# Impact of chia seed germination on its lipid composition and on its processability in water by an integrated process designed for the release of oil bodies

Éric Lacroux, Muriel Cerny, Jean-François Fabre, Romain Valentin, Othmane Merah

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Éric Lacroux, Muriel Cerny, Jean-François Fabre, Romain Valentin, Othmane Merah. Impact of chia seed germination on its lipid composition and on its processability in water by an integrated process designed for the release of oil bodies. LWT - Food Science and Technology, 2025, 223, pp.117802. 10.1016/j.lwt.2025.117802. hal-05069002

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# Impact of chia seed germination on its lipid composition and on its processability in water by an integrated process designed for the release of oil bodies

Éric Lacroux<sup>a</sup>, Muriel Cerny<sup>a</sup>, Jean-François Fabre<sup>a</sup>, Romain Valentin<sup>a</sup>, Othmane Merah<sup>a,b,*</sup>

## ARTICLE INFO

### Keywords:

Salvia hispanica

Reserve mobilization

Lipid droplets

Germination

Oilseeds

## ABSTRACT

Chia seeds, source of w3, w6 w9 and mucilage for food ingredients, are important for cardiovascular diseases prevention. A process for releasing the mucilage to recover the oil bodies from seeds has already been developed. The aim of this study was investigation the ability of seeds to be degummed, obtain a native lipid emulsion and examine the effect of germination, before or after degumming, on the reserves' composition. Proximate and lipid composition were determined at three times during germination (1, 24 and 48h). Results confirmed that degumming has no-effect on the seeds germination ability. The oil bodies release remains effective regardless of the germination stage, demonstrating the robustness of the process for recovering lipids. Degumming has no-effect on the fractionation of chia seeds whatever the timing of application. The mobilization of lipid reserves increased as germination progressed. The lipid content of germinated seeds declined, the proportion of triglycerides failed and higher proportions of partial glycerides and free fatty acids are formed. Lipids became more prevalent in the pellet, reducing oil bodies yield released into the upper emulsified phase after 48h of germination. This is the first report on the impact of germination on the integrated lipid release process in chia.

## 1. Introduction

Since time can be counted, man has exploited plants in various forms for a wide range of uses: food, pharmaceuticals, cosmetics, industry and livestock. To meet these needs, man has sought to use other plant species (Liu et al., 2022). These uses have sometimes required the elimination of toxic compounds, mainly by genetic means, as in the case of rapeseed to limit glucosinolates and erucic acid. However, in recent years, more and more studies have focused on exploiting germination to eliminate anti-nutritive substances present in seeds, but also to obtain new, richer or healthier nutrient compositions (Aguilar et al., 2019; Cao et al., 2024; Lan et al., 2023; Liu et al., 2022; Win et al., 2018; Yuan et al., 2024).

Numerous studies have been carried out to investigate the impact of sprouting on changes in the nutritional quality of cereals, pseudocereals and legumes. Lan et al. (2023) presented a state-of-the-art review of nutritional changes in sprouted quinoa seeds and their food applications. New insights into the components and bioactives during germination of edible seeds have highlighted the importance of germinated cereal and legume seeds as functional food supplements with undeniable

health benefits (Liu et al., 2022). López-Martínez et al. (2017) had already addressed the interest of cooking and sprouting on legume bioactive compounds and benefits for human health, through increased nutrient bioavailability and palatability, but also through the elimination of anti-nutritional substances.

Among the most studied species, quinoa has attracted interests due to nutritional profile for the based cereal industries. After 24 and 48h of germination, starch and protein levels inducing therefore an increase of amino acids (Guardianelli et al., 2022). This was accompanied with the augmentation of fibers, free fatty acids, tocopherols and phenolic compounds (Guardianelli et al., 2022; Vera et al., 2019). A similar difference was observed between germinated and native seeds in other chenopodiaceae (Jan et al., 2018). Moringa is an example for the studies on seed germination to produce sprouts (Nunez-Gastelum et al., 2023). It has been shown that sprouts, due to their nutrient and phytochemicals (mostly glucosinolates) are interesting to produce functional food and enrich the conventional ones (Coello et al., 2020, 2021; Leon-Lopez et al., 2019). In legumes, on the other hand, a decrease in amino acids, phenolic compounds and anti-nutritional compounds was observed

<footnote><sup>a</sup> Laboratoire de Chimie Agro-industrielle, LCA, Université de Toulouse, INRAE, Toulouse, France</footnote>

<footnote><sup>b</sup> Université Paul Sabatier, IUT A, Département Génie Biologique, 32000, Auch, France</footnote>

<footnote>* Corresponding author. Laboratoire de Chimie Agro-industrielle, LCA, Université de Toulouse, INRAE, Toulouse, France.</footnote>

<footnote>E-mail address: othmane.merah@ensiacet.fr (O. Merah).</footnote>
(Chinma et al., 2022). In species with high lipid reserves, a decrease in oil content and an increase in free fatty acids were noted (Dong et al., 2023; Keawkim & Na Jom, 2022; Tonguç et al., 2023; Yaniv et al., 1998). Comparable results have been observed in brown rice (Beaulieu et al., 2022).

Variation in released compounds is influenced by germination conditions, notably temperature (Cabrera-Santos et al., 2021; Gill et al., 2018; González Belo et al., 2014; Nemzer & Al-Taher, 2023). The growing interest in chia (Salvia hispanica) is linked to its nutritional qualities. Indeed, this annual Lamiaceae native to Latin America is rich in omega-3 and omega-6 fatty acids, fiber, protein, sterols, tocopherols, vitamins and other compounds (Gravé et al., 2019, 2023; Knez Hrnčić et al., 2019; Kulczyński et al., 2019; Zettel & Hitzmann, 2018). Few studies have addressed the impact of germination on the modification of nutrient composition. Ghafoor et al. (2022) studied the effect of germination, roasting and boiling on lipid and phenolic composition, with a decrease in oil content with germination. Cabrera-Santos et al. (2021) studied the effect of imbibition and germination on lipid composition at different temperatures in chia for agronomic interests. This species also contains high levels of mucilage with high added value for the food industry (Gravé et al., 2019). Apart from the study by Ghafoor et al. (2022) for food purposes, there are no studies on the impact of germination on the modification of the composition of compounds of interest and mainly lipids in chia.

In our previous studies on oilseeds, we developed a versatile and robust integrated process dedicated to the aqueous extraction of oil bodies (Cassen et al., 2022; Fabre et al., 2023). This is an eco-compatible biorefinery within the framework of the circular economy for the release in emulsified form of oil bodies (OB) from different oilseeds. This process allows the recovery in aqueous phase of oleosomes which are the main constituents of oil-rich seeds while retaining their native integrity. This integrated process uses different stages of mechanical treatment of seeds in water. In the case of mucilaginous seeds such as chia, it integrates a mucilage removal step using the synergic action of ultrasound and hydrodynamic forces (Fabre et al., 2015a, 2015b, 2020). This degumming in water makes it possible to obtain an extract rich in mucilaginous polysaccharides while minimizing its protein content but above all retaining the integrity and germinative capacity of the seeds. The degummed seeds are then processed in three successive stages. They are crushed in water and then the medium is homogenized in high pressure equipment before being separated into fractions during centrifugal decantation. The fractions thus obtained are a concentrated emulsion rich in OB, a mineralized aqueous phase enriched in water-soluble molecules and a sedimentary residue rich in lignocellulosic fibers. So far, no study has examined the effect of germination and degumming on modifications of active compounds and lipids in particular in an integrated process.

The presented work, carried out during projects gathering several French laboratories (AIC Coligrai and InterCarnot 3BCAR/Qualiment Obeinn Projects), aims at observing the influence of the degumming on the germination of chia seeds and conversely the effect of this germination on the processability of the seeds in order to release their oil-bodies. Our objectives are to examine the impact of germination of whole or degummed seeds on the lipid composition of chia seeds and on each fraction of the integrated process of OB recovery. To achieve these objectives, we opted for a simplified and reproducible germination procedure. The chemical analyses were performed on seeds at different stages of germination, with degumming before or after germination.

# 2. Materials and methods

## 2.1. Materials

The chia seeds studied are of the ORURO variety, a variety released and grown in France since 2013, adapted to the climatic conditions of the south of France. Seeds were supplied by PANAM (Villemur sur Tarn,

France), the seed company behind the registration of this new variety. The lot used is from the 2019 organic harvest, and is numbered: 119 AQ01 P1U5KG.

Cyclohexane and TBME (tert-butyl methyl ether) were purchased from Sigma Aldrich (St Quentin Fallavier, France). TMSH (Trimethyl sulfonium hydroxide) and MSHFBA (Methyl trimethylsilyl heptafluoro butyramide) were purchased from Macherey-Nagel (Hoerdt, France).

## 2.2. Germination of native and degummed chia seeds

Several germination trials of native chia seeds were carried out under various conditions before determining a protocol to use. The aim was to apply a simple, reproducible germination protocol. These trials were carried out by varying the incubation temperature and the seed/water ratio used for seed imbibition. The protocol chosen for germinating chia seeds was as follows:

- The mass of seed to be germinated was supplemented with three times its mass in water (2.25 times in the case of seeds already degummed and dried) and spread out in a single layer in a tray.

