# Analyzing the miraculin content of Beninese Sisrè berries (Synsepalum dulcificum Schumach. & Thonn.) and exploring its thermal behavior

Médérice Sènanmi Béhanzin, Adrien Servent, Romain Kapel, Christian Mertz, Ifagbémi Bienvenue Chabi, Manuel Dornier, Polycarpe Kayodé

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Médérice Sènanmi Béhanzin, Adrien Servent, Romain Kapel, Christian Mertz, Ifagbémi Bienvenue Chabi, et al.. Analyzing the miraculin content of Beninese Sisrè berries (Synsepalum dulcificum Schumach. & Thonn.) and exploring its thermal behavior. Journal of Agriculture and Food Research, 2025, 23, pp.102221. 10.1016/j.jafr.2025.102221. hal-05450773

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# Analyzing the miraculin content of Beninese Sisrè berries (*Synsepalum dulcificum* Schumach. & Thonn.) and exploring its thermal behavior

Médérice Sènanmi Béhanzin<sup>a,b</sup>, Adrien Servent<sup>b,c,*</sup> <sup>ID</sup>, Romain Kapel<sup>d</sup>, Christian Mertz<sup>b,c</sup>, Ifagbémi Bienvenue Chabi<sup>a</sup>, Manuel Dornier<sup>b,D</sup>, Polycarpe Kayodé<sup>a,**</sup> <sup>ID</sup>

## ARTICLE INFO

### Keywords:

- Miracle fruit

- Sisrè berry

- Miraculin extraction

- Thermal stability

## ABSTRACT

The pulp of the miracle fruit (*Synsepalum dulcificum*) has the ability to change the acidic taste of ingested foods into a sweet taste. This functionality is due to miraculin, a glycoprotein presents in the pulp of the fruit. Miraculin is low in calories and can be used for the development of stabilized natural sweetener for food. This study aimed to extract and quantify the miraculin level in the pulp of Sisrè (*Synsepalum dulcificum*) collected from various localities of Benin and explore its thermal behavior. The aim was to generate preliminary quantitative data on the termosensitivity of miraculin. The beninese miracle fruits were characterized by an average mass value of 0.9 g, a length of 1.66 cm and a thickness of 0.84 cm. In dry basis, the pulp represented 12.8 % of the fruit while the seed was preponderant representing 73.8 % of the fruit. Overall, the miracle fruits studied exhibited size and average mass 35 % lower than values reported in literature. Likewise, the pulp content is 33 % lower than those obtained in the literature. However, the miraculin concentration in the pulp was higher than those reported by other authors amounting to 4.8 g·kg⁻¹ DB. Heat treatments led to significant losses of miraculin. In the raw pulp (a<sub>w</sub> = 0.97), 70.5 % and 71.9 % of miraculin were lost after isothermal treatments for 5 min at 70 °C and 80 °C respectively. The intensity of degradation was slightly lower when the pulp was lyophilized up to a 0.31 a<sub>w</sub> with 66.8 % lost recorded at 70 °C (5 min). The findings highlight the potential of Beninese Sisrè as a natural sweetener, while also providing valuable insights for developing stabilization processes to obtain high-quality functional products.

## 1. Introduction

*Synsepalum dulcificum* (Schumach. and Thonn.) is a plant native of West Africa, with origins ranging from Ghana to Congo, including Togo, Benin, and Nigeria [1–3]. For centuries, the populations of these countries have used various parts of the plant as food. *S. dulcificum* produces fruits known as miracle fruit or miracle berry, measuring about 2 cm long and 1 cm wide, with a red color and oblong shape at maturity [1,3, 4]. In the Beninese local Fon language, the fruits are called “Sisrè”. A study carried out in Florida showed that the fruit yield of *S. dulcificum* plants varied from 0.73 to 2.76 kg·tree⁻¹·year⁻¹ as a function of morphotypes [4]. Achigan-Dako et al. [1] conducted research on the genetic characteristics and domestication of *S. dulcificum* plants in Benin which

resulted in a reduction in the production time of first fruits from 7 to 3 years since planting. However, there is a limited documentation on the nutritional and technological characteristics of the Sisrè berries in Benin.

Nkwocha et al. [5] conducted a study to determine the proximate composition and micronutrients of *S. dulcificum* pulp. According to Ref. [3], the Sisrè berry is an interesting source of phenolic compounds, tocopherols, and lutein. These components are known for their powerful antioxidant capacity and significant health benefits including the protection against chronic diseases. The Sisrè berry also has the ability to sweeten acidic foods when consumed [6], thanks to the miraculin which converts the sour taste of acidic foods into a sweet taste by binding to taste buds [6,7]. Miraculin is a glycoprotein with functional properties

<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> LRGP, Univ. Lorraine, Nancy, France</footnote>

<footnote>* Corresponding author. QualiSud, Univ. Montpellier, CIRAD, Institut Agro Montpellier, IRD, Univ. Avignon, Univ. La Réunion, France.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: adrien.servent@cirad.fr (A. Servent), kayodepolycarpe@gmail.com (P. Kayodé).</footnote>
that make it of particular interest to processors and consumers. It is sought after by food and pharmaceutical industries as additives to improve the taste of low-calorie foods or certain medications.

