# Combining sensory profiling and metabolomic approach to better understand the origins of bitter perception in faba bean (Vicia faba L. minor) fractions

Adeline Karolkowski, Christine Belloir, Christophe Martin, Géraldine Lucchi, Emmanuelle Meudec, Nicolas Sommerer, Emilie Bouzidi, Loïc Levavasseur, Christian Salles, Loïc Briand

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Adeline Karolkowski, Christine Belloir, Christophe Martin, Géraldine Lucchi, Emmanuelle Meudec, et al.. Combining sensory profiling and metabolomic approach to better understand the origins of bitter perception in faba bean (Vicia faba L. minor) fractions. Science Talks, 2024, 11, pp.100379. 10.1016/j.sctalk.2024.100379. hal-04977374

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# Combining sensory profiling and metabolomic approach to better understand the origins of bitter perception in faba bean (Vicia faba L. minor) fractions

Adeline Karolkowski<sup>a,b,*</sup>, Christine Belloir<sup>a</sup>, Christophe Martin<sup>a,c</sup>, Géraldine Lucchi<sup>a,c</sup>, Emmanuelle Meudec<sup>d,e</sup>, Nicolas Sommerer<sup>d,e</sup>, Emilie Bouzidi<sup>f</sup>, Loïc Levavasseur<sup>b</sup>, Loïc Briand<sup>a</sup>, Christian Salles<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Faba bean

Bitterness

Sensory profiling

Metabolomic approach

## ABSTRACT

Faba beans are promising alternatives to animal proteins due to environmental, food functional and nutritional benefits. However, they are characterised by off-flavours, notably off-notes and bitterness. Research on pulses and pulse-based products has largely focused on the involvement of volatile compounds in off-notes whereas the molecules responsible for pulse bitterness have not been studied as much. To better understand the role of non-volatile compounds in faba bean bitterness, different cultivars and air-classified fractions (flour, starch and protein) were investigated for their taste properties. Firstly, 21 trained panellists evaluated the bitter intensity of gels containing faba bean fractions. Bitter differences were highlighted according to the type of fractions and cultivars. Secondly, an untargeted metabolomics approach based on UHPLC-DAD-HRMS (ultra-high-performance liquid chromatography-diode array detector-high-resolution mass spectrometry) analysis was carried out for profiling the non-volatile content of the fractions. Thirdly, the sensory data was correlated with the metabolomic data resulting in the tentative identification of 42 phytochemical compounds (mainly alkaloids). Fourthly, the bitterness of highlighted compounds was studied using an *in vitro* cellular-based assay. Linking sensory attributes with the chemical content of pulse-based products allows to target compounds responsible for bitterness to propose strategies improving their flavour and the consumer acceptability.

Video to this article can be found online at https://doi.org/10.1016/j.sctalk.2024.100379.

<footnote><sup>a</sup> Centre des Sciences du Goût et de l'Alimentation, CNRS, INRAE, Institut Agro, Université de Bourgogne, F-21000 Dijon, France</footnote>

<footnote><sup>b</sup> Groupe Soufflet-Invivo, 10400 Nogent-sur-Seine, France</footnote>

<footnote><sup>c</sup> INRAE, PROBE Research Infrastructure, ChemoSens Facility, F-21000 Dijon, France</footnote>

<footnote><sup>d</sup> SPO, Université de Montpellier, INRAE, Institut Agro, 34000 Montpellier, France</footnote>

<footnote><sup>e</sup> INRAE, PROBE Research Infrastructure, PFP Polyphenol Analysis Facility, 34060 Montpellier, France</footnote>

<footnote><sup>f</sup> Vivien Paille (Groupe Avril), 59300 Valenciennes, France</footnote>