- The tray of seeds was placed to germinate in a dark chamber (0.24 m³) at a temperature of between 20 and 25 °C and a humidity of between 70 and 80 %.

Humidity in the chamber was achieved by the presence of a tray containing a 2L volume of a saturated ammonium sulfate solution. Temperature and humidity were controlled and monitored during germination using a data logger (LOG32 TH, TFA Dostmann, Wertheim, Germany).

## 2.3. Aqueous integrated processing of oleaginous seeds

### 2.3.1. Degumming of native chia seeds

For the mucilage removal, a LIXEA FORMULATOR C 80-500-PI tubular reactor designed by SinapTec (Lezennes, France) was used. It features 4 × 500 W ultrasonic generators. Each is connected to a ring of 8 transducers placed around a DN80 cylinder 50 cm long and with a volume of 3.1 L. The power of the generators can be modulated by NEXTGEN 2001 control software. A stream of compressed air circulates to cool the generators, enabling them to operate at temperatures of up to 80 °C. A Broyce Control M1ARM asymmetrical timer has also been added to this tubular reactor, enabling it to operate in pulsed mode. A pump (CR4-60, Grundfos, Bjerringbro, Danemark) with a nominal flow rate of 100 L/min was connected to the tubular reactor for use in recirculation mode. This increases the total volume treated from 3.1L to 6.5L. Treatment was then carried out on a medium made up of 5 % (w/v) chia seeds (325g seeds and 6175g water) for 15 min with recirculation of the medium via the pump, at 100 % ultrasonic power and with a pulsation of 5 s ON/20 s OFF. At the end of the treatment, the total medium is collected, and the separation between the seeds and the aqueous mucilage extract is carried out by filtration on 1 mm and then 0.5 mm stainless steel sieves.

The mucilage-free seeds are then spread out in a single layer in aluminum trays and dried for 48 h in a ventilated oven at 50 °C. The mucilaginous extract was stored at -20 °C or freeze-dried.

### 2.3.2. Recovery of oil bodies

The fractionation according to the integrated process developed (Cassen et al., 2022) was subject to some adjustments (% of seeds in the medium, grinding time) due to the hardness of chia seeds. The treated aqueous media were made up of 15 % (w/v) seeds (native or degummed and dried) in demineralized water. The required quantity of seeds was added to the volume of water under the action of a high-shear rate crushing device (Silverson L4RT, Silverson Machines Ltd., Waterside, UK) operating at maximum speed (5200–6000 rpm) for two 10 min periods separated by a break of 15 min.
The crushed material was directly homogenized using a high-pressure homogenizer (Lab 1000, APV, Evreux, France). Three successive cycles were performed at 50, 100 and 350 bars, reducing particle size and refining the resulting emulsion, while increasing OBs release efficiency. The resulting homogenate was then stored overnight at 4 °C before fractionation.

The homogenate was distributed into 500 mL centrifuge bottles and separated into 3 fractions (oil in water triglycerides emulsion, aqueous phase and pellet residue) by centrifugation (Sigma 6K15 Fisher Bioblock Scientific, Illkirch, France) at 10,000×g for 10 min at 4 °C. Three-phase separation was carried out in such a way as to avoid material transfer from one phase to another. To achieve this, the upper emulsion in the form of a creamy cake was carefully collected by passing the aqueous phase through a 100 µm mesh nylon filter. The aqueous phase thus isolated was again mixed with the residue by vigorous agitation and a second 10-min centrifugation at 10,000×g and still at 4 °C isolated a little more of the upper emulsion fraction which is added to that from the first centrifugation.

The various fractions obtained will be characterized in order to determine their physico-chemical properties and thus evaluate their qualities.

## 2.4. Analysis and characterization methods

### 2.4.1. Observations with a binocular magnifying glass

To monitor germination and determine the size of chia seeds, binocular observations were made. A Nikon SMZ1500 binocular magnifier, (Nikon France S.A.S, Champigny Sur Marne, France) was used. It was equipped with a Nikon DMX 1200 digital camera connected to a computer. Chia seed size was determined using Lucia G software. Image analysis was performed using LUCIA G Software, Version 4.7, developed by Laboratory Imaging s.r.o. (Prague, Czech Republic).

### 2.4.2. Refractive index

The refractive index was determined on mucilage extract using a RFM340-M refractometer (Bellingham & Stanley Ltd, Tunbridge Wells, UK). Refractive index determination was repeated 3 times to obtain a representative mean value.

### 2.4.3. Measurement of OB size distribution

The size distribution of oil bodies in emulsions was determined using a Malvern Mastersizer 3000 laser light scattering instrument coupled to a HydroS sample handling unit (Malvern, UK). A fraction of the sample was diluted in the analysis medium and stirred at 3000 rpm. Continuous ultrasonic treatment with 80 % tip displacement was performed until the droplet aggregates were disintegrated. Various measurements were taken during ultrasonic treatment. The average value obtained is finally recorded when stabilized.

### 2.4.4. Determination of the composition of each fraction

Dry matter content was determined according to the French NF V03-909 standard. The amount of water and volatile compounds was determined by difference.

Mineral content was determined in accordance with French standard NF V 03-322 by calcining the dry matter of the sample in an oven at 550 °C for 12 h.

Total nitrogen content was determined by elemental analysis on approximately 10 mg of delipidated dry matter. Protein content was calculated with a nitrogen-to-protein conversion factor of 5.39 determined after amino acids complete analysis of chia Oruro seeds. The amino-acid composition of the proteins was determined with a Bio-chrom 20+ amino acid analyzer (Biochrom Ltd., Cambridge, UK) equipped with a 200 × 4.6 mm column + precolumn system with sodium-based ion-exchange resins.

The polysaccharide content of the mucilage was determined by the difference between the dry matter and the protein and mineral content,

assuming that the lipid content of the mucilage extract tends towards zero.

The lipid content of the various samples was determined by soxhlet. Seeds, pellet residues and OB emulsions samples were oven-dried at 103 °C for 24 h, then ground (coffee grinder or knife mill) prior to lipid extraction. Extraction on 10-15 g of dried and ground sample was carried out using 150 mL cyclohexane in a balloon flask with a Soxhlet apparatus (VWR international, Fontenay-sous-Bois, France) for 6h, in accordance with French standard NF V03-908. The solvent was then removed using a rotary evaporator at 60 °C and 100 mbar. The extracts obtained were placed in an oven at 103 °C for 15 min to completely remove the solvent and perform a gravimetric measurement.

## 2.5. Chromatography

### 2.5.1. Fatty-acid composition determination by gas chromatography (GC)

The fatty acid profile of lipids was determined by gas chromatography (Varian 3900) coupled with a flame ionization detector. Lipids (15 mg approx.) were first solubilized in 1 mL of TBME (tert-butyl methyl ether) and then methylated with a trimethyl sulfonium hydroxide solution (0.2M in methanol) according to TMSH method (standard NF EN ISO 12966-3). The resulting fatty acid methyl esters (FAMES) were analyzed on a "CP-Select CB for FAME fused silica WCOT" column (50 m, 0.25 mm i.d., 0.25 µm film thickness). Injection of 1 µL, using the following parameters: injector in split mode, ratio of 1/100 at 250 °C for 55 min; carrier gas was helium, at a flow rate of 1.2 mL/min; the oven temperature was initially set at 185 °C for 40 min, then increased to 250 °C at a rate of 15 °C/min and maintained at 250 °C for 11 min; the temperature of the flame ionization detector was set at 250 °C. FAMES were identified by comparison with a mixture of commercial standards, Supelco 37 Component FAME Mix (Sigma Aldrich, St Quentin Fallavier, France). Supelco 37 is a FAME mix, enabling the retention time of each methylated fatty acid peak to be matched with the retention time of one of the 37 fatty acid methyl esters present in this commercial standard mix. Retention time data from the Supelco 37 Component FAME mix standard solution are thus used for calibration, quantification and identification of individual peaks in oil samples.

### 2.5.2. Glyceridic composition determination by gas chromatography (GC)

Analysis of glyceride composition was carried out by gas chromatography coupled to a flame ionization detector using a PerkinElmer apparatus (Norwalk, Connecticut, USA). In 10 mg of lipids, 100 µL of an internal standard stock solution (Heptadecane at 50 mg/mL in cyclohexane) was added, then topped up with 10 mL cyclohexane. Then 160 µL of this solution was added to 40 µL of MSHFBA (Methyl trimethylsilyl heptafluoro butyramide), silylating the acid and alcohol functions in the medium to obtain better-defined peaks. Finally, the resulting solution was heated for 3 min at 103 °C.

The resulting products were analyzed on a DB5HT Agilent column (15 m, 0.32 mm i.d., 0.25 µm film thickness). On-column injection of 1 µL. Carrier gas was helium. The oven temperature was initially set at 55 °C for 0.5 min, then increased to 80 °C at a rate of 45 °C/min, then increased to 360 °C at a rate of 10 °C/min and maintained at 360 °C for 16 min. The temperature of the flame ionization detector was set at 365 °C. Compounds were identified by comparing retention times with reference standards, and quantified by internal calibration with heptadecane. The standard used is a mixture of linoleic acid, linolenic acid, monolein, monolinolein, diolein, triolein, trilinolein and trilinolenin from Sigma-Aldrich (Saint Louis, USA).