In fact, miraculin can be present in several forms, monomeric, dimeric and tetrameric (Fig. 1). According to Kurihara et al. [8], in monomeric form which is its denatured and reduced form, it has a molecular weight of 25 kDa and consisting of a 220 amino acid chain that is stabilized by 3 intrachain disulfide bonds. The protein is N-glycosylated by glycans representing amount 13.9 % w/w [9] containing mainly the N-acetylglucosamine, mannose, xylose and fucose [10]. This monomeric form is not active. It also has a dimeric form which results from the union of two monomers by an interchain disulfide bond. According to Kurihara et al. [8] and Paladino et al. [10], the association of two miraculin dimers by weak bonds, forms a tetramer. So, the miraculin dimer and tetramer have a molecular weight of approximately 50 and 100 kDa respectively and are highly active. Miraculin's histidine 29 and histidine 59 are strongly involved in the protein ability to modify the taste [10].

A few studies have been conducted to isolate and purify miraculin. Kurihara and Beidler [11,12] isolated miraculin from the Sisrè berry using ion exchange chromatography, determined its molecular weight and established the mechanism by which, the molecule induced the modification of sour taste into sweet taste. Brouwer et al. [13] later purified the glycoprotein on a Sepharose column. Theerasilp et al. and Theerasilp & Kurihara [9,14] performed purification and full characterization of miraculin, to established the amino acid sequence and structure of the molecule. Kurihara [8] extracted, purified, and quantified miraculin content in Sisrè pulp, while [15] cloned and sequenced the cDNA encoding miraculin. In recent years, various studies focused on the improvement of miraculin extraction conditions [16] and on methods to purify the glycoprotein [16,17]. Due to the low quantity of miraculin in Sisrè berry and the difficulties in cultivating the tree outside its natural environment [18], studies focused on production and accumulation of miraculin from transgenic lettuce leaves [18], tomatoes [19], strawberries [20]. Recombinant miraculin has also been produced from bacterial or fungal cultures such as *Escherichia coli* [21], *Aspergillus oryzae* [22], *Saccharomyces cerevisiae* [23], and other yeasts [24]. There is a growing interest for safe natural food ingredients by food industry and consumers. Therefore, preservation and stabilization of Sisrè

extracts by fruit coating and impregnation techniques have been conducted in West Africa with the result that sweetness induction was preserved for 4 months after impregnation of gelatin and tapioca with miraculin extract, and sweetness induction was preserved for 3 months with the extract previously combined with sodium benzoate and refrigeration [25]. There are discrepancies in methods available to extract miraculin. The extraction methods in the various publications involve one to three washing operations with distilled water, and most use 0.5 M NaCl as the extraction solvent [4,14,16,18], with Tris-HCl added in some cases [17]. However, the operating conditions differ mainly in terms of solvent pH, centrifugation conditions and number of extractions using NaCl. The extraction methods had to be reviewed in order to understand the choice of the different operating conditions, identify and select the most appropriated method for our study.

Moreover, there is poor information regarding the thermosensitivity of miraculin. Although there is a general statement that miraculin is heat sensitive [13,26,27], no quantitative data exist in literature about its thermosensitivity. Obtaining quantitative data on the thermal behavior of miraculin, at least at temperatures in the range of those applied during conventional stabilization processes such as pasteurization, could help to define postharvest processes pathways for miraculin preservation in the pulp. In the present study, the methods of miraculin extraction were screened and the thermal behavior of native miraculin in the pulp of Sisrè berries (*Synsepalum dulcificum*) collected in natural environment of various localities of Benin was explored.

## 2. Material and methods

### 2.1. Plant material

About 11 kg of fresh Sisrè berries were collected in the period of December 2022 to January 2023 in the localities of Sey (6° 45' 14" N, 2° 06' 13" E) and Sèhouè (6° 55' 33" N, 2° 16' 16" E), in Republic of Benin. The fruits were packed in polyethylene bags, immediately transported to the laboratory where they were sorted to separate the damaged berries and freezed at -20 °C during 1 month in average. Then, the seeds were separated manually from the pulp in frozen state without removing the skin, using mortar and pestle. The pulp with skin was then packed in polyethylene bags and stored at -20 °C during around 1 month for

<figure><img src="image_3.png" /><figcaption>Fig. 1. Structure of miraculin polymers (adapted from Kurihara et al., 1992 and Paladino et al., 2008).</figcaption></figure>
further analysis. According to the study by Osseyi et al. [25], these storage conditions should not significantly affect the miraculin content of the products.

Fruit dimensions were measured as described by Ref. [4] using a caliper to measure the length and the thickness in the middle of Sisrè in triplicate on 15 fruits randomly selected from the stock of Sisrè berries. The weight of thousand fruits was determined on frozen fruit samples in triplicate. To determine the weight of the different part of Sisrè, a sample of 20 fruits was selected randomly and thawed at around 25 °C. The pulp and skin were manually separated from the seed. Each part of the fruit was weighed and the operation was repeated twice on two other batches of 20 fruits. The average values were reported for each part of the fruit.

