# Enzymatic crosslinking of rapeseed albumins: kinetic monitoring, physical characteristics, and functional property enhancements

Romain Faure, Romain Kapel, Philippe Marchal, Ghislain Génin, Sophie Beaubier

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## To cite this version:

Romain Faure, Romain Kapel, Philippe Marchal, Ghislain Génin, Sophie Beaubier. Enzymatic crosslinking of rapeseed albumins: kinetic monitoring, physical characteristics, and functional property enhancements. Food Hydrocolloids, 2025, 172, pp.112100. 10.1016/j.foodhyd.2025.112100. hal-05400665

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https://hal.science/hal-05400665v1

Submitted on 5 Dec 2025

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# Enzymatic crosslinking of rapeseed albumins: kinetic monitoring, physical characteristics, and functional property enhancements

Romain Faure, Romain Kapel, Philippe Marchal, Ghislain Génin, Sophie Beaubier*

Université de Lorraine, CNRS, LRGP, F-54000, Nancy, France

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## ARTICLE INFO

### Keywords:

- Rapeseed albumins

- Crosslinking

- Microbial transglutaminase

- SE-HPLC monitoring

- Gelling properties

- Emulsifying properties

## ABSTRACT

In response to the growing demand for plant-based and functional food ingredients, this study investigated the enzymatic crosslinking of rapeseed albumins (RA) using microbial transglutaminase (MTGase) to enhance their functional properties. A novel size-exclusion chromatography method was proposed to monitor the crosslinking kinetics, enabling the identification of RA dimers, trimers, and polydisperse products, as well as the residual native RA amounts. Differential scanning calorimetry revealed a slight reduction in denaturation temperature, and surface hydrophobicity increased steadily over time, indicating structural rearrangement. Crosslinked RA (CRA) exhibited a least gelation concentration of only 3 % compared to over 15 % for native RA, and formed stable gels with improved mechanical properties (elastic modulus of 500 Pa and critical strain of 4). In a mayonnaise-type model system, CRA significantly improved emulsion stability compared to native RA by reducing droplet size, thickening emulsion texture, and preserving structure over 30 days of storage. The enzymatic crosslinking process offers a promising strategy to valorize RA as a high-performance texturizing agent. These results contribute to the innovative applications of RA in plant-based emulsions and gels, contributing to the development of cleaner-label, protein-enriched foods.

## 1. Introduction

The food industry is increasingly seeking novel, plant-based ingredients to meet consumer demand for natural, sustainable alternatives. In this context, plant proteins appear to be a promising ingredient for formulating emulsions, gels, and foams in products such as plant-based milks, mayonnaises, and meat analogues (Melvin Tan & Buckow, 2023). However, native plant proteins often exhibit inferior techno-functional properties, such as solubility, gelling, emulsifying, and foaming, compared to animal counterparts, limiting their use in textured food systems.

To address these limitations, various protein modification processes have been explored, which can be mainly categorized as physical, chemical, or biological (Akharume et al., 2021). Chemical processes for modifying plant proteins generally include glycation, phosphorylation, acylation, scintillation, and deamidation. These processes require chemicals that are often hazardous or incompatible with food applications. They also result in the release of toxic or non-food-grade secondary by-products, limiting their use for food applications.

Traditionally, plant proteins have been modified using physical

transformation processes, mainly involving heating or high-pressure treatments. More recently, innovative physical techniques, such as ultrasound, pulsed electric fields, and ultraviolet radiation, have gained attention (Nasrabadi et al., 2021). Given that physical treatments often require high energy inputs and chemical processes are typically unsuitable for food-related applications, biological processes, especially enzymatic modifications, seem to be the most promising processing routes. These methods offer food compatibility, operate under mild conditions, and allow for targeted improvements in protein functionality. Enzymatic transformations primarily include proteolysis and crosslinking. Proteolysis involves the use of a protease to cleave the peptide bonds within proteins, resulting in a mixture of protein fragments, peptides, and free amino acids, depending on the specific protease employed and the applied operating conditions (Beaubier et al., 2021). Enzymatic crosslinking enables the formation of covalent bonds between protein chains, resulting in a complex mixture of higher-molecular-weight crosslinked products that are frequently polydisperse in size and possess distinctive characteristics (Djoullah et al., 2018).

Microbial transglutaminase (MTGase; E.C. 2.3.2.13) is the only food-

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: sophie.beaubier@univ-lorraine.fr (S. Beaubier).</footnote>
grade crosslinking enzyme currently in widespread use (Akharume et al., 2021). It catalyzes the acyl transfer reaction between the γ-carboxamide group of glutamine residue and the ε-amino group of lysine residue in the protein chain by creating an ε-(γ-glutamyl)-lysine isopeptide bond (Buchert et al., 2010; Heck et al., 2013). Transglutaminase crosslinking is a process that has been industrially well-established in meat processing and has attracted increasing attention for its potential application to plant proteins. Indeed, interest in this area has steadily grown in the literature over the last decade (Miwa, 2020). Numerous references studied the improvement of plant protein functionalities through crosslinking and particularly the improvement of the gelling properties of plant proteins, either through enzymatic or heat-induced gelation of crosslinked aggregates (Djoullah et al., 2015; Liu et al., 2024; Pinterits & Arntfield, 2008). The increase in size of the crosslinked products compared to native proteins facilitates the formation and stabilization of three-dimensional networks necessary for gel formation. However, most studies to date have focused only on the influence of the substrate pretreatment (native, thermally denatured, chemically denatured, etc.) and/or the operating conditions (temperature, pH, enzyme concentration, ionic strength, etc.) on the final functionality (e.g., gel strength, emulsion and foam stability, surface hydrophobicity, digestibility, etc.) (Liu et al., 2024; Shand et al., 2008; Wang et al., 2024; M. Wu et al., 2016; Zhou et al., 2023). The reaction kinetics and the influence of the operating conditions on the kinetic mechanisms and the crosslinked product compositions obtained are never studied in the literature. This gap is mainly due to a lack of suitable analytical tools to monitor reaction kinetics and product evolution, which limits the in-depth understanding of the enzymatic crosslinking process and its control and optimization for industrial applications.