## 2.6. Statistical analyses

Analyses of variance were performed to compare the compositions of the different fractions obtained after processing. This was performed by using the ANOVA procedure of SAS software. For the components of each fraction, the means were compared using Duncan's test with a
probability of 5 %.

## 3. Results

### 3.1. Effect of degumming process on germination and seeds lipid content

The germination results for whole and degummed seeds showed that mucilage removal as performed in our trial has quite no impact on the rate of germinated seeds. Although the germination rate of degummed seeds was slightly lower, this difference is not statistically significant (Fig. 1). Germination speed, defined as the time required for 50 % of seeds to germinate, was 28h for degummed seeds and 31h for whole seeds.

We opted for the simplest, most reproducible germination procedure possible in a dark chamber, so as not to be affected by changes in light conditions. After many trials carried out by varying the incubation temperature and the seed/water ratio used for seed imbibition, we chose 1h, 24h and 48h as the 3 germination stages studied. Fig. 2 shows photographs of native chia seeds at these different germination times according to our protocol.

Before examining the impact of germination, we determined the oil content of native, dried and degummed seeds. The relative oil content of seeds increases, as expected, by 4 % when moisture is removed to obtain dried seeds, and further increases when mucilaginous polysaccharides are removed from the seed surface, reaching 7 % more in degummed seeds than in native seeds (Fig. 3).

### 3.2. Effect of germination stage on mucilage extract and proximate composition

Pre-treatment of seeds by ultrasound-assisted degumming was carried out on native seeds as well as on seeds at three selected germination stages. The following table (Table 1) gives the composition of the mucilaginous extracts obtained using the same protocol on the different seeds.

The total mass percentage of extract in relation to the initial mass of treated seeds increases up to 24h of germination, after which the extracted mass appears almost constant up to 48 h. The mass of protein extracted during ultrasonic degumming treatment increases with the pre-germination time. These proteins are extracted by diffusion during ultrasonic treatment of the seeds in aqueous phase. Similarly, the mineral content of this mucilaginous polysaccharide extract increases with germination time. The sugar content of the total extract dry matter only decreases as the extract mass increases. The total mass of polysaccharides in the extract is constant whatever the germination stage of the seeds before treatment.

<figure><img src="image_2.png" /><figcaption>Fig. 1. Germination rate of native and degummed seeds.</figcaption></figure>

### 3.3. Effect of stage of germination on seeds lipid composition

Three times (1h, 24 h and 48h) of germination have been tested, native seed have been taken as control.

As expected the lipid content decreased significantly with the time of germination. Indeed, the proportion of lipids falls by more than a quarter in seeds 48 h after germination, compared with ungerminated seeds (Fig. 4).

Surprisingly, the fatty acids composition presented almost no significant modifications during seed germination (Table 2). This was the case for unsaturated fatty acids. In contrast, saturated fatty acids, particularly arachidic acid increased threefold after 1h after starting germination and falled three times at 24h. In the same way, stearic acid increased slightly at 1h and decreased by more than quarter at 24h.

During germination free fatty acids increased by nearly 21 % within the first hour of germination (rising from 5.3 % to 6.4 %). Similarly, monoglycerides rose from 0 (ungerminated seeds) to 0.7 % for seeds 24 h after germination (Table 3). This was more pronounced for diglycerides, which tripled at 48 h compared to both ungerminated seeds and at 1 h of germination. The triglycerides showed, in contrast, the opposite trend. Indeed, at 48h the total triglycerides content falled by more 4 % compared to ungerminated seeds.

### 3.4. Impact of germination on chia seeds processing for oil bodies recovery

In our integrated process, high-shear grinding of dried degummed seeds, followed by high-pressure homogenization and centrifugation, separated three phases: an aqueous phase, an emulsion upper phase and a pellet phase. Nutrients were assessed at three different stages of germination (1, 24 and 48 h) using two different methods. In the first case, seed germination was carried out before degumming, and in the second case, degumming was carried out on native seeds before starting the germination protocol on degummed seeds. For the two protocols studied, the continuation of the treatment was the same: the seeds were crushed in an aqueous medium, homogenized at high pressure and the fractions separated by centrifugal decantation.

Developed Methodology is displayed in figure below (Fig. 5).

#### 3.4.1. First protocol of seeds processing: germination before degumming

In this first protocol the germination of the seeds at different stages occurs before their degumming. The degummed seeds are then dried before being treated by grinding and high-pressure homogenization to a level of 15 % of seeds in an aqueous medium. Centrifugal separation then makes it possible to separate the emulsion of oil bodies, an aqueous phase and a lignocellulosic pellet.

##### 3.4.1.1. Impact of germination stage on recovered oil bodies emulsion.
When the germination time increases the proportion of the fraction of emulsified oil bodies recovered decreases and the dry matter content of this emulsified fraction also decreases (Table 4). However, the lipid content in the dry matter of this fraction remains constant whatever the germination stage. Protein content of emulsion rises slightly for germination times of 24 h or less, then falls for germination times of 48 h.

The analysis of the lipid profile of emulsions from chia seeds at different stages of germination showed that up to 24 h of germination the lipid profile remains overall similar while at 48 h the triglyceride content decreased and an increase in free fatty acids and glycerol partial esters was observed (Table 5).

Analysis of the fatty acids profile present in these emulsions of lipid bodies, however, demonstrated a similar composition whatever the stage of germination (Fig. 6).

##### 3.4.1.2. Impact of germination stage on the lignocellulosic pellet fraction.
As the germination time increased from 1 to 24 h, the proportion of lignocellulosic pellet mass separated during fractionation decreased
<figure><img src="image_3.png" /><figcaption>Fig. 2. Pictures of native chia seeds at different germination stages.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Fig. 3. Measured lipid content of Chia seeds.</figcaption></figure>

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

Characteristics of mucilaginous extract as a function of the germination stage of treated seeds.

<table><thead><tr><th rowspan="2">Trait</th><th rowspan="2">Ungerminated seeds</th><th colspan="3">Germinated seeds</th><th rowspan="2">LSD</th></tr><tr><th>1h</th><th>24h</th><th>48h</th></tr></thead><tbody><tr><td>Mucilaginous Extract (% of initial seed mass)</td><td>7.5 ± 0.1<sup>b</sup></td><td>7.8 ± 0.2<sup>b</sup></td><td>9.2 ± 0.2<sup>a</sup></td><td>8.7 ± 0.7<sup>a</sup></td><td>0.7</td></tr><tr><td>Polysaccharide Content % of extract D.M.</td><td>89.3 ± 1.7<sup>a</sup></td><td>88.5 ± 0.9<sup>a</sup></td><td>82.8 ± 0.5<sup>b</sup></td><td>78.1 ± 0.5<sup>c</sup></td><td>1.2</td></tr><tr><td>% of initial seed mass</td><td>6.7 ± 0.1<sup>b</sup></td><td>6.9 ± 0.2<sup>b</sup></td><td>7.6 ± 0.2<sup>a</sup></td><td>6.8 ± 0.5<sup>b</sup></td><td>0.3</td></tr><tr><td>Protein Content (% of extract D.M.)</td><td>4.3 ± 0.2<sup>c</sup></td><td>4.4 ± 0.5<sup>c</sup></td><td>8.0 ± 0.2<sup>b</sup></td><td>11.4 ± 0.2<sup>a</sup></td><td>0.8</td></tr><tr><td>Mineral Content (% of extract D.M.)</td><td>6.4 ± 1.7<sup>b</sup></td><td>7.1 ± 0.9<sup>b</sup></td><td>9.2 ± 0.5<sup>a</sup></td><td>10.5 ± 0.5<sup>a</sup></td><td>0.8</td></tr></tbody></table>

(Table 6). The dry matter and lipid (except for 24h) contents of the pellets were significantly higher during germination compared with the dry seed. The protein content of the sediment pellet increased by 6 % and decreases by 7 % at 24 and 48 h after imbibition, compared with dry seed.

Up to 24 h of germination lipid profile in the sedimentary pellet remained overall the same. On the other hand, after 48 h the triglycerides content decreased, while the diglycerides, monoglycerides and free fatty acids content increased (Table 7).

<figure><img src="image_5.png" /><figcaption>Fig. 4. Measured lipid content of degummed Chia seeds at each germination stage studied.</figcaption></figure>

It could be seen in Fig. 7 that lipid enrichment of the lignocellulosic pellet with a longer germination time favored saturated fatty acids. Thus, between a pellet from native seeds and a pellet from seeds germinated for 48 h, the saturated fatty acid content rose from 17.6 % to 27.4 %, while the polyunsaturated fatty acid content fell from 71.8 % to 56.6 %. At the same time, monounsaturated fatty acids in the sedimentary pellet increased by almost 50 %.