## 2.2. Proximate analysis and main characteristics of the pulp

After rapid thawing of the pulp at around 25 °C, the measurement of the total soluble solids content of the pulp was carried out using a refractometer (ATC refractometer 0-32 % Brix, Brouwland, Belgium) at room temperature. Dry matter was determined as described by the AACC method (1984) and the total ash content was determined by the AOAC method (1993) on the pulp, skin and seed. The pH was measured on the fresh pulp and on the extracts of miraculin using an InoLab digital pH meter (WTW series 730, Weilheim, Germany) calibrated with buffers at pH 4.0 and 7.0. The titratable acidity was measured on the fresh pulp and expressed in citric acid equivalent following the AOAC method (1984). Water activity was measured with an AquaLab $a_w$-meter (Aw Meter 4 TE, Decagon, Germany).

Total proteins content of Sisrè pulp powder was determined by Kjeldhal method according to Jung et al. [28]. Protein content of the crude extracts of miraculin was determined by both Kjeldhal method and the bicinchoninic acid assay (BCA) method. In the Kjeldhal method, protein content was estimated by multiplying the organic nitrogen content by the protein conversion factor of miraculin ($k_{miraculin} = 6.02 \text{ kg}_{prot} \cdot \text{kg}_{N}^{-1}$) calculated from its 220 AA sequence (UNIPROT database; P13087 - MIRA_SYNDU) and its molecular weight.

The BCA method measurement was carried out on a 96-well micro-plate. The absorbance was measured using a spectrophotometer (Spark 10M, Tecan, Austria) at 562 nm. Protein concentration was determined using the equation of the calibration curve plotted from the 0 µg. mL⁻¹ to 1000 µg. mL⁻¹ concentration range of Bovine Serum Albumin.

The total polyphenol content (TPC) was determined on the fresh pulp, skin, seed, pulp powders and on crude extracts according to the modified method of [29]. Gallic acid was used as a standard. The absorbance was measured at 760 nm and the results are expressed in mg Gallic acid equivalent per g of dry matter.

## 2.3. Experimental design

As a pilot investigation, the Sisrè pulp was first submitted to a temperature of 90 °C during 5 min to screen the thermal sensitivity of native miraculin in the pulp. In this respect, the frozen Sisrè pulps were crushed with a Thermomix grinder before the heat treatment, which was conducted in an open container combined with an induction hob and a thermometer in order to control the temperature. The freeze-dried samples (Freeze dryer SMH15, Usifroid, Spain) were reduced to powder using a grinder at 3000 rpm for 15 s (Grindomix GM200, Retsch, Germany). Samples from unheated and heated pulps were subjected to both reversed phase high performance liquid chromatography analysis (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Next, experiments at 70 °C and 80 °C during 5 min were performed (i) on frozen crushed pulp under the same conditions as before at an $a_w = 0.97$ (Fig. 2), (ii) on freeze-dried pulp ($a_w = 0.31$) packaged in vacuum-packed polyethylene bags and heated in the water bath. After the heat treatment, the bags were immediately immersed in an ice bath for 1 min. All heat-treated samples were stored at -20 °C until extraction and analysis. Replications of heat treatment were applied with frozen pulp heated at 70 °C for 5 min. Variation between the same treatments in terms of miraculin losses were consistently below 10 % indicating acceptable repeatability of the treatments applied.

<figure><img src="image_4.png" /><figcaption>Fig. 2. Experimental design of heat treatment on the pulp of the Sisrè fruit.</figcaption></figure>
## 2.4. Sample extraction for miraculin analysis (reference method)

Miraculin extraction was carried out as described by Ref. [16] with some modifications. A first extraction with water was performed by adding 40 mL of distilled water to 6 g of pulp freeze-dried powder weighed in a centrifuge tube. The mixture was homogenized for 2 min by vortex mixer and centrifuged (Avanti J-E Centrifuge, Beckman Coulter, USA) at 12 000×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 adjusted at pH 6.8 with HCl was added to the pellet. Next, homogenization followed by centrifugation were carried out under the same conditions as previously. The supernatant representing the crude miraculin extract was collected in a tube and stored at 4 °C for further analysis. All extraction procedures were performed at room temperature.

## 2.5. Optimization of extraction method

Due to the presence of a residual amount of protein in the extraction pellets, various modifications were applied to the initial extraction method to enhance the recovery of miraculin. Consequently, four additional methods, inspired by different approaches reported in the literature, were implemented in addition to the reference method. These extraction methods were tested in parallel under identical conditions on the same day using 6 g of lyophilized pulp.

**Method 1:** the reference method.

**Method 2:** three successive extractions were performed using 0.5 M NaCl at pH 6.8 without prior washing. The supernatants from the three extractions were pooled to form the crude miraculin extract.

**Method 3:** three successive washes with distilled water was realized and discarded, followed by three successive extractions using 0.5 M NaCl at pH 6.8. The last three supernatants were pooled to form the crude miraculin extract.

**Method 4:** which included a second water washing step to further remove impurities from the sample before NaCl extraction.

**Method 5:** The concentration of the NaCl extraction solvent was reduced to 100 mM to improve the outcomes of subsequent analyses like native electrophoresis.

These five extracts were analysed using RP-HPLC, size-exclusion chromatography, and assessed under reducing conditions using SDS-PAGE.

