661 at 25 °C and PP100 at 35 °C compared to the other three strains studied (Figure 4a). The production of diacetyl by LAB has been reported to be strain-dependent, as the presence of enzymes varies between species and strains [55]. Lpb. plantarum fermented fruit juices have previously been reported to produce higher concentrations of diacetyl compared to unfermented juices [56,57]. Further, Lpb. plantarum strains have been reported to produce diacetyl in a complex medium containing glucose as a carbon source, where Lev. brevis strains did not produce diacetyl and Lpb. plantarum strains produced higher concentrations of diacetyl when the complex medium was supplemented with citrate [58]. Since citrate and glucose co-metabolism was not investigated in the current study, diacetyl was assumed to be produced either from glucose or glucose and aspartic acid. The presence of aspartic acid catabolic enzymes has been reported in Lpb. plantarum and P. pentosaceus strains by a genomic study [59]. Therefore, in the current study, aspartic acid catabolism may also lead to pyruvate accumulation in LP100 and PP100 strains, in addition to glucose metabolism, explaining the higher concentration of diacetyl in these strains at 35 °C. Interestingly, PD661 produced higher concentration of diacetyl at 25 °C compared to PD5733 at either 25 or 35 °C. It is obvious that the activity of enzymes can differ between various strains of the same LAB species [28,59].
The concentration of m/z 91.072 (t.i. 2,3-butanediol) was significantly (p < 0.05) higher after 7 days of fermentation at 25 °C by PD661 compared to the other five strains studied (Figure 4b). 2,3-butanediol can be produced from acetoin using the enzyme diacetyl acetoin reductase (DAR). Acetoin is synthesised from α-acetolactate, an intermediate in diacetyl synthesis. α-acetolactate is an unstable intermediate that is decarboxylated enzymatically to yield acetoin. Acetoin can also be synthesised from diacetyl using the enzyme DAR. The 2,3-butanediol can subsequently be reoxidised into acetoin by enzyme 2,3-butanediol dehydrogenase (BDH) [52,53].
The higher production of diacetyl and 2,3-butanediol after fermentation by PD661 is explained by higher DAR enzyme activity and lower BDH enzyme activity. However, an in-depth genomic study in relation to flavour-forming pathways is required to confirm the presence and the activity of DAR and BDH enzymes in all strains studied.
The concentration of m/z 89.060 (t.i. ethyl acetate and acetoin) was significantly (p < 0.05) higher after 7 days of fermentation at 35 °C by LU5335 compared to the other five strains studied at both temperatures (Figure 4c). Based on the RT of the main and fragment ions checked in the fastGC-PTR-ToF-MS for standards and samples (Table 6), the m/z 89.060 detected by PTR-ToF-MS was likely to be mainly ethyl acetate and acetoin (a small signal was observed for ethyl butanoate). However, there were differences between LAB strains in the contribution of ethyl acetate and acetoin for m/z 89.060; the ethyl acetate signal was dominant in LB672 fermentation, the acetoin signal was dominant in the fermentations carried out by LP100 and PP100, and for the LU5335, PD661, and PD5733 fermentations, ethyl acetate and acetoin (small signal) signals were dominant.

3.2.3. Other Specific VOCs

The concentration of m/z 49.011 (t.i. methanethiol) was significantly (p < 0.05) higher after 7 days of fermentation at 35 °C by LB672 compared to the other five strains studied (Figure 5a). Methanethiol, which contributes to the flavour of meat [60] and cheese [61,62], can be produced by LAB from methionine via transamination reactions or demethiolation, or from the enzymatic breakdown of methional [45,63,64,65].
The concentration of m/z 71.085 (t.i. 3-methyl butanol fragment) was significantly (p < 0.05) higher after 7 days of fermentation at either 25 or 35 °C by PD661, LU5335, or PD5733 compared to the other three strains studied (Figure 5b). 3-Methyl butanol, which is an important flavour compounds in cheese [61,62], can be produced via the enzyme-mediated transamination of leucine [45,46].
The concentration of m/z 131.105 (t.i. 3-methyl butyl acetate), which is formed by a reaction between 3-methyl-butanol and acetyl CoA, was significantly (p < 0.05) higher after 7 days of fermentation at 35 °C by LU5335 compared to the other five strains studied at both temperatures (Figure 5c).
The concentration of m/z 115.112 (t.i. 2-heptanone), which is another important ketone in cheese [66], was significantly (p < 0.05) higher at 7 days of fermentation at either 25 or 35 °C by LP100 compared to the other strains at all temperature and time points (Figure 5d). 2-Heptanone is synthesised from octanoic acid. Octanoic acid is first oxidised into β-ketoacid via several β-oxidation steps using a range of enzymes, which is then decarboxylated into 2-heptanone with one less carbon atom [63,67].
The concentration of m/z 85.070 (t.i. 2-methyl-2-butenal and 3-methyl-2-butenal) was significantly (p < 0.05) higher after 7 days of fermentation at 35 °C for PP100, followed by LP100 at 35 °C compared to the other four strains studied (Figure 5e). 2-methyl-2-butenal and 3-methyl-2-butenal can be produced by LAB from isoleucine and leucine, respectively [68].

4. Discussion

The DM used was developed based on published studies and refined through several LAB growth trials. It contained glucose, peptone, and an amino acid mixture (leucine, isoleucine, phenylalanine, glutamic acid, aspartic acid, threonine, and methionine), sodium acetate, vitamins, and minerals at minimal concentrations needed for the optimum growth of LAB. The influence of seven LAB strains (LB672, LD677, PD661, LP100, PP100, PD5733, and LU5335) on the relative concentrations of VOCs in the DM was investigated in this study, as VOC production by LAB is strain-dependent [28,69].
All LAB strains except LD677 grew well in the developed DM, based on pH, OD600 values, and the visual inspection of the turbidity of the culture medium. As LAB’s nutritional needs are strain-dependent, the better overall growth of all strains may have been obtained if the DM was optimised for each LAB strain separately. This could have provided further information on the generation of VOCs for each LAB strain in relation to the composition of the DM.
