# Sweet taste induced by miraculin from Sisrè pulp (Synsepalum dulcificum Schumach. & Thonn.) from Benin as affected by heat treatment

Médérice Sènanmi Béhanzin, Adrien Servent, Laurent Adinsi, Charmelle Cyria Dahou, Ifagbémi Bienvenue Chabi, Polycarpe Kayodé, Manuel Dornier

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Médérice Sènanmi Béhanzin, Adrien Servent, Laurent Adinsi, Charmelle Cyria Dahou, Ifagbémi Bienvenue Chabi, et al.. Sweet taste induced by miraculin from Sisrè pulp (Synsepalum dulcificum Schumach. & Thonn.) from Benin as affected by heat treatment. Applied Food Research, 2026, 6 (1), pp.101646. 10.1016/j.afres.2025.101646. hal-05447231

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# Sweet taste induced by miraculin from Sisrè pulp (*Synsepalum dulcificum*) Schumach. & Thonn.) from Benin as affected by heat treatment

Médérice Sènanmi Béhanzin<sup>a,b</sup>, Adrien Servent<sup>b,c</sup>, Laurent Adinsi<sup>a,d</sup>, Charmelle Cyria Dahou<sup>a</sup>, Ifagbémi Bienvenue Chabi<sup>a</sup>, Polycarpe Kayodé<sup>a,*</sup>, Manuel Dornier<sup>b,* ID</sup>

## ARTICLE INFO

Keywords:

Miraculin

Heating

Sweetness inducing

Sweetness persistence

## ABSTRACT

This work focused on the effect of heating treatments on the sweetening property of miraculin contained in Sisrè, the fruit of *Synsepalum dulcificum*, from Benin. The aim was to define suitable conditions for stabilizing the fruit pulp, at a lower cost while preserving its functional properties. The fruits, collected in the locality of Sèhouè in the Republic of Benin, were frozen whole, pulped and then stored at -20 °C before processing and analyzing. Heat treatments ranging from 40 to 70 °C were applied to the fresh pulp for 5 min. Sensory tests were performed on the heated pulp samples to test the functionality of miraculin and determine the remanence time of the induced sweet taste after treatment at different temperatures. Reverse-phase and steric-exclusion chromatography, in association with electrophoresis were performed to analyze the miraculin content of the pulps, and the generated data were compared with the sensory results. Sensory analysis showed that the sweetening effect of the native pulp before heat treatment lasted at least >1 h 30 min. A significant loss of functionality after exposure of the pulp to temperature values above 50 °C was observed. The results of analyses also showed that from 50 °C, the miraculin concentration decreased significantly. At temperature values above 50 °C, miraculin completely lost its functionality even if the pulp still contained miraculin, probably in its monomeric form that are not active. The intensity and remanence of the sweetening effect of miraculin depended on its concentration. These results demonstrated that the pulp of Sisrè cannot be stabilized by thermal pasteurization and that drying at low temperatures between 40 and 50 °C could be a good technological option for preserving its functionality.

## 1. Introduction

*Synsepalum dulcificum*, is a plant native to West and Central Africa (Tchokponhoué et al., 2020). Its fruit also called Sisrè in the local Fon language of Benin, is known for its unusual ability to transform the sour taste of food into a sweet taste after consumption of its pulp. Sisrè contains approximately 80 % of water (Béhanzin et al., 2025), and is highly perishable fruit if stored at room temperature (Doddawad et al., 2022). Its property of modifying sour taste into sweetness is due to the presence of miraculin in the fruit pulp. Miraculin is a glycoprotein exhibiting a sweetening potency approximately 400,000 times greater than that of sucrose, on a molar basis (Gibbs et al., 1996; Rodrigues et al., 2016; Swiader et al., 2019). Although not inherently sweet,

miraculin activates sweet taste receptors under acidic conditions, typically at pH below 6. Miraculin is found in non-active monomeric form and in dimeric and tetrameric forms, both active at acidic pH (Diyapaththugama et al., 2024). The sweetening effect induced by miraculin was reported to persist in the mouth for 30 min to 2 h (Asakura et al., 2011; Brouwer et al., 1968; Choi & Garza, 2020; Swiader et al., 2019). Miraculin is also low in calories (Choi & Garza, 2020) and does not cause dental caries (Faus, 2000), thus the fruit is attracting great interest from the food, cosmetic and pharmaceutical industries (Achigan-Dako et al., 2015) as a natural sweetener and taste enhancer ingredient (Andrade et al., 2019; Rodrigues et al., 2016; Wong & Kern, 2011). Some sensory studies have demonstrated the effect of miraculin on product perception with sour, bitter, sweet and salty tastes (Andrade

<footnote><sup>a</sup> Labio, DNSTA, Faculté des Sciences Agronomiques, Univ. Abomey-Calavi, Benin</footnote>

<footnote><sup>b</sup> QualiSud, Univ. Montpellier, CIRAD, Institut Agro Montpellier, IRD, Univ. Avignon, Univ. La Réunion, France</footnote>

<footnote><sup>c</sup> CIRAD, UMR QualiSud, F-34398 Montpellier, France</footnote>

<footnote><sup>d</sup> Laboratoire des Sciences et Technologie des Aliments et Bioressources et de Nutrition Humaine (LaSTABNH), Ecole des Sciences et Techniques de Conservation et de Transformation des Produits Agricoles, Univ. Nationale d'Agriculture, BP 114 Sakété, Benin</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: kayodepolycarpe@gmail.com (P. Kayodé), manuel.dornier@institut-agro.fr (M. Dornier).</footnote>
et al., 2019; Capitanio et al., 2011; Choi & Garza, 2021; Choi & Park, 2023; Hudson et al., 2018; Lipatova & Campolattaro, 2016). Particularly, it was reported a change from sour to sweet taste after consumption of miraculin, without significant change on other types of tastes. These properties of miraculin would help people suffering from obesity and type II diabetes with their low-calorie diets by replacing sugar with miraculin products (Chen et al., 2006; Choi & Garza, 2020; Rodrigues et al., 2016; Wong & Kern, 2011). Nowadays, there is an increasing demand of consumers for natural, economical and low-calorie sweeteners as a substitute for sugar (Doddawad et al., 2022). Sisrè is reportedly known as the only plant species able to naturally produce miraculin (Kurihara & Beidler, 1968), and therefore is the ideal resource to meet this need. In addition to the perishability of the fruit, there are challenges in extracting and purifying its native miraculin Brouwer et al. (1968); Gibbs et al. (1996). To date, the yield of miraculin obtained from the pulp has rarely exceeded 0.36 % of freeze-dried pulp (He, Tan, Abbasiliasi et al., 2015; Kurihara, 1992; Sun et al., 2006).

