# Exploring the factors contributing to texture loss in lentil cv. Anicia during cooking

Guilhem Maillard, Guy Della Valle, Camille Alvarado, Xavier Falourd, Bérengère Marais, Pascale Millart, Kevin Billet, Patrice Papineau, Anne-Laure Réguerre, Marie-Françoise Devaux, et al.

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## ► To cite this version:

Guilhem Maillard, Guy Della Valle, Camille Alvarado, Xavier Falourd, Bérengère Marais, et al. Exploring the factors contributing to texture loss in lentil cv. Anicia during cooking. Food Chemistry, 2025, 495, pp.146422. 10.1016/j.foodchem.2025.146422. hal-05358079

HAL Id: hal-05358079

https://hal.inrae.fr/hal-05358079v1

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# Exploring the factors contributing to texture loss in lentil cv. Anicia during cooking

Guilhem Maillard<sup>a,b,d</sup>, Guy Della Valle<sup>a</sup>, Camille Alvarado<sup>a,b</sup>, Xavier Falourd<sup>a,b</sup>,
Bérengère Marais<sup>a,b</sup>, Pascale Millart<sup>a,b</sup>, Kevin Billet<sup>c</sup>, Patrice Papineau<sup>a</sup>,
Anne-Laure Réguerre<sup>a,b</sup>, Marie-Françoise Devaux<sup>a</sup>, Laurent Chaunier<sup>a</sup>, Bruno Novales<sup>a,b</sup>,
Anne-Laure Chateigner-Boutin<sup>a</sup>, Nadim Tayeh<sup>d</sup>, Sophie Le Gall<sup>a,b,w</sup>

## ARTICLE INFO

### Keywords:

Pulse

Seed canning quality

Cooking time

Water absorption

Starch

Pectins

Cellular adhesion

## ABSTRACT

Lentils are gaining attention with the rise of plant-based diets, yet their consumption remains limited, partly due to poor canning quality. To identify the factors affecting canning performance in Anicia lentils, three seed batches differing in quality were analysed in the dry state, during soaking, and during cooking. Assessments included seed appearance, morphology, histology, composition, mechanical properties, and hydration behaviour.

High-quality seeds had a lower initial hydration rate during soaking and a lower moisture equilibrium during cooking (140 % vs. 180 %), linked to greater deformability. They contained less amorphous starch (10 % vs. 15 %), more pectic homogalacturonans (1.3 % vs. 1.1 %) in cotyledons, and fewer intercellular spaces. These findings clarify the roles of the lentil seed coat and cotyledons, particularly with regard to pectin and starch, in water absorption and texture loss during cooking.

## 1. Introduction

Like other pulses, lentil (Lens culinaris Medik.) can contribute to reduce both nitrogen inputs to agriculture and human meat consumption. Despite its added value for the crop rotations and its relatively high demand as a plant-based protein source, lentil production in France remains very low compared to cereal production or even compared to other grain legumes. Lentil is a niche crop, with most production being of green lentil of cv. Anicia. In addition, the seeds produced often have a soft texture and tend to burst during canning.

Genotype and also cultural conditions (such as soil and climate during cultivation, harvesting and storage practices) can affect seed composition and structure (including cell and tissue organization) (Erskine et al., 1985) and lead to variations in seed properties during

hydrothermal processes. Softening and bursting of seeds occur during hydrothermal processing (canning or cooking), depending on various factors such as temperature, process duration or water content (Anzaldua-Morales et al., 1996; Bhatty, 1989; Varoquaux et al., 1995). Variations in lentil texture also depend on seed properties, structure and composition (Bhatty, 1984, 1988, 1990; Pirhayati et al., 2011). In a study of 24 lentil genotypes, Erskine et al. (1985) also showed that seed weight correlated with cooking time. However, seed morphology and density were not considered.

During seed hydration, the initial surface of contact with water is the seed coat. Pulse seed coat can be partially or totally impermeable and hilum is often considered to be the main entry path for water (Miano & Augusto, 2018). Bhatty (1995) showed that water uptake variations between batches of lentil seeds with different cooking time disappear

Abbreviations: a, major axis radius; AIR, alcohol insoluble residue; Ara, arabinose residues; b, minor axis radius; CWP, cell wall polysaccharides; e, ellipsoid eccentricity; ESEM, environmental scanning electron microscope; F, force; Gal, galactose residues; GalA, galacturonic acid residues; h, height; m, mass; Man, mannose residues; MC, moisture content; MD, methylation degree; ME, moisture equilibrium; r, radius; HG, homogalacturonan; RMSE, root mean square error; S, surface; t, cooking or soaking time; V, volume; vᵢ, initial rate of water uptake; w, weight; Xyl, xylose residues; ε, deformation; ρ, density; σ, stress.

<footnote><sup>a</sup> INRAE, UR1268 BIA, F-44300 Nantes, France</footnote>

<footnote><sup>b</sup> INRAE, PROBE Research Infrastructure, BIBS Facility, F-44300 Nantes, France</footnote>

<footnote><sup>c</sup> INRAE, UR1268 BIA, F-35650 Rennes, France</footnote>

<footnote><sup>d</sup> UMR Agroécologie, INRAE, Université Bourgogne Europe, Institut Agro Dijon, Dijon, France</footnote>

<footnote>* Corresponding author at: INRAE, UR1268 BIA, F-44300 Nantes, France.</footnote>

<footnote>E-mail address: sophie.le-gall@inrae.fr (S. Le Gall).</footnote>
when dehulled, which suggest an effect of seed coat as a barrier to water absorption. In cowpea (Vigna unguiculata (L.) Walp.), varieties with thick seed coat have slower initial water uptake rate than varieties with thin seed coat (Sefa-Dedeh et al., 1979). However, Joshi et al. (2010) demonstrated that the water uptake capacity of lentil seeds correlated with the water permeability of seed coat rather than with its thickness. In pulses, seed coat composition (especially phenolic compounds) could play a role in reducing its permeability and increasing seed hardness (Vijayan et al., 2023), through compounds like lignin (Oliveira et al., 2014) or tannins (Demonsais et al., 2020; Kantar et al., 1996). However, the impact of tannins on water absorption has only been studied in the context of seed germination, and the role of lignin in seed coat permeability has not been evidenced. Additionally, according to canning industries, threshing harvester, by damaging seed coat, could also influence seed coat permeability by creating potential entry points for water thus increase water absorption and ultimately affect canning quality.

During hydrothermal treatment, once the seed coat has been crossed, water penetrates the cotyledons, and the rate of seed imbibition subsequently relies on its macromolecular constituents (Miano & Augusto, 2018). Lentil cotyledons are primarily composed of starch (39–45 % dry matter) and proteins (24–29 % dry matter), along with cell wall polysaccharides (4.5–6.7 % dry matter) (Tahir et al., 2011) forming structures that are likely to influence water absorption and cell cohesion during the hydrothermal process, thereby affecting the final texture.

During hydrothermal treatment and water uptake, starch melts and proteins denature (Abu-Ghannam, 1998; Deshpande & Bal, 2001). Starch granules are known for their capacity to absorb water during hydrothermal treatment (Aguilera et al., 2009). Previous studies on cereals have elucidated the intricate relationship between the water binding capacity of starch and its structure, encompassing crystalline and amorphous starch (Cai et al., 2014; Colonna et al., 1980; Huang et al., 2015; C. Li et al., 2020; H. Li et al., 2019; Lin et al., 2016). Chung et al. (2009) have shown that heat-moisture treatment affects lentil seed starch by decreasing granular swelling, crystallinity, gelatinization enthalpy and increasing the range and temperature of gelatinization.

Variations of water uptake can also be attributed to protein content. For instance, French green lentil having higher protein content absorbs more water at high temperature (85 °C) than other varieties (Nugget, Boomer) (Joshi et al., 2010). While the water binding capacity of proteins has been demonstrated to be consistent across various genotypes (C. Li et al., 2020), proteins can alter the water binding capacity of starch during hydrothermal treatment by promoting the formation of aggregates.

Finally, primary cell walls of seed cotyledons, rich in polysaccharides, may impact pulse texture. Indeed, cell walls are reported to influence the mechanical properties of plant tissues and their cohesion (Bidhendi & Geitmann, 2016; Daher & Braybrook, 2015). Notably, the middle lamella, mainly composed of pectins, plays a significant role in cell cohesion (Zamil & Geitmann, 2017). Regarding pulse cooking, particularly beans, cell wall can be involved in the hard-to-cook phenomenon, through the formation of cation mediated crosslinks between de-methylated homogalacturonans during cooking (Reyes-Moreno et al., 1993).

Few studies have been carried out to understand how cooking affects the texture of lentil seeds. In these studies, lentil seeds with rapid texture loss during cooking are considered of high quality. Their aim was to obtain fast-cooking lentils. In contrast, since the canning process requires lentils that do not burst or loose texture too rapidly upon cooking, here lentils with long cooking time are considered as high quality. Moreover, the way cooking affects the texture of lentil seeds is still poorly understood, as well as mechanisms involved. Therefore, the aim of this study is to investigate the factors influencing the softening and bursting of French green lentils during cooking. For this purpose, a large set of methods was applied to assess the level of seed organization and the composition elements that are involved in the hydration and

mechanical properties in lentil seeds during cooking. The contribution of seed coat and cotyledons was investigated, which was not simultaneously addressed previously. In this multi-faceted approach, we hypothesise that the seed biochemical composition may influence tissue structure, which in turn could affect water absorption and impact its mechanical properties.

## 2. Material & methods

### 2.1. Plant material

Three batches of French green lentil of Anicia variety, referred to as A, B and C and harvested in 2022, were provided by Cofigeo (F46-Capdenac) that evaluated the canning quality of these batches. A quality score from 1 (high quality, e.g. textural integrity after canning) to 4 (low quality, e.g. textural loss after canning) was attributed by visual assessment of canned lentil seeds. The canning quality score of these three batches is 1 for A and 4 for both B and C.

### 2.2. Hydrothermal behaviour

#### 2.2.1. Visual assessment of cooking quality

About 8 g of lentil seeds (a representative amount of about 250 seeds) from each batch were cooked in distilled water at 92 °C during 1 h, and were then carefully dried with paper towel, placed in Petri dishes and photographed (Camera 13MP Huawei PSM19).