Among plant proteins, oilseed proteins, especially those from rape-seed meal, are particularly interesting. Rapeseed albumins (RA), or napins, constitute 20–40 % of total rapeseed proteins (Malabat et al., 2003), the other main part being the globulins, or cruciferins (Crouch & Sussex, 1981). RA are rich in sulfur-containing amino acids, which is rare for a plant protein, and can be industrially produced as isolates (>90 % protein) with light color and high solubility across a broad pH range (Defaix, Kapel, & Galet, 2019). They also exhibit favorable functional properties with satisfactory emulsifying (Albe-Slabi et al., 2022; Tan et al., 2014) and good foaming properties (Lu et al., 2023; Shen et al., 2023). Nevertheless, RA are low molecular weight proteins (10–15 kDa) with a compact heterodimeric structure, a high isoelectric point (~11), and thermal stability (>100 °C) (Barciszewski et al., 2000; Perera et al., 2016). This significant structural stability of RA has been attributed to the presence of disulfide bridges, resulting in poor gelling capacities and a high resistance to proteolysis, particularly to gastric digestion (Bos et al., 2007), which can be an obstacle to RA industrial use. Previous studies have demonstrated that RA can be hydrolyzed by applying controlled enzymatic proteolysis (Beaubier et al., 2021), and optimal conditions were identified to improve the *in vitro* digestibility while preserving their functional properties (Beaubier et al., 2023). To the best of our knowledge, the potential of MTGase-mediated crosslinking for enhancing RA functionality remains unexplored.

The objectives of this study are threefold: (i), to meticulously investigate the enzymatic crosslinking reaction of RA using MTGase, (ii), to thoroughly characterize the resulting crosslinked RA (CRA) products in terms of molecular composition, thermal properties, and surface hydrophobicity, and (iii), to evaluate their functional capacities as gelling or emulsifying agents. The crosslinking reaction kinetics were monitored using an original SE-HPLC approach developed in this study. This is the first time that the kinetics of crosslinking product formation have been assessed. Functional capacities were assessed through rheological characterization of the resulting gel and a model plant-based mayonnaise system.

# 2. Materials and methods

## 2.1. Materials and chemicals

Rapeseed albumin (RA) isolate was provided by Avril Group (France). The isolate was produced from industrial rapeseed meal by selective extraction of albumins under acidic conditions and further purification using membrane processes. The purity of the RA isolate obtained was 99.6 % (according to the Kjeldahl method, N × 6.25, on a dry matter basis). The RA isolate solubility was: 99.4 ± 0.56 % at pH 3, 99.5 ± 0.76 % at pH 5, 98.7 ± 1.4 % at pH 7, and 98.3 ± 1.9 % at pH 9 (Albe-Slabi et al., 2022).

Microbial Transglutaminase (MTGase) was purchased from Creatives Enzymes (New York, United States). The preparation consists of 60 % enzyme and 40 % maltodextrin for a declared activity of 135 U·g⁻¹. It was kept in the dark, at room temperature.

O-phthalaldehyde (OPA) and protein standards for SE-HPLC column calibration were purchased from Sigma-Aldrich (St. Louis, USA). HPLC-grade acetonitrile and trifluoroacetic acid (TFA) were purchased from Fisher Scientific (Geel, Belgium). 8-Anilino-1-naphthalenesulfonic acid probe (ANS) was purchased from ThermoFisher (Waltham, MA, USA). All other chemicals and reagents used were of analytical grade.

## 2.2. Implementation of the enzymatic crosslinking reaction

The enzymatic crosslinking reaction protocol was adapted from Djoullah et al. (2015). Briefly, the native RA isolate was dissolved in phosphate buffer solution (PBS), pH 7, 50 mM, at a protein concentration of 0.5 % (w/v). Then, the protein solution was treated with 20 U·g⁻¹ of MTGase at 50 °C for 5 h. During the crosslinking reaction, RA samples were collected at 10, 30, 60, 120, and 300 min of reaction to study the kinetics. A control sample, named T₀, was collected at the reaction conditions, and before the addition of the enzyme. To stop the enzymatic reaction, all samples were heated at 80 °C for 2 min. The solution obtained after 300 min of reaction was freeze-dried to obtain a powder, noted CRA.

## 2.3. Analysis and monitoring of the crosslinking reaction

### 2.3.1. SDS-PAGE profile

The RA samples from the crosslinking reaction were analyzed using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis analysis (SDS-PAGE) following the Laemmli method (LAEMMLI, 1970). Before analysis, 30 µL of the RA samples were diluted in 30 µL of Laemmli buffer containing 5 % β-mercaptoethanol (v/v) and heated at 95 °C for 10 min. Molecular weight markers from 10 to 250 kDa (Precision PlussProtein Standards) were used (Bio-Rad, Hercules, USA). Ten microliters of prepared samples and markers were deposited into a 5 % polyacrylamide stacking gel and separated in a 15 % polyacrylamide resolving gel. After migration at 20 mA, the gels were stained with Coomassie Brilliant Blue and then destained overnight in 5 % acetic acid solution (v/v). All reagents and electrophoretic equipment were obtained from Bio-Rad (Hercules, USA).

