Abstract
There is growing demand for plant-based foods due to concerns about the impact of animal-based foods on the environment, human health, and animal welfare. However, many consumers still like to craft meals that resemble traditional meat or dairy-based products, owing to the enjoyment they get from eating foods with familiar flavour, texture, and preparation steps. To help meet this demand, there has been a dramatic increase in the availability of plant-based meat and dairy analogues. However, obtaining realistic meat or dairy flavour volatile organic compounds (VOCs) in the analogues has proven to be challenging. Fermentation via lactic acid bacteria (LAB) is a viable option for modifying the desired flavour profile of plant-based analogues. LAB utilize substrates for their energy and growth, and produce secondary volatile compounds, which are flavour VOCs or precursors, in addition to the main fermentation end products. The specific VOCs produced through fermentation are influenced by plant substrate compositions, LAB strains employed, and fermentation conditions used. Owing to the complexity of the compounds present in plant-based systems, it is difficult to relate the influence of substrate on fermentation VOCs produced. To better understand the production of fermentation VOCs in accordance with the compositions of plant substrate, a defined medium (DM) is required.
Therefore, the main aim of this thesis was to comprehensively analyze the VOCs profile produced by different LAB strains growing in different DM compositions. To achieve this, the DM was developed based on past literature and a series of LAB growth trials. The fermentation experiments were carried out either by changing the DM compositions or by using different commercial LAB strains under different fermentation (temperature and time) conditions.
In the first study, the developed DM was supplemented with the amino acids (AAs) L-leucine (Leu), L-isoleucine (Ile), L-phenylalanine (Phe), L-threonine (Thr), L-methionine (Met), or L-glutamic acid (Glu) either separately or combined to determine their impact on the VOCs produced by Levilactobacillus brevis WLP672 (LB672) at 25 oC. At the end of fermentation (16 days), VOCs were measured using headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). A total of 49 VOCs were detected, which were mainly either alcohols, acids, esters, sulfur-derived compounds, ketones or aldehydes. VOCs associated with the specific AAs added included: benzaldehyde, phenylethyl alcohol, benzyl alcohol with added Phe; methanethiol, methional, dimethyl disulfide with added Met; 3-methyl butanol with added Leu; and 2-methyl butanol with added Ile. A limitation of this initial trial was that only the VOC present at the end of fermentation (16 days) were measured. Hence, it is likely that some fermentation compounds were not detected as they may have been produced and metabolized or lost prior to measurement.
To enable the detection of VOC produced during fermentation, proton transfer reaction-time of flight-mass spectrometry (PTR-ToF-MS) was used in subsequent trials. PTR-ToF-MS is a quick, direct, non-invasive, and highly sensitive (parts per trillion by volume) online method that can be used to monitor the production of VOCs. As compound identification by PTR-ToF-MS is tentative, GC-MS and fastGC-PTR-ToF-MS can be used in tandem to support identifications.
In second and third studies, the VOCs produced by LB672 growing in a DM containing either different carbon sources (either glucose (DM), fructose (DMFr) or citrate (DMCi)) or fatty acids (FAs) additions (either oleic acid (DMO), linoleic acid (DML), or α-linolenic acid (DMLN)) under a range of fermentation conditions (time (at 0, 7, and 14 days) and temperature (at 25 and 35 oC)) were measured using PTR-ToF-MS, fastGC-PTR-ToF-MS, and GC-MS. The medium containing glucose (DM) generated a higher concentration of VOCs (m/z) after 7 days of fermentation, such as acetaldehyde (m/z 45.033) and ethyl acetate (m/z 89.060) at either 25 or 35 oC and methanethiol (m/z 49.011) at 35 oC compared to medium containing fructose (DMFr) at both temperature conditions. There was not a clear relationship between FAs and FA-derived VOCs, however, supplementation of DM with FAs altered the concentrations of VOCs produced from other components (glucose or AAs) in the DM, especially for linoleic supplemented DM (DML), which had higher concentrations of propanoic acid (m/z 75.043) at 35 oC, and acetaldehyde and ethyl acetate at 25 oC after 7 days of fermentation.
The ability of LAB to produce VOCs from various media is strain-dependent. In the fourth study, seven LAB strains (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)), which were readily available, food grade and not very fastidious in terms of nutrient requirements or growth temperatures, were grown in DM under a range of fermentation conditions (time (at 0, 7, and 14 days) and temperature (at 25 and 35 oC)) and the VOC they produced were assessed by PTR-ToF-MS, fastGC-PTR-ToF-MS, and GC-MS. As expected, VOCs produced differed depending on the LAB strains used, with the results being interpreted in conjunction with the scientific literature to indicate the likely presence or activity of metabolic enzymes across the LAB strains. For example: LP100 produced higher concentrations of diacetyl (m/z 87.043) at 35 oC and 2-heptanone (m/z 115.112) at either 25 or 35 oC compared to the other strains after 7 days of fermentation; the highest concentration of 3-methyl butanol (m/z 71.085) was obtained in PD661 ferment after 7 days of fermentation at 35 oC compared to the other strains; and methanethiol was higher in LB672 ferment after 7 days of fermentation at 35 oC compared to all other strains studied. Further, FAs (hexanoic (m/z 117.091), octanoic (m/z 145.123) and decanoic (m/z 173.154) acids) were higher in LU5335 ferments after 7 days at 35 oC compared to the other strains.
In the fifth study, the VOCs produced by three LAB strains (LB672, LP100, and PP100) growing singly in DM supplementation with individual AAs (either Leu, Ile, Thr, Met, Phe, Glu, or L-aspartic acid (Asp)) at 25 oC was investigated after either 0, 7, or 14 days. VOCs associated after LB672, LP100, and PP100 fermentation at 7 days in DM supplementation with specific AAs included: methanethiol, dimethyl disulfide (m/z 95.0003) with added Met; acetaldehyde with added Thr; and 2-methyl-2-butenal (m/z 85.064) with added Ile. However, no specific VOCs were enriched in the DM added with either Asp, Glu, Leu, or Phe.
In the final study, the effect of mixed LAB culture (LB672: LP100: PP100 = 1:1:1, v/v) on the VOC generation in the DM was examined at three time points (at 0, 7, and 14 days) at 25 oC. The concentration of VOCs detected in the mixed LAB fermentation was either higher or lower than that detected in monoculture fermentation. For example: the concentration of acetaldehyde was lower after 7 days in the DM fermented by the mixed LAB culture compared to its concentration by monoculture fermentation; and the concentration of acetic acid was higher in the DM fermented by the mixed culture compared to its monoculture fermentation. However, there were no significant (p>0.05) differences obtained between the mixed culture fermentation and LP100 monoculture fermentation.
Across the experiments, VOCs detected were dependent on the DM composition (AAs, carbon source, or FAs), the LAB strain (homo, hetero, or facultative heterofermentative) used, the fermentation temperature and time, and the different extraction and analysis techniques (online or offline) used.
The knowledge gained from this study will help scientists and food manufacturers better target the production of specific VOCs from plant-based substrates.