### 3.4.2. Second protocol of seeds processing: germination after degumming

In this second protocol the germination of the seeds at different stages occurred after their degumming. The seeds were degummed and dried for 48 h in a ventilated oven at 50 °C. Degummed seeds were supplemented with 2.25 times their mass in water and placed to germinate in a dark chamber at a temperature of between 20 and 25 °C and a humidity of between 70 and 80 %. Processing of oil bodies recovery took places after 1 h or 48h in these germination conditions. Seeds were treated by grinding and high-pressure homogenization to a level of 15 % of seeds in an aqueous medium. Centrifugal separation then made it possible to separate the emulsion of oil bodies, an aqueous phase and a lignocellulosic pellet.

#### 3.4.2.1. Impact of germination stage on recovered oil bodies emulsion.
When the germination time increased the proportion of the fraction of emulsified oil bodies recovered decreased but, unlike the process using germination before degumming, the dry matter content of this emulsion did not decrease with germination time, and in fact tended to increase. However, the lipid content in the dry matter of this fraction remained constant whatever the germination stage (Table 8).
<table><caption>**Table 2**
Composition of fatty acids in lipids of chia seeds at different stages of germination.</caption><thead><tr><th rowspan="2">Fatty acid (%)</th><th colspan="2">Ungerminated seeds</th><th colspan="3">Germinated seed</th><th rowspan="2">LSD</th></tr><tr><th>Native</th><th>Degummed</th><th>1h</th><th>24h</th><th>48h</th></tr></thead><tbody><tr><td>C16 : 0 Palmitic acid</td><td>7.9 ± 0.2<sup>a</sup></td><td>8.1 ± 0.2<sup>a</sup></td><td>8.1 ± 0.1<sup>a</sup></td><td>8.0 ± 0.1<sup>a</sup></td><td>7.9 ± 0.2<sup>a</sup></td><td>0.5</td></tr><tr><td>C18 : 0 Stearic acid</td><td>3.5 ± 0.2<sup>a</sup></td><td>4.0 ± 0.3<sup>a</sup></td><td>3.9 ± 0.2<sup>a</sup></td><td>2.9 ± 0.1<sup>b</sup></td><td>2.6 ± 0.1<sup>b</sup></td><td>0.4</td></tr><tr><td>C20 : 0 Arachidic acid</td><td>0.5 ± 0.1<sup>b</sup></td><td>0.3 ± 0.1<sup>b</sup></td><td>1.5 ± 0.3<sup>a</sup></td><td>0.4 ± 0.1<sup>b</sup></td><td>0.4 ± 0.1<sup>b</sup></td><td>0.2</td></tr><tr><td><strong>Total Saturated FA</strong></td><td><strong>11.9 ± 0.4<sup>b</sup></strong></td><td><strong>12.4 ± 0.4<sup>b</sup></strong></td><td><strong>13.5 ± 0.2<sup>a</sup></strong></td><td><strong>11.3 ± 0.1<sup>bc</sup></strong></td><td><strong>10.9 ± 0.1<sup>c</sup></strong></td><td>0.3</td></tr><tr><td>C18 : 1n9 Oleic acid</td><td>7.6 ± 0.1<sup>a</sup></td><td>7.6 ± 0.2<sup>a</sup></td><td>7.7 ± 0.1<sup>a</sup></td><td>7.7 ± 0.1<sup>a</sup></td><td>7.6 ± 0.1<sup>a</sup></td><td>0.1</td></tr><tr><td>C18 : 1n7c Vaccenic acid</td><td>0.8 ± 0.0<sup>a</sup></td><td>0.8 ± 0.0<sup>a</sup></td><td>0.8 ± 0.0<sup>a</sup></td><td>0.8 ± 0.0<sup>a</sup></td><td>0.8 ± 0.0<sup>a</sup></td><td>0.0</td></tr><tr><td><strong>Total Monouns. FA</strong></td><td><strong>8.4 ± 0.1<sup>a</sup></strong></td><td><strong>8.4 ± 0.3<sup>a</sup></strong></td><td><strong>8.5 ± 0.1<sup>a</sup></strong></td><td><strong>8.5 ± 0.1<sup>a</sup></strong></td><td><strong>8.4 ± 0.1<sup>a</sup></strong></td><td>0.2</td></tr><tr><td>C18 : 2n6 Linoleic acid</td><td>21.9 ± 0.1<sup>a</sup></td><td>22.1 ± 0.1<sup>a</sup></td><td>22.2 ± 0.1<sup>a</sup></td><td>21.9 ± 0.1<sup>a</sup></td><td>22.0 ± 0.2<sup>a</sup></td><td>0.3</td></tr><tr><td>C18 : 3n3 a Linolenic acid</td><td>57.2 ± 0.4<sup>a</sup></td><td>57.0 ± 0.7<sup>a</sup></td><td>56.4 ± 0.3<sup>a</sup></td><td>57.2 ± 0.3<sup>a</sup></td><td>57.4 ± 0.5<sup>a</sup></td><td>1.1</td></tr><tr><td><strong>Total Polyuns. FA</strong></td><td><strong>79.1 ± 0.4<sup>a</sup></strong></td><td><strong>79.1 ± 0.7<sup>a</sup></strong></td><td><strong>78.6 ± 0.1<sup>a</sup></strong></td><td><strong>79.1 ± 0.2<sup>a</sup></strong></td><td><strong>79.3 ± 0.2<sup>a</sup></strong></td><td>0.6</td></tr><tr><td><strong>Total</strong></td><td><strong>100.0 ± 0.1<sup>a</sup></strong></td><td><strong>99.9 ± 0.1<sup>a</sup></strong></td><td><strong>100.0 ± 0.1<sup>a</sup></strong></td><td><strong>100.0 ± 0.1<sup>a</sup></strong></td><td><strong>100.0 ± 0.1<sup>a</sup></strong></td><td>0.4</td></tr></tbody></table>

<table><caption>**Table 3**
Profile of lipids in chia seeds at different germination stages.</caption><thead><tr><th rowspan="2">Trait</th><th rowspan="2">Ungerminated seeds</th><th colspan="3">Germinated seeds</th><th rowspan="2">LSD</th></tr><tr><th>1h</th><th>24h</th><th>48h</th></tr></thead><tbody><tr><td>Free fatty acids</td><td>5.3 ± 0.7<sup>b</sup></td><td>6.4 ± 0.2<sup>a</sup></td><td>6.4 ± 0.1<sup>a</sup></td><td>6.6 ± 0.2<sup>a</sup></td><td>0.6</td></tr><tr><td>Monoglycerides</td><td>ND</td><td>ND</td><td>0.7 ± 0.1<sup>a</sup></td><td>0.6 ± 0.2<sup>a</sup></td><td>0.2</td></tr><tr><td>Diglycerides</td><td>1.2 ± 0.1<sup>b</sup></td><td>1.0 ± 0.1<sup>b</sup></td><td>1.4 ± 0.3<sup>b</sup></td><td>3.6 ± 2.0<sup>a</sup></td><td>0.8</td></tr><tr><td><strong>Triglycerides</strong></td><td><strong>93.6 ± 0.1<sup>a</sup></strong></td><td><strong>92.6 ± 0.2<sup>a</sup></strong></td><td><strong>91.6 ± 0.4<sup>ab</sup></strong></td><td><strong>89.4 ± 2.0<sup>b</sup></strong></td><td><strong>1.6</strong></td></tr></tbody></table>

Within each line, means with the same letter are not statistically different based on Duncan's test at 0.05 probability level. LSD: Least significant difference. N.D.: Not detected.

<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Applied Methodology in this study.</figcaption></figure>

As with previous protocol, analysis of lipid composition of oil bodies emulsions showed a rapid mobilization of lipid reserves during germination.

### 3.4.2.2. Impact of germination stage on the lignocellulosic pellet fraction.
The mass proportion of lignocellulosic pellet separated during fractionation remained quite the same whatever the germination time (Table 10). The dry matter content of the pellets remained constant but on the other hand the lipid content increased with germination time (see Table 11).

<table><caption>**Table 4**
Proximate composition of the emulsion fraction of germinated seeds before degumming.</caption><thead><tr><th rowspan="2">Trait</th><th rowspan="2">Ungerminated<br>Seeds</th><th colspan="3">Germinated seeds</th><th rowspan="2">LSD</th></tr><tr><th>1h</th><th>24h</th><th>48h</th></tr></thead><tbody><tr><td>Emulsion<br>(relative %)</td><td>9.4</td><td>9.8</td><td>7.7</td><td>6.8</td><td></td></tr><tr><td>Dry Matter (%)</td><td>39.5 ± 0.9<sup>a</sup></td><td>35.6 ±<br>0.1<sup>b</sup></td><td>30.9 ±<br>0.1<sup>c</sup></td><td>30.2 ±<br>0.4<sup>c</sup></td><td>0.9</td></tr><tr><td>Lipids (% DM)</td><td>87.1 ± 0.1<sup>b</sup></td><td>84.5 ±<br>0.6<sup>c</sup></td><td>86.4 ±<br>2.3<sup>bc</sup></td><td>92.1 ±<br>0.3<sup>a</sup></td><td>0.6</td></tr><tr><td>Proteins (%<br>DM)</td><td>12.4 ± 0.4<sup>b</sup></td><td>13.0 ±<br>0.7<sup>ab</sup></td><td>13.9 ±<br>0.2<sup>a</sup></td><td>9.6 ±<br>1.0<sup>c</sup></td><td>0.8</td></tr><tr><td>Minerals (%<br>DM)</td><td>1.8 ± 0.3<sup>a</sup></td><td>1.7 ±<br>0.1<sup>a</sup></td><td>1.7 ±<br>0.1<sup>a</sup></td><td>1.2 ±<br>0.3<sup>b</sup></td><td>0.3</td></tr></tbody></table>

Within each line, means with the same letter are not statistically different based on Duncan's test at 0.05 probability level. LSD: Least significant difference.