## 2.6. RP-HPLC analysis of miraculin

The quantification of miraculin was carried out by RP-HPLC according to the method used by Ref. [4] with some modifications. A C18 100A column 250 × 4.6 mm × 5 µm (Kinetex, Phenomenex, USA) was used. The column was equilibrated with miliQ water containing 0.1 % trifluoroacetic acid (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 (v/v) 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 and the absorbance were measured at 214, 280 and 325 nm using a Diode-Array Detector (DAD) (1200 series, Agilent, USA) at 30 °C.

### • Identification of miraculin peak

As the pure miraculin standard is not commercially available, an identification of the miraculin peak was carried out on the basis of three characteristics of the molecule: its retention time (5–12 min) as reported by Refs. [4,16] using RP-HPLC C18, then the proteins absorbance wavelength [4,14,16,17] and finally its thermal sensitivity properties as

postulated by Refs. [13,26,27]. It is well established that at 280 nm, all the aromatic substances absorb (phenolics and proteins) while at 325 nm proteins do not show significant absorbance [30]. On the 280 nm chromatogram, peaks with a signal at 325 nm were assigned to phenolic compounds on the basis of their UV spectrum. Peaks at 280 nm with no signal at 325 nm, on the other hand, were considered to be proteins. Furthermore, the length of the optical path being constant, the ratio of peak areas at 280 nm is therefore proportional to the ratio of concentrations. This ratio was therefore used, with reference to the thermosensitivity of miraculin, to determine the percentage of miraculin loss, between the Sisrè pulp sample previously heated and the unheated one. According to the study on protein denaturation conducted by Ref. [31], the main assumption is that the denatured miraculin is not extractable through the standard protocol used.

### • Determination of miraculin concentration

The miraculin concentration was calculated based on data relating to the peak previously identified by applying Beer Lambert's law at 214 nm [32], the wavelength at which proteins are more sensitive and interference from solvents and biological buffer components is limited [30]. Since the molar extinction coefficient at 280 nm was not available, the one at 214 nm was determined. The molar extinction coefficient of miraculin ε at 214 nm was calculated by adding those of the amino acids composing its sequence and that of the 220 peptide bonds [33]. The length of the optical path was 1 cm and the molar extinction coefficient was expressed in equivalent monomer ε₂₁₄ = 478 999 AU·mol⁻¹·L·cm⁻¹

To validate the quantification method, a repeatability test was performed. The miraculin extraction process was repeated three times using the same sample of the same freeze-dried pulp powder, generating three crude miraculin extracts. Each extract was analysed separately by HPLC. The coefficient of variation was calculated.

## 2.7. SDS-PAGE analysis

Primarily to RP-HPLC analysis and to verified the presence of miraculin in our samples, a Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed on the crude miraculin extracts. A standard consisting of Precision Plus Protein™ Standards size marker mix (BIO-RAD Catalog #161–0363) was used. Samples of unheated and heated pulp extract of miraculin were introduced in the SDS-PAGE plate wells. By doing so, it would be expected a decline in the intensity of the band of miraculin on heated sample; based on the statement concerning thermosensitivity of miraculin as reported by Refs. [13,26].

The acrylamide gel used was composed of a 15 % resolving gel and a 5 % stacking gel. A volume of 12.5 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. Then, 10 µL of Precision Plus Protein™ Standards size marker mix (Catalog #161–0363, BioRad, USA) was also placed in a well. Electrophoresis was carried out at 10 mA for 40 min on the stacking gel and 20 mA for the separation gel for approximately 30 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 Coomassie blue R250 staining solution (50 % ethanol v/v, 10 % acetic acid v/v and 1 % Coomassie blue w/v) and placed under stirring for 30 min. The colored gel was rinsed with osmosis water and subjected to weak destaining overnight with an osmosis water solution containing 5 % acetic acid (v/v).

## 2.8. Size exclusion chromatography

Size-exclusion chromatography was performed using a Proteins column 250 × 4.6 mm × 5 µm (KW-802.5 Shodex, Japan). A 20 µL
aliquot of crude extract was injected into the column and eluted with an isocratic elution of 0.1 M phosphate buffer (pH 7.0) at a flow rate of 1.0 mL/min at 30 °C. The total runtime was 20 min, and the absorbance was monitored at 280 nm using a DAD.

## 2.9. Statistical analysis

Descriptive statistics (means, standard deviations, variation etc.) were carried out with Excel 2016 (Microsoft, USA).

# 3. Results and discussion

## 3.1. Main characteristics of sisrè berries

The average values obtained for the length and the largest thick of the miracle berries are 1.66 ± 0.04 cm and 0.84 ± 0.02 cm respectively. Demesyeu et al. [4] reported an average length of 1.99 cm and a thickness of 1.18 cm for Sisrè berries from USA. He et al. [3] reported a length of 2.00 cm and 1.88 cm respectively for fruits from Malaysia and Benin. The average weight of the Beninese Sisrè fruit is 0.90 g, which was lower than values reported by Refs. [4,34] who reported values of 1.39 g and 1.09 g respectively for fruits from Florida in the USA and Benin. Indeed, the size and average mass of the our Sisrè berry were approximately 35 % lower than those obtained by these authors, probably due to the genetic makeup of the crop or the growing environmental conditions.