A possible solution to overcome this constraint would be to use the
pulp directly as miraculin source. This urges for the development of
appropriate technique to stabilize the Sisrè pulp. The most widely used
methods for Sisrè pulp stabilization include freezing at -12 °C, which
preserves miraculin activity for at least one month (Osseyi et al., 2008)
as well as freeze-drying or freezing at -29 °C (Doddawad et al., 2022).
However, these methods require significant technical, financial and
energy resources. Alternative stabilization methods such as conven-
tional drying or pasteurization may be worth exploring due to their
comparatively lower cost and resource requirements. However,
although it has been reported that miraculin is heat-sensitive (Brouwer
et al., 1968; Choi & Garza, 2020) no precise quantitative data could be
found on the impact of temperature on this glycoprotein's concentration
in the pulp as well as on its sensorial functionality. This study is there-
fore designed to evaluate the effects of heat treatments on the func-
tionality of miraculin of Sisrè pulp. Both sensory evaluation methods
and instrumental measurements were used to assess the glycoprotein
structure modifications and functional property.

## 2. Materials and methods

2.1. Plant material

The miracle berries were collected from Sèhouè markets in Benin
over a period of 4 months (December 2023 to March 2024). The berries
were sorted and washed 3 times with water. They were then left to drain
on stainless steel racks for 20 min and chilled down to 16 °C. These
operations lasted 2 – 4 h depending on the quantity of fruits processed.
They were weighed in polyethylene bags and kept whole at –20 °C until
the end of collection period. The seeds were manually removed from the
frozen fruits and the pulp with the skin was returned to the freezer at
–20 °C for at least 24 h before undergoing the crushing process at
22,000 – 26,000 rpm with meat crusher (Kesun / PY-8960, Jiangsu,
China). The crushed material was collected in a large container and
mixed with a spatula to homogenize the pulp samples. The various
following experiments were performed using this homogeneous stock.

## 2.2. Sample preparation

The crushed pulp was divided into 750 g batches distributed in 5
sealed aluminum-polyethylene heat-resistant bags and heated in a water
bath at 40, 45, 50, 60 and 70 °C for 5 min. The heated pulp was then
immediately immersed in an ice-cold water bath for 5 min to stop the
heat treatment.

The temperature interval (40 – 70 °C) used in the experiment was
chosen based on previous investigations which revealed the loss of
miraculin content at 70 °C (Béhanzin et al., 2025) and a total loss of pulp
functionality recorded at the same temperature thanks to a preliminary
sensory test with 5 participants.

The lowest temperature was chosen according to the ambient tem-
perature (30 – 40 °C) prevailing in the Sisrè growing environment
(Akinmoladun et al., 2020). At the end of heating, for each temperature,
2.5 g of heated pulp (representing among 3–5 whole fruit’s pulp) was
weighed in a polyethylene film, rolled to form a ball and frozen at –20
°C to constitute the portions to be used for the sensory analysis. Like-
wise, a sample of 2.5 g of unheated pulp was prepared as described
above and was used as a control for the various investigations. This pulp
quantity was chosen to ensure that panelist will consume a sufficient
quantity of miraculin during sensory analysis. The remaining pulp was
stored at –20 °C for instrumental analyses.

## 2.3. Sensory analysis

2.3.1. *Ethical assessment and consent*

The protocol used for this study was approved by the ethics com-
mittee of the University of Abomey-Calavi. Informed consent was ob-
tained from the participating panelists. They were informed that their
participation was entirely voluntary, and they could withdraw from the
panel at any time.

2.3.2. *Panel selection test and training*

A total of fifteen panelists were recruited based on their familiarity
with the basic tastes (sweet, sour, salty and bitter). The panelists first
attended four sessions, on two consecutive days, aiming to test their
ability to discriminate the sour, bitter, sweet and salty taste. Six dilutions
of the same taste were prepared at different concentrations following the
method used by Maraval et al. (2018). For sour taste, the dilutions were
obtained from citric acid solution (food grade anhydrous citric acid,
TTCA Co., China) with concentrations ranging from 0.16 to 0.48 g·L⁻¹.
Quinine sulfate solution (Pharmaquick, ABT SA, Cotonou, Benin) with
concentrations from 1.4 to 8.4 mg·L⁻¹ was considered for bitter taste
while, sweet taste was evaluated from sucrose solution (white powdered
sugar Bio Daddy®, Cristalco, Paris, France) with concentrations varying
from 1.88 to 11.20 g·L⁻¹. Salty taste was evaluated from sodium chlo-
ride solution (iodine-free, anti-caking table salt, Le Guérandais®, SCA
Les Salines de Guérande, France) with concentrations between 0.24 and
1.40 g·L⁻¹. All solutions were prepared using bottled drinking still water
(FIFA de Ste Luce, ETE SA, Cotonou, Benin) with a pH of 5.3. The six
dilutions of the same taste coded with a random three-digit number,
were presented in one session to each panelist who must rank them from
the least concentrated to the most concentrated while recognizing the
taste. Based on the results, only 9 panelists (5 males and 4 females; age
range: 22–30 years; mean age: 27 years) were selected to participate in
the second step of sensory analysis. Subsequently, the nine panelists
were semi-trained during two sessions on how to (i) define clearly each
taste, (ii) use the scoring scale correctly, and (iii) improve their ability to
discriminate between tastes. For evaluation of each taste, panelists
received 10 mL of each solution simultaneously. A glass of water was
provided, so that the panelists could rinse their mouth between tasting
each sample. Both steps of sensory analysis were carried out in the
morning, with panelists arriving on an empty stomach and drinking at
least 250 mL of water before starting the tests, to avoid stomach irrita-
tion by the tested solutions. All samples in selection test and in the
following main tests were presented in random order, and testers rated
the intensity of each solution by putting a mark on a 10-cm unstructured
linear scale anchored at the ends (minimum rating at the left end to
maximum rating at the right end).

2.3.3. *Experimental design 1: effect of miraculin on the four basic tastes*

The solution concentrations were chosen as the maximum used for
panelist selection by Maraval et al. (2018) and were also included in the
interval of sour, bitter, sweet and salty solution used by Bartoshuk et al.
(1969) to test miraculin activity in relation to these tastes. A citric acid
solution at 0.6 g·L⁻¹ (pH 2.59), a quinine sulfate solution at 12 mg·L⁻¹
(pH 5.54), a sucrose solution at 16 g·L⁻¹ (pH 5.24) and a sodium
chloride solution at 2 g·L⁻¹ (pH 6.07) were prepared the day before testing sessions and stored at 4 °C.