Fermentation reactions happen in real-time, where compounds can be produced and then consumed during fermentation, which means that during fermentation, compounds that could have been produced are subsequently converted into other compounds and hence are not present at the end of fermentation. To address this challenge, real-time PTR-ToF-MS was used to determine the VOCs produced by LAB strains in the DM overtime. A disadvantage of PTR-ToF-MS is that it is a one-dimensional analytical technique (i.e., no separation of VOCs occurs), meaning that unambiguous identification is not always possible. This limitation in part can be overcome by coupling PTR-ToF-MS with fastGC to improve compound identification and the use of complimentary analysis by GC-MS to support VOC identification [32,33,34]. Therefore, to support the identification of VOCs detected by PTR-ToF-MS, this study used fastGC-PTR-ToF-MS and HS-SPME-GC-MS (Table 3, Table 4, Table 5 and Table S1).
The heatmap analysis highlighted differences between the VOCs produced by different LAB strains after 7 days of fermentation at either 25 or 35 °C (Figure 1 and Figure 2, respectively). The DM used in this study had the same composition for all the LAB strains fermentation, but the VOCs produced varied among LAB strains incubated at either 25 or 35 °C. This result demonstrates that the presence of enzymes and/or enzyme activity varied among these strains.
As well as supporting growth, amino acids serve as the building blocks for important flavour compounds [64]. Though the amino acids methionine, leucine, and isoleucine were present in the DM in the same concentration, the concentrations of amino acid-derived VOCs such as methanethiol, 3-methyl butanol, 3-methyl-2-butenal, and 2-methyl-2-butenal differed based on the LAB strain, suggesting that the activity of methionine, leucine, and isoleucine catabolic enzymes varied between the LAB strains used in this study.
It is clear that at 35 °C as opposed to 25 °C, there was a significant increase in the concentration of specific VOCs (Figure 4 and Figure 5). For example, the concentration of diacetyl was higher in LP100 fermentation at 35 °C; esters (isoamyl acetate and ethyl acetate) were higher in LU5335 ferment at 35 °C; the concentration of higher alcohol, 3-methyl butanol, was higher in PD661 and LU5335 ferments at 35 °C; and the sulphur VOC, methanethiol, was higher in LB672 ferment at 35 °C. This demonstrates that temperature influenced the VOC generation by these LAB strains in the DM.
In addition to the seven LAB strains used in the current study, strains such as Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lactobacillus acidophilus, Lacticaseibacillus rhamnosus, Lactobacillus helveticus, and Bifidobacterium lactis, have been reported in plant-based fermentation studies [70]; therefore, further study using different commercial LAB strains is required.

5. Conclusions

The generation of fermentation VOCs by six (seven) commercial LAB strains growing in a DM was analysed using PTR-ToF-MS, HS-SPME-GC-MS, and fastGC-ToF-MS. The use of PTR–ToF–MS enabled the discovery of differences in VOCs produced between LAB strains (either homo, hetero, or facultative heterofermentative) and fermentation temperature (at either 25 or 35 °C). GC-MS-based techniques such as HS-SPME-GC-MS and fastGC-PTR-ToF-MS supported the identification of compounds detected by direct PTR-ToF-MS. Overall, differences between the relative concentrations of VOCs produced by LAB strains in the DM suggest the presence or differing activity of various enzymes.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/applmicrobiol5010033/s1: Table S1: The VOCS (m/z) detected by PTR-ToF-MS during LAB fermentation in defined medium that significantly (p < 0.05) distinguished between different LAB strains (S), fermentation time (0 and 7 days) (T), and temperature (either at 25 or 35 °C) (Temp) and their interaction effects; Table S2: Heatmap compounds (m/z) at 25 °C; Table S3: Heatmap compounds (m/z) at 35 °C.

Author Contributions

S.R.—methodology, investigation, formal analysis, data curation, writing—original draft, and writing—review and editing; I.K.—methodology, formal analysis, data curation, and writing—review and editing; P.S.—conceptualisation, methodology, writing—review and editing, and supervision; E.B.—formal analysis and data curation; F.B.—methodology, writing—review and editing, and supervision; P.B.—conceptualisation, methodology, writing—review and editing, and supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Accelerating Higher Education Expansion and Development (AHEAD) operation (AHEAD/PhD/R3/Agri/394), a world bank-funded project; the Ministry of Education, Sri Lanka; University of Otago doctoral scholarship; the University of Otago postgraduate publishing bursary. Catalyst: Seeding funding was provided by the New Zealand Ministry of Business, Innovation and Employment and administered by the Royal Society Te Apārangi. This study was partly carried out within the ON Foods—Research and innovation network on food and nutrition Sustainability, Safety and Security—Working ON Foods and received funding from the European Union Next-Generation EU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR)—MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.3—D.D. 1550 11/10/2022, PE00000003). This manuscript reflects only the authors’ views and opinions; neither the European Union nor the European Commission can be considered responsible for them.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applmicrobiol 05 00033 g001
Figure 1. Heatmap visualisation and hierarchical clustering analysis of m/z (VOCs) produced by different LAB strains (Table 1) based on the log 2-transformed average concentration (ppbV) of each m/z. Fermentation was carried out in the DM for 7 days at 25 °C. The green colour represents a higher abundance, while the red colour indicates a lower abundance. The flavour VOCs represented in the heatmap are numbered according to the numbers in Table S2.
Figure 1. Heatmap visualisation and hierarchical clustering analysis of m/z (VOCs) produced by different LAB strains (Table 1) based on the log 2-transformed average concentration (ppbV) of each m/z. Fermentation was carried out in the DM for 7 days at 25 °C. The green colour represents a higher abundance, while the red colour indicates a lower abundance. The flavour VOCs represented in the heatmap are numbered according to the numbers in Table S2.
