Flavor of Meat Substitutes

Biotechnology could prove to be a game changer in the production of natural flavors, helping to improve the organoleptic properties of vegan meat substitutes. Chemical analysis plays a key role in this context, particularly solvent free stir-bar sorptive extraction (SBSE) of flavor compounds from the fermentation broth followed by thermal desorption and GC-MS.

By: Guido Deußing

The Starting Point: Industry and science are working hard to match taste and aroma of plant-based meat substitutes as closely as possible to the original. Researchers at the University of Hohenheim, Germany are convinced that biotechnology plays a central role in the production of natural flavorings. Stöppelmann et al. [1-3] have developed a biotechnological fermentation process that specifically produces meat-like flavors from plants by fermenting yellow onions (Allium cepa L.) in submerged cultures with selected basidiomycetes.

A Brief Overview of the details: The ingredients for the biotechnological fermentation were the mycelium of a sulphur polypore (Laetiporus sulphureus) and finely chopped yellow onions (Allium cepa L.). Onions provide free, sulfur-containing amino acids such as cysteine and methionine, which are important for the Maillard reaction and thus precursors of meaty flavor. The fungal mycelium uses the sugar contained in onions as a fermentation substrate, i.e., as food, and metabolizes the molecules on offer from the onion into buttery creamy ketones, fatty meaty aldehydes, and alcohols. As Stöppelmann et al. report, the fermentation broth exudes meaty and tallowy-fatty odors characteristic of fried meatballs.

Tracking Meat Aroma: In the development and production processes, it is important to know both the composition of the aroma profile of the fermentation broth, and the aroma compounds that form the main characteristics of the intended meat-like flavor. In order to qualify and quantify the olfactory impression, Stöppelmann et al. conducted a sensory test with the help of trained panelists combined with chemical analysis of the fermentation broth with the characteristic fried meatballs odors. The researchers from the University of Hohenheim used thermal desorption gas chromatography (TD-GC) coupled to mass spectrometry (MS) and olfactory detection (O). TD-GC-MS/O analysis serves to qualify or semi-quantify aroma-active compounds. To this end, however, the aroma compounds and their precursors must first be extracted from the fermentation broth and converted into an analyzable form. The researchers chose stir bar sorptive extraction (SBSE) based on the GERSTEL Twister®, more specifically performing multiple SBSE (mSBSE) by inserting two Twister stir bars into the sample vial: One submerged in the sample, the other attached to the vial wall in the headspace above the sample.

Results of the SBSE-GC-MS/O Analysis: SBSE-GC-MS/O analysis of the fermented and unfermented onion main culture revealed a total of 62 odor-active regions in the chromatogram, of which 14 were detectable exclusively in the unfermented raw culture, eleven only in the fermented onion main culture, and 37 in both. The most frequently detected compound classes were aldehydes, followed by sulfur compounds, ketones, and lactones. Green and fatty odor impressions, attributable to aldehydes such as pentanal and undecanal, were reduced by fermentation. Also identified in the fermentation broth were (E,Z)-2,4-decadienal (fatty, talc-like) and benzothiazole (sulfur-containing, broth-like); these odor impressions dominated the sensory impression of the fermentation broth.

Key aroma compounds identified by GC/O: To determine how the sulfur polypore influences the aroma profile of the fermented onion main culture, Stöppelmann et al. conducted a semi-quantitative determination of the relevant odor-active compounds based on both an internal standard (2-methylthiophene) and a comparative determination with standard solutions of the aroma compounds to be determined. It was deemed necessary to determine the odor activity values (OAV). Olfactory detection in parallel with mass spectrometry played an important role in this study, as it enables the perception of odor impressions relevant to the aroma profile, even when the mass spectrometer does not record a signal due to insufficient sensitivity.

Conclusion: Stöppelmann et al. found that both the fermented and the unfermented onion main culture contained twelve compounds with OAVs greater than one, suggesting,, in the absence of synergistic effects, that these compounds contribute to the overall aroma profiles. Fermentation led to a shift in the aroma profile, with three alkadienals, which were not present in onions exhibiting the highest OAV in the fermented main culture. The aldehydes in question were: (E,E)-nonadienal (fatty, fried), (E,Z)-2,4-decadienal (fatty, talc-like), and (E,E)-2,4-decadienal (fatty, fried). Further investigation using automated headspace solid-phase microextraction GC-MS (HS-SPME-GC-MS; GERSTEL MPS, Agilent 8890 GC and 5977B Inert Plus MSD) revealed that (E,Z)-2,4-decadienal had the highest OVA in the fermentation solution with a "pie-like" aroma impression.

Final Comments: In their search for the reaction mechanism, Stöppelmann et al. discovered that linolenic acid, which is found in onions, was a potential precursor. This was confirmed experimentally by adding linolenic acid. According to the researchers, L. sulphureus enzymatically alters the fatty acid during fermentation, thereby shaping the aroma impression. In addition to (E,Z)-2,4-decadienal, they identified the sulfurous and broth-like odorant benziothiazole, which also contributes to the meaty, savory aroma.

Literature references

[1] Stöppelmann F, Chan LF, Hildebrand G, Hermann-Ene V, Vetter W, Rigling M, Zhang Y (2024). Molecular decoding a meat-like aroma generated from Laetiporus sulphureus-mediated fermentation of onion (Allium cepa L.). Food Research International 192: 114757. https://doi.org/10.1016/j.foodres.2024.114757

[2] Stöppelmann F, Zhang Yangyan (2025). Natürliche Fleischaromen durch Pilzfermentation: Basidiomyceten-Allium-Systeme im Scale-up und Anwendungstest. Deutsche Lebensmittel-Rundschau 121,8: 321-328

[3] Stöppelmann F, Chanc LF, Liang J, Greiß M, Lehnert AS, Pfaff C, Langen-berg T, Zhu L, Zhang Y (2023). Generation of Meaty Aroma from Onion (Allium cepa L.) with Polyporus umbellatus: Fermentation System, Sensory Profile, and Aroma Characterization. Journal of Agriculture and Food Chemistry 71: 13054-13065. https://www.doi.org/10.1021/acs.jafc.3c03153