<footnote>* Corresponding authors at: Centre des Sciences du Goût et de l'Alimentation, CNRS, INRAE, Institut Agro, Université de Bourgogne, F-21000 Dijon, France.
E-mail addresses: adeline.karolkowski@inrae.fr (A. Karolkowski), christian.salles@inrae.fr (C. Salles).</footnote>
<figure><img src="image_2.png" /><figcaption>Fig. 1. Interests of faba beans (Vicia faba L. minor) [1,2].</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>Fig. 2. Faba bean market in France (2014-2020) [3].</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Fig. 3. Sensory profile of faba bean protein concentrates (intensity/10) and potential compounds responsible for pulse off-flavours, in particular bitterness [4–6].</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig. 4. Combination of sensory and untargeted metabolomic approaches to identify the non-volatile compounds responsible for faba bean bitterness.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 5. Faba bean seed transformation using air-classification.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 6. Schematic representation of the material and method used to identify non-volatile compounds responsible for faba bean bitterness.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 7.** Biplot representation of the PCA (centred reduced variables, Pearson correlation, $\alpha$ = 5.0%) of the detected compound areas in the negative mode and the perceived bitterness (as a supplementary variable in light green) of the 9 faba bean fractions. The compounds positively correlated with bitterness are related to variables in blue for the linear model and in pink for both the linear and logarithmic models, whereas the black highlighted compounds correspond to the mis-dereplicated data of the positively correlated compounds. S: starch fraction; F: flour; P: protein fraction – the number after the fraction corresponds to the cultivar (1, 2, or 3) [7].</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 8.** Biplot representation of the PCA (centred reduced variables, Pearson correlation, $\alpha$ = 5.0%) of the detected compound areas in the positive mode and the perceived bitterness (as a supplementary variable in light green) of the 9 faba bean fractions. The compounds positively correlated with bitterness are related to variables in blue for the linear model, in dark green for the logarithmic model and in pink for both the linear and logarithmic models, whereas the black highlighted compounds correspond to the mis-dereplicated data of the positively correlated compounds. S: starch fraction; F: flour; P: protein fraction – the number after the fraction corresponds to the cultivar (1, 2, or 3) [7].</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Fig. 9. Concentration-response curve for TAS2R16 stimulated by vicine and TAS2R16 and TAS2R42 stimulated by soyasaponin βb [8].</figcaption></figure>

### CRediT authorship contribution statement

**Adeline Karolkowski:** Writing – review & editing, Writing – original draft, Visualization, Validation, Resources, Investigation, Formal analysis, Conceptualization. **Christophe Martin:** Validation, Supervision, Resources, Methodology. **Géraldine Lucchi:** Validation, Supervision, Resources, Methodology. **Emmanuelle Meudec:** Writing – review & editing, Validation, Supervision, Resources, Methodology, Investigation. **Nicolas Sommerer:** Writing – review & editing, Supervision, Resources, Project administration, Conceptualization. **Emilie Bouzidi:** Resources. **Loïc Levavasseur:** Project administration. **Loïc Briand:** Writing – review & editing, Project administration, Funding acquisition. **Christian Salles:** Visualization, Validation, Supervision, Resources, Methodology, Investigation.

### Data availability

The authors do not have permission to share data.

### Acknowledgments

INRAE, Regional Council of Bourgogne Franche-Comté and the European Regional Development Fund (FEDER) are thanked for the equipment funding.

The PhD of Adeline Karolkowski was funded by Groupe Soufflet-Invivo (France), Groupe Avril (France), and ANRT (National Agency for Research and Technology, France) (ANRT-CIFRE 2019/1744).

### Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Adeline KAROLKOWSKI and Loïc LEVAVASSEUR were employed by the Groupe Soufflet-Invivo and Emilie BOUZIDI was employed by the Groupe Avril at the time of this experiment.

### References

[1] R. Amarowicz, A. Troszynska, N. Barylko-Pikielna, F. Shahidi, Polyphenolics extracts from legume seeds: correlations between total antioxidant activity, total phenolics content, tannins content and astringency, J. Food Lipids 11 (4) (2004) 278–286, https://doi.org/10.1111/j.1745-4522.2004.01143.x.

[2] Khrisanapant, Kebede, Leong, Oey, A comprehensive characterisation of volatile and fatty acid profiles of legume seeds, Foods 8 (12) (2019) 651, https://doi.org/10.3390/foods8120651.