#### 2.2.2. Cooking time distribution

A Mattson-cooker type apparatus (Mattson, 1946; Sabadoti et al., 2020) was used to measure the cooking time of lentil seeds. Twenty-four lentil seeds were cooked in distilled water at 92 °C and subsequently individually crushed by 100-g spikes. The proportion of crushed lentil seeds was recorded throughout the cooking process to generate a cooking time distribution. This distribution was then fitted by a sigmoid function according to:

$$p = \frac{1}{1 + e^{-b*(t-c)}} \quad (1)$$

where p is the proportion of crushed lentil seeds, t the cooking time, b and c, respectively the slope of the curve and the characteristic cooking time at inflexion point.

This experiment was carried out three times per batch (i.e. 72 seeds per batch) and the mean and standard deviation of b and c values were calculated. An analysis of variance was made to evaluate the difference between the three batches, regarding the characteristic cooking time. A Tukey test with Holm correction was used to evaluate which batch was different from the other and Cohen's d was used to examine the effect size.

### 2.3. Hydration kinetics

#### 2.3.1. Seeds fully immersed in water

For each batch, 5 g ± 0.04 g of lentil seeds were cooked in distilled water at 92 °C ± 3 °C at 17 different times between 2 and 70 min (Julabo water baths TW8). Once cooked, seeds were carefully dried with paper towel and weighed. The moisture content (expressed in water percentage on dry matter) was calculated from weight and initial moisture content. Measurements were performed in triplicate and the mean and standard deviation of moisture content were calculated. The hydration kinetic was also measured in triplicate for lentils soaked in distilled water at room temperature (21 °C) during 15, 45, 75, 105, 165, 225, 285, 405 and 585 min.

$$MC_t = \frac{w_t*(MC_0 + 1)}{w_0} - 1 \quad (2)$$
where $MC_t$ is the moisture content of cooked lentils (in dry basis), $w_t$ the weight of cooked lentil seeds, $w_0$ the initial weight of lentils and $MC_0$ the initial moisture content (in dry basis), calculated as follow:

$$MC_0 = \frac{w_{\text{raw}}}{w_{\text{dried}}} - 1 \qquad (3)$$

where $w_{\text{raw}}$ is the weight of a grinded powder of lentil seeds (blade grinder IKEA Tube-Mill Control) and $w_{\text{dried}}$ is the weight of this powder after 72 h of drying at 70 °C under vacuum (Heraeus Vacuum Oven VT6025).

Hydration kinetics were fitted by Peleg's model (Peleg, 1988):

$$MC_t = MC_0 + \frac{t}{k_1 + k_2t} \qquad (4)$$

where $MC_t$ is the moisture content of cooked lentils, $MC_0$ the initial moisture content, t the cooking (or soaking) time, $k_1$ and $k_2$ are model parameters which values allow to calculate moisture equilibrium (ME) and initial rate of water uptake at $t_0$ ($v_i$) as:

$$ME = MC_0 + \frac{1}{k_2} \qquad (5)$$

$$v_i = \frac{1}{k_1} \qquad (6)$$

Finally, cooking times were determined for each batch to obtain moisture contents of 100 %, 110 %, 120 %, 130 % and 140 % (total wet basis).

### 2.3.2. Seeds lying on wet cotton bed

For all three batches, 5 intact seeds and 5 dehulled seeds were conscientiously laid down on wet cotton in order to avoid any contact between hilum and cotton. Cotton was beforehand hydrated by 25 mL of distilled water at 21 °C and contained in 9 cm diameter petri dishes. Seeds mass was measured every two hours and moisture content was calculated as above. Mean and standard deviation were calculated from the 5 seeds. This experiment was performed to check whether hilum is the main entry for water.

## 2.4. Mechanical behaviour

The mechanical behaviour of lentil seeds from batches of different quality was determined at given moisture content (blue crosses on Fig. S2), by using results from hydration kinetics (2.3). For each moisture content, reached after cooking at 92 °C ± 3 °C in distilled water, ten seeds were individually compressed between two parallel plates (10 mm diameter). Mechanical trials were carried out down to 0.3 mm height at a speed of 5 mm/min, using a universal testing machine (Adamel-Lhomargy Model DY34B, MTS Systems, F-94 Créteil). The height and diameter of each seed were measured with a Vernier calliper before compression. Stress-deformation curves $\sigma = f(\epsilon)$ were drawn after calculating:

$$\sigma = \frac{F}{\pi * r^2} \qquad (7)$$

$$\epsilon = \frac{\text{top plate displacement}}{h} \qquad (8)$$

where $\sigma$ is the compression stress, F the force, r the lentil radius, $\epsilon$ the deformation and h the lentil height.

For each individual curve ($\sigma = f(\epsilon)$), the apparent modulus, deformation and stress at the rupture point and area under the curve, for deformation between 0 and 0.6, were computed; rupture point and modulus were manually chosen after the observation of each curve. For these 4 properties, mean and standard deviation were calculated. Analysis of variance followed by an HSD Tukey test were carried out for

each moisture content to investigate the difference between batches.

## 2.5. Seed morphology

Numerical images of samples were acquired by a camera tri-CCD (Hitachi Kokusai Electric Inc. - HV-F202GV) with wide angle lens (Myutron LS2528-F f25mm F2.8 F mount, Alliance Vision) placed at the level of a hole above a lighting box prototype (Devaux et al., 2008) in which the sample is placed and indirectly lightened by two LED strips (180 LED 3014, 8.6 lm/LED) arranged on both sides below the sample and intensity at 1548 lm. Intensities of obtained images are coded on 256 levels and dimensions are 1200 × 1600 pixel, at a distance of 24 cm. Camera shutter was 16,000 ns.

Lentil seeds were arrayed on a blue circular plate with 35 holes. For each batch (A, B and C), 10 images of 35 seeds were made, thus 350 different seeds were studied for each batch. Seeds on each image were individualized by creating a mask of regions of interest determined by removing blue pixels having a value superior to a threshold of 140. To fill remaining blue pixels on seeds, the Matlab function “imfill” that uses an algorithm based on morphological reconstruction (Soille, 1999) was used on an inverted image of the mask. Remaining noise on the mask of the blue plate was removed by size filtering of the elements.

Because images are taken from above, areas of objects represent the larger slice of seeds, that is the middle slice assuming an oblate shape of seeds (this assumption is consistent considering obtained width-to-length ratio on seed images that is near of 1 so object are circular). Surface was chosen to describe seed size and width-to-length ratio to describe shapes. Seed surface is relevant considering the hypothesis of an impact of seed surface in contact of water on the water absorption rate. Seeds density was measured by the volume displacement of 10 g of lentil seeds put into 15 mL of water, leading to a density of 1.28, 1.32 and 1.25 respectively for seeds from batches A, B and C (table S4). The mean major axis radius for 35 seeds was calculated as the mean of width and length of each seed. The minor axis radius (height) was calculated assuming an oblate shape, from mass (measured for each 35 seeds assay), major axis radius and previously measured densities. Minor axis radius, volume and surface of seeds were calculated by the following formula:

$$b = \frac{\frac{m}{\rho}}{\frac{4}{3}\pi * a^2} \qquad (9)$$

$$V = \frac{4}{3} * \pi * a^2 * b \qquad (10)$$

$$S = 2\pi a^2 + \frac{\pi b^2}{e} * \ln \frac{1 + e}{1 - e} \qquad (11)$$

$$e = \frac{\sqrt{a^2 - b^2}}{a} \qquad (12)$$

where V is the volume, a is the major axis radius, b is the minor axis radius, m is the mass, $\rho$ is the density, S is the surface and e is the ellipsoid eccentricity.

Because homoscedasticity prerequisite was not met, Kruskal-Wallis test followed by pairwise Wilcoxon test were performed to investigate the differences between the three batches. In order to better preserve the size and shape of the seeds, these characteristics were measured on images with no erosion of the remaining blue edges around seeds.

## 2.6. Biochemical composition

### 2.6.1. Plant material preparation

For tannins measurements, raw seeds from all batches were manually dehulled using tweezers and scalpel and seed coat was grounded with a blade grinder (IKEA Tube-Mill Control) and vacuum dried at 40 °C (Heraeus Vacuum Oven VT6025). Seed coat was grinded at low
temperature in liquid nitrogen. Measurements of starch, cell wall polysaccharides and proteins were made on cotyledons, seed coats and whole seeds for raw, soaked and cooked lentil seeds. The soaking time was 2 h at 21 °C and the cooking time was 20, 15 and 10 min at approximately 92 °C (Julabo water baths TW8), respectively for A, B and C batches. The dehulling of lentils seeds was made by chocking seeds with a pestle in a mortar for raw seeds, and by removing seed coat by hand for soaked and cooked seeds. Plant material (seed coat, cotyledon and whole seed for raw, soaked and cooked lentil seeds) was ground with a blade grinder (IKA Tube-Mill Control) and vacuum dried at 40 °C (Heraeus Vacuum Oven VT6025). For starch and cell wall polysaccharides, alcohol insoluble residue (AIR) was grinded again with a ball grinder (MP Fastprep-24). For pectins methylation, samples were prepared by the same way except that lentils were dehulled using a tong and scalpel instead of a mortar.

### 2.6.2. Proteins measurement

The protein amount was measured by using organic (carbon and nitrogen) elemental analyser (CNS Vario Cube, Elementar) from 5 mg of milled raw seeds that was encapsulated in a tin sheet. Flour reference provided by Bipea laboratory association (n°2/352 – January 2004) was used as nitrogen standard and control and acetanilide (3 mg) was used as carbon standard. Nitrogen in the samples were freed by combustion in an oven at 1080 °C under oxygen and helium flow. The gas was purified through a reduction tube at 850 °C. The amount of nitrogen was measured by a TCD detector and converted in equivalent protein content with a conversion factor of 5.36 (Mariotti et al., 2008).

### 2.6.3. Tannins measurement

Five ±0.25 mg of grinded seed coat powder was solubilized in 3.1 mL of solvent (butanol-HCl:ferric ammonium sulfate = 30:1). Standards and samples solutions were incubated 3 h at 95 °C then cooled and filtered with butanol resistant filter (Teflon PTFE) and absorbance at 550 nm was measured and tannins amount was calculated by comparing samples to standards results (standard curve from 0 to 100 µg/mL of procyanidin in butanol-HCl:ferric ammonium sulfate solvent).