### 2.3.2. Fractionation using size exclusion chromatography

Analysis of the RA samples from the crosslinking reactions were assessed by size-exclusion chromatography (SE-HPLC) method adapted from (Defaix, Aymes, et al., 2019). Briefly, 10 µL of the sample was injected into a Biosep-SEC-s-3000 column (300 × 7.8 mm, 5 µm) from Phenomenex (Torrance, California, USA) kept at 35 °C. The column was connected to a Shimadzu model SLC-40 system equipped with a photodiode array detector SPD M40 from Shimadzu (Kyoto, Japan). The mobile phase consisted of 45 % acetonitrile, 54.9 % ultrapure water, and 0.1 % trifluoroacetic acid (v/v/v) and was set at a flow rate of 1 mL min⁻¹ for 30 min. All RA samples were filtered using 0.22 µm regenerated cellulose membranes before analysis. UV detection was recorded
at 214 nm for RA and CRA products. Data collection and analysis were performed using LabSolutions software (Shimadzu, Kyoto, Japan).

The column was calibrated using 4 protein standards: aprotinin (6.5 kDa), cytochrome C (12.4 kDa), carbonic anhydrase (29 kDa), and bovine serum albumin (66 kDa), eluted under the same conditions. The retention time for each known molar mass (MMx) of protein standard was determined using the chromatogram and the LabSolutions software (Shimadzu Corporation, Japan). The calibration equation (MMx vs. Trx) was obtained by performing linear regression using Excel (Eq. (1), R² = 0.97), as:

$$MM_x = 10^{-0.422 \times Tr_x + 4.14} \quad (\text{Eq. 1})$$

where MMx is the molar mass of the point ‘x’ of the chromatogram (g/mol) and Trx is the retention time of the point ‘x’ of the chromatogram, in the calibration range.

The final reaction kinetic point (300 min) was further fractionated by SE-HPLC under the conditions above-mentioned, and fractions were collected every 10 s from 5 to 10 min of the elution time. The collected fractions were then analyzed by SDS-PAGE as previously described. The fractionation was repeated 4 times to collect enough samples. Before SDS-PAGE analysis, the fractions were dried and resuspended in Laemmli buffer.

### 2.3.3. Degree of crosslinking by the OPA method

The measurement of the free amino acids to calculate a degree of crosslinking was assessed by the OPA method, according to the protocol of (Dinnella et al., 2002), with some modifications. OPA reagent was prepared by dissolving 80 mg of o-phthalaldehyde (OPA) in 2 mL of ethanol. 50 mL of tetraborate buffer (pH 9.5, 0.1 M) and 10 mL of 10% SDS (v/v) were added to a 100 mL volumetric flask. The OPA solution in ethanol and 0.2 µL of 2-mercaptoethanol were added to the volumetric flask. The OPA reagent was filled up to 100 mL with deionized water.

Fifteen microliters of RA samples were mixed with 2 mL of OPA reagent. After 2 min of reaction, the absorbance at 340 nm of the samples was measured.

To quantify the ammonia formed during the crosslinking reaction, three hundred microliters of the RA samples were transferred to centrifugal test tubes AMICON 0.5 mL, 3 kDa, and centrifuged at 5000×g for 1 h. The permeates were analyzed by the OPA method above-mentioned.

The degree of crosslinking was then calculated as follows:

$$DR_{(t)}(\%) = \left( 1 - \frac{Abs_{(t)} - Abs_{p(t)}}{Abs_{(0)} - Abs_{p(0)}} \right) * 100 \quad (1)$$

where DR(t) was the degree of crosslinking at the reaction time t, Abs(t) was the absorbance of the crosslinked RA sample at the reaction time t, Abs(0) was the absorbance of the native RA sample at the reaction conditions, Abs(p(t)) was the absorbance of the permeate of the crosslinked RA sample at the reaction time t, and Abs(p(0)) was the absorbance of the permeate of the native RA sample at the reaction conditions.

## 2.4. Physicochemical characteristics of crosslinked rapeseed albumin products

### 2.4.1. Surface hydrophobicity ($H_0$)

Surface hydrophobicity of RA samples from the crosslinking reaction was measured using the ANS-probe method adapted from (Kato & Nakai, 1980). An 8 mM ANS solution was prepared by dissolving ANS in PBS buffer (pH 7, 50 mM). The RA samples were diluted to concentrations between 0.05 % and 0.5 % solution with PBS buffer (pH 7, 50 mM). Twenty microliters of the ANS solutions were mixed with 4 mL of the diluted protein sample. The fluorescence spectra of the mixture were measured between 400 and 700 nm (emission wavelength) for an excitation wavelength of 390 nm using a Fluorolog FL3-222 spectrophotometer (Horiba Jobin Yvon, Palaiseau, France). Then, the areas under fluorescence intensity curves were plotted against protein

concentrations, and the slope was identified as the surface hydrophobicity coefficient, $H_0$.

### 2.4.2. Differential scanning calorimetry (DSC)

Differential scanning calorimetry (DSC) was performed using a VP-DSC (MicroCal, Northampton, MA). Data were analyzed by a Windows-based software package. All DSC experiments were achieved with a protein concentration of 5 g.L⁻¹ in PBS (50 mM, pH 7). The reference cell was filled up with PBS (50 mM, pH 7). The DSC scan rate was set to 10 °C.min⁻¹ from 20 °C to 120 °C, for all experiments.

## 2.5. Gelling properties

### 2.5.1. Least gelling concentration (LGC)

Least gelling concentration (LGC) was evaluated following the method of (O’Kane et al., 2005). RA and CRA suspensions were prepared at concentrations from 1.5 % to 15 % of proteins (w/v) in distilled water. After the complete dissolution of the powder, the pH of the suspension was adjusted to 7.0 by adding 1 M NaOH solution. Then, the suspensions were poured into test tubes. The tightly sealed test tubes were heated at 95 °C for 30 min in a water bath. After 30 min, test tubes were allowed to cool down at room temperature for 1 h and then kept at 6 °C overnight. The next day, the test tubes were inverted, and the samples that did not flow were considered to have gelled.