The test was carried out twice at different days. At each session, the 4 solutions were tested monadically. For each solution, 10 mL were presented to panelist to drink progressively and evaluate the intensity. Thereafter, 2.5 g of unheated fresh pulp was served to panelist in order to chew and spread over their entire tongue and to rate the sweet taste intensity.

This test was conducted to evaluate the effect of miraculin on taste modalities other than sourness. Subsequent tests focused on characterizing the pulp, determining the remanence time of the effect and assessing the impact of heat on the functional stability of miraculin.

### 2.3.4. Experimental design 2: miraculin sweetness and remanence time

The citric acid solution with a concentration of 0.01 M and pH = 2.3 was prepared according to Capitanio et al. (2011) the day before the test and kept refrigerated at 4 °C.

The test was carried out twice on two separate days. For the first evaluation, each panelist received 10 mL of citric acid solution and rated sweetness, acidity and a potential third taste (to be specified if detected). In second evaluation, a 2.5 g frozen ball of crushed unheated fresh pulp was presented to panelist to chew and spread over their entire tongue and to rate the intensity of the three aforementioned taste. Thereafter, 10 mL of citric acid solution was presented every 30 min (t₀, initial time) until 2 h 30 min. In total, 6 samples of the same citric acid solution were presented and the intensity of the three tastes was rated after tasting of each solution. The duration of 2 h 30 min was chosen based on preliminary tests conducted with 5 participants, in which the presence of miraculin effect was recorded over 3 h.

To determine the remanence time, defined as the time required to reach 50 % loss of the induced sweet taste intensity (I₁/₂) from its initial value at t₀ (first citric acid solution tasting time after pulp ingestion), the 'half-life' (t₁/₂), was calculated by linear interpolation between the two adjacent sweet intensity values surrounding I₁/₂. The same approach was applied to the sour taste, but in this case, the time corresponding to a 50 % recovery of the perceived sourness intensity—comparing pre- and post-ingestion levels at t₀—was estimated.

### 2.3.5. Experimental design 3: impact of heat on the functional properties of miraculin and remanence time

A citric acid solution with a concentration of 0.01 M and pH = 2.3 was prepared the day before the test and kept refrigerated at 4 °C.

Pulps previously heated to 40, 45, 50, 60 and 70 °C were used for this experiment. One day was devoted for testing each heated pulp. Two repetitions were performed for each heated pulp on random days, resulting in a total of 10 days for the five pulps. For the first evaluation, each panelist received 10 mL of citric acid solution and rated sweetness, acidity and a potential third taste (to be specified if detected). In second evaluation, 2.5 g frozen ball of heated pulp was presented to panelist to chew and spread over their entire tongue and to rate the intensity of the three aforementioned taste. Thereafter, 10 mL of citric acid solution was presented every 30 min (t₀, initial time) until 2 h 30 min. In total, 6 samples of the same citric acid solution were presented and the intensity of the three tastes was rated after tasting of each solution.

The remanence time was determined as described above for each of the heated pulps.

## 2.4. Analytical measurements

Instrumental analyses were carried out on the freeze-dried powders of the different pulp samples generated for the sensory analysis. These were fresh freeze-dried pulp (untreated pulp), coded PNT, and fresh pulp heated at 40, 45, 50, 60 and 70 °C and then freeze-dried, coded PT40, PT45, PT50, PT60 and PT70 respectively.

### 2.4.1. Sample extraction for miraculin analysis

Miraculin extraction was carried out as described by He, Tan, Lai et al. (2015) with some modifications (Béhanzin et al., 2025). A first extraction with water was performed by adding 40 mL of distilled water to 6 g of pulp powder weighed in a centrifuge tube. The mixture was homogenized for 2 min by vortex and then centrifuged (Beckman Coulter, Avanti J-E Centrifuge) at 12,000 x g for 30 min. The supernatant was discarded and a second extraction with distilled water was carried out on the pellet obtained. After homogenization and centrifugation under the same conditions, the supernatant was discarded and 30 mL of extraction solvent consisting of 0.5 M NaCl at pH 6.8 adjusted with HCl was added to the pellet. The mixture was homogenized and centrifuged under the same operating conditions as above. The supernatant representing the crude miraculin extract was collected in another centrifuge tube and stored at 4 °C for further analyses. All extraction procedures were performed at room temperature.

### 2.4.2. High performance liquid chromatography analysis

The quantification of miraculin was carried out by RP-HPLC according to the method used by Demesyeux et al. (2020) with some modifications (Béhanzin et al., 2025). A C₁₈ column 250 × 4.6 mm, 5 µm (Kinetex, Phenomenex, USA) was used. The column was equilibrated with milliQ water containing 0.1 % trifluoroacetic acid TFA (phase A) 95 % for 5 min and then 20 µL of sample extract was injected. A concentration gradient from 5 to 70 % of acetonitrile (v/v) containing 0.1 % TFA (phase B) was applied for elution for 35 min followed by an increase to 100 % acetonitrile for 10 min. Phase A was then used to reequilibrate the column 95 % for 10 min. The total duration of the analysis was 60 min with a flow rate of 0.7 mL·min⁻¹ and the absorbance were measured at 214, 280 and 325 nm using a Diode-Array Detector (1200 series, Agilent, USA) at 30 °C.

### 2.4.3. Determination of miraculin concentration

The miraculin concentration was calculated based on data relating to the miraculin peak identified by applying Beer Lambert's law at 214 nm (Béhanzin et al., 2025; Defaix et al., 2019), wavelength at which proteins are more sensitive and interference from solvents and biological buffer components is limited (Noble, 2014). The molar extinction coefficient of miraculin ε at 214 nm used for calculation was 478 999 mol⁻¹·L·cm⁻¹.

### 2.4.4. SDS-PAGE analysis

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed on the crude miraculin extracts under reducing (breaking of disulfide bonds) and non-reducing conditions (preservation of disulfide bonds) according to Defaix et al. (2019) and Béhanzin et al. (2025) with slight modification. A pre-casted Novex™ 4 - 20 %, Tris-glycine Plus WedgeWell™ Gel (1.0 mm × 10 well) was used. Under reducing conditions, 15 µL of extracts previously diluted ½ in Laemmli buffer mixed with 50 µL β-mercaptoethanol and heated in a dry bath at 95 °C for 5 min, were deposited in the wells. Under non-reducing conditions, 50 µL of extracts were added to 950 µL of Laemmli buffer and heated in a dry bath at 95 °C for 5 min before an aliquot of 15 µL of this mixture was deposited in the wells.