As regards with lipid composition, it evolved in the same way with increasing germination time. Triglycerides content fell, while partial esters and free fatty acids increased with germination time.

### 3.5. Impact of germination stage on the size of oil bodies

Table 12 and Figs. 8 and 9 present the size of oil bodies of emulsion obtained from chia seeds germinated before and after degumming. The oil bodies released in the emulsions present almost the same size whatever the process used, except at 48h. The curves are representative of many tests on several different volumes of emulsions and that the device analyses all the droplets that passed in front of the lasers. The Dx 50 values given in the table are those of the representative curves.

## 4. Discussion

The objectives of this study were the investigation of the impact of germination of whole or degummed seeds on the lipid composition of chia seeds, on each fraction composition at the end of the integrated process of OB recovery. To do this, a simplified and reproducible
<table><caption>**Table 5**
Lipids profile in the emulsion fraction of germinated seeds before degumming.</caption><thead><tr><th rowspan="2">Glycerides Composition (%)</th><th rowspan="2">Ungerminated Seeds</th><th colspan="3">Germinated seeds</th><th rowspan="2">LSD</th></tr><tr><th>1h</th><th>24h</th><th>48h</th></tr></thead><tbody><tr><td>Free fatty acids</td><td>7.1 ± 0.1<sup>b</sup></td><td>5.8 ± 0.1<sup>d</sup></td><td>6.4 ± 0.2<sup>c</sup></td><td>8.7 ± 0.2<sup>a</sup></td><td>0.5</td></tr><tr><td>Monoglycerides</td><td>0.2 ± 0.0<sup>b</sup></td><td>0.1 ± 0.1<sup>b</sup></td><td>0.2 ± 0.1<sup>b</sup></td><td>0.5 ± 0.2<sup>a</sup></td><td>0.1</td></tr><tr><td>Diglycerides</td><td>3.6 ± 0.1<sup>b</sup></td><td>3.0 ± 0.4<sup>bc</sup></td><td>2.8 ± 0.1<sup>c</sup></td><td>5.8 ± 1.5<sup>a</sup></td><td>0.4</td></tr><tr><td>Triglycerides</td><td>89.1 ± 0.2<sup>b</sup></td><td>91.1 ± 0.6<sup>a</sup></td><td>90.6 ± 0.4<sup>a</sup></td><td>85.1 ± 1.8<sup>c</sup></td><td>0.6</td></tr></tbody></table>

Within each line, means with the same letter are not statistically different based on Duncan's test at 0.05 probability level. LSD: Least significant difference.

<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Nature of FA in oil bodies emulsion at each germination time.</figcaption></figure>

<table><caption>**Table 6**
Proximate composition of the lignocellulosic pellet fraction of germinated seeds before degumming.</caption><thead><tr><th rowspan="2">Trait</th><th rowspan="2">Ungerminated<br>Seeds</th><th colspan="3">Germinated seeds</th><th rowspan="2">LSD</th></tr><tr><th>1h</th><th>24h</th><th>48h</th></tr></thead><tbody><tr><td>Pellet (relative<br>%)</td><td>30.7</td><td>25.8</td><td>23.5</td><td>25.0</td><td></td></tr><tr><td>Dry Matter<br>(%)</td><td>35.4 ± 0.9<sup>a</sup></td><td>32.1 ±<br>0.9<sup>c</sup></td><td>33.5 ±<br>0.2<sup>b</sup></td><td>32.5 ±<br>1.4<sup>bc</sup></td><td>1.2</td></tr><tr><td>Lipids (% DM)</td><td>11.9 ± 1.1<sup>d</sup></td><td>14.7 ±<br>2.3<sup>c</sup></td><td>22.8 ±<br>3.2<sup>a</sup></td><td>18.1 ±<br>1.7<sup>b</sup></td><td>1.0</td></tr><tr><td>Proteins (%<br>DM)</td><td>21.5 ± 1.0<sup>b</sup></td><td>21.3 ±<br>0.6<sup>b</sup></td><td>27.0 ±<br>1.5<sup>a</sup></td><td>20.1 ±<br>0.7<sup>c</sup></td><td>1.2</td></tr><tr><td>Minerals (%<br>DM)</td><td>4.6 ± 0.1<sup>a</sup></td><td>4.8 ±<br>0.2<sup>a</sup></td><td>4.4 ±<br>0.1<sup>bc</sup></td><td>4.1 ±<br>0.1<sup>b</sup></td><td>0.3</td></tr></tbody></table>

Within each line, means with the same letter are not statistically different based on Duncan's test at 0.05 probability level. LSD: Least significant difference.

germination procedure was used at different germination stages that occur before or after the degumming step.

### 4.1. Effect of degumming process on germination and seeds lipid content

The effect of degumming on seed germination has been examined

and results have shown no significant effect on germination rate. Our results showed no significant differences between the two types of seeds (Fig. 1). The slight observed difference is probably due to process effect. Geneve et al. (2017) have investigated the effect of mucilage removal on germination of chia seed. They have observed a difference in germination rate between degummed and intact seeds which confirm the observed results in our study. Indeed, in our process developed for mucilage seeds, ultrasounds are used to extract the mucilage. This method of extracting polysaccharides involves creating cavitation bubbles. The collapse of these bubbles creates domes of high temperature and high pressure, causing the tegument and mucilage break up (Fabre et al., 2015a, 2015b, 2020). These bubbles have probably caused lesions on the embryo of some seeds leading to slight decrease of germination rate of degummed seeds compared to native ones. Geneve et al. (2017) have found no significant differences between native and degummed seeds of chia for the germination rate which is in line with the observed results in our study. Moreover, the mucilage-free seeds germinated earlier that intact ones. This can be explained by the degumming process during which the seeds are placed in an aqueous medium and shearing the mucilaginous polysaccharides located on the surface of the seeds using ultrasound, but without destructuring the seeds beforehand. The circulation of water under pressure causes the seeds to become deeply imbibed. This results in hydropriming, which in turn leads to rapid seed germination (Sharma et al., 2014; Gonçalves et al., 2024; Muhie et al.,

<table><caption>**Table 7**
Lipids profile in the emulsion f the lignocellulosic pellet fraction of germinated seeds before degumming.</caption><thead><tr><th rowspan="2">Glycerides Composition (%)</th><th rowspan="2">Ungerminated Seeds</th><th colspan="3">Germinated seeds</th><th rowspan="2">LSD</th></tr><tr><th>1h</th><th>24h</th><th>48h</th></tr></thead><tbody><tr><td>Free Fatty Acids</td><td>7.7 ± 0.0<sup>b</sup></td><td>6.1 ± 0.1<sup>c</sup></td><td>6.4 ± 0.2<sup>c</sup></td><td>14.1 ± 0.0<sup>a</sup></td><td>1.2</td></tr><tr><td>Monoglycerides</td><td>0.3 ± 0.0<sup>b</sup></td><td>0.3 ± 0.1<sup>b</sup></td><td>0.2 ± 0.0<sup>b</sup></td><td>1.5 ± 0.5<sup>a</sup></td><td>0.4</td></tr><tr><td>Diglycerides</td><td>3.9 ± 0.0<sup>b</sup></td><td>3.6 ± 0.1<sup>b</sup></td><td>3.1 ± 0.9<sup>b</sup></td><td>7.1 ± 0.3<sup>a</sup></td><td>1.0</td></tr><tr><td>Triglycerides</td><td>88.2 ± 0.1<sup>b</sup></td><td>90.1 ± 0.1<sup>a</sup></td><td>90.3 ± 0.6<sup>a</sup></td><td>77.3 ± 0.4<sup>c</sup></td><td>1.7</td></tr></tbody></table>

Within each line, means with the same letter are not statistically different based on Duncan's test at 0.05 probability level. LSD: Least significant difference.
<figure><img src="image_8.png" /><figcaption>Fig. 7. Nature of FA in pellet phase after processing seeds at each germination time.</figcaption></figure>

**Table 8**

Proximate composition of oil bodies emulsion of seeds germinated after degumming.