### 2.5.2. Rheological tests

A 5 % CRA gel (w/v) at pH 7.0 was prepared as described previously. The gel was then gently removed from the test tube and subjected to dynamic oscillatory rheological measurements. The rheological properties of the CRA gels were determined using an ARES rheometer (TA Instruments, DE, USA). A parallel plate geometry (25 mm diameter) was used to carry out a frequency sweep and critical strain measurement. Parallel plates were equipped with needles to prevent the gel from slipping during rheological tests.

#### 2.5.2.1. Frequency sweep measurement.
Frequency sweep measurement was carried out at a fixed strain of 0.003 (within the linear viscoelastic domain, preliminary determined) and for an angular frequency sweep from 0.01 to 100 rad s⁻¹. Elastic and viscous moduli (G' and G'', respectively) were recorded against the angular frequency of deformation (ω).

#### 2.5.2.2. Critical strain measurement.
Critical strain measurement was assessed at a fixed deformation frequency of 1 rad s⁻¹ and for a strain sweep from 0.001 up to the critical strain. Elastic and viscous moduli were recorded against strain, and critical strain was defined as the intersection point between the elastic G' and viscous G'' moduli curves. From these measurements, the yield stress of the gel is calculated to evaluate the strength of a gel by multiplying the critical strain by the complex modulus G* at the G' and G'' crossing point.

## 2.6. Emulsifying properties in model plant-based mayonnaise system

### 2.6.1. Preparation of model emulsions

CRA products were used in a simplified mayonnaise-type model system. CRA powder was suspended with 0.16 g of NaCl in distilled water. A spatula point of sodium azide was added to avoid microbial contamination during storage. The pH of the suspension was adjusted to 3.5 by adding 5 M HCl, and the suspension was completed up to 5.7 mL. Then, the suspension was vigorously stirred with progressive addition of colza oil up to an oil/water ratio of 80/20 (w/w) using an Ultra-Turrax® homogenizer (IKA, Staufen im Breisgau, Germany) at 10,000 rpm for 5 min. The final protein concentration of the emulsion was 0.5 % (w/v). Emulsification process was conducted at room temperature. Control emulsion was realized with native RA isolate following the same
protocol. Each emulsion was prepared in triplicate and analyzed once.

### 2.6.2. Particle size distribution

The particle size distributions of the CRA and RA emulsions were determined using a MasterSizer 3000 (Malvern Instruments Ltd, Malvern, Worcestershire, UK), equipped with a wet sample dispersion unit (Malvern Hydro MV, UK). The background and sample integration times were 5 s and 20 s, respectively. Ten measures were recorded for each sample with a delay time of 10 s between measurements. The obscuration of the laser was between 5 % and 15 % during measurements. The optical properties were defined as refractive index 1.47 (colza oil) and 1.33 (dispersant water) and absorption index 0.001. The percentile values $D_x(10)$, $D_x(50)$, and $D_x(90)$ shown are the mean of the percentile values of three emulsion analyses. Each percentile value from one emulsion analysis is calculated from the mean of the last five particle size distribution measurements out of ten.

### 2.6.3. Viscosity measurement

Flow curves of the native and CRA emulsions were measured by a flow sweep test. The emulsions were placed under an AR-G2 stress-controlled rheometer (TA Instruments, DE, USA) equipped with a parallel plate geometry (40 mm). Shear stress sweep was applied from 0.1 to 100 Pa, and the apparent viscosity measured was plotted against the applied shear stress.

## 2.7. Statistical analysis

All experiments were conducted in triplicate, and the data are presented as the mean (n = 3) ± standard deviation (SD). The data were subjected to a one-way analysis of variance (ANOVA) after testing for normality and homogeneity of variance. A “Tukey test” ($p \le 0.05$) was performed to identify significant differences among samples. Values were considered significantly different if the p-value was <0.05. The results of the statistical analysis are represented by letters, and samples with the same letters are not significantly different.

# 3. Results and discussion

## 3.1. Monitoring crosslinking reaction of rapeseed albumins using size measurements and spectrophotometric method

### 3.1.1. SDS-PAGE profiles

The enzymatic crosslinking of RA with MTGase was initially assessed by monitoring the molecular size evolution using SDS-PAGE. Fig. 1 presents the SDS-PAGE profiles obtained throughout the reaction, from time 0 ($T_0$) to 300 min, under both non-reducing (Fig. 1A) and reducing conditions (Fig. 1B). At $T_0$, corresponding to the untreated RA isolate, a broad and intense band appeared around 15 kDa, which was in adequation with the reported molecular weight of RA (Perera et al., 2016). This dominant band represents the native albumin proteins.

Additionally, faint bands were observed at approximately 20 and 25 kDa in the same lane, corresponding to minor residual amounts of rapeseed globulin subunits (Schwenke et al., 1998).

The enzymatic crosslinking of RA by MTGase resulted in the appearance of a diffuse band in the upper half of the gel (Fig. 1A). This smear-like band is characteristic of the MTGase-mediated crosslinking of plant proteins (Ikura et al., 1980; Lu et al., 2023; Ramirez-Suarez, 2003; Sun & Arntfield, 2011), and indicates that RA are a suitable substrate for MTGase, which can be crosslinked. The polydisperse nature of this band, from 50 to 150 kDa, reflects the formation of crosslinked protein aggregates of varying sizes. In addition to the diffuse region, distinct bands were observed around 25 kDa and 37 kDa. These bands likely correspond in size to two- and three-times RA monomer size, respectively. From this result, the hypothesis that those bands could correspond to a dimer and a trimer of RA, formed through covalent crosslinking between RA monomers, can be formulated. However, validation of this hypothesis would necessitate subsequent analysis.