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Figure 2. Heatmap visualisation and hierarchical clustering analysis of m/z (VOCs) produced by different LAB strains (Table 1) based on the log 2-transformed average concentration (ppbV) of each m/z. Fermentation was carried out in the DM for 7 days at 35 °C. The green colour represents a higher abundance, while the red colour indicates a lower abundance. The VOCs represented in the heatmap are numbered according to the numbers in Table S3.
Figure 2. Heatmap visualisation and hierarchical clustering analysis of m/z (VOCs) produced by different LAB strains (Table 1) based on the log 2-transformed average concentration (ppbV) of each m/z. Fermentation was carried out in the DM for 7 days at 35 °C. The green colour represents a higher abundance, while the red colour indicates a lower abundance. The VOCs represented in the heatmap are numbered according to the numbers in Table S3.
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Figure 3. Mean concentrations (ppbV) of m/z 47.049 (t.i. ethanol) (a) and m/z 57.070 (t.i. butanol fragment) (b) across the DM fermented by different LAB strains at either 25 (Applmicrobiol 05 00033 i001) or 35 (Applmicrobiol 05 00033 i002) °C after 7 days. Values are presented as mean ± standard error (n = 4). Different superscript lowercase letters represent significant differences between the DM fermented by different LAB strains according to Tukey’s test at p < 0.05.
Figure 3. Mean concentrations (ppbV) of m/z 47.049 (t.i. ethanol) (a) and m/z 57.070 (t.i. butanol fragment) (b) across the DM fermented by different LAB strains at either 25 (Applmicrobiol 05 00033 i001) or 35 (Applmicrobiol 05 00033 i002) °C after 7 days. Values are presented as mean ± standard error (n = 4). Different superscript lowercase letters represent significant differences between the DM fermented by different LAB strains according to Tukey’s test at p < 0.05.
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Figure 4. Mean concentrations (ppbV) of m/z 87.043 (t.i. diacetyl) (a), m/z 91.072 (t.i. 2,3-butanediol) (b), and m/z 89.060 (t.i. ethyl acetate and acetoin) (c) across the DM fermented by different LAB strains at either 25 (Applmicrobiol 05 00033 i001) or 35 (Applmicrobiol 05 00033 i002) °C after 7 days. Values are presented as mean ± standard error (n = 4). Different superscript lowercase letters represent significant differences between the DM fermented by different LAB strains according to Tukey’s test at p < 0.05.
Figure 4. Mean concentrations (ppbV) of m/z 87.043 (t.i. diacetyl) (a), m/z 91.072 (t.i. 2,3-butanediol) (b), and m/z 89.060 (t.i. ethyl acetate and acetoin) (c) across the DM fermented by different LAB strains at either 25 (Applmicrobiol 05 00033 i001) or 35 (Applmicrobiol 05 00033 i002) °C after 7 days. Values are presented as mean ± standard error (n = 4). Different superscript lowercase letters represent significant differences between the DM fermented by different LAB strains according to Tukey’s test at p < 0.05.
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Figure 5. Mean concentrations (ppbV) of m/z 49.011 (t.i. methanethiol) (a), m/z 71.085 (t.i. 3-methyl butanol) (b), m/z 131.105 (t.i. isoamyl acetate) (c), m/z 115.112 (t.i. 2-heptanone) (d), and m/z 85.070 (t.i. 2-methyl-2-butenal and 3-methyl-2-butenal) (e) across the DM fermented by different LAB strains at either 25 (Applmicrobiol 05 00033 i001) or 35 (Applmicrobiol 05 00033 i002) °C after 7 days. Values are presented as mean ± standard error (n = 4). Different superscript lowercase letters represent significant differences between the defined medium fermented by different LAB strains according to Tukey’s test at p < 0.05.
Figure 5. Mean concentrations (ppbV) of m/z 49.011 (t.i. methanethiol) (a), m/z 71.085 (t.i. 3-methyl butanol) (b), m/z 131.105 (t.i. isoamyl acetate) (c), m/z 115.112 (t.i. 2-heptanone) (d), and m/z 85.070 (t.i. 2-methyl-2-butenal and 3-methyl-2-butenal) (e) across the DM fermented by different LAB strains at either 25 (Applmicrobiol 05 00033 i001) or 35 (Applmicrobiol 05 00033 i002) °C after 7 days. Values are presented as mean ± standard error (n = 4). Different superscript lowercase letters represent significant differences between the defined medium fermented by different LAB strains according to Tukey’s test at p < 0.05.
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Table 1. LAB strains used in the study.
Table 1. LAB strains used in the study.
AbbreviationLAB StrainsCulture FormSource
LB672Lev. brevis WLP672LiquidWhite labs, USA
LD677Lb. delbrueckii WLP677LiquidWhite labs, USA
PD661P. damnosus WLP661LiquidWhite labs, USA
LP100Lpb. plantarum LP100Lyophilised strain powderBioagro, Italy
PP100P. pentosaceus PP100Lyophilised strain powderBioagro, Italy
PD5733P. damnosus 5733LiquidWyeast, USA
LU5335Len. buchneri 5335LiquidWyeast, USA
Table 2. The mean pH and OD600 of samples after 14 days of fermentation by different LAB strains in the DM.
Table 2. The mean pH and OD600 of samples after 14 days of fermentation by different LAB strains in the DM.