### 2.6.4. Alcohol insoluble residue (AIR) preparation

Nearly 500 mg of ground plant material for whole seed and cotyledon or 100 mg for seed coat were weighed. Extractions were performed using an accelerated solvent extractor (ASE™ 350, Thermo Scientific™ Dionex™) with an ethanol solution (80 % ethanol at 100 °C, at flow rate of 2 mL/min (as described in Le Gall et al. (2016), during 15 min for cotyledons and whole seeds and 1 min for seed coats.

### 2.6.5. Starch content measurement

The starch amount was determined after enzymatic hydrolysis from 5 ± 0.5 mg of AIR and glucose measurement by High-Performance Anion Exchange Chromatography (Thermo Fisher Scientific ICS-6000) as described in Le Gall et al. (2016).

### 2.6.6. Cell wall polysaccharides measurement

Cell wall polysaccharides were determined as described in Lahaye et al. (2020) by measurement of neutral and acid monosaccharides after acid hydrolysis. According to Anton et Barett (2004) methanol esters were determined after saponification of AIR. Degree of methyl esterification are expressed as MD corresponding to molar ratio of methanol on uronic acids.

## 2.7. Solid-state NMR measurements

Raw lentil seeds were manually grinded in a mortar then put in a desiccator (Sanplatec Dry Keeper) with saturated solution of NaCl at 20 ± 0.1 °C to reach a water activity of 0.75 because moisture contents impact starch structuration (Paris et al., 1999). Three assays for each batch were carried out: 124.4 ± 2.1 mg for batch A, 125.2 ± 4 mg for

batch B, 123.1 ± 4.5 mg for batch C of grinded seeds were packed in 4 mm diameter rotors. A Bruker AVANCE NEO 400 WB spectrometer operating at a proton frequency of 400.13 MHz and a carbon frequency of 100.62 MHz was used for all NMR spectra acquisition. A double resonance ¹H/X CPMAS 4 mm probe connected on a high power-level amplifier was used. The magic angle spinning (MAS) rate was set at 12 kHz and each acquisition was acquired at regulated temperature (293 K). The experiments were conducted using a 90° proton pulse of 2.85 ± 0.05 µs. ¹³C CPMAS spectra were acquired using a contact time of 1.5 ms, 2048 accumulations and a recycling delay of 8 s. Spectra chemical shifts were referenced using Glycine. For spectral deconvolution, chemical shifts, width and area at half height of the ¹³C peaks were determined using a least-square fitting method available in the Peakfit® software (Systat software Inc., USA). The peak at about 102 ppm corresponding to single helices, three peaks at 100 to 98 ppm corresponding to double helices of A-type polymorph and two peaks at 96 to 93 ppm corresponding to amorphous starch (Paris et al., 1999, 2001). Spectra were decomposed using this template to determine different peaks area that were used to calculate the relative proportions of single helices, double helices and amorphous starch.

## 2.8. Differential scanning calorimetry

Lentil seeds from all three batches were cooked in water at 92 °C ± 3 °C during 2, 4, 10 and 35 min. Cooked and raw lentils were manually grinded in a mortar. About 10 mg of grinded seeds were weighed precisely and put with 40 µL of water in a hermetic inox capsule heated in parallel of a pressure capsule filled of 50 µL of water, in a differential scanning calorimeter DSC Q100 (TA Instruments). The heating rate was 3 °C/min, heating from 25 °C to 120 °C then cooling at 25 °C/min and repeating this loop once, with a sampling interval of 1 pt./s. For each assay, moisture content of grinded seeds was measured in desiccant scale MB90 (OHAUS) in order to calculate the dry weight of materiel put in the capsule. Measurements were made in triplicate for raw seeds and cooked seeds at 2 and 4 min and only one assay was made at 10 and 35 min (considering non-existent or very few endothermic peaks at these cooking time). Endothermic peak temperature and gelatinization enthalpy were obtained by integrating manually peaks on thermograms and dividing by the amount of dry starch obtained after biochemical analysis.

## 2.9. Microstructure

### 2.9.1. Optical microscopy

Lentil seeds of all three batches were cooked in water at 92 °C ± 3 °C for 10 and 35 min. Raw seeds and cooked seeds were cut twice with a razor blade, perpendicularly to the hilum, to obtain a central slice of the seeds. Seeds slices were prepared for observation by optical microscopy by fixing in 0.5 % (v/v) glutaraldehyde and 3 % formaldehyde in 0.1 M phosphate buffer (pH 7.4) during 24 h at 4 °C. Seeds slices were then rinsed 3 times (5 min each) in 0.1 M phosphate buffer and 5 times in deionized water. Samples were dehydrated by a graded aqueous ethanol series and embedded in London Resin White acrylic (LRW) as described in Chateigner-Boutin et al. (2014). Semi-thin sections (1 µm) were prepared using an ultramicrotome (Leica EM UC7) and sections were stained (triple coloration) with naphthol blue black (5 % in 7 % acetic acid) during 10 min, acridine orange (1 % in ultrapure water) during 5 min and Lugol's iodine (20 % in ultrapure water) during 2 min. The sections were then observed using an optical microscope (Leica DM2500 LED, magnification of 10). A minimum of two sections from two different seeds were examined per modality/condition (raw seeds, 10 min and 35 min cooked seeds, for batches A, B and C). Because the differences in cell structures seemed higher between batches A and B for raw and 35 min cooked seeds, 3 more seed samples were examined for these 4 modalities. The images were acquired in the same area for all samples.
## 2.9.2. Electronic microscopy

### 2.9.2.1. External observations.
External surface of seed coat of whole raw lentil seeds was observed with an environmental scanning electron microscope (ESEM) (Quattro S, Thermo Fisher Scientific) under classical mode, with approximately a pressure of 100 Pa, a voltage of 10 kV, a working distance of 6 ± 0.5 mm and a magnification of 50, then 500.

### 2.9.2.2. Internal observations.
Lentil seeds were cooked in water at approximately 92 °C during 2, 10 and 35 min. Raw seeds and cooked seeds (2, 10 and 35 min) were cut twice with a razor blade to obtain a central slice. Slices were directly observed with the ESEM. For raw seeds, observations were made under classical mode, with approximately a pressure of 100 Pa, a voltage of 8 kV, a magnification of 250 and a working distance of 7 ± 0.5 mm. For cooked seeds, observations were made with the environmental mode of the ESEM, that allows to observe wet samples, with approximately a pressure of 900 Pa, a voltage of 10 kV, a magnification of 350 and a working distance of 5 ± 0.5 mm. For each modality, a minimum of two seed samples were observed.

## 2.10. Statistical analyses

All calculations for modelling and statistical tests were performed in R version 4.2.2 using RStudio software. Non-linear modelling for sigmoidal fit was conducted using *nls()* function from the "stats" package. Linear modelling were carried out with the *lm()* function from base R. Package "car" was used for analyses of variance (type II). Assumptions of data independence, residual normality and homoscedasticity were verified using the *durbinWatsonTest()*, *shapiro.test()* and *Bartlett.test()* functions, respectively. When ANOVA assumption were not met, the non-parametric *Kruskal.test()* function was used instead. Post-hoc comparison were conducted using Tukey's tests via *glht()* function from the "multcomp" package, from which the group letters displayed on figures are derived.

# 3. Results

Three batches of French green lentils (cv. Anicia) from the same harvest year, evaluated for their ability to be canned without loss of texture, were characterized in terms of hydration and mechanical properties, composition (proteins and polysaccharides) and tissue structure. The results section presents the different analyses carried out to identify the seed characteristics that influence the canning quality of lentil seeds.

## 3.1. Validation of a method for assessing cooking quality of lentils

### 3.1.1. Visual assessment after cooking concord with industrial assessment

After cooking for 1 h in water at 92 °C, most lentil seeds from batch A were darker and browner than seeds from batch B and C, they remained intact whereas many lentil seeds from B and C were damaged, with torn and broken seed coats revealing cotyledons and burst cotyledons. Cooked lentil seeds are shown in supplementary data (see Fig. S1). These results were in coherence with the industrial assessment.

### 3.1.2. Cooking time distribution by Mattson-cooker can be used as reliable cooking quality assessment

Results from Mattson-cooker showed that lentil seeds from batches B and C, considered of lower canning quality, need less time in water at 92 °C to be cooked than lentil seeds from batch A (Fig. 1). Little difference was found between lentil seeds from batches B and C. Lentil seeds from batch B displayed narrower distribution with the shortest mean cooking time (28 min). In contrast, lentil seeds from batch A showed a broader distribution, with the longest mean cooking time (52.4 min). Lentil seeds from batch C had an intermediate behaviour,

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Mattson-Cooker cooking time distributions in water at 92 °C of lentil seeds from three batches A (■), B (○) and C (♦) and their fitting by sigmoid function (eq. 1) ($r^2 \ge 0.82$). The black triangle symbol shows, for each assay, the characteristic cooking time. The grey dashed line is at a proportion of crushed lentils of 0.5.</figcaption></figure>

closer to batch B (mean cooking time: 32.1 min).

Interestingly, sigmoid models fit the cooking time distribution of lentil seeds with $r^2$ between 0.82 and 0.98 and root mean square error (RMSE) between 0.17 and 0.04. Details of these parameters and characteristic cooking time are shown in table S1 in supplementary data. According to the Tukey test, characteristic cooking time values were significantly lower for batches B and C than for batch A, whereas no difference was found between batch B and batch C.

## 3.2. Hydration kinetics differ according to cooking quality

The kinetics of water uptake during cooking and soaking were different from batch to batch (Fig. 2). At large cooking and soaking time values, hydration kinetics reached a plateau (Fig. 2a and Fig. 2b). After 70 min cooking in water at 92 °C, lentil seeds from batch A reached a moisture content of 141 % (dry weight) while seeds of batches B and C rose to 179 % and 174 %, respectively. After 595 min of soaking in water at 22 °C, lentil seeds from batch A reached a moisture content of 122 % while seeds of batches B and C rose to 117 % and 115 %, respectively. By looking at experimental values upon cooking (points on Fig. 2a), the water uptake of lentil seeds from batch A was lower than those of batches C and B at the very beginning of the kinetics. A significant difference can be observed between moisture content of experimental values at 2 min and 4 min between all three batches. When soaked, lentil seeds from batch A were less hydrated than those from batches C and B, except, finally, at the plateau.