Three microliters of Precision Plus Protein™ Standards size marker mix (BIO-RAD Catalog #161–0363), allowing determination between 10 and 250 kDa, were also placed in a well for the both reducing and non-reducing conditions. Electrophoresis was carried out in a Mini Gel Tank, Invitrogen by Thermo Fisher Scientific (REF A25977) at constant voltage 200 V for 55 min (until the colored indicator contained in the deposited sample reached the base of the resolving gel). The plate-gel assembly was placed in an osmosis water bath to remove the gel from the mold. It was then placed in a tray and covered with a Ready Blue™ Protein Gel Stain solution (Sigma-Aldrich, USA) and left overnight away from light. The next morning, the staining solution was removed and the colored gel was rinsed with osmosis water and subjected to weak
destaining for few minutes or hours with an osmosis water solution containing 5 % acetic acid (v/v) if the gel is too much colored.

On a black-white image, framed around the 25 kDa band, where a total of around 9000 pixels were considered, a greyscale analysis was carried out using Chat GPT (OpenAI). This consists in evaluating the distribution of light intensities for each image pixel, considering 0 for black and 255 for white. The number of pixels in the image corresponding to each gray level was counted, and the values of the darkest pixels (minimum values) and brightest pixels (maximum values) were determined for each image. The images treated corresponded to the results of SDS-PAGE under reducing conditions for the PNT, PT40, PT45, PT50, PT60 and PT70 samples. As the band is darker than the gel, the darkest values represent the band and the lightest the gel background, the latter being identical for all processed images. Between 3 and 4 levels of gray were identified for the different images. Percentages of pixels below 100 (darker tones) were considered to compare band intensities between samples. The values obtained were called indexes of band intensity (IBI).

### 2.4.5. Native electrophoresis

A non-denaturing electrophoresis was carried out according to the protocol described in the manufacturer's manual (Pub. No. MAN0014610; ThermosFisher Scientific; NuPAGE Bis-Tris & Bolt Bis-Tris Plus Gels - BJ, s. d.) on the concentrates (×5) of the different extracts. Twenty microliters (20 µL) of extracts were added to 20 µL of Tris-Glycine Native Sample Buffer (×2) Novex™ and 25 µL of this mixture were loaded in the Pre-casted NuPAGE™ 10 % Bis-Tris Gel (1.0 mm × 15 well). Three µL of Precision Plus Protein™ Standards size marker mix (BIO-RAD Catalog #161-0363) was also placed in a well. Electrophoresis was carried out in a Mini Gel Tank, Invitrogen by Thermo Fisher Scientific (REF A25977) at constant voltage 200 V for 55 min. To avoid any heating of samples or running buffer during the analysis, the tank was placed in an ice bath and the Native Running buffer (×1) was pre-cooled to 4 °C before starting of the analysis.

### 2.4.6. Size exclusion chromatography (SEC)

Size-exclusion chromatography was performed according to Kamoun et al. (2003) with some modification. A Proteins KW-802.5 column (250 × 4.6 mm, 5 µm; Shodex, Japan) was used. A 20 µL aliquot of crude extract was injected into the column and eluted with a 0.1 M phosphate buffer (pH 7.0) at a flow rate of 1.0 mL·min⁻¹. The total runtime was 20 min, and absorbance was monitored at 280 nm using a diode-array detector (1200 series, Agilent, USA).

## 2.5. Statistical analysis

The data collected were processed with MS-Excel and the analysis of variance was performed and pair comparison with Fisher test using X1stat 2019.2.2 statistical software (p < 0.05).

# 3. Results and discussions

## 3.1. Sweet taste inducing capacity of miraculin on various tastes

After consumption of Sisrè fresh pulp, sweetness induction was significantly observed for sour, bitter and salty solutions (Table 1).

<table><caption>**Table 1**
Sweet taste induction (sensory scores) capacity of fresh Sisrè pulp after ingestion of various solutions.</caption><thead><tr><th>Sweet taste induction</th><th>Sour solution</th><th>Bitter solution</th><th>Sweet solution</th><th>Salty solution</th></tr></thead><tbody><tr><td>Before fresh pulp</td><td>0.0 ± 0.0<sup>b.s</sup></td><td>0.0 ± 0.0<sup>b</sup></td><td>3.5 ± 1.9<sup>a</sup></td><td>0.0 ± 0.0<sup>b</sup></td></tr><tr><td>After fresh pulp</td><td>5.1 ± 2.9<sup>a</sup></td><td>1.5 ± 1.9<sup>a</sup></td><td>4.7 ± 1.6<sup>a</sup></td><td>1.9 ± 1.9<sup>a</sup></td></tr></tbody></table>

Sweetness induction was more pronounced from sour solution while the consumption of fresh pulp did not increase the sweetness of the sweet solution.