DM Inoculated
with Differ LAB Strains		Initial pHat 25 °Cat 35 °C
pHOD600pHOD600
LB6725.67 ± 0.009 Aa4.47 ± 0.03 Cb1.40 ± 0.005 Ba4.44 ± 0.018 Db1.20 ± 0.04 Bb
LD6775.66 ± 0.002 Aa5.62 ± 0.003 Aa0.05 ± 0.003 Ea5.63 ± 0.003 Aa0.04 ± 0.005 Ea
PD6615.65 ± 0.005 Aa5.22 ± 0.035 Bb0.70 ± 0.01 Da5.69 ± 0.06 Aa0.70 ± 0.008 Da
LP1005.65 ± 0.01 Aa3.95 ± 0.1 Db2.40 ± 0.05 Aa4.02 ± 0.008 Eb2.32 ± 0.005 Aa
PP1005.66 ± 0.005 Aa4.47 ± 0.001 Cc1.31 ± 0.015 Ba4.86 ± 0.055 Cb0.75 ± 0.02 Db
PD57335.67 ± 0.003 Aa5.09 ± 0.065 Bb1.15 ± 0.015 Ca4.94 ± 0.06 Cb0.98 ± 0.008 Cb
LU53355.68 ± 0.006 Aa5.08 ± 0.03 Bc0.74 ± 0.005 Da5.32 ± 0.005 Bb0.70 ± 0.045 Da
Values are the means ± standard error of 2 replicates. Values with different superscript uppercase letters (A–E) in the column (either pH or OD600) are significantly different according to Tukey’s test at p< 0.05. Values with different superscript lowercase letters (a–c) in the row (either pH or OD600) are significantly different according to Tukey’s test at p< 0.05.
Table 3. VOCs detected after 14 days of fermentation by different LAB strains (either LB672, LP100, PP100, PD661, PD5733 or LU5335) in the DM using HS-SPME-GC-MS at 25 and 35 °C.
Table 3. VOCs detected after 14 days of fermentation by different LAB strains (either LB672, LP100, PP100, PD661, PD5733 or LU5335) in the DM using HS-SPME-GC-MS at 25 and 35 °C.
NoVOCsFormulaRTRI. CalRI. Litat 25 °Cat 35 °C
Acids
1Acetic acidC2H4O215.2914671449✓✓
2Butyric acidC4H8O219.6316461625✓✓
3Hexanoic acidC6H12O224.4418621846✓✓
4Octanoic acidC8H16O228.7620352060✓✓
5Decanoic acidC10H20O232.7021542276✓✓
Alcohols
62-PropanolC3H8O3.07934927✓✓
7EthanolC2H6O3.16941932✓✓
82-PentanolC5H12O6.6911341119✓✓
91-ButanolC4H10O7.2711581142✓✓
102/3-Methyl-1-butanolC5H12O8.8612201208/1209✓✓
113-Methyl-3-buten-1-olC5H10O9.9912631248✓✓
122-HeptanolC7H16O11.7813321320✓✓
13HexanolC6H14O12.6713651355✓✓
142,3-ButanediolC4H10O217.4415541543✓✓
151-OctanolC8H18O17.8615711557✓✓
16MentholC10H20O19.8116531637✓✓
172-UndecanolC11H24O21.5917311717✓×
18Benzyl alcoholC7H8O25.1418951870✓✓
19Phenylethyl alcoholC8H10O25.8519301906✓✓
202-TridecanolC13H28O25.9019331903✓✓
21P-cresolC7H8O29.4520512080✓✓
Aldehydes
22ButanalC4H8O2.75911877✓✓
232-Methyl butanalC5H10O2.90922914✓✓
243-Methyl butanalC5H10O2.96926918✓✓
252-Methyl-2-butenalC5H8O6.1711141095✓✓
263-Methyl-2-butenalC5H8O8.7712161215✓✓
272-Methyl pentanalC6H12O13.661403-✓✓
28BenzaldehydeC7H6O17.1515421520✓✓
29BenzeneacetaldehydeC8H8O20.0316631640✓✓
Esters
30Ethyl acetateC4H8O22.61901888✓✓
31Isoamyl acetateC7H14O26.8111391122✓✓
32Octanoic acid ethyl esterC10H20O214.8114481435✓✓
33Decanoic acid ethyl esterC12H24O219.7716521638✓✓
342-Phenylethyl acetateC10H12O223.8918361813✓✓
35Dodecanoic acid ethyl esterC14H28O224.3018561841✓✓
Furans
36FurfuralC5H4O215.7214841461×✓
372-FuranmethanolC5H6O220.4016791660✓✓
Ketones
38AcetoneC3H6O1.97823819✓✓
39DiacetylC4H6O23.84989979✓✓
402-HeptanoneC7H14O8.2911981182✓✓
41AcetoinC4H8O211.0013021284✓✓
Sulphur compounds
42Dimethyl disulphideC2H6S25.7310951077×✓
43MethionalC4H8OS15.4714741454✓✓
44Cyclohexyl isothiocyanateC7H11NS20.6116871667✓✓
453-(methylthio)-1-propanol (methionol)C4H10OS21.6417341719✓✓
Pyrazine
46PyrazineC4H4N29.0812281212✓✓
Unknown compounds
47Unknown 1 4.92 ✓✓
48Unknown 25.05×✓
49Unknown 3 6.04✓✓
50Unknown 412.53✓✓
✓: VOCs detected at given temperature. ×: VOCs not detected at given temperature.
Table 4. The VOCS (m/z) detected by PTR-ToF-MS during LAB fermentation in the DM that ANOVA analysis determined significantly (p < 0.05) distinguished between different LAB strains (S), fermentation time (0 and 7 days) (T), temperature (either at 25 or 35 °C) (Temp), and their interaction effects.
Table 4. The VOCS (m/z) detected by PTR-ToF-MS during LAB fermentation in the DM that ANOVA analysis determined significantly (p < 0.05) distinguished between different LAB strains (S), fermentation time (0 and 7 days) (T), temperature (either at 25 or 35 °C) (Temp), and their interaction effects.