Variations of moisture content with cooking or soaking time for batches A, B and C were fitted by Peleg model (Fig. 2).

For soaked lentil seeds (Fig. 2b), Peleg model fits well for all the three batches (with $r^2 > 0.985$, see table S2), except for moisture equilibrium of batch A that could be overestimated. Batches B and C curves are characterized by a high initial hydration rate. The initial rate of water uptake during soaking is lower for batch A (0.6%/min) than C (1.1%/min) and lower for batch C than B (1.6%/min) (table S2). The values of moisture content at 585 min for experimental measurements between all three batches are close (about 1.3–1.4, see Fig. 2a and table S2).

For cooked lentil seeds, Peleg's model fits well for the batch A but less for B and C in spite of high $r^2$ values (0.992, 0.959 and 0.975, respectively). Actually, B and C kinetics display singularities compared to that of batch A: experimental measurements deviate from model value at about 2 min and moisture content increases sharply at 35 min (Fig. 2a and Fig.S2)). These singularities suggest that structural changes may have occurred in the seeds. Unlike soaking, the model coefficients values (see table S2), shows that water uptake initial rate during cooking is slightly higher for batch A (13%/min) than B and C (10%/min). The
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Hydration kinetics (% dry weight) of lentil seeds, for three batches A (■), B (●) and C (♦), by cooking in water at 92 °C (2a) or by soaking in water at 21 °C (2b) and fitted by the Peleg model (eq.3, r² ≥ 0.96 for all kinetics). Each point of kinetics represents the mean value, and error bars represent standard deviation calculated on three assays for Fig. 2a and b. Solid, dashed and dot-dashed lines represent models for respectively batches A, B and C (Fig. 2a, b).</figcaption></figure>

beginning of the kinetics looks very similar (Fig. 2a), which may be due to a poor fitting of the model at the beginning of the kinetics and experimental values should be kept in mind. The equilibrium value of water uptake is lower for batch A (169 %) than for batches B (233 %) and C (226 %).

All kinetics displayed downward concave shape, suggesting that hilum is not the main water pathway as described by Miano & Augusto, 2018 but this assumption is based on pulses, not specifically on lentils. Moreover, they propose downward concave shape for lentils but sigmoid shape for green lentils, such as those studied here. This was checked by comparing hydration kinetics of dehulled and intact seeds lying on a wet cotton (Fig. S3). Indeed, no significant difference of initial hydration rate was found within a single batch (~0.5 %/min for all batches, whether dehulled or not, as indicated in table S2). The values of moisture content at different hydration times in the kinetics do not differ significantly for all three batches. Finally, these results confirm that hilum is not the main water pathway for hydration of Anicia lentil seeds.

### 3.3. Mechanical behaviour differs according to moisture content and cooking quality

For each value of moisture content from 100 % to 140 %, mean curves of compression stress in function of deformation σ (ε) display an elasto-plastic behaviour. After a first stage (ε ≤ 0.2) due to the increase of seed-plate contact area, such behaviour is characterized by a linear stress increase until a maximum value (at ε ≈ 0.4) before a plateau sometimes preceded by a slight decrease, and a final increase at sample densification typically for deformation above 50 % (Fig. 3a, b, c). Due to the wide variability of curve shapes among the same batch and moisture

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Stress-deformation mean curves σ = f(ε) for moisture content from 100 % (light blue) to 140 % (dark blue) for batches A (3a), B (3b) and C (3c) and mean deformation value at rupture point of the σ = f(ε) curves, for three batches A (■), B (●) and C (♦), in function of moisture content reached for cooking time deduced from hydration kinetics measurements (3d). Mean values are calculated from individual curves and are represented by largest symbols; error bars are standard deviation. Individuals values are represented by small symbols. A slight abscissae shift was introduced between the three batches for more readability of standard deviation bars (3d). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
content, mean curve are presented to better account for the general shape, and its change with water content. This behaviour is characteristic of soft materials, especially “semi-solid” foods, and the stress decrease may be due to internal rupture of some structural entity or tissue. At a 100 % moisture content, the general shape of the curve for batch A shows some difference with batches B and C, with a marked stress increase, reflecting a stronger seed resistance to compression. This difference, still observable for a moisture content of 110 %, decreases, and is no more detected when increasing moisture content from 120 % to 140 %, where curve shapes are very similar for the three batches (Fig. 3b). Among the four mechanical properties studied (see table S3), variations in deformation at rupture $\epsilon_r$ with moisture content showed the most significant difference between batches (Fig. 3d). Concerning others parameters (compression stress at rupture point, modulus, and area under the curve), no significant difference between batches was found but values decreased as moisture content increased (table S3). Lentil seeds from batch A show a larger $\epsilon_r$ value than seeds from batches B and C; this difference is significant from a moisture content of 120 % and it is more important when moisture content increase. This result shows that seeds from batch A are more deformable during cooking than lentils from batch B and C. It provides an explanation for the least bursting and broken cotyledons for the seeds of this batch (see 3.1.1).

## 3.4. Before cooking, seed morphology differs between batches and visual appearance does not show seed coat integrity failure

The measured width-to-length ratio of lentil seeds from all three batches is around 0.95. Lentil seeds from all the three batches are then

nearly circular with similar width and length.

Average density of lentil seeds, measured by water immersion, is 1.28, 1.35 and 1.25 for batches A, B and C, respectively (table S4). This result is consistent with the value found by Joshi et al. (1.3 g/mL) (Joshi et al., 2010). Surface area of lentil seeds were then deduced from seed density, weight and radius. Lentil seeds from batch A are significantly smaller (38.25 mm²) than C (41.68 mm²) itself smaller than B (44.17 mm²) (table S4). Details of mean, standard deviation and statistical analysis results for surface, surface-to-volume ratio and width-to-length ratio are available in supplementary data (table S4).

No differences between batches was observed, by scanning electron microscopy, on the lentil seed coat from high or low-quality batches, nor any abrasion marks, faults or pores that hypothetically could impact water absorption rate (data not shown), suggesting that seed coat integrity is not involved in the water absorption differences between batches.

## 3.5. Cell wall polysaccharides, tannins, and starch structure may have a role in differences in cooking resistance observed between batches of whole lentils

### 3.5.1. Differences of composition in whole seeds

Raw lentil seeds contain nearly 40.5–42.8 % starch, 21.2–23.0 % proteins and 12.3–14.7 % cell wall polysaccharides (Table 1). No significant differences were observed considering starch and protein contents. In contrast, the content of cell wall polysaccharides is significantly higher in batch A than in batch B, itself higher than batch C (respectively 14.72 % vs 12.27 % and 13.92 % (Table 1)). The monosaccharide

<caption>**Table 1**</caption>

Mean, standard deviation and Tukey's test results for major compounds of whole lentil seeds, cotyledons (starch, proteins and cell wall polysaccharides (CWP)) and seed coat (proteins, cell wall polysaccharides and tannins) of raw, soaked or cooked lentil seeds from batches A, B and C.

Results are expressed as percentage of dry weight. No difference between batches at 5 % threshold Tukey's test are indicated by same lowercase letter. Results are highlighted in grey when a significant difference is observed between lentil seeds from batch A and from both batches B and C. Starch was not measured in seed coat. Tannins were measured only for raw seed cot. These non-available data were indicated by “NA” in table.