<table><thead><tr><th rowspan="2">Trait</th><th rowspan="2">Ungerminated Seeds</th><th colspan="2">Germinated seeds</th><th rowspan="2">LSD</th></tr><tr><th>1h</th><th>48h</th></tr></thead><tbody><tr><td>Emulsion (relative %)</td><td>9.4</td><td>8.2</td><td>7.1</td><td></td></tr><tr><td>Dry Matter (%)</td><td>40.0 ± 0.9<sup>b</sup></td><td>40.8 ± 0.2<sup>b</sup></td><td>43.2 ± 0.4<sup>a</sup></td><td>1.0</td></tr><tr><td>Lipids (% DM)</td><td>87.0 ± 0.0<sup>b</sup></td><td>90.3 ± 2.1<sup>a</sup></td><td>90.2 ± 5.2<sup>a</sup></td><td>0.9</td></tr><tr><td>Proteins (% DM)</td><td>12.4 ± 0.4</td><td>N.A.</td><td>N.A.</td><td></td></tr><tr><td>Minerals (% DM)</td><td>1.8 ± 0.3<sup>a</sup></td><td>2.0 ± 0.1<sup>a</sup></td><td>1.9 ± 0.1<sup>a</sup></td><td>0.3</td></tr></tbody></table>

Within each line, means with the same letter are not statistically different based on Duncan's test at 0.05 probability level. N.A.: Not available. LSD: Least significant difference.

**Table 9**

Lipids profile of oil bodies emulsion of seeds germinated after degumming.

<table><thead><tr><th rowspan="2">Glycerides (%)</th><th rowspan="2">Ungerminated Seeds</th><th colspan="2">Germinated seeds</th><th rowspan="2">LSD</th></tr><tr><th>1h</th><th>48h</th></tr></thead><tbody><tr><td><strong>Free Fatty Acids</strong></td><td><strong>7.1 ± 0.1<sup>b</sup></strong></td><td><strong>5.0 ± 0.1<sup>c</sup></strong></td><td><strong>12.7 ± 0.1<sup>a</sup></strong></td><td><strong>1.1</strong></td></tr><tr><td>Monoglycerides</td><td>0.2 ± 0.0<sup>b</sup></td><td>0.3 ± 0.0<sup>b</sup></td><td>0.6 ± 0.1<sup>a</sup></td><td>0.2</td></tr><tr><td>Diglycerides</td><td>3.6 ± 0.0<sup>b</sup></td><td>2.4 ± 0.0<sup>c</sup></td><td>5.0 ± 0.0<sup>a</sup></td><td>0.9</td></tr><tr><td>Triglycerides</td><td>89.1 ± 0.2<sup>b</sup></td><td>92.4 ± 0.1<sup>a</sup></td><td>80.8 ± 0.1<sup>c</sup></td><td>1.8</td></tr></tbody></table>

Within each line, means with the same letter are not statistically different based on Duncan's test at 0.05 probability level. LSD: Least significant difference.

**Table 10**

Proximate composition of lignocellulosic pellet fraction of seeds germinated after degumming.

<table><thead><tr><th rowspan="2">Trait</th><th rowspan="2">Ungerminated seeds</th><th colspan="2">Germinated seeds</th><th rowspan="2">LSD</th></tr><tr><th>1h</th><th>48h</th></tr></thead><tbody><tr><td>Pellet (relative %)</td><td>30.7</td><td>30.4</td><td>29.4</td><td></td></tr><tr><td>Dry Matter (%)</td><td>35.4 ± 0.9<sup>a</sup></td><td>35.0 ± 0.6<sup>a</sup></td><td>35.1 ± 0.5<sup>a</sup></td><td>0.9</td></tr><tr><td>Lipids (% DM)</td><td>12.0 ± 1.1<sup>b</sup></td><td>12.7 ± 0.2<sup>b</sup></td><td>21.6 ± 0.9<sup>a</sup></td><td>2.6</td></tr><tr><td>Proteins (% DM)</td><td>21.5 ± 1.0</td><td>N.A.</td><td>N.A.</td><td></td></tr><tr><td>Minerals (% DM)</td><td>4.6 ± 0.1<sup>a</sup></td><td>4.6 ± 0.1<sup>a</sup></td><td>4.1 ± 0.1<sup>b</sup></td><td>0.2</td></tr></tbody></table>

Within each line, means with the same letter are not statistically different based on Duncan's test at 0.05 probability level. N.A.: Not available. LSD: Least significant difference.

2024). This result clearly shows that our process enables the mucilage to be extracted without altering germination, thus opening up the possibility of using the seeds for various other processes or for agricultural use (after being dried), such as sowing. Moreover, removing mucilage is

**Table 11**

Lipids profile of lignocellulosic pellet fraction of seeds germinated after degumming.

<table><thead><tr><th rowspan="2">Glycerides (%)</th><th rowspan="2">Ungerminated Seeds</th><th colspan="2">Germinated seeds</th><th rowspan="2">LSD</th></tr><tr><th>1h</th><th>48h</th></tr></thead><tbody><tr><td>Free Fatty Acids</td><td>7.7 ± 0.0<sup>b</sup></td><td>7.3 ± 0.5<sup>b</sup></td><td>13.3 ± 0.1<sup>a</sup></td><td>1.1</td></tr><tr><td>Monoglycerides</td><td>0.3 ± 0.1<sup>b</sup></td><td>0.3 ± 0.1<sup>b</sup></td><td>0.6 ± 0.1<sup>a</sup></td><td>0.2</td></tr><tr><td>Diglycerides</td><td>3.9 ± 0.0<sup>b</sup></td><td>4.5 ± 0.2<sup>a</sup></td><td>4.9 ± 0.1<sup>a</sup></td><td>0.5</td></tr><tr><td>Triglycerides</td><td>88.2 ± 0.1<sup>a</sup></td><td>87.9 ± 0.3<sup>a</sup></td><td>81.2 ± 0.1<sup>b</sup></td><td>0.9</td></tr></tbody></table>

Within each line, means with the same letter are not statistically different based on Duncan's test at 0.05 probability level. LSD: Least significant difference.

**Table 12**

Size of oil bodies obtained from chia seeds germinated before and after degumming.

<table><thead><tr><th rowspan="2">Chia OB Emulsion</th><th rowspan="2">Seeds Germinated BEFORE Degumming</th><th colspan="2">Seeds Germinated AFTER Degumming</th></tr><tr><th>Size, Dx (50), µm</th><th>Size, Dx (50), µm</th></tr></thead><tbody><tr><td>Ungerminated seeds</td><td>2.01</td><td></td><td>2.01</td></tr><tr><td>1h Germination</td><td>2.05</td><td></td><td>2.22</td></tr><tr><td>24h Germination</td><td>2.19</td><td></td><td>—</td></tr><tr><td>48h Germination</td><td>1.88</td><td></td><td>2.35</td></tr></tbody></table>

mandatory in the present process in order to obtain OBs with water extraction.

Fig. 3 presents content of oil in native, dried and degummed seeds. The observed significant differences between dried and native seeds are, as expected, due to moisture removing. Those noticed between native and degummed (and dried) are due to both moisture and mucilage removing. This result agrees with already published reports (Fabre et al., 2020) in camelina (Camelina sativa), another mucilaginous seed. Ghafoor et al. (2022) studied effect of roasting (6 min at 120 °C) on chemical composition of Chia seed. They have observed an increase of oil content nearly 2 %. This is in line with the observed result in our study. The difference is due the used method in the two studies. Indeed, we have dehydrated the seeds during 48h at 50 °C which allows to reduce the water moisture of the seed by 4 %. The roasting method used the study of Ghafoor et al. (2022) is shorter even at higher temperature and therefore the water removed was less important. The evolution of the imbibition of chia seeds has also been studied by Muñoz et al. (2012) who examined physical and morphological traits of seeds and the characterization of the release of mucilage during hydration of chia grains. They found that mucilage represented almost 3 % of the seed's
<figure><img src="image_9.png" /><figcaption>Fig. 8. Size of droplets in emulsions from native or germinated chia seeds (1h, 24h and 48h) before degumming.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>Fig. 9. Size of droplets in emulsions from chia seeds germinated after degumming (1h or 48h).</figcaption></figure>

weight. This result confirms the findings of our study (Fig. 3). In their study, Muñoz et al. (2012), did not examine the germination of chia seeds.

## 4.2. Effect of germination stage on mucilage extract and proximate composition

The effect of germination on changes in chemical and nutritional composition has been investigated in many species (Lan et al., 2023). Little work has been done on this aspect in *Salvia hispanica*, with the exception of two recent studies. Cabrera-Santos et al. (2021) studied the effect of two treatments (soaking and germination) on seed composition at different temperatures with the aim of determining cardinal temperatures for chia seeds. Ghafoor et al. (2022) studied the effects of three processes (boiling, roasting and germination) on the composition of chia seeds for nutritional purposes. To date, no study has investigated the effect of different germination stages on the mucilaginous extract.

Table 1 shows the proximal composition of mucilage extract on chia after the germination process. The levels of protein, ash and total mucilage mass extract increase significantly. The sugar content, on the other hand, fell by 10 %. These results are an artefact due to water absorption. Indeed, when considering the level of sugar content on total

dry matter basis, no differences were noticed (Table 1) except at 24h.

The mass extract of mucilage compared with native seeds increases during germination by 0.3 and 1.7 % at 1 h and 24 h, respectively (Table 1). The proportions of mucilage extracted in our study are higher than in previous reports. Numerous studies have examined mucilage extraction methods and their optimization. Yields vary considerably (4.9–8 %) depending on the variety, the method used and the unit in which the results are presented (Campos et al., 2016; Muñoz et al., 2012; Nayani & Rao, 2020). The extraction rate was highest at 24 and 48 h. These stages correspond in fact to the maximum imbibition rate of the seed. Furthermore, during our process, the seeds remain in the water for a sufficiently long time under pressure, allowing greater penetration of the water into the seed and therefore greater release of proteins and soluble minerals (Fabre, Lacroux, Cerny, & Mouloungui, 2015, 2020).