Nom/zSum FormulaIdentificationp Value
STTempS×TS×TempT×TempS×T×Temp
126.016C2H2+Common fragment<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
227.025C2H3+ <0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
328.031C2H4+ <0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
431.018CH2OH+Formaldehyde fragment<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
534.996H2SH+Hydrogen sulphide<0.0001<0.00010.010<0.0001<0.00010.017<0.0001
641.039C3H5+ <0.0001<0.0001<0.0001<0.0001<0.00010.0730.209
742.010 <0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
843.018C2H3O+Common fragment0.005<0.00010.3890.4680.3930.4810.055
943.054C3H7+Propanol fragment 1<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
1047.049C2H6OH+Ethanol 1<0.0001<0.0001<0.0001<0.0001<0.0001<0.00010.001
1149.011CH4SH+Methanethiol<0.0001<0.0001<0.0001<0.0001<0.00010.901<0.0001
1253.006 <0.0001<0.0001<0.0001<0.0001<0.0001<0.00010.002
1357.036C3H4OH+ <0.0001<0.00010.050<0.00010.6020.4120.806
1457.070C4H9+1-Butanol fragment 1<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
1559.049C3H6OH+Acetone1<0.0001<0.0001<0.0001<0.0001<0.00010.858<0.0001
1663.009CO2H3O+ <0.0001<0.00010.166<0.00010.4070.9750.059
1764.005 <0.0001<0.0001<0.0001<0.00010.0080.009<0.0001
1871.085C5H11+3-Methyl-butanol fragment 1,2<0.0001<0.0001<0.0001<0.0001<0.00010.005<0.0001
1978.967CH2S2H+ <0.00010.008<0.0001<0.0001<0.00010.0020.001
2081.016 <0.0001<0.0001<0.0001<0.0001<0.00010.001<0.0001
2185.066C5H8OH+2-Methyl-2-butenal 1 and
3-Methyl-2-butenal 1		<0.0001<0.0001<0.0001<0.0001<0.00010.001<0.0001
2287.043C4H6O2H+Diacetyl 1,2<0.0001<0.00010.004<0.0001<0.00010.282<0.0001
2389.060C4H8O2H+Ethyl acetate 1,2,3 and Acetoin 1,2,3<0.0001<0.00010.6820.0010.2390.9180.161
2491.027C3H6OSH+Methyl thiolacetate/Mercaptoacetone<0.0001<0.00010.009<0.0001<0.00010.007<0.0001
2591.072C4H10O2H+2,3-Butanediol 10.0010.0060.0780.0560.1390.3450.130
2695.004C2H6S2H+Dimethyl disulphide1<0.00010.052<0.0001<0.0001<0.00010.001<0.0001
2795.093 <0.0001<0.0001<0.0001<0.0001<0.0001<0.00010.001
2897.063C6H8OH+2,5-Dimethylfuran/Cyclohexen-2-one<0.0001<0.0001<0.00010.002<0.0001<0.0001<0.0001
2997.106C7H13+ <0.0001<0.0001<0.0001<0.00010.0050.4150.029
3099.119C7H15+2-Heptanol fragment 1<0.0001<0.0001<0.0001<0.00010.0010.013<0.0001
31103.074C5H10O2H+C5 esters and acids (i.e., pentanoic acid/3-methyl-butanoic acid)<0.0001<0.00010.037<0.00010.2040.7610.207
32105.046C4H8OSH+Methional 1<0.0001<0.00010.1850.4590.2670.182<0.0001
33107.066C4H10OSH+ Methionol 10.013<0.00010.4690.0250.0740.7050.061
34107.107 <0.0001<0.0001<0.0001<0.00010.001<0.00010.006
35109.059C7H8OH+Benzyl alcohol 1<0.0001<0.00010.074<0.00010.1360.1380.105
36111.099 0.001<0.00010.2220.0020.3890.3060.399
37115.112C7H14OH+2-Heptanone 1,2<0.0001<0.00010.025<0.00010.7960.1470.977
38117.091C6H12O2H+Hexanoic acid 1<0.0001<0.00010.5620.0120.1250.6060.036
39119.093C6H14SH+ <0.00010.0020.0110.0620.1670.9630.022
40121.119 <0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
41123.117C9H15+ <0.00010.0150.4340.0050.2410.8400.432
42126.967 <0.0001<0.0001<0.0001<0.0001<0.00010.111<0.0001
43127.050 0.007<0.00010.0980.0890.4360.6080.831
44131.105C7H14O2H+Isoamyl acetate 1<0.0001<0.00010.137<0.00010.6450.8480.415
45133.117C7H16O2H+ <0.00010.0180.0100.0760.0200.6690.027
46135.100C6H14O3H+ <0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
47135.134 <0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
48139.137 <0.0001<0.0001<0.0001<0.00010.0010.0020.001
49143.137C9H18OH+Nonanal/Nonanone<0.0001<0.00010.019<0.00010.0010.612<0.0001
50145.123C8H16O2H+Octanoic acid 1<0.0001<0.0001<0.0001<0.0001<0.00010.006<0.0001
51163.077C10H10O2H+ 0.0010.0020.8240.0010.0680.5930.122
52173.154C10H20O2H+Decanoic acid 1<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001<0.0001
53201.185C12H24O2H+Decanoic acid ethyl ester 1<0.0001<0.0001<0.0001<0.00010.005<0.00010.020
1: m/z that HS-SPME-GC-MS identified. 2: m/z identified by fastGC-PTR-ToF-MS and/or injection of pure standard. 3: Ethyl acetate signal dominant in LB672 fermented samples, acetoin signal dominant in LP100 and PP100 fermented samples, ethyl acetate and acetoin (small) signals dominant in LU5335, and PD661 and PD5733 fermented samples based on fastGC-PTR-ToF-MS.
Table 5. The mean concentration (ppbV) of selected VOCs (m/z) after 7 days of fermentation by different LAB strains in the DM at either 25 or 35 °C. Values are presented as mean ± standard error (n = 4).
Table 5. The mean concentration (ppbV) of selected VOCs (m/z) after 7 days of fermentation by different LAB strains in the DM at either 25 or 35 °C. Values are presented as mean ± standard error (n = 4).