According to Paladino et al. (2008), miraculin modifies sour taste into sweet taste via its interaction with the taste receptor T1R2–T1R3, a G-protein coupled receptor (located on the inner face of the plasma membrane). Indeed, at acidic pH, miraculin adopts a so-called open conformation that exposes histidine residues (His-29 and His-59) and creates a basic region on its surface. This basic region interacts electrostatically with the negative cavity of the receptor, which triggers its activation and induces the perception of sweet taste in the presence of sour food. Miraculin therefore behaves as an agonist in the presence of a low pH solution (Misaka, 2013) and as an antagonist at neutral pH. At low pH, the complete activation of the receptor sends signals to the brain, through a cascade of internal chemical reactions, indicating that the consumed acidic compound is “sweet”. Miraculin would therefore only deceive the brain about the real nature of the acidic food consumed. Therefore, the high intensity of the sweetness perceived in the acidic solution compared to the other tastes tested would be distinctively attributable to it inherent low pH. This result was consistent with data reported by Lipatova and Campolattaro (2016), (Rodrigues et al., 2016), (Misaka, 2013), (Capitanio et al., 2011), (Koizumi et al., 2011) who indicated that the functionality of miraculin is pH-dependent. A 40 % decrease in the initial acidity of the sour solution was recorded after consumption of miraculin possibly, because of a masking of the acidity by the sweet taste. Such a phenomenon is well known when adding sugar to an acidic drink such as lemonade. The sweetness intensity of the sweet solution in the presence of miraculin did not increase significantly certainly because of the neutral pH of the solution not allowing the adequate miraculin's conformation to be reached in order to stimulate the receptors. In addition, the fact that the sweet taste receptor being already coated by miraculin would have prevented the sugar from associating with it as is the case for other sweeteners (aspartame, saccharin, etc.) or sweetening proteins once the miraculin is attached to the receptor (Koizumi et al., 2011; Misaka, 2013). Capitanio et al. (2011), also reported an unchanged sweet intensity of the sucrose solution after administration of miraculin. Furthermore, a decrease in the perceived salty taste after consumption of a salt solution in the presence of miraculin was highlighted without inducing sweetness. The statement was that miraculin have caused a sweet taste by acting on saliva, which, in turn, would have suppressed the salinity. Nevertheless, it was added that this theory, if effective, should normally affect the bitterness of the bitter solution tested, which was not the case in this study. This difference was therefore attributed in response to the fact that sweetness would have more stable suppressive effects on salinity at low concentrations, than on bitterness as suggested by Keast and Breslin (2003). Unlike these studies, for salty and bitter tastes, a significant sweetness was recorded in the present experiment. Since these solutions had almost a neutral pH (6 and 5.5 respectively), therefore it might not lead to an induction of sweet taste in the presence of miraculin. The literature has not revealed this kind of phenomenon to our knowledge, which suggests that in-depth studies could be carried out to explain the mechanism by which this reaction is obtained with NaCl and quinine in the presence of miraculin. But, (Capitanio et al., 2011) showed that the sweetness was induced by binary mixtures of these tastes with a citric acid solution. This result raises a question regarding the state of other taste receptors after addition of miraculin to the tongue and the processing of information by the nervous system in the identification of perceived tastes. Two main ideas have long been debated regarding the strategy used by the taste system to represent basic tastes. One suggesting that basic tastes are represented by successive populations of nerve fibers specific to a sensory quality (labeled lines) (Yarmolinsky et al., 2009) and the other by populations of neurons sensitive to several basic tastes (code combination) (Lemon & Katz, 2007). But in 2015, a study by Reiter et al. (2015) suggested that the taste system generates representations of individual chemical substances, not basic taste

<footnote>* Letter's comparison was done in column (p < 0.05).</footnote>
categories. The sensory information elicited by a taste stimulus would therefore be encoded by the activation of several different fibers representing a taste quality.

The following sections focused on the sweetening effect induced by miraculin in the presence of the acid solution.

## 3.2. Profiling the sensory characteristics of the various heated pulps studied

Preliminary analyses helped establishing the sensory profile of the heated Sisrè pulp. The panelists found that the fresh pulp had an acidic, sweet and slightly bitter taste. The fresh pulp and all the heated pulps were found to have similar acidic and bitter tastes (Table 2). The fresh pulp and those heated at 40 °C (PT40) and 45 °C (PT45) showed comparable levels of sweetness, which were higher than the sweetness intensity observed in pulps heated to 50, 60, and 70 °C. It was also observed that all heated pulps exhibited a visually brown hue after heating, whereas unheated pulp was pink. These differences could be attributed to Maillard reaction at the highest temperatures inducing the browning observed in heated pulp and the drop in sweetness; owing to the modification of reducing sugars by the heat treatment. As Manayay and Ibarz (2010) reminded, this non-enzymatic reaction is very favored during heating of foods containing reducing sugars and amino compounds. Finally, it cannot be excluded that enzymatic browning reactions may also occur in the pulp after thawing (Manayay & Ibarz, 2010).

## 3.3. Remanence time of the sweet taste induced by miraculin from fresh sisrè pulp

After ingestion of 2.5 g of fresh pulp by the panelists, the intensity of the sweet taste, which was almost zero at the starting point following the ingestion of acidic solution, increased to 7 on a scale of 10. In the opposite, the intensity of the sour taste, initially at the level of 7, dropped to around 2 (Fig. 1a). This perception corresponds to miraculin's typical functional property of imparting a sweet taste to acidic foods. Thirty min after pulp consumption, the intensity of the sweet taste decreased gradually to reach the level 2 after 120 min. Conversely, the intensity of the acidic taste reached the level 5.5 after 90 min. Thereafter, the intensities of both tastes remained almost constant up to 150 min. These results showed that under the present experimental conditions, ingestion of the Sisrè pulp increased the perceived sweetness following the consumption of acidic solution by a factor 70 and the intensity of perceived acidic taste was 3 times lower than the intensity perceived before eating Sisrè pulp.

From the experiment, it clearly appeared that the sweetening effect lasted for at least >2 h 30 min. However, the sweetness had decreased by half after about 1 h 30 min and the sour taste increased by half after around 1 h 10 min Fig. 2. Based on the time required to reach 50 % loss of the induced sweet taste intensity ($I_{1/2}$) at $t_0$ (immediately after pulp ingestion), referred to the half-life ($t_{1/2}$), our experiment suggested that the remanence time of the sweet taste induced by native miraculin in Sisrè pulp was 1 h 30 min. This remanence time was similar to the one (1

<table><caption>**Table 2**
Sensory characteristics (scores) of the tested Sisrè pulps.</caption><thead><tr><th>Taste</th><th>Fresh pulp</th><th>PT40</th><th>PT45</th><th>PT50</th><th>PT60</th><th>PT70</th></tr></thead><tbody><tr><td>Sour taste</td><td>5.3 ± 3.4 <sup>a*</sup></td><td>4.3 ± 3.0 <sup>a</sup></td><td>5.2 ± 2.7 <sup>a</sup></td><td>5.3 ± 3.0 <sup>a</sup></td><td>5.9 ± 2.4 <sup>a</sup></td><td>5.2 ± 2.8 <sup>a</sup></td></tr><tr><td>Sweet taste</td><td>3.8 ± 2.2 <sup>a</sup></td><td>3.4 ± 2.2 <sup>a</sup></td><td>3.2 ± 1.8 <sup>a</sup></td><td>1.5 ± 1.3 <sup>b</sup></td><td>1.0 ± 1.6 <sup>b</sup></td><td>1.3 ± 1.6 <sup>b</sup></td></tr><tr><td>Bitter taste</td><td>0.5 ± 0.8 <sup>a</sup></td><td>0.5 ± 0.8 <sup>a</sup></td><td>1.1 ± 1.5 <sup>a</sup></td><td>0.8 ± 0.9 <sup>a</sup></td><td>1.1 ± 1.1 <sup>a</sup></td><td>1.0 ± 1.0 <sup>a</sup></td></tr></tbody></table>

PT## = Pulp treated at ## °C for 5 min.

h) reported by Asakura et al. (2011)) and 1 – 2 h reported by Brouwer et al. (1968) and (Gibbs et al., 1996).