SDS-PAGE analysis under reducing conditions (Fig. 1B) showed two distinct bands at the lower region of the gel, corresponding to the two subunits that constitute the RA heterodimer (So & Duncan, 2021). In the later time points of the reaction, the band associated with the ~10 kDa subunit progressively faded, while the intensity of the second subunit band remained relatively stable. This differential behavior suggests that one of the RA subunits is more actively involved in the crosslinking reaction, potentially due to a higher content and/or greater accessibility of reactive lysine and glutamine residues. The kinetic evolution of the MTGase crosslinking appeared to reach a plateau after approximately 10 min of reaction, as indicated by minimal changes in the intensity of the major identified bands. This observation has also been made in SDS-PAGE analyses of MTGase-mediated crosslinking in pea (Djoullah et al., 2015) and rapeseed (Lu et al., 2023) proteins.

While SDS-PAGE analyses effectively confirm the occurrence of protein crosslinking, it lacks the resolution and quantitative capability required for accurate kinetic monitoring. In contrast, size-exclusion high-performance liquid chromatography (SE-HPLC) offers a more precise and quantitative approach, making it a valuable tool for detailed monitoring of the crosslinking reaction catalyzed by MTGase.

### 3.1.2. Fractionation using size-exclusion chromatography

#### 3.1.2.1. Analyses of chromatograms and identification of RA crosslinked products.
SE-HPLC was employed as a complementary analytical technique to monitor the reaction kinetics. It provides high resolution and quantitative information on molecular weight distribution, allowing a more accurate assessment of reaction progress and the characterization of crosslinked products. Chromatograms at 214 nm from the SE-HPLC analysis of RA crosslinking reaction samples are shown in Fig. 2. Fractionation yielded chromatograms (Fig. 2A and B) featuring a dominant peak at a retention time of 7.5 min, present at $T_0$, corresponding to native RA (Defaix, Aymes, et al., 2019). In addition, several peaks

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** SDS-PAGE profiles of rapeseed albumins (RA) in the course of the crosslinking reaction by MTGase at pH 7, 50 °C under (A) non-reducing and (B) reducing conditions.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Size exclusion chromatography monitoring of the MTGase-mediated crosslinking reaction of Rapeseed Albumins (RA): (A) Full chromatograms at 214 nm of the kinetic monitoring; (B) identification of the crosslinked products on chromatograms of the kinetic monitoring between 5 min and 10 min elution time; (C) fractionation of the end-time (300 min) sample from RA crosslinking reaction and SDS-PAGE analysis of the collected fractions. The elution conditions were 10 µL injection volume, 1 mL min⁻¹ flow rate with water/acetonitrile/TFA mix (55/44.9/0.1, v/v/v) buffer. The calibration equation obtained by linear regression was: $MMx = 10^{-0.422e^{Trx+4.14}}$, and the determination coefficient (R²) was 0.97.</figcaption></figure>

emerged at lower retention times, indicating higher molecular weight crosslinked products. Two defined peaks, with retention times of 6.9 and 6.6 min, respectively, were identified as RA dimers and trimers. A broad peak spanning 5.5 and 6.5 min was attributed to polydisperse crosslinked RA aggregates. The chromatograms also demonstrated minor peaks with retention times greater than 8 min. These peaks correspond to protein fragments and phenolic compounds, which are not involved in the crosslinking reaction, as their shape and intensity remained unchanged throughout the reaction.

To confirm the identification of the crosslinked products observed in the chromatograms, the SEC column was calibrated, and the resulting calibration equation was then used to estimate the molecular weights of the eluted products. Based on this analysis, the estimated molecular weights of native RA, RA dimers, and RA trimers were determined to be 9.4 kDa, 18.1 kDa, and 23.8 kDa, respectively. A slight discrepancy was observed between the expected molecular weights and those calculated from the calibration curve. However, the dimer calculated size was close to double that of native RA, and the trimer slightly below triple. This discrepancy is likely since size exclusion chromatography separates molecules based on their hydrodynamic radius rather than exact molecular weight. Assuming a globular conformation for RA, the crosslinking of two or three monomers would result in a hydrodynamic radius between two and three times that of the monomer, which is consistent with the observed elution behavior. Consequently, these results support the identification of the RA dimer and RA trimer observed in both the SE-HPLC chromatograms and SDS-PAGE profiles.

Finally, to ensure that each chromatographic peak corresponds to a unique compound that has been identified in both the previous analysis and the SDS-PAGE analysis section, further fractionation of the final crosslinking reaction sample (300 min) was assessed. It was subjected to size exclusion fractionation, and the fractions collected were analyzed by SDS-PAGE, as presented in Fig. 2C. The results confirmed that the compounds identified in this section using SE-HPLC calibration, as well as in the SDS-PAGE analysis (see section 3.1.1), correspond to a single, distinct, and integrable chromatographic peak.

### 3.1.2.2. Kinetic monitoring of the identified crosslinked products.
The identification of dimers, trimers, and polydisperse crosslinked products

has previously been established in the field of casein crosslinking by transglutaminase using low-pressure chromatographic techniques developed over the past two decades (Lauber et al., 2000; Raak & Corredig, 2022). This identification is commonly employed to calculate a degree of polymerization, based on the ratio of chromatographic peak areas, which serves as a valuable indicator for assessing the progression of the enzymatic reaction. In comparison, the application of SE-HPLC for monitoring MTGase-mediated crosslinking in plant proteins is more recent and less frequently reported. However, the formation of crosslinked products has been documented in various systems, including sesame proteins (Anuradha & Prakash, 2009) and rice protein-casein complexes (He et al., 2021). Renzetti et al. (2012) also analyzed the enzymatic crosslinking of brown rice flour and qualitatively tracked changes in protein profiles over the reaction using SE-HPLC. These studies, both from dairy and plant protein systems, have contributed to the development of a quantitative approach to monitoring crosslinking kinetics. This approach relies on the analysis of chromatographic peak evolution corresponding to previously identified crosslinked products, providing a tool to accurately track the progress of the reaction.