<table><thead><tr><th>Tissue</th><th>Treatment</th><th>Batch</th><th>Starch</th><th>Proteins</th><th>CWP</th><th>Tannins</th></tr></thead><tbody><tr><td rowspan="24">Whole seed</td><td rowspan="3">Raw</td><td>A</td><td>41.99±1.60<sup>a</sup></td><td>22.83±0.99<sup>a</sup></td><td>14.72±0.18<sup>c</sup></td><td>NA</td></tr><tr><td>B</td><td>40.48±1.79<sup>a</sup></td><td>22.97±0.55<sup>a</sup></td><td>12.27±0.25<sup>a</sup></td><td>NA</td></tr><tr><td>C</td><td>42.79±1.30<sup>a</sup></td><td>21.24±0.03<sup>a</sup></td><td>13.92±0.09<sup>b</sup></td><td>NA</td></tr><tr><td rowspan="3">Soaked</td><td>A</td><td>41.00±1.55<sup>a</sup></td><td>21.18±1.17<sup>a</sup></td><td>14.77±0.04<sup>c</sup></td><td>NA</td></tr><tr><td>B</td><td>45.14±1.05<sup>b</sup></td><td>22.28±0.12<sup>a</sup></td><td>11.46±0.15<sup>b</sup></td><td>NA</td></tr><tr><td>C</td><td>50.60±0.43<sup>c</sup></td><td>20.81±1.07<sup>a</sup></td><td>6.88±0.20<sup>a</sup></td><td>NA</td></tr><tr><td rowspan="3">Cooked</td><td>A</td><td>44.89±0.65<sup>a</sup></td><td>21.06±1.01<sup>a</sup></td><td>14.36±0.05<sup>c</sup></td><td>NA</td></tr><tr><td>B</td><td>46.51±0.80<sup>a</sup></td><td>21.82±1.11<sup>a</sup></td><td>12.04±0.24<sup>b</sup></td><td>NA</td></tr><tr><td>C</td><td>51.09±1.73<sup>b</sup></td><td>20.71±0.42<sup>a</sup></td><td>7.31±0.20<sup>a</sup></td><td>NA</td></tr><tr><td rowspan="24">Cotyledon</td><td rowspan="3">Raw</td><td>A</td><td>48.45±4.88<sup>a</sup></td><td>24.13±0.52<sup>a</sup></td><td>10.52±0.52<sup>b</sup></td><td>NA</td></tr><tr><td>B</td><td>49.47±6.36<sup>a</sup></td><td>26.05±0.29<sup>a</sup></td><td>7.69±0.46<sup>a</sup></td><td>NA</td></tr><tr><td>C</td><td>42.44±2.95<sup>a</sup></td><td>25.50±0.83<sup>a</sup></td><td>14.61±0.13<sup>c</sup></td><td>NA</td></tr><tr><td rowspan="3">Soaked</td><td>A</td><td>47.77±0.72<sup>a</sup></td><td>22.83±0.74<sup>a</sup></td><td>6.35±0.10<sup>b</sup></td><td>NA</td></tr><tr><td>B</td><td>47.83±1.82<sup>a</sup></td><td>24.21±0.59<sup>ab</sup></td><td>6.65±0.16<sup>b</sup></td><td>NA</td></tr><tr><td>C</td><td>56.09±1.16<sup>b</sup></td><td>24.58±0.67<sup>b</sup></td><td>4.42±0.14<sup>a</sup></td><td>NA</td></tr><tr><td rowspan="3">Cooked</td><td>A</td><td>51.16±0.23<sup>a</sup></td><td>21.96±0.24<sup>a</sup></td><td>8.05±0.23<sup>c</sup></td><td>NA</td></tr><tr><td>B</td><td>48.88±0.66<sup>a</sup></td><td>24.41±0.61<sup>b</sup></td><td>6.22±0.21<sup>b</sup></td><td>NA</td></tr><tr><td>C</td><td>54.84±1.77<sup>b</sup></td><td>22.46±1.15<sup>a</sup></td><td>4.92±0.24<sup>a</sup></td><td>NA</td></tr><tr><td rowspan="24">Seed coat</td><td rowspan="3">Raw</td><td>A</td><td>NA</td><td>8.85±1.41<sup>a</sup></td><td>54.70±2.46<sup>a</sup></td><td>11.87±0.72<sup>b</sup></td></tr><tr><td>B</td><td>NA</td><td>7.97±0.34<sup>a</sup></td><td>55.04±1.67<sup>a</sup></td><td>9.83±0.18<sup>a</sup></td></tr><tr><td>C</td><td>NA</td><td>7.86±0.39<sup>a</sup></td><td>57.74±1.52<sup>a</sup></td><td>9.36±0.28<sup>a</sup></td></tr><tr><td rowspan="3">Soaked</td><td>A</td><td>NA</td><td>8.41±0.24<sup>b</sup></td><td>53.50±1.88<sup>a</sup></td><td>NA</td></tr><tr><td>B</td><td>NA</td><td>7.16±0.23<sup>a</sup></td><td>59.11±1.92<sup>b</sup></td><td>NA</td></tr><tr><td>C</td><td>NA</td><td>8.16±0.32<sup>b</sup></td><td>54.21±2.73<sup>ab</sup></td><td>NA</td></tr><tr><td rowspan="3">Cooked</td><td>A</td><td>NA</td><td>11.11±0.21<sup>b</sup></td><td>59.22±1.04<sup>ab</sup></td><td>NA</td></tr><tr><td>B</td><td>NA</td><td>10.67±0.11<sup>b</sup></td><td>64.30±1.97<sup>b</sup></td><td>NA</td></tr><tr><td>C</td><td>NA</td><td>9.96±0.21<sup>a</sup></td><td>57.76±1.99<sup>a</sup></td><td>NA</td></tr></tbody></table>
composition of cell wall polysaccharides revealed that the arabinoxose residues (Ara) were significantly higher in batch A than in batch B (respectively 2.73 % and 2.45 %) (but not C (2.54 %)) while other monosaccharides showed no significant differences (Table 2). Cellulose content, as a trend, is higher in batch A (6.43 %) than in batches B (5.30 %) and C (4.98 %). These values are higher than those found in literature that are about 3 % (Srivastava & Vasishtha, 2013) but literature values were measured using a different method and on a different variety.

After cooking, cell wall polysaccharides amount in whole seeds of the batch A remained significantly higher than in batches B and C (respectively 14.36 % vs 12.04 % and 7.31 % with trends observed for Ara and galactose (Gal) residues, while no differences were observed in terms of starch and protein contents (Table 1). Batch C displayed a higher amount of starch (51.09 %) than batches A (44.89 %) and B (46.51 %) and this was also observed in soaked whole seeds (with respectively 41 %, 45.14 % and 50.6 % for batches A, B and C). The amount of starch is consistent with what was previously measured in literature (Brummer et al., 2015) for raw green lentils (about 42 %) but less for cooked seeds (up to 50 % against 41 %), maybe due to more leaching of other compounds because seeds were cooked in higher volume of water. But, as batches were cooked under same conditions, they remain comparable.

After soaking, while the protein contents showed no difference among batches, the cell wall polysaccharides content in the batch A was significantly higher than in batch B, which was also higher than in batch C (respectively 14.77 %, 11.46 % and 6.88 %) (Table 1) mainly due to

the Ara residue (Table 2). Once more, non-starch glucose was not detected for batch C (Table 2). After soaking, the amount of starch was significantly lower in batch A compared to the batches B and C (respectively 41.00 % vs 45.14 % and 50.60 %) (Table 1).

Results are consistent with the reported composition of cell walls isolated from raw lentil seeds var. Eston with slow and fast cooking behaviours (Bhatty, 1990) with galacturonic acid (GalA) and Ara displaying higher amount in slow cooking seeds.

### 3.5.2. Differences of composition in cotyledons

On raw cotyledons, after dehulling, results showed same trends among batches as those observed in raw whole seeds. While no differences were observed in term of starch and protein contents, batch A had a significantly higher content of cell wall polysaccharides than batch B but not than batch C that display even higher amount of cell wall polysaccharides (Table 1). This is explained by a higher amount of Ara and GalA residues in batch A compared to batch B (for Ara and GalA respectively: 2.33 % and 1.32 % for batch A, 2.02 % and 1.15 % for batch B while batch C contained a similar amount of Ara (2.30 %) than batch A and a lower amount of GalA (1.11 %)) (Table 2). Pectins from batch A had significantly higher degree of methylation (15.58 %) than in batch B (13.15 %), batch C being intermediate (14.24 %). Surprisingly, batch C had significantly higher amount cellulose (6.69 %) than batches A and B (1.57 % and 1.33 %) but this large difference was not confirmed on soaked or cooked seeds where cellulose was not detected. And this

<caption>**Table 2**</caption>

Mean, standard deviation and Tukey's test results for cell wall polysaccharides composition and pectins methylation degree (MD) for whole seeds, cotyledons and seed coat of raw, soaked or cooked lentil seeds form batches A, B and C.

Results are expressed as percentage of dry weight. No difference between batches at 5 % threshold Tukey's test are indicated by same lowercase letter. Results are coloured in grey when a significant difference is observed between lentil seeds from batch A and from both batches B and C. The amount of rhamnose, galacturonic acid, arabinoxose and galactose, varying together, reflects the amount of pectins (that are made of these compounds). The methylation degree of pectins was only measured for raw cotyledons and raw seed coat. These non-available data were indicated by "NA" in table. Some non detected values were indicated by "ND" in table.