From the structural point of view, the pulp content of the Sisrè studied was about 30.9 % wet basis and 12.82 % dry basis (DB). It was 1.4 times lower than that of the Sisrè berries from Florida evaluated by Ref. [35] (18.07 % DB) and 1.6 times lower than that reported by Ref. [3] (20.42 % DB) for berries from Malaysia. The skin and seed portion of the fruit were 13.44 and 73.75 % DB respectively (Table 1). The values of skin weight are lower than those obtained by Refs. [3,35], 15.47 % DB. In contrast, weight value obtained for the seed of fruit from Benin is higher than ones reported by Refs. [3,35], 66.01 % and 64.11 % DB respectively.

The pH of the pulp was 2.44. Its titratable acidity was 1.61 g of citric acid equivalent/100 g and its total soluble solid content was 13.9 g.100 g⁻¹. The protein contents determined on the freeze-dried pulps of both unheated and heated (90 °C/5 min) samples were similar amounting to 7.41 and 7.36 g.100 g⁻¹ DB respectively. These values were similar to the one obtained by Ref. [5]. The TPC of the fresh pulp without skin was 29.7 mg GAE.g⁻¹. That result was slightly lower than that obtained by Ref. [3] of 32.46 mg g⁻¹ in Sisrè berries from Malaysia, but higher than value (16.95 mg g⁻¹) reported by Ref. [35] for Sisrè berries from Florida. Likewise, the values obtained for the TPC in the skin (47.71 mg GAE.g⁻¹ DB) and the seed (4.49 mg GAE.g⁻¹ DB) were lower than those obtained respectively by Refs. [3,35] with 73.79 and 59.54 mg GAE.g⁻¹ DB in skin and 13.52 and 18.55 mg GAE.g⁻¹ DB in seed respectively. However, these data confirmed that the skin of Sisrè was richer in polyphenols than the pulp. The differences observed were most likely explained by the difference between the origins of the Sisrè berry, and so the climatic conditions, the variety of the fruits, the growing conditions, the levels of maturity or ripening of the fruits.

<table><caption>**Table 1**
Physicochemical properties of miracle fruit and its various components.</caption><thead><tr><th>Parameters</th><th>Pulp</th><th>Skin</th><th>Seed</th></tr></thead><tbody><tr><td>Weight ratio (%, dry basis)</td><td>12.82 ± 0.59</td><td>13.44 ± 0.63</td><td>73.75 ± 0.89</td></tr><tr><td>Weight ratio (%, wet basis)</td><td>30.86 ± 1.13</td><td>23.95 ± 1.17</td><td>45.19 ± 0.64</td></tr><tr><td>Dry matter (%)</td><td>13.73 ± 0.15</td><td>20.47 ± 0.46</td><td>64.62 ± 0.19</td></tr><tr><td>Ash (%, dry basis)</td><td>0.65 ± 0.01</td><td>0.49 ± 0.08</td><td>0.35 ± 0.31</td></tr><tr><td>Total phenolic contents (mg GAE.g<sup>-1</sup> DB)</td><td>29.72 ± 1.16</td><td>47.51 ± 5.92</td><td>4.49 ± 0.30</td></tr></tbody></table>

## 3.2. Characteristics of the extracts

The pH values for our pulp extracts (unheated and heated pulp) obtained by using the reference extraction method, were comparable around 3 and similar to that reported by other studies [8,14,16,17]. With respect to the residues resulting after the extraction, it was possible to quantify as much as 4.78 g.100 g⁻¹ and 4.28 g.100 g⁻¹ DB of protein contents in the pellets of the unheated pulp and the heated pulp respectively. Comparing these values with those of freeze-dried pulp, it appeared that only 35.49 % and 41.85 % protein was extracted for unheated and heated pulp respectively. This result indicated that miraculin was not the only protein accumulated in Sisrè or that otherwise the extraction carried out did not allow all of the proteins to be extracted and could therefore be optimized.

The extracts from heated pulp exhibited lower protein value (0.17 g. L⁻¹) than that of the extract from the unheated sample of 0.28 g. L⁻¹ possibly because a denaturation of the proteins occurred during heating. Denaturation may occasion a decrease of protein solubility during the extraction process. The material balance was around 64 %, which would indicate a loss of proteins, probably due to the washing step during extraction. It should be noted that the total proteins content of the unheated pulp extract was lower than values of 0.33 g. L⁻¹ and 0.36 g. L⁻¹ reported by Refs. [14,36] respectively.

## 3.3. Miraculin analytical procedure

Because no miraculin standard was available on market, miraculin quantification or monitoring was particularly challenging. In this study, an original methodology to identify miraculin tentatively was proposed. In a first qualitative step, a SDS-PAGE analysis was carried out to ensure of miraculin presence. Next, HPLC analysis was carried out and the cleaning of polyphenol peak was performed using chromatogram at 325 nm to compare with the one obtained at 280 nm. Finally, the thermal sensitivity of miraculin announced by Brouwer et al. [13] and Choi & Garza [26] was used as a guide to determine the differences (area decrease/peak disappearance) between peaks in the polarity zone and select the potential miraculin peak.