To assess the applicability of the SE-HPLC approach, the mass balance of the products of interest was assessed by analyzing the total chromatographic area, which remained constant throughout the reaction. This result indicated that all RA crosslinked products remained soluble and were consistently detectable within the chromatograms. These findings confirm that the SE-HPLC approach is suitable for monitoring the reaction. Consequently, the kinetics of both native RA and its crosslinked products can be reliably quantified through chromatogram integrations, assuming that each peak corresponds to a specific compound identified previously by SDS-PAGE analysis.

The kinetics of RA and its crosslinked products were assessed by calculating the area ratios between the RA or crosslinked products and the total chromatographic area of protein compounds present in the chromatogram. This enabled the plotting of the relative percentages of residual native RA and its crosslinked products obtained throughout the reaction. Fig. 3 exhibits the kinetics of native RA, RA dimers, RA trimers, and RA polydisperse crosslinked products over reaction time. To ensure accurate mass balance, the potential contribution of rapeseed globulins, which were present initially, was subtracted from the area of each
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Kinetics of the MTGase-mediated crosslinking reaction of rapeseed albumins: evolution of residual native RA (Rapeseed Albumins) content (primary axis) and formation of RA dimers, trimers, and polydisperse crosslinked products (secondary axis) based on SE-HPLC analysis.</figcaption></figure>

crosslinked product. This approach maintained a mass balance of over 95 % throughout the reaction.

The kinetics of the crosslinking reaction (Fig. 3) and the corresponding SE-HPLC chromatograms (Fig. 2B) indicate that the reaction did not result in complete crosslinking of RA under the experimental conditions applied. This outcome can be attributed to two main factors: (i) the gradual decrease in available crosslinking sites as the reaction progressed, and/or, (ii) the degradation of the transglutaminase at 50 °C. Indeed, in these conditions, a 60 % loss of activity after 2 h was previously reported (Djoullah et al., 2015). The crosslinking kinetics also revealed the continuous formation of all identified crosslinked products, with no conversion between them, suggesting that distinct formation mechanisms were involved. Under the conditions of the experiment carried out, the final product mixture consisted of approximately 54 ± 0.5 % native RA, 14 ± 0.5 % RA polydisperse crosslinked products, 7 ± 0.5 % RA trimers, and 21 ± 0.3 % RA dimers, and was obtained after approximately 1 h.

Consequently, SE-HPLC has proven to be a reliable and quantitative tool for monitoring the enzymatic crosslinking process of RA by transglutaminase. This monitoring approach can be applied to model, simulate, and optimize this enzymatic process. For instance, it can be used to assess the kinetics of crosslinking under varying operational conditions to minimize industrial costs.

### 3.1.3. Degree of crosslinking by the OPA method

The OPA method is typically used to calculate a degree of crosslinking by an absorbance ratio or an equivalent lysine concentration ratio, which is determined using a calibration curve. This method is analogous to the OPA spectrophotometric method used to calculate the degree of hydrolysis in proteolysis experiments (Nielsen et al., 2001). Similar to the degree of hydrolysis, the degree of crosslinking is an effective means of evaluating the progress of the enzymatic crosslinking reaction. However, the calculation of the degree of crosslinking is less accurate. This is due to the increasing size of crosslinked products, which can render amines of the protein chain inaccessible. Additionally, the by-product of the crosslinking reaction is ammonia, which contributes to the measured absorbance.

Absorbance measurements of RA samples mixed with the OPA reagent showed no significant variation over time, indicating that the absorbance values remained constant throughout the reaction. This observation is consistent with the known transglutaminase crosslinking mechanism, which dictates that the disappearance of a free amine group from the glutamine side chain results in the release of a free amine group

into the medium (Buchert et al., 2010; Heck et al., 2013). Several studies have reported that the OPA absorbance values required a correction to account for interferences to yield meaningful kinetic data (Dinnella et al., 2002; Djoullah et al., 2015). Despite the implementation of the correction method, the OPA method failed to reflect any detectable changes in absorbance values (free amine concentrations) during the reaction. This unexpected result suggests that the OPA method may lack the sensitivity required to monitor crosslinking kinetics under the specific experimental conditions used, or with RA as a substrate. One potential explanation for this phenomenon is that the enzymatic reaction produced a limited number of crosslinks, resulting in a small amount of free amine in the medium, which is insufficient to produce a measurable change in absorbance. Consequently, the corrected OPA signal remained low and unchanged throughout the reaction, indicating that this method is not suitable for kinetic monitoring of RA crosslinking by transglutaminase under these conditions.

## 3.2. Characterization of physicochemical properties of crosslinked products

### 3.2.1. Differential scanning calorimetry (DSC)

Enzymatic crosslinking by transglutaminase modifies the physicochemical properties of plant proteins by covalently linking protein chains. Such structural modifications can alter, increase, or even decrease these properties depending on the extent and nature of the crosslinking. Among these properties, the thermal denaturation temperature of proteins is an important parameter to be aware of, as it directly influences their behavior during processing. RA are known for their high thermal stability, with reported denaturation temperatures ranging from 100 °C to 110 °C (Nioi et al., 2012; J. Wu & Muir, 2008; Yang et al., 2014).

DSC analysis of both native and CRA products is shown in Fig. 4A. The thermogram of native RA revealed a denaturation temperature of 99 °C, which is consistent with previous findings (Nioi et al., 2012), who reported a value of 100 °C. The slight difference may be attributed to variations in protein concentration between the two studies (5 % (w/v) in this study versus 7 % (w/v) in the previous one).