## 3.4. Modification of miraculin during heat treatments

Since miraculin is polymorphic (Paladino et al., 2008), analyses were performed to monitor its modifications and behavior under various heat treatment conditions. Quantification of miraculin by RP-HPLC revealed that modifications occurred in its concentration as a result of heat treatments.

The chromatographic profiles obtained at 280 nm for the different heated samples (40 to 70 °C) clearly showed the thermosensitivity of miraculin Fig. 3 as previously revealed by Brouwer et al. (1968) and Choi and Garza (2020). The slight variations in retention time observed, were attributed to the complex chemical composition of the extract, to the column itself and to the elution gradient. However, this drifting did not affect the peak identification or its area calculation. Miraculin concentration decreased from 3.07 g·kg⁻¹ in sample heated at 40 °C to 2.17 g·kg⁻¹ in pulp heated at 70 °C (Table 3). High temperatures resulted in low miraculin recovery, and the degradation was more pronounced at 60 and 70 °C. This result was in agreement with those obtained previously (Béhanzin et al., 2025).

Qualitative modifications in miraculin were monitored as well by performing SDS-PAGE and native electrophoresis analysis. In SDS-PAGE under reducing conditions (addition of 2-mercaptoethanol), a 25 kDa band corresponding to the molecular weight of miraculin in its monomeric form (Kurihara, 1992) was observed. This result was in accordance with data reported by Demesyeux et al. (2020); Duhita et al. (2009); He, Tan et al. (2015) and Ito et al. (2007) performing SDS-PAGE under reducing and non-reducing conditions to confirm the presence of miraculin in their purified samples. The authors found only miraculin monomer at 25 kDa under reducing conditions and miraculin dimer at 50 kDa under non-reducing conditions. According to the IBI (Index of Band Intensity), there is a general trend in the band intensity to decrease from the unheated pulp sample (Indexe of band intensity = 11.2 %) to highly heated samples. The band has almost disappeared in the pulp heated at 70 °C (Table 3). Based on the thermosensitivity of miraculin and the molecular weight of its monomer, we could partially confirm the presence of miraculin in our samples. Furthermore, the presence of a single band in the samples would suggest that miraculin is the predominant protein in the extracts.

In SDS-PAGE under non-reducing conditions, indeed allowing the preservation of disulfide bonds (Rotoli & Caradonna, 2019), a band at 50 kDa (Fig. 4) corresponding to the molecular weight of miraculin in its dimeric form (Kurihara, 1992) was observed, thus corroborating the works of Demesyeux et al. (2020), Duhita et al. (2009); and Ito et al. (2007). Contrary to the findings of these authors who did not find miraculin monomers under non-reducing conditions, a band at 25 kDa were observed in the studied samples. This would suggest, on the one hand, that the tetrameric form of miraculin was probably destructured by the SDS which can break weak bonds of proteins thus giving dimers which were observed on the gel. On the other hand, there are monomers already present in both unheated and heated samples (example of the samples heating at 40 °C and 70 °C), which would explain the presence of the bands at 25 kDa in non-reducing conditions.

In native electrophoresis, smears and a band at 100 kDa was observed and showed the same trend of decreasing intensity, starting from the unheated sample to the most heated sample (Fig. 5). This native electrophoresis showed the presence of a protein in the samples (more visible in the unheated sample) which weighted 100 kDa. According to Wittig et al. (2006) and Wittig and Schägger (2009), native electrophoresis allow to reveal native molecular weight of proteins. Thus, it could be supposed that this band at 100 kDa represent the tetrameric form of miraculin.

Furthermore, the size-exclusion chromatography (SEC) in which the reference mix proteins used ranged from 100 kDa to 25 kDa was

<footnote>* Letter's comparison was done in line ($p < 0.05$).</footnote>
<figure><img src="image_3.png" /><figcaption>Fig. 1. Intensities of sweet and sour tastes induced by unheated and heated Sisrè pulp after citric acid solution consumption: (a) unheated pulp; (b) pulp heated for 5 min at 40 °C; (c) at 45 °C; (d) at 50 °C; (e) at 60 °C and (f) at 70 °C.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Fig. 2. Half-life time ($t_{1/2}$) of sweet and sour tastes induced by unheated (30 °C assigned to the unheated pulp according to ambient temperature in the harvesting area) and heated pulps.</figcaption></figure>

performed. The aim was to confirm the different molecular weight of
miraculin present in the samples (100 kDa for tetrameric form, 50 kDa
for dimeric form and 25 kDa for monomeric form). The chromatogram at
210 nm displayed protein with 100 kDa molecular mass around 5–6 min,
50 kDa around 8–9 min and lower molecular weight proteins and other
compounds around 10 min. Optimizing the chromatographic conditions
could provide a more accurate link between retention times and molar
masses as suggested by Kammoun et al. (2003).

In all samples (unheated and heated) the presence of a protein with a
molecular weight of approximately 100 kDa corresponding to the mo-
lecular weight of miraculin in its tetrameric form (Kurihara, 1992) was
observed (data not shown). Furthermore, the peak area of this protein
decreased from the unheated sample to the heated ones. Considering the
results of the SDS-PAGE analysis under reducing conditions which
designated miraculin as the major protein, this protein observed at 100
kDa would therefore correspond to the tetramer of miraculin. The
decrease in peak area from the unheated pulp to the most heated one
confirmed the thermosensitive nature of miraculin.

3.5. *Impact of heat treatments on tastes intensity and remanence time*

At time $t_0$, pulps heated at 40 and 45 °C induced a sweetness intensity
levels just below 7 representing that of unheated pulp (the reference).
Their sweetness levels were 6.7 and 5.8 respectively (Figs. 1b& 1c). The
sweetness intensity induced by pulp heated at 50 °C was only half of the
reference (Fig. 6). These sweetness intensities were higher than the
maximum intensity of 4.98 induced in an unsweetened lemonade after
consumption of miracle fruit (Rodrigues et al., 2016). The pulps heated
at temperatures above 50 °C did not induce any sweetness in the pres-
ence of sour solution at time $t_0$. Based on the sweetness inducing ca-
cacity, the pulps were classified into 3 groups. The unheated pulp and
pulps heated at 40 °C and 45 °C were assigned to group 1 with the
highest sweetness intensity, the pulp heated at 50 °C belonged to group
2 having an average sweetness intensity and pulps heated at 60 °C and
70 °C fell within group 3 which exhibited no sweetness capacity.