The results of the SDS-PAGE analysis showed clear bands on the polyacrylamide gel (Fig. 4), corresponding to that of the 25 kDa size marker and the molecular weight of miraculin, for the unheated pulp extract. However, no band was visible for the heated pulp extract. This could be explained by a denaturation of the protein resulting in a conformational change following the heat treatment applied to this sample. This result confirmed the presence of miraculin in the crude extracts.

The HPLC identification of the miraculin peak was carried out based on the chromatograms at 280 and 325 nm while the determination of the concentration was carried out based on the chromatograms at 214 nm. The chromatograms of the unheated and heated pulp extracts showed the same pattern at 280 nm. Five peaks, numbered 1 to 5, were identified at 280 nm in the range of miraculin retention time (5–12 min) and therefore as potential miraculin peaks (Fig. 5a).

Only peaks 2, 4 and 5 did not absorbed at 325 nm (Fig. 5b), and were therefore assigned to protein peaks. However, comparing the unheated sample to the heated one (Fig. 6), it was observed an increase in the intensity of peak 2 while intensity of peaks 4 and 5 decreased. This increase in intensity of peak 2 could also justify the increase in the polyphenol content subsequently observed in the heated pulp samples, which would probably make it a polyphenol. Furthermore, referring to the thermosensitivity of miraculin [13,26,27], it can be assumed that the peak corresponding to that of miraculin was either peak 4 or peak 5.

Comparing the ratios of peak areas between the heated pulp sample (90 °C) and the unheated one (Table 2), it was observed that only peak 4 loosened up to 70 % of its intensity while peak 2 and 5 presented rather a gain of their intensities. This result definitively, confirmed that peak 2 could not be miraculin peak and either discarded peak 5. In addition, by
<figure><img src="image_5.png" /><figcaption>Fig. 3. Size Exclusion Chromatography chromatogram at 210 nm wavelength of unheated pulp extracts for five extraction methods tested in view of optimization (with. Method 1: 2 washing +1 extraction; Method 2: 3 extraction without washing; Method 3: 3 washing +3 extractions; Method 4: 3 washing +1 extraction and Method 5: 2 washing +1 extraction with NaCl 100 mM).</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 4. Image of the polyacrylamide gel resulting from SDS-PAGE analysis on the crude extracts. a) Standard protein used with markers of sizes ranging from 250 kDa to 10 kDa; b, c, d and e) crude extract of unheated pulp; f and g) crude extract of pulp heated to 90 °C; 5 min.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 5. Unheated pulp extract chromatogram at 280 and 325 nm wavelength used for cleaning of polyphenols peaks (peaks numbered from 1 to 5 in the order of elution).</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Fig. 6. Chromatograms at 280 nm of unheated pulp and heated (90 °C, 5 min) crude extracts showing the miraculin peak 4 (peaks numbered from 1 to 5 in the order of elution).</figcaption></figure>

<table><caption>**Table 2**
Evolution of in miraculin concentration at 214 nm according to heat treatments at different water activity (Aw).</caption><thead><tr><th rowspan="3">N° of peak</th><th colspan="6">Frozen pulp a<sub>w</sub> = 0.97</th><th colspan="6">Freeze-dried pulp a<sub>w</sub> = 0.31</th></tr><tr><th colspan="2">70 °C</th><th colspan="2">80 °C</th><th colspan="2">90 °C</th><th colspan="2">70 °C</th><th colspan="2">80 °C</th></tr><tr><th>C/C<sub>0</sub></th><th>Evolution</th><th>C/C<sub>0</sub></th><th>Evolution</th><th>C/C<sub>0</sub></th><th>Evolution</th><th>C/C<sub>0</sub></th><th>Evolution</th><th>C/C<sub>0</sub></th><th>Evolution</th></tr></thead><tbody><tr><td>2</td><td>2.78</td><td>↑</td><td>2.63</td><td>↑</td><td>2.09</td><td>↑</td><td>1.55</td><td>↑</td><td>2.17</td><td>↑</td></tr><tr><td>4</td><td>0.29</td><td>↓</td><td>0.28</td><td>↓</td><td>0.30</td><td>↓</td><td>0.33</td><td>↓</td><td>0.23</td><td>↓</td></tr><tr><td>5</td><td>1.26</td><td>↑</td><td>1.20</td><td>↑</td><td>1.13</td><td>↑</td><td>0.76</td><td>↓</td><td>0.81</td><td>↓</td></tr></tbody></table>

comparing the chromatograms at 280 nm of the unheated sample and the heated one, it was observed that this peak 4 decreased in intensity at the level of the heated sample (Fig. 6). Since miraculin is known to be heat-sensitive, peak 4 was therefore the best candidate peak for miraculin. In addition, the maximum of absorption of this peak was 276 nm, which was conventional for proteins (280 nm).

### 3.4. Reproducibility of the analytical procedure

Miraculin determination was performed on a lyophilized Sisrè's pulp to check up the reproducibility of extraction and analytic measuring method at 280 and 214 nm. The variation coefficient obtained was 4.7 % and 11.9 % at 214 nm and 280 nm respectively. Since the first value was inferior to 10 %, it showed a great confidence in the way that the manipulations were carried out.

### 3.5. Optimization of extraction method

First, an SDS-PAGE performed on the extracts from the five methods revealed a band at 25 kDa, corresponding to the molecular weight of miraculin, for the first four methods. However, no band was observed for method 5, where the concentration (100 mM) of the extraction NaCl solvent was reduced. This suggests that the solvent concentration was likely insufficient for effective miraculin extraction.