<table><thead><tr><th>Tissue</th><th>Treatment</th><th>Batch</th><th>Rhamnose</th><th>Arabinose</th><th>Xylose</th><th>Mannose</th><th>Galactose</th><th>Non-starch glucose*</th><th>*Including Cellulose</th><th>Galacturonic acid</th><th>MD of Pectins</th></tr></thead><tbody><tr><td rowspan="12">Whole seed</td><td rowspan="3">Raw</td><td>A</td><td>0.30±0.03<sup>a</sup></td><td>2.73±0.11<sup>b</sup></td><td>1.42±0.06<sup>b</sup></td><td>0.17±0.01<sup>a</sup></td><td>1.16±0.13<sup>a</sup></td><td>6.67±1.64<sup>a</sup></td><td>6.43±1.64<sup>a</sup></td><td>2.31±0.36<sup>a</sup></td><td>NA</td></tr><tr><td>B</td><td>0.26±0.02<sup>a</sup></td><td>2.45±0.04<sup>a</sup></td><td>1.45±0.14<sup>b</sup></td><td>0.24±0.03<sup>a</sup></td><td>1.01±0.06<sup>a</sup></td><td>4.84±0.72<sup>a</sup></td><td>5.30±0.72<sup>a</sup></td><td>2.02±0.28<sup>a</sup></td><td>NA</td></tr><tr><td>C</td><td>0.25±0.01<sup>a</sup></td><td>2.54±0.1<sup>ab</sup></td><td>1.12±0.04<sup>a</sup></td><td>0.24±0.04<sup>a</sup></td><td>1.23±0.11<sup>a</sup></td><td>6.61±2.07<sup>a</sup></td><td>4.98±0.42<sup>a</sup></td><td>2.01±0.4<sup>a</sup></td><td>NA</td></tr><tr><td rowspan="3">Soaked</td><td>A</td><td>0.31±0.07<sup>a</sup></td><td>2.78±0.06<sup>b</sup></td><td>1.27±0.1<sup>a</sup></td><td>0.22±0.08<sup>a</sup></td><td>0.64±0.04<sup>a</sup></td><td>7.40±0.5<sup>b</sup></td><td>4.72±0.50<sup>a</sup></td><td>2.16±0.08<sup>a</sup></td><td>NA</td></tr><tr><td>B</td><td>0.27±0.03<sup>a</sup></td><td>2.63±0.06<sup>b</sup></td><td>1.39±0.09<sup>a</sup></td><td>0.23±0.03<sup>a</sup></td><td>0.55±0.05<sup>a</sup></td><td>4.07±0.98<sup>a</sup></td><td>5.63±0.98<sup>a</sup></td><td>2.31±0.16<sup>a</sup></td><td>NA</td></tr><tr><td>C</td><td>0.24±0.04<sup>a</sup></td><td>2.30±0.17<sup>a</sup></td><td>1.29±0.04<sup>a</sup></td><td>0.20±0.03<sup>a</sup></td><td>0.57±0.01<sup>a</sup></td><td>ND</td><td>ND</td><td>2.29±0.09<sup>a</sup></td><td>NA</td></tr><tr><td rowspan="3">Cooked</td><td>A</td><td>0.30±0.05<sup>a</sup></td><td>2.77±0.13<sup>b</sup></td><td>1.29±0.10<sup>a</sup></td><td>0.20±0.03<sup>a</sup></td><td>0.94±0.06<sup>b</sup></td><td>6.36±1.14<sup>b</sup></td><td>4.96±1.14</td><td>2.49±0.25<sup>a</sup></td><td>NA</td></tr><tr><td>B</td><td>0.31±0.04<sup>a</sup></td><td>2.47±0.18<sup>ab</sup></td><td>1.37±0.15<sup>a</sup></td><td>0.36±0.03<sup>b</sup></td><td>0.82±0.04<sup>ab</sup></td><td>4.75±3.46<sup>a</sup></td><td>3.20</td><td>2.07±0.07<sup>a</sup></td><td>NA</td></tr><tr><td>C</td><td>0.27±0.02<sup>a</sup></td><td>2.43±0.04<sup>a</sup></td><td>1.37±0.07<sup>a</sup></td><td>0.19±0.05<sup>a</sup></td><td>0.77±0.05<sup>a</sup></td><td>ND</td><td>ND</td><td>2.28±0.23<sup>a</sup></td><td>NA</td></tr><tr><td rowspan="12">Cotyledon</td><td rowspan="3">Raw</td><td>A</td><td>0.29±0.07<sup>a</sup></td><td>2.33±0.15<sup>b</sup></td><td>0.35±0.06<sup>a</sup></td><td>0.19±0.04<sup>a</sup></td><td>0.85±0.38<sup>a</sup></td><td>5.22±0.45<sup>a</sup></td><td>1.57±1.09<sup>a</sup></td><td>1.32±0.09<sup>b</sup></td><td>15.58±1.15<sup>b</sup></td></tr><tr><td>B</td><td>0.26±0.03<sup>a</sup></td><td>2.02±0.09<sup>a</sup></td><td>0.30±0.05<sup>a</sup></td><td>0.22±0.04<sup>a</sup></td><td>0.66±0.27<sup>a</sup></td><td>3.21±1.56<sup>a</sup></td><td>1.33±0.99<sup>a</sup></td><td>1.15±0.12<sup>a</sup></td><td>13.15±1.10<sup>a</sup></td></tr><tr><td>C</td><td>0.31±0.03<sup>a</sup></td><td>2.30±0.12<sup>b</sup></td><td>0.37±0.03<sup>a</sup></td><td>0.24±0.07<sup>a</sup></td><td>1.05±0.07<sup>a</sup></td><td>9.23±2.17<sup>b</sup></td><td>6.69±2.17<sup>b</sup></td><td>1.11±0.09<sup>a</sup></td><td>14.24±0.45<sup>ab</sup></td></tr><tr><td rowspan="3">Soaked</td><td>A</td><td>0.28±0.03<sup>a</sup></td><td>2.32±0.04<sup>c</sup></td><td>0.36±0.03<sup>a</sup></td><td>0.15±0.03<sup>a</sup></td><td>0.50±0.01<sup>a</sup></td><td>1.34±0.94<sup>a</sup></td><td>ND</td><td>1.40±0.01<sup>b</sup></td><td>NA</td></tr><tr><td>B</td><td>0.28±0.03<sup>a</sup></td><td>2.22±0.04<sup>b</sup></td><td>0.37±0.05<sup>a</sup></td><td>0.16±0.04<sup>a</sup></td><td>0.49±0.09<sup>a</sup></td><td>1.82±0.78<sup>a</sup></td><td>0.89±0.21</td><td>1.33±0.02<sup>ab</sup></td><td>NA</td></tr><tr><td>C</td><td>0.24±0.04<sup>a</sup></td><td>1.98±0.02<sup>a</sup></td><td>0.28±0.03<sup>a</sup></td><td>0.15±0.04<sup>a</sup></td><td>0.49±0.03<sup>a</sup></td><td>ND</td><td>ND</td><td>1.27±0.06<sup>a</sup></td><td>NA</td></tr><tr><td rowspan="3">Cooked</td><td>A</td><td>0.31±0.05<sup>a</sup></td><td>2.42±0.1<sup>a</sup></td><td>0.42±0.04<sup>a</sup></td><td>0.16±0.02<sup>a</sup></td><td>0.74±0.02<sup>c</sup></td><td>2.60±1.92<sup>a</sup></td><td>2.77±1.92</td><td>1.46±0.10<sup>a</sup></td><td>NA</td></tr><tr><td>B</td><td>0.30±0.02<sup>a</sup></td><td>2.10±0.07<sup>a</sup></td><td>0.40±0.08<sup>a</sup></td><td>0.24±0.03<sup>b</sup></td><td>0.59±0.02<sup>a</sup></td><td>1.30±0.48<sup>a</sup></td><td>ND</td><td>1.31±0.10<sup>a</sup></td><td>NA</td></tr><tr><td>C</td><td>0.25±0.05<sup>a</sup></td><td>2.13±0.22<sup>a</sup></td><td>0.37±0.03<sup>a</sup></td><td>0.20±0.02<sup>ab</sup></td><td>0.66±0.04<sup>b</sup></td><td>ND</td><td>ND</td><td>1.31±0.03<sup>a</sup></td><td>NA</td></tr><tr><td rowspan="12">Seed coat</td><td rowspan="3">Raw</td><td>A</td><td>0.46±0.05<sup>a</sup></td><td>4.72±0.31<sup>a</sup></td><td>8.81±0.4<sup>a</sup></td><td>0.44±0.05<sup>a</sup></td><td>1.22±0.15<sup>a</sup></td><td>29.15±0.19<sup>a</sup></td><td>27.28±1.40<sup>a</sup></td><td>9.92±0.86<sup>a</sup></td><td>17.75±2.62<sup>a</sup></td></tr><tr><td>B</td><td>0.47±0.03<sup>a</sup></td><td>4.77±0.17<sup>a</sup></td><td>9.62±0.32<sup>b</sup></td><td>0.60±0.05<sup>b</sup></td><td>1.32±0.16<sup>a</sup></td><td>28.72±1.23<sup>a</sup></td><td>26.82±1.23<sup>a</sup></td><td>9.86±1.26<sup>a</sup></td><td>16.07±1.31<sup>a</sup></td></tr><tr><td>C</td><td>0.51±0.04<sup>a</sup></td><td>5.14±0.04<sup>a</sup></td><td>9.56±0.22<sup>b</sup></td><td>0.64±0.06<sup>b</sup></td><td>1.64±0.01<sup>b</sup></td><td>30.03±0.49<sup>a</sup></td><td>27.47±0.49<sup>a</sup></td><td>10.33±0.95<sup>a</sup></td><td>16.26±1.11<sup>a</sup></td></tr><tr><td rowspan="3">Soaked</td><td>A</td><td>0.51±0.02<sup>a</sup></td><td>4.26±0.32<sup>a</sup></td><td>8.50±0.41<sup>a</sup></td><td>0.53±0.11<sup>a</sup></td><td>1.47±0.01<sup>b</sup></td><td>28.72±1.11<sup>ab</sup></td><td>26.32±1.11<sup>a</sup></td><td>9.51±0.79<sup>a</sup></td><td>NA</td></tr><tr><td>B</td><td>0.42±0.07<sup>a</sup></td><td>4.58±0.27<sup>a</sup></td><td>10.83±0.32<sup>c</sup></td><td>0.40±0.1<sup>a</sup></td><td>0.96±0.15<sup>a</sup></td><td>31.49±1.63<sup>b</sup></td><td>30.35±1.63<sup>b</sup></td><td>10.44±0.48<sup>a</sup></td><td>NA</td></tr><tr><td>C</td><td>0.43±0.02<sup>a</sup></td><td>4.55±0.32<sup>a</sup></td><td>9.74±0.5<sup>b</sup></td><td>0.47±0.09<sup>a</sup></td><td>1.04±0.18<sup>a</sup></td><td>27.31±1.63<sup>a</sup></td><td>26.18±1.63<sup>a</sup></td><td>10.67±0.44<sup>a</sup></td><td>NA</td></tr><tr><td rowspan="3">Cooked</td><td>A</td><td>0.43±0.06<sup>a</sup></td><td>4.58±0.11<sup>a</sup></td><td>9.63±0.11<sup>a</sup></td><td>0.67±0.03<sup>a</sup></td><td>3.71±0.16<sup>b</sup></td><td>29.56±0.19<sup>a</sup></td><td>27.30±0.19<sup>a</sup></td><td>10.56±0.14<sup>a</sup></td><td>NA</td></tr><tr><td>B</td><td>0.51±0.03<sup>a</sup></td><td>5.02±0.10<sup>b</sup></td><td>11.50±0.35<sup>b</sup></td><td>0.58±0.05<sup>a</sup></td><td>3.34±0.17<sup>a</sup></td><td>33.81±1.05<sup>b</sup></td><td>29.87±1.05<sup>b</sup></td><td>9.52±0.67<sup>a</sup></td><td>NA</td></tr><tr><td>C</td><td>0.53±0.03<sup>a</sup></td><td>4.74±0.25<sup>ab</sup></td><td>10.19±0.52<sup>a</sup></td><td>0.58±0.12<sup>a</sup></td><td>3.14±0.09<sup>a</sup></td><td>29.28±1.07<sup>a</sup></td><td>27.09±1.07<sup>a</sup></td><td>10.23±0.65<sup>a</sup></td><td>NA</td></tr></tbody></table>
difference was not observed on whole seeds, nor on seed coats (Table 2). However, as no difference was found between batches A and B that are high and low quality, cellulose amount was no longer investigated.

After cooking, significant differences were observed in terms of starch and protein contents, with the starch contents in batch A higher than in batch B (respectively 51.16 %, 48.88 %) but lower than C (54.84 %) and the protein contents in the batches A and C significantly lower than in batch B (respectively 21.96 %, 22.46 % and 24.41 %) (Table 1). Significant differences of cell wall polysaccharides contents are observed in cooked cotyledons with higher amount of Ara, GalA and Gal residues in batch A than in batches B and C (respectively for A, B and C: 2.42 %, 2.10 % and 2.13 % for Ara, 1.46 %, 1.31 % and 1.31 % for galacturonic acid, 0.74 %, 0.59 % and 0.66 % for Gal) (Table 2).

After soaking, batch C is significantly higher than batches A and B in terms of starch content (as observed in the whole seeds). In cotyledons, a significant difference between cell wall polysaccharides in batches A and C is observed (Table 1) as it was in whole seed (respectively 6.35 % and 4.42 %) but not between A and B (6.65 % for B). This is mainly due to Ara and GalA residues (respectively for A, B and C, for cell wall polysaccharides: 5.02 %, 4.84 % and 4.42 %, for Ara: 2.32 %, 2.22 % and 1.98 %, for GalA: 1.40 %, 1.33 % and 1.27 %) (Table 2).