Total protein content of the mucilage extract increased by more than 2.5 times along the germination process of chia seeds (Table 1). Germination is a complex phenomenon involving several processes of neo-synthesis and catabolism. As a result, macromolecule content fluctuates according to a number of factors (Lan et al., 2023; Paucar-Menacho et al., 2018). Ghafoor et al. (2022) has reported a slight increase in protein content in germinated seed compared to raw seed. An increase of protein level has been observed in germinated seeds of pigeon pea
(Chinma et al., 2022), flaxseed (Dong et al., 2023) and Chickpea (Ferreira et al., 2019). In Quinoa, an increase in protein content has been observed in several studies (Lan et al., 2024; Paucar-Menacho et al., 2018; Suarez-Estrella et al., 2020). This increase is thought to be due, particularly in the first three days of germination, to new syntheses of catalytic proteins (Chinma et al., 2022; Ferreira et al., 2019; Khalil et al., 2007; Sattar et al., 2017) but also to the consumption of carbohydrates by respiration during the first few days of this process (Lan et al., 2023). This same physiology could be involved during chia seeds germination leading to an increase of protein content. The increase of ash content also supports this explanation (Table 1).

The ash content of the mucilage extract coming from ungerminated chia seeds was 6.4 %. This level increased by 10.9, 43.8 and 68.9 % after 1, 24 and 48 h of germination (Table 1). This result is in line with what has previously been observed in chia (Ghafoor et al., 2022), legume seeds (Chinma et al., 2022; Ferreira et al., 2019); Chenopodiaceae (Jan et al., 2018; Lan et al., 2023) and degumming flax (Dong et al., 2023). As already explained, in our degumming process, the seeds are immersed in water with a regular flow of pressurized water. This allows the water to penetrate deep into the seed, leading to an increase in the liberation of proteins and soluble minerals. In addition, previously germinated seeds (1h, 24h or 48h) have already been in contact with water during the germination period, before being dried and then brought back into contact with water during degumming. The diffusion of minerals and proteins was thus initiated during this first imbibition, which explains why degumming leads to an extract that becomes increasingly rich in proteins and minerals the longer the germination time.

### 4.3. Effect of stage of germination on seeds lipid composition

During germination, the lipid content fell considerably (Fig. 4). This drop was 5.3 % on the first day and twice as much on the second day (Fig. 4). The same trend was observed for lipid profile (Table 3). The decrease in triglycerides is logically accompanied by an increase in di- and monoglycerides during germination (Table 3), which results in an increase in free fatty acids (Table 3). Surprisingly, little change was observed in the fatty acids composition at any stage of germination (Table 2). No study has examined the variation in lipid profile and free fatty acid composition during germination. The work of Ghafoor et al. (2022) showed a variation in fatty acid composition with an increase in all fatty acids except linoleic acid, which showed a decrease. This difference with our results is probably due to several factors. The genotype used is not the same. Moreover, Ghafoor et al. (2022) examined the impact of roasting, boiling and germination on seed composition. In this study, germination was carried out after 4 days at 20 °C. Cabrera-Santos et al. (2021) reported that the highest germination rate was observed at temperatures above 20 °C, which is the case in our study. However, in our study no significant differences in fatty acid composition were observed during the germination stages examined (Table 2). The germination stages are too far apart compared with the study conducted by Cabrera-Santos et al. (2021). In Brassicaceae, fatty acid levels vary little in the first few days and increase after 4 days (Yaniv et al., 1998). Few differences have been observed in fatty acid composition between several Quinoa genotypes at different periods of germination (Vera et al., 2019). Keawkim and Na Jom (2022) noted an increase in free fatty acids in germinated *Plukenetia volubilis* seeds compared to ungerminated seeds. This result is confirmed in our study. The increase in free fatty acids, mono and diglycerides resulting from the degradation of triglycerides (Table 3). This is expected and can be explained by the catabolic activities of lipases which lead to the degradation of triglycerides into di, monoglycerides and free fatty acids (Dong et al., 2023; Kumar et al., 2021; Vera et al., 2019). Marin et al. (2020), have reported that upregulation of lipase activity in coffee seed germination also supports this explanation. In fact, during germination of chia seeds, the catabolic activity of endolipases leads to the release of fatty acids with the formation of mono and diglycerides. This perfectly

demonstrates the hydrolysis of triglycerides thus providing the energy necessary for seed germination. A more detailed analysis of the data shows that free fatty acids and partial esters of glycerol seem to be mobilized first, as their content drops slightly during the first hour of germination, before triglyceride hydrolysis provides new partial esters and free fatty acids. A change in FA composition has been already observed during imbibition (from 1 to 5h after hydration) in the study of Cabrera-Santos et al. (2021) supports the proposed explanation.

The maintenance of fatty acid profiles can be explained by a continuous supply (until depletion of lipid reserves at the end of the heterotrophic stage of germination) of fatty acids from lipase activity. The fatty acid profile is specific to each species and genotype and is maintained during the germination period examined in our study. The comparisons observed are mainly due to different germination stages and different genotypes.

### 4.4. Impact of germination on chia seeds processing for oil bodies recovery

Numerous studies have addressed the processes for obtaining oil bodies. Fabre et al. (2023) have summarized this literature. Different processes have been used to extract oleosomes from different oilseeds species (Fabre et al., 2023). These processes involve several steps. A preliminary pre-soaking stage, which is not mandatory depending on the species, followed by crushing (using different tools). This is followed by a washing stage involving the use of different solutions to preserve the structure of the oil bodies (Fabre et al., 2023). Other unit operations have been added to this general process to facilitate the separation of the phases obtained. In sunflower, for example, lipases have been added to in the process to obtain free fatty acids, has resulted in improving the produced emulsion (Cassen et al., 2022). For other species, the seeds were dehulled (rapeseed (Ntone et al., 2020), or shelled (sunflower (Cassen et al., 2022; Karefyllakis et al., 2019),). Our process, based on three steps (high shear-crushing, high pressure homogenization and separation by centrifugation) has already been used on flax and sunflower seeds. In flax, prior degumming has also been introduced into a process (Fabre et al. 2015a, 2015b). In chia, the impact of imbibition (Cabrera-Santos et al., 2021) or in the switch between the end of the heterotrophic phase of germination and the seedling stage (Ghafoor et al., 2022) have been studied. To date, no study has examined the impact of both factors on emulsion processes with oleosomes. Two protocols of seeds processing have been tested with germination before or after degumming. Seeds degummed or not were dried, crushed and homogenized at high pressure to a level of 15 % seeds in an aqueous medium. Centrifugal separation then produces three phases: a fat emulsion, an aqueous phase and a lignocellulosic pellet.

#### 4.4.1. Impact of germination stage on recovered oil bodies emulsion

As the germination stages progress, the proportion of the fraction of emulsified fats recovered decreases, as well as the dry matter content of this fraction regardless degumming. However, the lipid content of the dry matter of this fraction remains constant whatever the germination stage. A slight increase is noted at 48h after imbibition (Tables 4 and 9). The protein content of the emulsion increases slightly in the first 24 h after imbibition, then decreases thereafter (48 h) for seed germinated before degumming.

In contrast, for seeds germinated after degumming, the lipid composition of the fat emulsions showed a rapid mobilization of reserve lipids during germination, starting with the consumption of free fatty acids and partial esters before the hydrolysis of triglycerides. Moreover, the values obtained were higher than those obtained for seeds degummed after germination. Furthermore, degummed seeds germinate quickly than native seeds (at least 3 h early), This time delay also has repercussions in terms of lipid degradation activity and reserve remobilization allowing more free fatty acids and less triglycerides (Table 9). Indeed, during the process developed in this study, the seeds were soaked for sufficient time in water under ultrasonic treatment, which
allowed the water to penetrate deep into the seed. This step occurring
before germination it results in earlier mobilization of reserves and
therefore earlier germination.

A decrease in lipid content has already been reported in germinated
seeds of several species (Dong et al., 2023; Jan et al., 2018; Vera et al.,
2019; Yaniv et al., 1998), including chia (Cabrera-Santos et al., 2021).
This decrease is attributed to the mobilization of lipids for the produc-
tion of energy required for germination (Dong et al., 2023; Yaniv et al.,
1998). The increase in proteins observed could be attributed to the
mobilization of catalytic proteins, including lipases. The drop-in pro-
teins 24 h after imbibition is probably due to their degradation (Dong
et al., 2023). These authors observed an increase in free amino acids in
germinating linseed, confirming this hypothesis.

Analysis of the lipid profile of chia seed emulsions at different times after imbibition shows that up to 24 h, the lipid profile remains broadly similar, whereas at 48 h the triglyceride content decreases and an increase in free fatty acids and mono- and diglycerides is observed (Table 5).