Nom/zLB672PD661LP100PP100PD5733LU5335
at 25 °Cat 35 °Cat 25 °Cat 35 °Cat 25 °Cat 35 °Cat 25 °Cat 35 °Cat 25 °Cat 35 °Cat 25 °Cat 35 °C
126.016167.048 ± 1.87441.51 ± 7.46521.94 ± 14.33809.748 ± 37.026.42 ± 8.0432.447 ± 4.8814.709 ± 5.0764.365 ± 38.27432.918 ± 3.19637.032 ± 6.32408.468 ± 8.04847.47 ± 72.77
227.02580.096 ± 1.17191.378 ± 4.39270.533 ± 23.04376.513 ± 23.3414.404 ± 4.6814.874 ± 2.257.521 ± 2.2230.175 ± 17.55205.566 ± 2.94287.241 ± 4.45195.772 ± 3.35387.008 ± 31.54
328.0318.817 ± 0.1621.155 ± 0.5127.7 ± 3.0235.906 ± 1.641.554 ± 0.272.562 ± 0.131.156 ± 0.173.816 ± 1.4921.056 ± 0.3229.6 ± 0.1619.782 ± 0.2736.692 ± 2.36
431.018928.421 ± 8.452132.831 ± 30.712193.987 ± 21.773378.789 ± 78.38113.72 ± 2.24132.123 ± 3.1662.939 ± 15.28165.598 ± 93.392105.244 ± 12.262959.667 ± 32.482006.605 ± 33.323231.393 ± 83.87
534.9967.906 ± 0.2514.439 ± 1.067.596 ± 1.8314.935 ± 2.5012.348 ± 0.620.692 ± 0.082.408 ± 0.230.758 ± 0.032.71 ± 0.193.876 ± 0.726.139 ± 0.344.801 ± 1.06
641.03921.498 ± 0.4253.09 ± 1.58601.747 ± 118.91822.194 ± 66.5031.464 ± 0.4656.122 ± 2.8216.502 ± 2.7473.972 ± 4.08238.258 ± 6.52236.99 ± 5.23293.942 ± 9.25516.946 ± 32.39
742.01205.561 ± 2.28547.972 ± 10.35750.214 ± 51.631102.089 ± 76.2448.238 ± 13.6854.497 ± 7.6125.718 ± 7.45103.207 ± 57.47541.102 ± 4.52827.993 ± 8.69512.99 ± 11.151178.195 ± 131.83
843.0184158.139 ± 62.529122.072 ± 343.9620261.619 ± 12219.108616.588 ± 327.426452.574 ± 201.496037.236 ± 340.363937.451 ± 294.383808.769 ± 771.346128.931 ± 68.918725.072 ± 116.736255.702 ± 108.3411864.271 ± 1221.21
943.0545.054 ± 1.1418.602 ± 1.92289.246 ± 26.12519.05 ± 10.560 ± 00 ± 00 ± 02.77 ± 1.62165.123 ± 5.13145.644 ± 5.6212.367 ± 6.92327.327 ± 14.86
1047.04917,447.171 ± 176.6644,863.183 ± 1176.3351,191.95 ± 3319.0382,907.442 ± 4413.17286.84 ± 33.86935.419 ± 85.42498.008 ± 296.582407.504 ± 2051.6944,565.668 ± 369.8266,142.439 ± 1101.3241,885.022 ± 781.0988,784.653 ± 4937.75
1149.011182.917 ± 13.60459.487 ± 31.3349.417 ± 5.948.268 ± 3.96210.152 ± 16.33171.222 ± 41.0154.535 ± 2.3629.212 ± 3.7851.715 ± 1.3115.122 ± 16.458.243 ± 3.5363.686 ± 6.42
1253.0069.648 ± 0.1326.73 ± 0.48104.944 ± 11.88164.801 ± 12.268.502 ± 0.5112.591 ± 0.413.941 ± 0.6516.672 ± 2.2652.793 ± 1.1666.852 ± 1.1059.265 ± 1.90123.647 ± 11.11
1357.0365.489 ± 0.1011.73 ± 0.39138.128 ± 70.24145.162 ± 12.156.711 ± 0.0714.952 ± 0.765.347 ± 0.2914.073 ± 0.3335.028 ± 1.0744.354 ± 1.2640.768 ± 1.56102.011 ± 5.78
1457.0713.976 ± 0.5633.623 ± 1.16558.761 ± 11.311080.518 ± 32.6510.042 ± 0.6530.006 ± 2.259.744 ± 0.1620.545 ± 0.7177.261 ± 3.79236.018 ± 5.82200.739 ± 7.39527.166 ± 34.32
1559.04924.784 ± 3.4932.257 ± 4.61421.171 ± 68.79534.178 ± 27.502206.881 ± 16.401745.93 ± 45.96332.084 ± 15.63847.678 ± 23.83159.598 ± 3.26388.981 ± 2.7149.138 ± 6.58382.086 ± 164.52
1663.00982.849 ± 1.97127.534 ± 30.44107.423 ± 4.06111.562 ± 34.9515.177 ± 0.4717.316 ± 0.5113.164 ± 1.4212.978 ± 4.18128.245 ± 6.2797.983 ± 6.32106.979 ± 5.34121.257 ± 7.12
1764.0051.989 ± 0.123.025 ± 0.21.245 ± 0.171.453 ± 0.441.212 ± 0.050.969 ± 0.10.615 ± 0.090.383 ± 0.031.829 ± 0.061.726 ± 0.131.516 ± 0.081.502 ± 0.23
1871.08510.255 ± 0.5321.316 ± 0.63570.456 ± 11.351106.131 ± 32.594.368 ± 0.249.914 ± 0.635.146 ± 0.1610.824 ± 0.17370.814 ± 10.82332.782 ± 8.55475.144 ± 17.27819.329 ± 47.8
1978.9675.29 ± 0.5320.076 ± 2.171.37 ± 0.52.901 ± 0.786.06 ± 0.5221.371 ± 6.392.336 ± 0.213.435 ± 0.331.372 ± 0.126.38 ± 0.841.961 ± 0.162.234 ± 0.45
2081.0163.575 ± 0.1711.114 ± 0.187.418 ± 0.819.752 ± 2.770.208 ± 0.030.309 ± 0.070.357 ± 0.030.521 ± 0.178.684 ± 0.398.228 ± 0.537.232 ± 0.4311.237 ± 0.71