In this section, relative peak area corrected by volume of extract and sample weight (peak area multiplied by volume and divided by sample weight) was used as a basis for comparison between the different extraction conditions tested. HPLC chromatograms at 280 nm further indicated that method 2 (three successive extractions with NaCl, without washing) yields the highest corrected peak area of miraculin of 8351.3 mAU.min⁻¹, followed by method 3 (three washes and three successive extractions with NaCl) 799.1 mAU.min⁻¹. Method 1 (the reference method, with two washes and one NaCl extraction) showed a corrected peak area of miraculin of 603.1 mAU.min⁻¹, and method 4 (three washes and one NaCl extraction) presents the lowest corrected peak area 543.6 mAU.min⁻¹.

However, the baseline resulting from the analysis of the extract of

method 2 did not return completely after the peaks as for the reference method. This was likely due to the presence of numerous compounds in the extract, which may hinder accurate readings by the detector, as unresolved peaks could be masking others. As a result, it was not possible to confidently asserted that the area of the miraculin peak in this extract corresponds exclusively to the protein. Consequently, method 2 led to inaccurate quantification of miraculin.

In contrast, method 3 successfully purified the extract, resulting in fewer impurities in the protein polarity zone and well-resolved peaks. This method demonstrated that multiple washes effectively purify the sample, leading to a 1.6-fold increase in miraculin corrected peak area compared to the reference method. However, the experimentation was more time-consuming and less cost-efficient.

Regarding method 4, the addition of one more wash step compared to the reference method (which used two washes) removed more impurities from the extract, yet the miraculin corrected peak area was almost identical to that of the reference method.

Furthermore, in size-exclusion chromatography the reference mix proteins used ranged from 100 kDa to 25 kDa in view of having the different molecular weight of miraculin (100 kDa for tetrameric form, 50 kDa for dimeric form and 25 kDa for monomeric form). The chromatogram displayed protein with 100 kDa molecular mass around 5–6 min, 50 kDa around 8–9 min and other molecular weight protein and other compound around 10 min. It revealed that both method 4 and the reference method yield nearly identical concentrations and higher than other methods, as indicated by the 210 nm peak corresponding to compounds with a molecular weight of approximately 100 kDa (Fig. 3), representing the miraculin tetramer (active form). Therefore, the reference method proved to be the most practical in terms of extraction efficiency, concentration yield, and cost-effectiveness.

### 3.6. Quantification of miraculin in crude extracts

The miraculin concentration then calculated from the area of the identified miraculin peak (peak 4) revealed values of 960 and 290 mg. L⁻¹ of extract for the crude extract from the unheated pulp and from the pulp heated to 90 °C respectively. Considering that the extraction yield
was 100 %, this result indicated a native miraculin content of 4.8 g. kg⁻¹ of freeze-dried pulp in Sisrè of Benin. This value was similar to that reported by Ref. [8] of 3.6 g. kg⁻¹ of freeze-dried pulp, and higher than that obtained by Ref. [16] of 1.9 g. kg⁻¹ of freeze-dried pulp and [18] of 0.3 g. kg⁻¹ of fresh fruit equivalent approximately to 1.7 g. kg⁻¹ of freeze-dried pulp. But compared to the total protein content 7.36 g. 100 g⁻¹ in the unheated freeze-dried pulp, it turned out that the amount of miraculin measured represented only 6.5 % of the total protein.

### 3.7. Impact of heat treatments

The TPC of heated pulp (90 °C/5 min) was of 30.7 ± 6.5 mg GAE.g⁻¹ DB, higher than the unheated pulp TPC of 26.1 ± 3.4 mg GAE.g⁻¹ DB. Among the possibles causes of this increase, cell degradation due to heating had facilitated phenolic compound extraction. Additionally, the short heating time (5 min) might not have allowed the maximum level of polyphenol release to be reached. As [37] postulated, duration of heating impacted increasing (same temperature, short time) or decreasing (same temperature, long time) of polyphenols concentration. So, if the pulp was heated for a longer period, a decrease in the total phenolic content or values almost equal to those of the unheated pulp would likely have been observed. For instance Ref. [38], reported an increase in total phenolic compounds in *S. aromaticum* seeds that were heated (roasted over medium heat for 5 min under continuous agitation in a thick-bottomed pan). Indeed, according to the trials conducted by Ref. [39] on spices (*Aframomum citratum* and *Tetrapleura tetraptera* fruits), an increase in temperature and roasting time significantly enhanced the phenolic content of both spices up to a certain level (optimal zone). The opposite effect was observed when both factors were increased beyond this critical level. Xu et al. [40] reported that thermal treatment applied to plant-based foods caused the evaporation of intracellular water, triggering chemical reactions that can alter the lignocellulosic structure (with lignin covalently bound to cellulose in the cell walls) and promoted protein denaturation, potentially leading to greater availability of plant phenolic compounds. Indeed, thermal and/or enzymatic treatments could break both non-covalent and cova- lent associations of polyphenols with food macromolecules.