In cotyledons, results show higher amount of pectins, with higher methylation degree in raw cotyledons, for batch A than batches B and C, whether on raw, soaked or cooked seeds.

### 3.5.3. Differences of composition in seed coat

Raw seed coat contained nearly 7.8–8.8 % of proteins and 54.5–57.7 % of cell wall polysaccharides. While no significant differences were observed considering total cell wall polysaccharides and proteins, the tannin content was significantly higher in batch A than in batches B and C (11.87 %, 9.83 % and 9.36 %) (Table 1). Considering monosaccharide composition, xylose (Xyl) and mannose (Man) residues were significantly lower in batch A than in batches B and C (respectively for A, B and C, for Xyl: 8.81 %, 9.62 % and 9.56 %; for Man: 0.44 %, 0.60 % and 0.64 %) (Table 2).

The observed differences persisted after cooking for Xyl residues, while a significant difference was observed for Gal residues (respectively for A, B and C: 1.47 %, 0.96 % and 1.04 %) with significantly higher quantities in batch A compared to batches B and C (Table 2). For batch B, cellulose amount was significantly higher (29.87 %) than for batches A and C (27.30 % and 27.09 %) and this was also observed for soaked seed coats (30.35 % for batch B, 26.32 % for batch A and 26.18 % for batch C) but not in raw seeds coat (Table 2). These results are consistent with those found by Sharma et al., 2022 (33 %).

After soaking, the observed differences in respectively raw and cooked seed coat persisted with significantly lower amount of Xyl residues (respectively for A, B and C, 8.50 %, 10.83 % and 9.74 %) and inversely higher amount of Gal residues in batch A compared to batches B and C (1.47 %, 0.96 % and 1.04 % respectively) (Table 2).

### 3.5.4. Starch structure and behaviour differs among batches

As expected, the gelatinisation enthalpy $ΔH_g$ of starch decreased from about 11 J/g, as cooking time increased until 35 min where no residual enthalpy was observed (Fig. 4). Slight differences between the three batches were observed, with batch A displaying the lowest $ΔH_g$ value and starch from batch B exhibiting the highest, except at 2 min of cooking (Table 3). Curves at 10 min of cooking indicated minimal intact starch remaining. Starch from batch A exhibited the highest gelatinisation peak temperature (72 °C), while starch from batch B (67 °C) showed the lowest, at 0, 2, and 4 min of cooking. Results obtained for $ΔH_g$ of starch in lentil seeds is similar to previous measurement between 10 J/g and 13.5 J/g, but the higher temperature of gelatinization suggests that crystalline structure in starch of A seeds is more ordered (Chung et al., 2010; Hoover & Manuel, 1996; L. Li et al., 2019).

Lentil structure was investigated by solid-state NMR for raw seeds of all batches. Signals associated with starch and proteins are identified on

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** thermogram of raw and cooked lentil seeds at 2, 4, 10 and 35 min for batches A, B and C. Heat flow values are shown as relative value to avoid curves overlap and facilitate figure reading. For raw seeds and cooked seeds at 2 and 4 min, the most representative curve of the three assays was chosen.</figcaption></figure>

all NMR spectra as referenced in literature (Nahar et al., 2024). These spectra provide unique insights into the spatial structure of starch. Specifically, the proportions of double helices (sometimes referred as the degree of crystallinity (Paris et al., 1999)), single helices, and amorphous regions were determined through spectral deconvolution of the signal associated with the C1 carbon of the glucosidic units (90–110 ppm), as illustrated in Fig. S4. The results shown in Table 3 highlight that seeds from batch A contains significantly less amorphous starch (10 % against 15 %) and simple helices starch (28 % against 31 % and 32 %) and more double helices starch (62 % against 54 % and 53 %) than seeds from the other batches.
<caption>**Table 3**</caption>

starch endothermic peak temperature and gelatinization enthalpy of raw and cooked lentil seeds at 2, 4, 10 and 35 min and proportion of amorphous starch, double helices and simple helices of raw lentil seeds for batches A, B and C. As NMR measurement were only performed on raw seeds, non-available data for cooked seeds were indicated by “NA” in table.

<table><thead><tr><th>Time (min)</th><th>Batch</th><th>Peak temp. (°C)</th><th>ΔH (J/g)</th><th>Amorphous starch (%)</th><th>Double helices (%)</th><th>Simple helices (%)</th></tr></thead><tbody><tr><td rowspan="4">0</td><td>A</td><td>72.71 ± 0.07</td><td>9.75 ± 0.11</td><td rowspan="4">10 ± 1</td><td rowspan="4">62.2 ± 2</td><td rowspan="4">28 ± 1</td></tr><tr><td>B</td><td>67.33 ± 0.29</td><td>11.10 ± 0.30</td></tr><tr><td>C</td><td>69.28 ± 0.51</td><td>11.24 ± 0.48</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="4">2</td><td>A</td><td>72.20 ± 0.24</td><td>11.96 ± 2.30</td><td rowspan="4">NA</td><td rowspan="4">NA</td><td rowspan="4">NA</td></tr><tr><td>B</td><td>68.80 ± 0.54</td><td>8.80 ± 0.20</td></tr><tr><td>C</td><td>71.38 ± 1.69</td><td>5.74 ± 1.42</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="4">4</td><td>A</td><td>76.34 ± 1.30</td><td>4.89 ± 0.58</td><td rowspan="4">NA</td><td rowspan="4">NA</td><td rowspan="4">NA</td></tr><tr><td>B</td><td>70.04 ± 1.09</td><td>6.72 ± 0.26</td></tr><tr><td>C</td><td>71.98 ± 0.11</td><td>6.28 ± 1.82</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="3">10</td><td>A</td><td>76.75</td><td>3.49</td><td rowspan="3">NA</td><td rowspan="3">NA</td><td rowspan="3">NA</td></tr><tr><td>B</td><td>76.90</td><td>5.38</td></tr><tr><td>C</td><td>74.27</td><td>3.24</td></tr><tr><td rowspan="3">35</td><td>A</td><td>NA</td><td>0</td><td rowspan="3">NA</td><td rowspan="3">NA</td><td rowspan="3">NA</td></tr><tr><td>B</td><td>NA</td><td>0</td></tr><tr><td>C</td><td>NA</td><td>0</td></tr></tbody></table>

### 3.6. Cell cohesion differs between batches after cooking

#### 3.6.1. Larger intercellular lacunas appear in low quality batches than high quality one

Optical microscopy of raw lentil seeds, allows to visualize starch granules, proteins and cell walls in cotyledons, by triple coloration (Fig. 5a). Sectioning resulted in the loss of some starch granules, as shown by the presence of white holes. No obvious difference in surface area occupied by protein or starch was noticed between lentil seeds form batches A, B or C. After 10 min cooking, the hydration of the seed is visible, beginning from the edges, with swollen starch granules that occupy the largest part of border cells. After 35 min cooking, the hydration reached the core of the seed with all cells containing swollen starch granules. Intercellular spaces of different sizes appeared in the cotyledons. For most seeds, the size and frequency of these intercellular spaces were higher for lentil seeds from batches B and C than A (Fig. 5 & Fig. S5). These results are consistent with the reported presence of more cell separation in cooked Eston lentil with rapid cooking behaviour compared to slow cooking samples (Bhatty, 1990).

#### 3.6.2. Differences of appearance of internal architecture appears at 10 min (moisture content 80 %) cooking between high and low-quality batches

The degradation of cellular structure during cooking has been confirmed by scanning electron microscopic observations (Fig. 5b). On raw lentil seeds of all batches, the sections of cotyledons show mainly starch granules in the intracellular matrix. At 2 min of cooking, same observations can be made but starch granules begin to swell. At 35 min cooking, no more individual starch granules are observed and, instead, whole cells are revealed. Cells are spaced by holes and do not adhere together. At 10 min of cooking, an intermediate observation is made

with more or less granular starch, partially swollen, and some cells visible with variable spacing and adhesion between them. Lentil cells and granules from batch A appear more similar to those observed at 2 min of cooking: most starch granules partially swelled and cells not yet visible. Whereas those from batch B and C look more like what can be observed at 35 min of cooking: large proportion of starch granule swollen and letting cells appear; less cell cohesion. These observations suggest that cellular adhesion between lentil cells from batch B and C seems to be lost faster than for lentil cells from batch A.

## 4. Discussion

### 4.1. Macrostructure & hydrothermal behaviour

In contrast to batch A, batches B and C are overcooked and present texture and appearance defects after 1 h of cooking. Using a Mattson-cooker allowed ranking batches by their characteristic cooking time $t_c$, with B and C being cooked faster than A, which confirms and ascertains visual observations. Thus, faced with observation and empirical grading, Mattson-cooker appears as a relevant method to assess the cooking quality of lentil seeds. Compared to other methods (sensory method, tactile method, spread area ratio) (Wood, 2017), it offers the advantage of providing quantitative data and considering the intra-batch variability, while being fast and simple to carry out.

Regarding values of deformation at rupture point observed on stress-deformation curves, cooked lentil seeds from high quality batch A were found to be more deformable than lentil seeds from batches B and C, which appears consistent with the larger characteristic cooking time observed in Mattson-cooker. In addition, lentil seeds from batch A display a slower water absorption than seeds from batches B and C and a different hydration behaviour. So, differences in cooking quality concords with characteristic cooking time, hydration kinetics and seed deformation, which validates the link between texture, mechanical behaviour and hydration.

Furthermore, our results show that differences in initial rate of water absorption between batches are not due to defects in seed coat integrity. Seed coat in Eudicots is mainly made of cell wall polysaccharides (cellulose, hemicelluloses and pectins) and lignin. Considering cellulose amount, difference with high quality batch was found for one of the low quality batches but not the other. It is thus difficult to conclude on its potential role in hydration behaviour of the seed. Lower amount of heteroxylans (Xyl residues) were found in high quality batch while higher amount of tannins observed in high quality batch. However, these differences did not lead to a difference of initial hydration rate. Hydration initial rate and moisture equilibrium between dehulled seeds and intact seeds show that the role of seed coat as barrier for water penetration is negligible, since intact seeds performed similar to dehulled seeds, despite their hilum was not in contact with wet cotton. These results show that lentil hydration is not ruled by seed coat permeability and that hilum is not the main pathway for water unlike in other pulses (Miano & Augusto, 2018).