2185.0660.667 ± 0.041.813 ± 0.181.760 ± 0.202.977 ± 0.404.007 ± 0.087.929 ± 0.282.836 ± 0.1312.255 ± 0.390.836 ± 0.021.861 ± 0.080.956 ± 0.042.4 ± 0.18
2287.0432.494 ± 0.112.522 ± 0.0719.148 ± 3.184.047 ± 0.6910.271 ± 0.3730.118 ± 0.894.348 ± 0.2122.055 ± 2.132.624 ± 0.092.446 ± 0.092.821 ± 0.082.657 ± 0.07
2389.06107.588 ± 1.68583.1 ± 11.55513.225 ± 74.50662.711 ± 77.4521.932 ± 1.6336.727 ± 2.056.935 ± 0.8015.053 ± 1.39768.849 ± 27.8834.338 ± 15.05688.761 ± 13.901323.605 ± 88.72
2491.0273.1 ± 0.1614.864 ± 0.694.058 ± 2.113.108 ± 1.061.036 ± 0.031.64 ± 0.070.723 ± 0.031.06 ± 0.084.727 ± 0.24.062 ± 0.355.73 ± 0.323.626 ± 2.13
2591.0724.743 ± 0.1927.938 ± 1.74288.497 ± 51.59137.918 ± 10.331.117 ± 0.183.118 ± 0.40.907 ± 0.0716.874 ± 14.3425.936 ± 0.8576.375 ± 7.4224.001 ± 0.9679.585 ± 1.29
2695.00410.25 ± 0.8137.968 ± 4.047.437 ± 1.906.718 ± 1.3710.963 ± 1.4732.705 ± 7.265.798 ± 0.67.747 ± 0.473.966 ± 0.3113.75 ± 1.525.264 ± 0.455.799 ± 0.89
2795.0931.81 ± 0.0712.486 ± 0.4323.106 ± 10.4835.172 ± 1.930.244 ± 0.040.893 ± 0.310.248 ± 0.060.27 ± 0.0810.958 ± 0.2824.895 ± 0.539.809 ± 0.5337.52 ± 1.52
2897.0630.963 ± 0.0910.732 ± 0.682.777 ± 2.340.478 ± 0.060.732 ± 0.041.093 ± 0.150.444 ± 0.020.424 ± 0.050.484 ± 0.042.646 ± 0.430.66 ± 0.031.806 ± 0.79
2997.1060.044 ± 0.020 ± 00.241 ± 0.050.364 ± 0.090.294 ± 0.040.31 ± 0.060.055 ± 0.010.27 ± 0.040.1 ± 0.010.052 ± 0.020.092 ± 0.020.204 ± 0.05
3099.1190.059 ± 0.030.239 ± 0.010.025 ± 0.020.01 ± 0.010.027 ± 0.010.08 ± 0.020.041 ± 0.000.016 ± 0.010.012 ± 0.010.038 ± 0.0190.093 ± 0.020.135 ± 0.05
31103.0740.633 ± 0.070.997 ± 0.054.497 ± 2.243.697 ± 0.180.713 ± 0.070.747 ± 0.050.48 ± 0.020.675 ± 0.051.644 ± 0.062.076 ± 0.11.518 ± 0.054.072 ± 0.33
32105.0461.026 ± 0.061.22 ± 0.049.568 ± 4.362.927 ± 0.353.033 ± 0.684.07 ± 0.381.69 ± 0.34.448 ± 0.711.546 ± 0.11.62 ± 0.082.044 ± 0.104.597 ± 1.15
33107.06627.644 ± 1.29143.431 ± 10.15217.522 ± 151.9061.948 ± 6.591.192 ± 0.088.355 ± 1.111.498 ± 0.645.285 ± 2.4958 ± 0.56113.907 ± 2.9462.916 ± 2.64121.476 ± 25.12
34107.1072.129 ± 0.127.99 ± 0.5111.654 ± 3.3714.393 ± 0.670.222 ± 0.040.512 ± 0.060.188 ± 0.050.483 ± 0.175.652 ± 0.178.888 ± 0.36.233 ± 0.2013.897 ± 0.98
35109.0590.336 ± 0.022.186 ± 0.113.468 ± 2.032.352 ± 0.610.035 ± 0.020.136 ± 0.040.008 ± 0.010.08 ± 0.021.373 ± 0.052.024 ± 0.191.179 ± 0.13.182 ± 0.24
36111.0990.46 ± 0.022.347 ± 0.058.428 ± 6.185.95 ± 0.370.176 ± 0.020.187 ± 0.010.176 ± 0.030.217 ± 0.021.838 ± 0.054.268 ± 0.081.64 ± 0.096.77 ± 0.36
37115.1120.121 ± 0.030.204 ± 0.060.648 ± 0.020.938 ± 0.084.814 ± 0.163.831 ± 0.110.153 ± 0.030.277 ± 0.040.41 ± 0.040.676 ± 0.060.284 ± 0.010.77 ± 0.1
38117.0910.488 ± 0.031.118 ± 0.054.582 ± 2.781.807 ± 0.080.574 ± 0.030.513 ± 0.020.406 ± 0.020.403 ± 0.061.854 ± 0.151.738 ± 0.171.384 ± 0.113.8 ± 0.28
39119.0930.204 ± 0.010.368 ± 0.051.974 ± 0.811.592 ± 0.640.293 ± 0.010.248 ± 0.010.136 ± 0.020.258 ± 0.080.282 ± 0.020.608 ± 0.060.294 ± 0.041.642 ± 0.59
40121.1190.076 ± 0.010.448 ± 0.035.147 ± 1.8125.03 ± 3.210 ± 00 ± 00 ± 00.016 ± 0.012.278 ± 0.065.06 ± 0.172.35 ± 0.1411.913 ± 1.15
41123.1170.016 ± 0.010.067 ± 0.020.341 ± 0.20.183 ± 0.020.004 ± 0.000.049 ± 0.010.011 ± 0.010.037 ± 0.010.049 ± 0.010.079 ± 0.010.053 ± 0.010.162 ± 0.02
42126.9670.708 ± 0.051.55 ± 0.110.422 ± 0.020.449 ± 0.110.87 ± 0.071.09 ± 0.250.337 ± 0.010.428 ± 0.020.244 ± 0.010.549 ± 0.060.342 ± 0.020.368 ± 0.06
43127.050.126 ± 0.020.125 ± 0.020.21 ± 0.080.214 ± 0.030.137 ± 0.020.15 ± 0.010.098 ± 0.010.119 ± 0.020.153 ± 0.020.157 ± 0.010.14 ± 0.010.224 ± 0.02