Under identical experimental conditions, the thermogram of CRA product showed a denaturation temperature of 97 °C (Fig. 4A). This value, while slightly lower than that of native RA, is in line with expectations given the CRA composition, which consists of approximately 60 % native RA and 40 % crosslinked products. The small decrease in denaturation temperature suggests that the presence of crosslinked products could slightly reduce the thermal stability of the product. However, further structural analysis is necessary to definitively ascertain the reliability of this result and to determine which crosslinked products may be responsible for the observed decrease.

### 3.2.2. Surface hydrophobicity ($H_0$)

Surface hydrophobicity is another important physicochemical property of proteins, as it influences their behavior in aqueous solution, affecting the solubility and the ability to stabilize interfaces. This property plays a crucial role in the formation and stabilization of emulsions (oil-water interfaces) and foams (air-water interfaces). In the case of soy proteins, MTGase-mediated crosslinking has been shown to modify their three-dimensional structure, resulting in increased surface hydrophobicity of the protein nanoparticles (S. Wu et al., 2023).

In the present study, the evolution of RA surface hydrophobicity during the enzymatic crosslinking reaction was monitored and is shown in Fig. 4B. The surface hydrophobicity coefficient $H_0$ gradually and significantly increased during the reaction, indicating enhanced exposure of hydrophobic groups. This increase is likely due to conformational changes induced by the formation of covalent crosslinks, which may disrupt the native tertiary structure and expose hydrophobic regions that are normally buried within the protein structure. This structural rearrangement would allow greater accessibility for the ANS probe.
<figure><img src="image_8.png" /><figcaption>Fig. 4. Effect of the MTGase-mediated crosslinking reaction on the thermal denaturation of RA and CRA (A) and the evolution of the surface hydrophobicity of RA (B).</figcaption></figure>

A similar observation was reported for total rapeseed proteins crosslinked by transglutaminase, where an overall increase in surface hydrophobicity was associated with improved foaming capacity, depending on the duration of the enzymatic treatment, while maintaining good foam stability (Lu et al., 2023). These findings further support the role of structural rearrangements in modulating functional properties through enzymatic crosslinking.

### 3.3. Potential food applications of CRA products as texturizing agents

RA are known for their resistance to gastrointestinal digestion (Bos et al., 2007), which limits their suitability as primary protein sources in food products. Given the limited *in vitro* digestibility of RA isolate (Beaubier et al., 2023), CRA products would be more appropriate for use as functional ingredients rather than primary protein source, and therefore, incorporated at low levels to improve texture or stability in formulated foods. Consequently, the potential of CRA products as gelling and emulsifying agents was further investigated for their application in food systems.

#### 3.3.1. Effect of enzymatic crosslinking on gelling properties of rapeseed albumins

Fig. 5 presents the gelling properties of RA and CRA, including the least gel concentration (LGC) measurements of RA and CRA in Fig. 5A

and B, respectively, and the rheological characterization of CRA gels in Fig. 5C and D. LGC evaluations revealed a striking difference between native RA and CRA. While RA failed to form a gel after 30 min in a 95 °C water bath, even at a protein concentration as high as 15 % (Fig. 5A), CRA exhibited an LGC of approximately 3 % (w/v; of proteins) at pH 7.0 (Fig. 5B). This inability to gel of the native RA is well known and can be explained by their high thermal and structural stability (Nioi et al., 2012; J. Wu & Muir, 2008), making the applied heat treatment at 95 °C insufficient to induce denaturation and subsequent gelation.

In previous research, the LGC of the globulin fraction (the other main rapeseed proteins) from various plant sources typically ranged between 3 % and 15 % of proteins, with a median around 10 % (Zhu et al., 2024). Moure et al. (2006) reported LGC values for oilseed proteins depending on the extraction method, none falling below 6 %, and most around 15 %. Similarly, Ge et al. (2021) observed LGC values between 8 % and 10 % across eight Chinese bean species at pH 7. These comparisons underscore the remarkable gel efficiency of CRA, with one of the lowest LGCs reported for enzymatically modified plant proteins. The capacity of CRA to form gels at such low concentrations was unexpected, particularly given that their denaturation temperature (97 °C; Fig. 4A) remained close to that of native RA. This suggests that gelation is driven not only by the thermal unfolding of native RA, but by the presence of crosslinked products (RA dimers, trimers, and polydisperse crosslinked products). Nonetheless, further investigations are needed to identify

<figure><img src="image_9.png" /><figcaption>Fig. 5. Effect of the MTGase-mediated crosslinking reaction on the gelling properties of RA and CRA: Least gelling concentration of RA (A) and CRA (B); frequency sweep (C), and strain sweep (D) measurements of a 5 % CRA gel (w/v). Gels were heat-induced by heat treatment in a water bath at 95 °C for 30 min.</figcaption></figure>
which specific crosslinked compounds are primarily responsible for this enhanced gelling ability.

Rheological characterization of CRA gels was assessed at 5 % (w/v) protein concentration. This protein concentration was chosen for the gel to ensure accurate measurement of rheological parameters since the 3 % protein gel was not fully homogeneous. Frequency sweep test (Fig. 5C) showed that both elastic (G') and viscous (G") moduli were relatively insensitive to the frequency of deformation. In addition, G' consistently exceeded G" across the tested range, indicating solid-like, elastic behavior. The gel strength, defined as the mean value of G' on the frequency sweep, was approximately 500 Pa, which is higher than that of a reported 15 % RA protein gel (200 Pa at pH 7; Yang et al., 2014), and also stronger than 6 % soy protein gels crosslinked with MTGase (G' ~ 200 Pa; Wang et al., 2024). These results confirmed the interesting solid consistency of the CRA gels.