Comparison of results obtained during hydration of seeds, either disposed on wet cotton or fully immersed, allows to conclude that hydration rate is not directly related to seed surface in contact with water. Moreover, compared to large seeds, small seeds have higher specific surface, hence a larger total area per volume unit in contact with water, what should be in favour of a faster water absorption for small seeds. Here the effect of density, hence porosity, in water diffusion was not considered since seeds from three lentil batches have similar densities. However, seeds from batch A have smaller size than from batches B and C, and they present lower (soaking) or slightly lower (cooking) water absorption rate, (see part 3.2. Hydration kinetics). This suggests that water absorption is not governed by simple diffusion mechanisms alone, like in a homogeneous medium, and that other mechanisms (barrier effect, permeation) play a significant role at microstructural scale.

Consequently, the differences of hydration, mechanical and cooking
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Observations in optical microscopy (A) and scanning electron microscopy (B) with environmental mode of lentils seeds, for each batch and each cooking time. In optical microscopy, triple coloration highlights starch in blue/purple, proteins in cyan and cell wall in yellow/orange. Observations shown were chosen to be representative of others observations and are made at the border of the section in order to show both seed coat and interior of the cotyledon. MC = Layer of macrooclereid cells (palisade); OC = Osteoclereid cells (subepidermal layer); SPL = Spongy parenchyma layer; IS = Intercellular space; CW = Cell wall; SG = Starch granule; VB = Vascular bundle. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

behaviour between the three lentil batches may be due to differences in seed composition and/or internal structure.

## 4.2. *The role of cotyledons biopolymers*

Given their importance in water absorption for cereals, especially wheat, proteins could be involved in the difference in hydrothermal behaviour of lentil seeds, but this is unlikely for lentil since no significant difference in protein content was found between the batches

considered in this study, whether cotyledon was cooked or soaked. Therefore, the focus was shifted on other components.

First, differences in global amount of starch could explain differences in water absorption. Starch is known for its water absorption capacity, that differs according to temperature (Sangokunle et al., 2020). Nevertheless, batch C that displays the highest value in most of conditions studied (soaked and cooked except raw cotyledons) has an intermediate behaviour in term of water absorption initial rate during soaking, and similar behaviour with batch B in terms of initial rate during cooking
and moisture equilibrium for both soaking and cooking. However, differences in the fine structure of starch could affect water absorption. Amorphous starch absorbs more water and swell first when heated in water than simple helix and double helix starch, as hydrogen bonds in amorphous regions are disrupted (BeMiller, 2011; Trommsdorff & Tomka, 1995). Moreover, the higher value of gelatinisation temperature peak in batch A also suggests a more ordered crystalline structure compared to the starch in seeds from batches B and C. These structural features of starch, less amorphous and more ordered crystalline, contribute to its lower water absorption upon heating, and therefore to its difference of behaviour during cooking (Zhang et al., 2024).

The cell wall is primarily composed of pectins, hemicellulose and, to a lesser extent, cellulose. The main hemicelluloses in cotyledons cell walls of eudicots such as lentil, are xyloglucans (backbone of β(1,4)-linked glucose residues, most of which are substituted with Xyl and other residues), xylans (backbone of β(1,4)-linked Xyl residues) and mannans (backbone of β(1,4)-linked Man residues) (Rodrigues Mota et al., 2018). Still in eudicots, pectins are major constituents of cell wall (Rodrigues Mota et al., 2018), and are composed of two main domains: homogalacturonan (HG; α(1,4)-linked GalA residues) and rhamnogalacturonan I (RGI; repeats of the disaccharide α(1,4)GalA-α(1,2)Rha) that can be substituted, by different esters (methyl, acetyl), sugar (Xyl) for HG, or side chains (chains of Ara (arabinans), Gal (galactans) or both (arabinogalactans)) for RGI (Voragen et al., 2009). Thus, the amounts of GalA, Rha, Ara and Gal residues can be linked to pectins and a large proportion of Xyl residues to hemicelluloses. Our results showed significant differences in the total amount of the constitutive polysaccharides units in raw, cooked or even soaked cotyledons of high-quality seeds versus low quality batches. In particular, cotyledons from batch A would contain a larger amount of pectic HG than batches B and C.

Pectins are known to be involved in several mechanisms related to texture loss during hydrothermal treatment. Pectic HG are involved in the cell adhesion mechanism (Daher & Braybrook, 2015; Voragen et al., 2009). Our results show that raw cotyledons of high quality batch contained more GalA, and thus more HG, in their cell walls, than those of low quality batches, so their cells might adhere more strongly together. This mechanism explains the lower amount of intercellular spaces observed in the cooked cotyledons of high-quality seeds (A). In addition, larger intercellular spaces could be implied in the absorption of higher amount of intercellular water for lower quality seeds (B, C), since water can fill these spaces rapidly during cooking. The hydrothermal treatment of cowpeas was shown to induce cell wall softening by solubilising pectins, leading to the disorganisation of the middle lamella in the cotyledons and subsequently reducing seed firmness by the reduction of cell adhesion (Sefa-Dedeh et al., 1978). The strength of cell adhesion could be directly involved in mechanical behaviour leading to more deformable cells (A), whereas weaker adhesion would lead to cell wall rupture and intercellular spaces creation (B, C), as shown in Fig. 5. Clearly, cell adhesion impacts mechanical behaviour through the absorption of water and the weakening of tissue structure.

Measurements on raw cotyledons showed higher methylation degree (MD) for pectins from highest quality batch (A) than lowest quality batch (B). In beans, a pulse subject to hard-to-cook phenomenon, according to the pectin-cation-phytate model, pectic HG interact with cations released by the hydrolysis of phytate during cooking to form cross-links that strengthen the cell wall (Perera et al., 2023). These interactions need demethylated homogalacturonan domains (Grant et al., 1973), so according to this model, a low MD should contribute to seed hardness after cooking. However, we showed that lentil seeds are not hard-to-cook pulses. Indeed, the lentil batch having the highest MD pectins displayed the longest cooking time, suggesting that this model is not involved here. Other pectin-based mechanisms have been evoked to explain pulses softening during cooking, such as pectins degradation (Liu & Bourne, 1995). Non enzymatic mechanisms were described for degradation of pectins such as β-elimination (Liu & Bourne, 1995) or

acid hydrolysis. Accordingly, the MD could decrease acid hydrolysis of pectins but increase β-degradation (Krall & McFeeters, 1998). Enzymatic degradation of pectins could also occur during the early phases of cooking (J. Li et al., 2024), and methylation is known to protect pectins from enzymatic degradation (Wolf & Greiner, 2012). In this study, MD of pectins measured in lentil seeds is low and few differences between high and low quality batches were observed, what does not support this hypothesis. Additionally, other cell wall polymers, such as the galactan and arabinan side chains of RGI may also contribute to the mechanical properties of the cell wall (Zykwinska et al., 2005). These compounds can also impact water absorption, due to hydrophilic nature of arabinan (Larsen et al., 2011) or hydrophilic interaction of galactan with cellulose (Lopez-Sanchez et al., 2020).

In summary, our results show that seed water absorption is linked to both the proportion of amorphous starch and the amount of pectins in cotyledons, likely through their impact on cellular adhesion and intercellular spaces. However, the precise role of pectins (methylated HG and/or RGI side chains) in these processes remains to be elucidated.

## 5. Conclusions

Firstly, the Mattson cooker was shown to discriminate well between the performance of high and low texture quality batches of lentil after cooking, with higher characteristic cooking time for high quality batch than low quality batches. The more deformable seeds of the high quality batch corroborate these results (a more deformable seed enduring longer cooking time before rupture). In fact, the decrease in the value of deformation at rupture point with increasing moisture content highlighted the role of hydration on mechanical behaviour and thus on cooking quality. During cooking, the equilibrium moisture content of lentil seeds from low quality batches is higher than that of high quality batch, reflecting a higher absorption of water, impacting deformability and thus quality.

Neither seed morphology nor the barrier role of the seed coat could explain the differences in water absorption, despite the higher amount of seed coat tannins in the high quality batch.

Faster loss of cell adhesion and tissue structure during cooking of low quality batches has been shown. This let appear larger and more numerous intercellular gaps. These gaps are thought to be filled by water, contributing to higher water absorption, and thus differences in mechanical properties, such as a higher deformability. The stronger cell adhesion within the cotyledons could be related to the amount of pectins, a higher concentration of which was measured in high quality batch.

On these three batches, contrasted with respect to their canning quality, these results highlight the role of pectins (via the loss of cell adhesion) and amorphous starch on water absorption and mechanical properties. To go further, these results need to be confirmed on a wider range of batches, and the effect of genotype and growing conditions on these different properties should be investigated. The specific role of pectins structure in cell adhesion should also be determined. In the long term, these results will help to control the quality of lentil seeds during processing and to solve the problems encountered during canning.

## CRediT authorship contribution statement

**Guilhem Maillard:** Writing – original draft, Visualization, Software, Methodology, Investigation, Formal analysis. **Guy Della Valle:** Writing – review & editing, Supervision, Conceptualization. **Camille Alvarado:** Investigation. **Xavier Falourd:** Investigation. **Béréngère Marais:** Investigation. **Pascale Millart:** Investigation. **Kevin Billet:** Investigation. **Patrice Papineau:** Software. **Anne-Laure Réguerre:** Software. **Marie-Françoise Devaux:** Software. **Laurent Chaunier:** Resources. **Bruno Novales:** Resources. **Anne-Laure Chateigner-Boutin:** Writing – review & editing, Supervision, Conceptualization. **Nadim Tayeh:** Writing – review & editing, Supervision, Conceptualization. **Sophie Le**
Gall: Writing – review & editing, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

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

Acknowledgements

This study was conducted as part of a thesis funded by INRAE and the Pays-de-la-Loire region. Lentil seeds batches were provided by a French canning industrial named Cofigeo. Microscopic observations, solid-state NMR and biochemical analysis were performed on the BIBS instrumental platform (DOI: 10.15454/1.5572358121569739E12; http://www.bibs.inrae.fr/bibs_eng/, UR1268 BIA, BIISA, Phenome-Emphasis- FR ANR-11-INBS- 0012, PROBE infrastructure, CALIS infrastructure, Biogenouest).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2025.146422.

Data availability

Data will be made available on request.

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