Pulps of group 1 exhibited a remanence time exceeding 1 hour (Fig. 2). The remanence time for group 2 pulp was about 40 min. These
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** RP-HPLC chromatogram at 280 nm displaying the miraculin peak in the different heated Sisrè pulp extracts.</figcaption></figure>

results were similar to findings by Asakura et al. (2011) who reported that miraculin sweetening effect can last up to 1 h. It worthwhile to point out that although the group 3 pulps (heated to 60 and 70 °C) did not induce a sweet taste, a drop in the intensity of the perceived acidic taste was recorded for the citric acid solution. This intensity took around 18 and 13 min respectively (Fig. 2) to recover 50% of the initial acidic taste intensity of the same acid solution. Previous studies also reported that miraculin reduced sourness of unsweetened lemonade. In this beverage, miracle fruit induced the most reducing intensity of sourness compared to other sweeteners as sucrose or sucralose (Andrade et al., 2019; Rodrigues et al., 2016).

It is most likely that the taste modification observed in the present study is associated with miraculin modification as a response to heat treatment (Béhanzin et al., 2025). Our data suggested that heat treatments at temperatures above 50 °C for 5 min would lead to a significant impact on miraculin functionality. A loss in sweetness inducing capacity of miraculin was also reported after heating the pulp of *Synsepalum dulcificum* at 60 °C for 10 min NDA et al. (2021). Miraculin in Sisrè pulp showed heat sensitivity similar to that of curculin (a protein extracted from the fruit of *Curculigo latifolia* Dryand), which begins to lose its sweetening activity after heating at 50 °C for 1 h. Functionality of other sweetening proteins were reported to be heat sensitive. Particularly, it has been reported that thaumatin (extracted from the fruit of *Thaumatococcus danielii* Benth; also called katemfe fruit), retains its sweetening capacity after pasteurization. It was reported that mabinlin (extracted of

the seeds of *Capparis masaikai* Lev) and brazzein (isolated from the fruit of the African climbing shrub *Pentadiplandra brazzeana* Baillon), retain their sweetening functionality after heating at 80 °C for 1 and 4 h, respectively (Fry, 2012; Gibbs et al., 1996).

It should be noted that among the sweetening proteins (miraculin, thaumatin, brazzein, curculin, monellin, mabinlin II, pentadin), only miraculin (unsweetened) and curculin (sweetened) had a taste-modifying property and a persistent effect in the mouth without the need for repeated consumption (Farag et al., 2022; Kant, 2005; Y. Kurihara, 1992; Paladino et al., 2008).

The reduction in the duration of the effect induced by the pulp heated at 50 °C could be explained by the reduction in the amount of active miraculin remaining in the pulp following the partial denaturation resulting from the heating. According to Kurihara and Beidler (1969), reducing the concentration of the protein would also reduce the duration of its effect.

By examining the relationship between miraculin's concentration and the intensity of the sweet taste perceived by the panelists at time $t_0$, it appeared that a minimum miraculin content was required to obtain the sweetening effect (Fig. 7a). The presence of a plateau was expected but was not highlighted, probably due to the limited number of experimental points available. Furthermore, in the concentrations range studied, the persistence time ($t_{1/2}$) clearly increased with the concentration (Fig. 7b). This is consistent with the results obtained by Kurihara and Beidler (1969) who reported that the duration of the effect would be reduced if the miraculin concentration was also reduced. Our findings were in accordance with data reported by Diyapaththugama et al. (2024) who compared several sensory studies testing the effect induced

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Image of the polyacrylamide gel resulting from SDS-PAGE analysis in reducing conditions (a) and non-reducing conditions (b) on the crudes extracts: 1) Standard protein used with markers of sizes ranging from 10 kDa to 250 kDa; 2) and 5) extract from fresh pulp; 3) and 6) extract from pulp heated for 5 min at 40 °C; 4) and 7) at 70 °C.</figcaption></figure>

<table><caption>**Table 3**

Effect of heating on the characteristics of the Sisrè pulps.</caption><thead><tr><th>Parameters</th><th>Fresh pulp</th><th>PT40</th><th>PT45</th><th>PT50</th><th>PT60</th><th>PT70</th></tr></thead><tbody><tr><td>Induced sweetness intensity at t<sub>0</sub><sup>*</sup></td><td>7.1 ± 1.6<sup>**</sup></td><td>6.7 ± 1.5</td><td>5.8 ± 2.1</td><td>3.3 ± 2.1</td><td>0.1 ± 0.3</td><td>0.1 ± 0.2</td></tr><tr><td>Perceived sourness intensity at t<sub>0</sub><sup>*</sup></td><td>2.4 ± 1.8<sup>**</sup></td><td>2.1 ± 1.9</td><td>2.4 ± 2.2</td><td>3.6 ± 2.8</td><td>5.1 ± 2.3</td><td>4.4 ± 2.5</td></tr><tr><td>Time taken to drop to a sweet intensity equal to 2 (min)</td><td>≥ 150</td><td>≥ 150</td><td>≥ 150</td><td>30</td><td>0</td><td>0</td></tr><tr><td>Losses RP-HPLC (280 nm)</td><td>-</td><td>0</td><td>0</td><td>1.5 %</td><td>20.4 %</td><td>29.4 %</td></tr><tr><td>Losses SEC (210 nm)</td><td>-</td><td>0</td><td>0</td><td>11.8 %</td><td>32.4 %</td><td>36.3 %</td></tr><tr><td>Miraculin concentration in lyophilized pulp (g·kg<sup>-1</sup>)</td><td>3.09 ± 0.26<sup>***</sup></td><td>3.07</td><td>3.21</td><td>3.04</td><td>2.49</td><td>2.17</td></tr><tr><td>SDS-PAGE reducing conditions (25 kDa band)</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Indexes of band intensity (%)</td><td>11.2</td><td>10.9</td><td>17.3</td><td>13.3</td><td>2.9</td><td>0.0</td></tr></tbody></table>