44131.1050.344 ± 0.020.67 ± 0.030.855 ± 0.031.078 ± 0.110.206 ± 0.010.219 ± 0.020.213 ± 0.010.184 ± 0.062.190 ± 0.183.322 ± 0.612.756 ± 0.126.385 ± 1.02
45133.1170.025 ± 0.020.036 ± 0.022.152 ± 0.661.873 ± 0.960.046 ± 0.020.094 ± 0.010.034 ± 0.020.1 ± 0.070.142 ± 0.010.344 ± 0.020.152 ± 0.023.062 ± 1.52
46135.10.265 ± 0.022.334 ± 0.035.114 ± 2.895.545 ± 0.570.132 ± 0.010.12 ± 0.020.092 ± 0.010.139 ± 0.023.261 ± 0.115.336 ± 0.172.618 ± 0.0710.64 ± 0.58
47135.1340.017 ± 0.010.126 ± 0.049.817 ± 1.5628.434 ± 2.930 ± 00.011 ± 0.010.008 ± 0.010.045 ± 0.035.025 ± 0.196.919 ± 0.116.173 ± 0.4620.47 ± 1.62
48139.1370.231 ± 0.011.246 ± 0.042.396 ± 1.44.003 ± 0.230.192 ± 0.020.389 ± 0.060.233 ± 0.030.333 ± 0.030.834 ± 0.032.522 ± 0.120.684 ± 0.064.342 ± 0.22
49143.1370.217 ± 0.030.161 ± 0.040.574 ± 0.240.411 ± 0.160.883 ± 0.030.778 ± 0.400.256 ± 0.020.198 ± 0.030.225 ± 0.041.254 ± 0.040.204 ± 0.020.296 ± 0.05
50145.1230.218 ± 0.030.43 ± 0.050.69 ± 0.240.617 ± 0.080.264 ± 0.010.202 ± 0.020.176 ± 0.020.209 ± 0.030.634 ± 0.10.883 ± 0.100.574 ± 0.051.592 ± 0.14
51163.0770.113 ± 0.020.118 ± 0.020.324 ± 0.120.194 ± 0.020.09 ± 0.010.1 ± 0.010.131 ± 0.020.112 ± 0.020.17 ± 0.010.131 ± 0.020.155 ± 0.000.274 ± 0.04
52173.1540.189 ± 0.010.629 ± 0.110.667 ± 0.151.103 ± 0.080.072 ± 0.030.116 ± 0.010.059 ± 0.020.066 ± 0.0130.958 ± 0.181.692 ± 0.250.706 ± 0.0162.857 ± 0.27
53201.1850.046 ± 0.010.177 ± 0.020.145 ± 0.0220.195 ± 0.010.027 ± 0.010.034 ± 0.020.012 ± 0.010.026 ± 0.0140.15 ± 0.040.241 ± 0.020.15 ± 0.0140.186 ± 0.02
Table 6. Main and fragment ions checked for m/z 89.060 in fastGC-PTR-ToF-MS.
Table 6. Main and fragment ions checked for m/z 89.060 in fastGC-PTR-ToF-MS.
Compound NameMolecular FormulaMain/Fragment Ions Checked
m/zm/zm/zm/z
Ethyl acetateC4H8O289.06 (C4H8O2)H+61.028 (C2H4O2)H+43.018 (C2H3O)H+
Butyric acidC4H8O289.06 (C4H8O2)H+71.049 (C4H6O)H+43.054 (C3H7)H+29.039 (C2H5)H+
AcetoinC4H8O289.06 (C4H8O2)H+
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Rajendran, S.; Khomenko, I.; Silcock, P.; Betta, E.; Biasioli, F.; Bremer, P. The Use of Proton-Transfer-Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) to Determine the Volatile Organic Compounds (VOCs) Produced by Different Lactic Acid Bacterial Strains Growing in Defined Media. Appl. Microbiol. 2025, 5, 33. https://doi.org/10.3390/applmicrobiol5010033

AMA Style

Rajendran S, Khomenko I, Silcock P, Betta E, Biasioli F, Bremer P. The Use of Proton-Transfer-Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) to Determine the Volatile Organic Compounds (VOCs) Produced by Different Lactic Acid Bacterial Strains Growing in Defined Media. Applied Microbiology. 2025; 5(1):33. https://doi.org/10.3390/applmicrobiol5010033

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Rajendran, Sarathadevi, Iuliia Khomenko, Patrick Silcock, Emanuela Betta, Franco Biasioli, and Phil Bremer. 2025. "The Use of Proton-Transfer-Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) to Determine the Volatile Organic Compounds (VOCs) Produced by Different Lactic Acid Bacterial Strains Growing in Defined Media" Applied Microbiology 5, no. 1: 33. https://doi.org/10.3390/applmicrobiol5010033

APA Style

Rajendran, S., Khomenko, I., Silcock, P., Betta, E., Biasioli, F., & Bremer, P. (2025). The Use of Proton-Transfer-Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) to Determine the Volatile Organic Compounds (VOCs) Produced by Different Lactic Acid Bacterial Strains Growing in Defined Media. Applied Microbiology, 5(1), 33. https://doi.org/10.3390/applmicrobiol5010033

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