[3] Terres Univia, La filière de la féverole en France, Terres Univia press release, 2021, http://www.terresunivia.fr/.

[4] A. Karolkowski, C. Belloir, L. Briand, C. Salles, Non-volatile compounds involved in bitterness and astringency of pulses: a review, Molecules 28 (8) (2023), https://doi.org/10.3390/molecules28083298 Article 8.

[5] A. Karolkowski, C. Martin, E. Bouzidi, J.-F. Albouy, L. Levavasseur, L. Briand, C. Salles, Heat treatment, cultivar and formulation modify the sensory properties and consumer acceptability of gels containing Faba bean (Vicia faba L. minor) protein concentrates, Foods 11 (19) (2022), https://doi.org/10.3390/foods11193018 Article 19.

[6] W.S.U. Roland, L. Pouvreau, J. Curran, F. van de Velde, P.M.T. de Kok, Flavor aspects of pulse ingredients, Cereal Chem. J. 94 (1) (2017) 58–65, https://doi.org/10.1094/CCHEM-06-16-0161-FI.

[7] A. Karolkowski, E. Meudec, A. Bruguière, A.-C. Mitaine-Offer, E. Bouzidi, L. Levavasseur, N. Sommerer, L. Briand, C. Salles, Faba bean (Vicia faba L. minor) bitterness: an untargeted Metabolomic approach to highlight the impact of the non-volatile fraction, Metabolites 13 (8) (2023), https://doi.org/10.3390/metabo13080964 Article 8.

[8] A. Karolkowski, C. Belloir, G. Lucchi, C. Martin, E. Bouzidi, L. Levavasseur, C. Salles, L. Briand, Activation of bitter taste receptors by saponins and alkaloids identified in faba beans (Vicia faba L. minor), Food Chem. 426 (2023), 136548, https://doi.org/10.1016/j.foodchem.2023.136548.

### Further reading

[1] R. Ferrer-Gallego, J.M. Hernández-Hierro, J.C. Rivas-Gonzalo, M.T. Escribano-Bailón, Sensory evaluation of bitterness and astringency sub-qualities of wine phenolic compounds: synergistic effect and modulation by aromas, Food Res. Int. 62 (2014) 1100–1107, https://doi.org/10.1016/j.foodres.2014.05.049.

[2] L. Heng, J.-P. Vincken, G. van Koningsveld, A. Legger, H. Gruppen, T. van Boekel, J. Roozen, F. Voragen, Bitterness of saponins and their content in dry peas, J. Sci. Food Agric. 86 (8) (2006) 1225–1231, https://doi.org/10.1002/jsfa.2473.

[3] N. Ishibashi, K. Sadamori, O. Yamamoto, H. Kanehisa, K. Kouve, E. Kikuchi, H. Okai, S. Fukui, Bitterness of phenylalanine- and tyrosine-containing peptides, Agric. Biol. Chem. 51 (12) (1987) 3309–3313, https://doi.org/10.1080/00021369.1987.10868574.

**Adeline Karolkowski** studied food science in Nancy (France). In 2018, she graduated from ENSAIA (French National Agriculture and Food Engineering School), where she specialised in food formulation. At the same time, she obtained a Master 2 Degree in biotechnology, food processing, and design of experiments from the University of Lorraine. Then, for 2 years, she worked as an R&D engineer in a start-up incubated by AgroParisTech in Massy (France), using the fermentation process to make plant-based cheeses. In September 2023, she obtained her PhD (sensory, analytical and cellular skills), which focused on understanding the mechanisms involved in the formation of off-flavours in faba bean fractions obtained by air-classification. This work was carried out at the INRAE/CSGA (French National Research Institute for Agriculture, Food, and Environment/Centre for Taste and Feeding Behaviour) laboratory in Dijon (France) under the supervision of Dr. Christian Salles and Dr. Loïc Briand, in collaboration with the Groupe Soufflet-Invivo (Nogent-sur-Seine, France) and the Groupe Avril (Valenciennes, France). https://www.researchgate.net/profile/Adeline-Karolkowski.