<footnote>* Sensory scores.</footnote>

<footnote>** Calculated with $n = 18$.</footnote>

<footnote>*** Calculated with $n = 3$.</footnote>
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Image of the polyacrylamide gel resulting from native electrophoresis on crude extracts: 1) Standard protein used with markers of sizes ranging from 10 kDa to 250 kDa; 2) extract from the fresh pulp (30 °C); 3) extract from the pulp heated for 5 min at 40 °C; 4) at 45 °C; 5) at 50 °C; 6) at 60 °C; 7) at 70 °C.</figcaption></figure>

by different concentrations of miraculin in the presence of foods at acidic pH. Furthermore, Andrade et al. (2019) demonstrated that the ability of miraculin to induce sweet taste was dose-dependent. Thus, the authors established that a small amount of miraculin obtained from 150 mg of miracle fruit would induce a weak sweet taste of unsweetened lemonade while, miraculin contained in 300 mg of miracle fruit reduced sourness and induced a sweetness comparable to that of sucralose. In addition, the results of non-reducing SDS-PAGE and native electrophoresis showed the presence of all three forms of miraculin (monomer, dimer and tetramer) in the samples including pulp heated to 70 °C in which it was observed a loss of activity. It is most likely that heating at this temperature for 5 min would not break the disulfide bonds between the dimers (active form) but could have led to a loss of solubility during extraction (Béhanzin et al., 2025) due to irreversible changes of the protein tertiary/quaternary structures (resulting in a change in

conformation). Therefore, it could be assumed that the analysis measured miraculin monomers (non-active form) that were initially present in the sample before heating. Secondly, a miraculin concentration higher than that recorded in the pulp heated at 70 °C was found in the pulp heated to 50 °C which presented sweetening activity. In this case, it was assumed that the HPLC analysis could measure by coelution both non-active forms (monomers initially present in the sample) and active forms (dimers) which would be the remainder of undenatured proteins by heating at this temperature. For a sample (pulp heated to 40 °C) with a sweetening activity and remanence almost similar to that of unheated pulp, miraculin concentration is identical to that of pulp with average sweetening activity. On the basis of the above two hypotheses, the analysis would have enabled us to measure non-active forms and active forms that were little or not at all denatured at this temperature, which was relatively close to that of the fruit's development environment 30 – 40 °C (Akinmoladun et al., 2020). This would suggest that, in the HPLC assay, both active forms (dimers and tetramers) and non-active forms (monomers) would probably all be measured. Taken together, these results suggested that heating above 50 °C led to the denaturation of the protein, resulting in a probable conformational change. On the one hand, it prevented the protein from interacting with sweet taste receptors in the presence of acid (no functionality). On the other hand, it decreased protein solubility, leading to a reduction in the measured concentration in samples with no more sensorial functionality. Further investigations are needed to understand the loss of functionality and the behavior of the different forms of miraculin during fruit processing.

4. Conclusion

Miraculin induced a sweet taste for sour, bitter and salty solutions after consumption of the fresh pulp while it exhibited no sensorial effect in the presence of a sweet solution. The intensity and remanence of the sweetening effect of miraculin depended on its concentration. Overall, this study provides a detailed investigation into the temperature sensitivity of miraculin in Sisrè pulp by combining a sensory approach with an instrumental one. Focusing the study on five temperatures within the range of 40 to 70 °C, it demonstrated that the functionality of miraculin was preserved when the pulp was heated at 45 °C for 5 min. Miraculin began to lose its sweetening power at 50 °C and was sensorially inactivated from 60 °C. The results also showed that data generated by the sensory tests are in close agreement with those from the analytical measurements. Further work could be considered in order to better take

<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Influence of temperature on taste intensities as perceived by consumers (t = 0 min) after ingestion of unheated (30 °C assigned to the unheated pulp according to ambient temperature in the harvesting area) and heated pulps.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Relation between (a) sweet taste intensity (sensory scores), (b) sweetness remanence time ($t_{1/2}$) of each sample perceived by panelists and miraculin concentration as measured by RP-HPLC.</figcaption></figure>

into account the miraculin polymorphism monitoring the evolution of the 3 different forms of miraculin during pulp processing. The results of this study suggested that it is possible to stabilize Sisrè fresh pulp applying temperature below 45 °C during the process. Low temperature air drying should be for example an interesting option to produce dry powder from Sisrè berry that can be used as a natural sweetener. Such a process could be an alternative to the freezing and freeze-drying processes currently wide used but costly in terms of energy and logistics, and therefore difficult to implement in the Beninese context. Definitively, the conventional air-drying technique combined with adequate packaging, would be a simpler, more economical, easier to implement (low-tech) method for long-term preservation of the dried Sisrè pulp. Nevertheless, the impact of a long-term drying process on the sweetening power of the pulp needs to be investigated.

## Fundings

- Agence Nationale de la Recherche (France), Partenariat Académique Afrique-France (PeA) BIOVALOR project n°ANR-21-PEA2-0006.

- CIRAD, Montpellier (France).

## Ethical statement

The study did not involve animals. Sensory evaluation was conducted with the help of volunteers recruited at the campus of University of Abomey-Calavi in Benin. Ethical approval for the study was obtained from the ethics committee of the University of Abomey-Calavi.

## CRediT authorship contribution statement

**Médérice Sènani Béhanzin:** Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation.
**Adrien Servent:** Writing – review & editing, Visualization, Validation, Software, Methodology, Formal analysis, Data curation.
**Laurent Adinsi:** Writing – review & editing, Visualization, Validation, Methodology, Data curation, Conceptualization.
**Charmelle Cyria Dahou:** Writing – review & editing, Visualization, Investigation.
**Ifagbémi Bienvenue Chabi:** Writing – review & editing, Validation, Supervision, Methodology, Funding acquisition, Conceptualization.
**Polycarpe Kayodè:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.

**Manuel Dornier:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.

**Médérice Sènani Béhanzin:** Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis, Data curation. **Adrien Servent:** Writing – review & editing, Visualization, Validation, Software, Methodology, Formal analysis, Data curation. **Laurent Adinsi:** Writing – review & editing, Visualization, Validation, Methodology, Data curation, Conceptualization. **Charmelle Cyria Dahou:** Writing – review & editing, Visualization, Investigation. **Ifagbémi Bienvenue Chabi:** Writing – review & editing, Validation, Supervision, Methodology, Funding acquisition, Conceptualization. **Polycarpe Kayodè:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.

**Manuel Dornier:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.

## Declaration of competing interest

None of the authors of this study declares a conflict of interest.

## Acknowledgements

The authors would like to thank the PEA BIOVALOR, an academic partnership program between Université d'Abomey-Calavi (Benin), Université de Lorraine (France), Institut Agro Montpellier (France) and CIRAD (France) for funding most of this work in Benin and France. They would also like to thank CIRAD (France) for its financial support of the project and Isabelle MARAVAL for her contribution in the preparation of sensory analyses of this work.

## Data availability

Data will be made available on request.

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