No studies have been carried out on the lipid composition of chia
seeds during germination *sensu stricto*. The results obtained in our study
clearly suggest the hydrolysis of triglycerides to provide the energy
required for seed germination. The composition of ungerminated seeds
already shows a 'reserve' of free fatty acids, which are mobilized during
the first few hours after imbibition (1.3 % drop). The free fatty acid
content then increased at 24 and 48 h. A similar increase was observed
for partial glycerol esters. In contrast, a decrease in triglycerides was
observed from 48h after imbibition (Table 5). These results suggest the
hydrolysis of triglycerides to provide new partial esters and free fatty
acids. Lipid mobilization during germination occurs during the first 24 h
in lupin (Dagnia et al., 1992). This degradation is due to lipolytic activity
and the breakdown of triglycerides during germination (Dong et al.,
2023; Jan et al., 2018; Kumar et al., 2021; Vera et al., 2019). In the work
by Cassen et al. (2022), we used the same process with or without the
addition of lipase during homogenization of sunflower seeds. The result
was an almost complete release of fatty acids and glycerol. This result
also supports our hypothesis. In addition, numerous studies have re-
ported a continuous and significant increase in lipase activity in seeds
during germination (Kelly et al., 2011; Kumar et al., 2021; Vijayakumar
& Gowda, 2012).

However, analysis of the profile of fatty acids present in these lipid
emulsions shows a similar composition (Fig. 6) regardless of the
germination stage. This result is not in agreement with those found in
chia in soaked seeds (Cabrera-Santos et al., 2021) or in germinated seeds
(Ghafoor et al., 2022). These two studies are the sole that have found a
variation in fatty acid composition. These differences are due to several
factors. The genotype and experimental conditions are not the same. The
work by Cabrera-Santos et al. (2021) was carried out during the first 5 h
corresponding to the imbibition phase of germination. In contrast, the
work by Ghafoor et al. (2022) was carried out 72 h after imbibition,
which corresponds to the green cotyledons separated stage. In our study,
the fatty acid composition was measured in the interval between the two
studies corresponding to the start of imbibition (1h after watering) and
root protrusion (24-48h after watering) according to Geneve et al.
(2017). During this interval, the germinating seed first consumes the
free fatty acids available in the native seed for energy production
(Table 5) before activating the lipases that will break down the tri-
glycerides (Table 5). The result is an initial variation in the fatty acids
during the imbibition phase (corresponding to the degradation of the
free fatty acid reserve). Lipase activity is at its maximum during the
period of embryo growth (Kumar et al., 2021). In our study, this would
be between 24 and 48 h according to the lipid composition data
(Table 5). The second variation in lipid composition occurs at the end of
lipid reserve depletion. A slight variation in the composition of fatty
acids in germinated seeds compared with dry chia seeds was reported by
Ghafoor et al. (2022), signaling the start of reserve depletion. Between
these two phases of variation, the profile is not expected to change and is

the imprint of the species (Roche et al., 2016; Yaniv et al., 1998) or
genotype (Vera et al., 2019; Zemour et al., 2021).

4.4.2. Impact of germination stage on the lignocellulosic pellet fraction

The mass proportion of lignocellulosic pellets separated during
fractionation decreased with the advancement of the germination stages
(Table 6). The dry matter content of these pellets and the lipid content
increased with the germination time regardless the degumming step.
This lipid enrichment is correlated to the reduction in the mass of
emulsion recovered during fractionation. The protein content of the
sediment pellet is higher at 24 after imbibition for non-degummed
grains (Table 6) which probably corresponds to reserves mobilization
for seeds germination.

The profile of lipids in the sediment pellet is similar from the start of
imbibition until 24 h. However, after this stage, the triglycerides content
decreases, while the partial glycerol esters and free fatty acids content
increases, reflecting lipid hydrolysis by endogenous enzymes present in
the seeds (Jan et al., 2018). As the separation between the two fractions
(pellet and lipid emulsion) took place after the germination period, it
reflects what was seen in recovered oil bodies emulsion.

The lipid enrichment of the lignocellulosic pellet throughout
germination favours saturated fat chains and monounsaturated chains
(Fig. 7). A difference in composition between pellets from native seeds
and those from germinated seeds taken at 48 h shows that the saturated
fatty acid content rises from 17.6 % to 27.4 %, while that of poly-
unsaturated fatty acids falls from 71.8 % to 56.6 %. At the same time,
monounsaturated fatty acids in the sediment pellet increased by almost
50 %. This difference is most likely linked to the early stages of fatty acid
degradation, with a shift from polyunsaturated to monounsaturated and
then to saturated fatty acids in order to provide the energy required for
embryo development (Dong et al., 2023). This tendency was reported by
Yaniv et al. (1998) during germination of crambe and Sinapis seeds and
by K. S. Liu & Brown (1996) in soya. Moreover, the released saturated
fatty acids are probably trapped in sediment pellet. Indeed, our process
is carried out at 4 °C, including centrifugation. The saturated fatty acids
that were released solidified and became trapped in ligno-cellulosic
material of the pellets due to their melting points of 62.9, 69.6 and
75.4 °C for palmitic, stearic and arachidic acids, respectively (Cuvelier
et al., 2004).

4.5. Impact of germination stage on the size of oil bodies

The oil bodies present almost the same size whatever the process
used, except at 48h (Table 12, Figs. 8 and 9).

This study is the first report on oil bodies size during germination of
chia seeds. The size of the oleosomes released in our emulsions remains
statistically stable. Our process in water does not involve any heat
treatment (i.e. no heat treatment to denature the proteins). As a result,
the proteins are not degraded and the surfactants, the oleosins that
stabilize our oleosomes, remain perfectly functional, preserving the
integrity of the oleosomes and still fulfilling their stabilizing role. The
sizes of these oleosomes are maintained even at 48 h (Table 12). The
differences observed at 48h in seeds germinated before degumming, is
probably due to the fact that lipids were not yet fully mobilized. In
contrast, in the process with germination before degumming, the size of
oil bodies was slightly higher at 48h. the potential explanation may be
linked to early germination of seed and therefore the remobilization of
lipids has started earlier. A part of oil bodies was degraded to release
lipids and the rest of the oleosomes coalesce.

5. Conclusion

The aim of this work was to investigate the processability of chia
seeds for degumming and obtaining a native lipid emulsion and to
examine the effect of germination before or after degumming on the
composition of the products obtained. This work is the first report on the
impact of germination in the strict physiological sense on the integrated
process designed to release oil bodies. This process makes possible to
release the mucilage in order to perform an aqueous extract of the lipids
from the seed. The results confirm that degumming, while necessary for
the processing of seeds in water, has no influence on the germination
capacity of chia seeds in our process, enabling them to be germinated.
The release of OBs remains convincing regardless of the germination
stage, demonstrating the robustness of the integrated aqueous process
for recovering OBs. Degumming has no consequence on the aqueous
fractionation of chia seeds, whatever the time of application (before or
after germination). This result means that this fraction can be potentially
exploited in a number of industrial fields (functional foods, cosmetics,
etc.). The mobilization of reserve lipids increases as the germination
stages progress. As a result, the total lipid content of germinating seeds
decreases, the proportion of triglycerides falls and greater proportions of
partial glycerides and free fatty acids are formed. Lipids, particularly
saturated fatty acids, become more widespread in the pellet, reducing
the yield of OBs released in the upper emulsified phase regardless the
germination process and its stage. This study covered three times during
germination. It would be interesting to reproduce the same in-
vestigations at a 2-h step from 0 to 72 h after imbibition, even if the work
involved is colossal with our process. This work should be completed by
omics studies (metabolomics, lipidomics and proteomics) on each of the
fractions obtained in our process in order to determine the mechanisms
that occur in the oilseed during germination and in each of its phases.
The aim is to be able to identify the stages of the germination process,
thereby making it possible to obtain concentrates enriched in certain
molecules of interest.

CRediT authorship contribution statement

Éric Lacroux: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Conceptualization. Muriel Cerny: Writing – original draft, Validation, Resources, Methodology, Formal analysis, Data curation. Jean-François Fabre: Writing – original draft, Software, Formal analysis, Data curation. Romain Valentin: Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization. Othmane Merah: Writing – review & editing, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

Funding

This work was performed within the frame of two programs:

- Collective Incentive Action INRAE « Oil Bodies from germinated seeds », AIC-IB2021-COLIGRAI.

- Inter Carnot 3BCar/Qualiment Resourcing Project « Oil BodiEs for INNovative food products: from plant seed processing to digestive fate », IC2022-OBΕΙΝΝ.

The financial support of this work by INRAE and by the Carnot in-
stitutes Qualiment (https://qualiment.fr/) and 3BCAR (https://3bcar.
fr/) is gratefully acknowledged.

Declaration of competing interest

The authors declare the following financial interests/personal re-
lationships which may be considered as potential competing interests:
Merah reports was provided by University of Toulouse. If there are other
authors, they declare that they have no known competing financial in-
terests or personal relationships that could have appeared to influence
the work reported in this paper.

Acknowledgments

The authors are grateful to AGROFÜN and PANAM for supplying of
chia seeds of the ORURO variety.

We also thank the Laboratoire de Chimie de Coordination-CNRS
(Toulouse, France) for help to the elemental analyses.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2025.117802.

Data availability

Data will be made available on request.

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