**Christine Belloir** is an engineer at the Centre for Taste and Feeding Behaviour (CSGA) in Dijon, France, and specializes in the functional study and biochemistry of olfactory and taste receptors. Her PhD is in biochemistry and molecular biology.

**Christophe Martin** is an INRAE engineer, specialized in sensory analysis. His education background includes a qualification in fermentation and biological engineering and in food and industrial engineering. In 2007, he obtained a degree in food biochemistry from the Conservatoire National des Arts et Métiers (CNAM, Dijon, France), then, he specialized in sensory analysis and consumer testing. He has been working since 2010 for the ChemoSens platform of Centre des Sciences du Goût et de l'Alimentation (CSGA, Dijon, France). He is the author of 37 scientific articles, 1 patent, three book chapters (h-index: 16). https://www.researchgate.net/profile/Christophe-Martin.

**Géraldine Lucchi** is a research engineer at INRAE. She obtained a university degree in plant physiology in 1996 and specialized in mass spectrometry four years later. Until 2017, she contributed her expertise in proteomics to various public institutes (INRA, INSERM, university hospitals), before being recruited by the CSGA. She works on the ChemoSens platform, which develops methods and tools for the physico-chemical and sensory characterization of food. She is currently responsible for the mass spectrometry, olfactometry, and gustometry division.
**Emmanuelle Meudec** is an engineering assistant at INRAE/PFP (Polyphenol Analysis Facility). She is specialized in the analysis of polyphenols by mass spectrometric methods.

**Nicolas Sommerer** is a research engineer and head of INRAE/PFP (Polyphenol Analysis Facility). He is a specialist of mass spectrometry based analytical approaches. He contributed to research on plant proteomics, and plant and food metabolomics.

**Emilie Bouzidi** is the R&D manager of Vivien Paille (Groupe Avril) located in Valenciennes (France).

**Loïc Levavasseur** is the scientific director of the Group Soufflet-Invivo Research & Innovation Centre (CRIS) located in Nogent-sur-Seine (France).

**Loïc Briand's** research deals with the molecular mechanisms underlying the chemosensation of tasting and odorant molecules. He is interested in olfactory receptors and taste receptors (umami, sweet, and bitter), as well as sweet-tasting and taste-modifying proteins, such as miraculin and gurmarin. He is also investigating the impact of odorant-binding proteins on olfactory perception and the use of these proteins for the development of biosensors. More recently, his research has focused on the role of taste receptors located in other organs throughout the body. Dr. Loïc Briand received his PhD in biochemistry and molecular biology

from Nantes University in France. After a postdoc at Glasgow University in the UK, he became interested in olfaction and joined the INRAE centre of Jouy-en-Josas (Paris) as a Research Associate in 1999. In 2007, Loïc Briand joined the Centre for Taste, Smell and Feeding Behaviour (CSGA) in Dijon, France, and started his own research group. In 2021, he was appointed Director of the CSGA. Loïc Briand is a member of the European Chemoreception Research Organization (ECRO) board. He is the author/co-author of more than 70 peer-reviewed articles, more than 10 reviews in the chemosensory area, and over 12 book chapters.

**Christian Salles** is research director at INRAE. His main research field carries on in-mouth mechanisms leading to flavour release and perception with in vivo and in vitro approaches. He coordinated the development of chewing simulators dedicated to in vitro approaches of temporal flavour release and food breakdown studies in controlled eating conditions. He has coordinated several projects related to the reduction of salt content in food and in particular a European project (TeRiFiQ - from 1/2012 to 12/2015) on the reduction of salt, fat and sugar in food, with 17 European partners including 11 SMEs. These last years, he focused his research on the temporal release of sodium according to food matrix composition and oral condition. He is the author of 80 publications in peer-reviewed journals, 117 communications in scientific congresses (oral and poster) and the author of 53 book chapters. He is the editor of 2 books: Flavour, from food to perception (Wiley Blackwell, 2017) and Flavor: From Food to Behaviors, Wellbeing and Health (Woodhead Publishing, 2016).