Moreover, previous studies have reported that thermal treatment increased the total phenolic content in various foods, such as grapes [41], dates [42], dried apricots [43], and dried tomatoes [44].

The heat responses of native miraculin of Sisrè were examined at various temperatures (70 °C and 80 °C) in wet (a<sub>w</sub> = 0.97) and dry (a<sub>w</sub> = 0.31) pulps (Fig. 2). Moreover, the pulp was heated at 90 °C for 5 min. The concentration of miraculin drastically decreased from 8.02 g. kg⁻¹ in the unheated frozen pulp to 2.37 g. kg⁻¹ in heated frozen pulp at 70 °C and to 2.26 g. kg⁻¹ in heated frozen pulp at 80 °C (Fig. 7). The degra- dation was similar at both temperatures. The ratio of peak areas at 214 nm and the corresponding percentage loss between the unheated pulp extract and each of the pulp extracts heated to 70 and 80 °C, allowed to assess the impact of the heat treatments applied. At a water activity of 0.97, the losses were around 70 % for the 2 temperatures tested (70 and 80 °C) (Table 2).

The dry pulps (a<sub>w</sub> = 0.31) heated at 70 and 80 °C also exhibited lower levels of miraculin concentration. The impact of temperature was more significant in dry pulp compared to the frozen one. The loss in miraculin concentration increased about 16 % compare to 70 °C results when the temperature shifted from 70 to 80 °C corroborating the observation of [45] who reported that when the temperature increases by 10 °C near the temperature at which protein denaturation occurs, the rate of denaturation is high. Heat treatment at 90 °C, 5 min induced more reduction in miraculin concentration which dropped to 1.7 g. kg⁻¹ in the freeze-dried pulp.

The decrease in miraculin concentration at this temperature could be explained by a reduction in protein solubility due to heat-induced ag- gregation in the pulp, as observed in the case of Bovine Serum Albumin or β-lactoglobulin [45]. According to this author, during heating at

<figure><img src="image_9.png" /><figcaption>**Fig. 7.** Effect of heat treatment on miraculin concentration in miracle fruit pulp with various water activity (with = = = as the literature mean value of miraculin concentration in miracle fruit = 1.52 g. kg⁻¹). LPU = Lyophilized Pulp Unheated; FPH70 = Frozen Pulp Heated at 70 °C; FPH80 = Frozen Pulp Heated at 80 °C; LPH70 = Lyophilized Pulp Heated to 70 °C; LPH80 = Lyophilized Pulp Heated to 80 °C.</figcaption></figure>

temperatures of 60–70 °C, inter- or intra-molecular disulfide (S-S) bridges may form through irreversible exchanges, leading to these polymerizations.

Despite the decrease in miraculin concentration after heating the pulps, it remained higher than the average reported for fresh pulp in the literature [4,8,16,18] (Fig. 7).

These findings suggested that heating of dry pulp (low water activ- ity) would induce less loss of miraculin in Sisrè berry. Clearly, the results showed the thermosensitivity of miraculin at the different temperatures applied for the two water-activity of pulp used. However, further research would be necessary to understand the differences of behavior observed between the different heat treatments, and to verify whether the lyophilization process itself does not damage the miraculin structure.

### 4. Conclusion

The Beninese Sisrè berries turned out to be morphologically smaller than those studied by other authors weighing 30 % less than average weight reported. A significant level of total polyphenol was found in the pulp. Higher level of miraculin was found in the pulp in the Beninese Sisrè berries compared to literature although the Sisrè berries used were smaller than those described in the literature. Five extraction methods were compared for the quantitative determination of miraculin in pulp samples. The results showed that the method proposed by Ref. [16] provided the best compromise between extraction efficiency and ease of use.

The different heat treatments applied to the Sisrè pulp revealed that a pasteurization induced a reduction in miraculin contents of around 70 % in the pulps. Indeed, the study showed that under controlled conditions, about 70 % miraculin was denatured when it was exposed to heat treatment in the range of 70–80 °C during 5 min. In addition, a lower water activity limited the denaturation of miraculin in the pulp. How- ever, it will be necessary to determine the temperature range in which miraculin is not degraded in order to be able to propose low-cost and low-tech processes for Sisrè berry stabilization. This information will also be useful for the development of finished or semi-finished products in the Sisrè chain in Benin. Further studies are needed to complete this work. To begin with, the miraculin content of Sisrè berries from different Benin accessions should be characterised in order to better assess regional biodiversity. Finally, a full kinetic study of the effect of tem- perature on the sweetening properties of the berries at different water activities is essential to optimise stabilization processes such as drying.
CRediT authorship contribution statement

**Médérice Sènami Béhanzin:** Writing – original draft, Investigation, Formal analysis. **Adrien Servent:** Writing – review & editing, Validation, Methodology, Investigation, Formal analysis, Data curation. **Romain Kapel:** Writing – review & editing, Validation, Resources, Methodology, Investigation, Formal analysis, Data curation. **Christian Mertz:** Writing – review & editing, Formal analysis. **Ifagbémi Bienvenue Chabi:** Writing – review & editing, Conceptualization. **Manuel Dornier:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization. **Polcarpe Kayodé:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization.

Fundings

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

- CIRAD, Montpellier (France).

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), L'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.

Data availability

Data will be made available on request.

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