As illustrated in Fig. 5D, the critical strain of the CRA gels (defined as the maximum strain the gel can withstand without undergoing irreversible alterations) was measured at approximately 4. From this measurement, the yield stress of the CRA gel was calculated to evaluate its strength, with this value and the complex modulus G* (~80 Pa; at the G' and G" crossing point). It was measured at 320 Pa, which is consistent with the gel strength (500 Pa) measured by the frequency sweep test. Furthermore, the critical strain measured was notably higher than typical values reported for other plant protein gels. For example, Ge et al. (2023) presented values between 0.2 and 1 for 14 % legume protein gels, while Wang et al. (2024) found a critical strain of 1 for 6 % soy protein gels crosslinked by MTGase. This means that the MTGase-mediated crosslinking of RA not only confers gelling ability to proteins that are otherwise non-gelling under mild conditions, but also results in the formation of gels with notably high mechanical properties. This enzymatic transformation has the potential to open new pathways for the use of CRA as a promising texturizing ingredient for innovative food preparations, especially in plant-based applications.

### 3.3.2. Effect of crosslinking on emulsifying properties of rapeseed albumins

The following section examined the impact of enzymatic crosslinking on the emulsifying properties of RA. A preliminary functional screening (data not shown) suggested that CRA exhibited both thickening and emulsifying properties. As a result, their emulsifying potential was further explored using a simplified mayonnaise-type model system, without a texturizing agent. Fig. 6 presents the results of this study,

which include microscopic (Fig. 6A) and macroscopic (Fig. 6B) observations, particle size distributions (Fig. 6C), and flow curves (Fig. 6D) measurements.

At the macroscopic level, both CRA-based emulsions (CRAE) and native RA-based emulsions (NRAE) initially appeared visually similar, with a smooth, white appearance (data not shown). However, after 30 days of storage at 6 °C, visual inspection revealed signs of instability in the NRAE, notably through the appearance of visible fine oil droplets, whereas CRAE maintained a smooth, homogenous, and bright structure (Fig. 6B).

Microscopic observations (Fig. 6A) showed that the initial droplet size of CRAE was markedly smaller than in NRAE. This qualitative observation was quantitatively confirmed through particle size distribution analysis and the calculated Dₓ(10), Dₓ(50), and Dₓ(90) percentile values (Fig. 6C). At day 0, these values for NRAE were approximately 1.5 times higher than those of CRAE, indicating a more effective droplet dispersion when CRA was used as the emulsifier and presuming a better stability over time. After 30 days of storage at 6 °C, this discrepancy in size became even more pronounced. The percentile values for the NRAE increased significantly, whereas those for CRAE remained remarkably constant. Besides, after storage, those values for NRAE were approximately twice those of CRAE. These results highlight the superior physical stability of CRAE over time and the poor one of NRAE, and are consistent with macroscopic observations. This outcome also aligns with the well-established principle that emulsions with smaller droplet sizes tend to exhibit better stability (Mcclements, 2007). The enhanced stability of CRAE can be attributed to the higher surface hydrophobicity (H₀) of CRA resulting from enzymatic crosslinking. This increased surface hydrophobicity allows for more efficient protein adsorption at the oil-water interface, promoting the formation of smaller and more stable droplets, and reinforcing the emulsion structure during storage (Nasrabadi & Ghosh, 2024).

The flow curve measurements of NRAE and CRAE provide insights into their rheological behavior and allow for a qualitative evaluation of their texture based on their flow characteristics. At day 0, the NRAE and CRAE flow curves demonstrated a Newtonian plateau over a decade (viscosity linear domain), followed by a decrease in viscosity over another decade (Fig. 6D). While both emulsions exhibited similar viscosities within their respective linearity domains, their rheological behaviors were distinguished. Notably, the viscosity linearity range varied between the emulsions, with the end of the linear regime differing

<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Characterization of RA and CRA mayonnaise-type emulsions: macroscopic observations after 30 days of storage (A) of RA (left) and CRA (right) mayonnaise-type emulsions and microscopic observations (x800) of the droplets at initial time (B), of RA (left) and CRA (right) mayonnaise-type emulsions, particle size distributions (C), and flow curves (D) of RA and CRA mayonnaise-type emulsions at initial time (0th day) and after 30 days (30th day). The emulsions were kept under 6 °C during the 30 days of storage.</figcaption></figure>
greatly. CRAE exhibited a gradual decrease in viscosity beyond the plateau, while NRAE displayed a sharper decrease. This disparity at the end of the Newtonian plateau indicates a divergence in flow behavior between NRAE and CRAE once yield stress has been reached. Consequently, NRAE is expected to exhibit a more uniform and abrupt onset of flow (marked by a sudden decrease in viscosity), whereas CRAE is anticipated to show a delayed flow response (characterized by a gradual viscosity decrease), revealing a thickness effect of the crosslinked products. The flow curves obtained after 30 days of storage demonstrated a similar behavior to those at day 0, with a slight shift in the overall profiles. These observations are corroborated by the macroscopic flow behavior of NRAE and CRAE emulsions after 30 days of storage (Supplementary material 1). This finding highlights the distinct rheological and morphological properties of both emulsions, as reflected by differences in their droplet size distributions and flow rate profiles.

Typically, RA are not recognized as effective emulsifiers, especially when compared to rapeseed globulins (Tan et al., 2014). However, the current results demonstrate that enzymatic crosslinking significantly enhances the emulsifying capacity of RA, in addition to conferring a thickening property to RA. This was reflected in the formation of smaller oil droplets, improved long-term emulsion stability, and thickened emulsion texture. Consequently, the application of MTGase-mediated crosslinking may offer a promising strategy to leverage RA as a functional ingredient in emulsion-type food preparations such as plant-based mayonnaise.

## 4. Conclusion

In this study, it was demonstrated that RA can be effectively crosslinked by microbial transglutaminase, resulting in significant changes in their physicochemical and functional properties. Through a novel SE-HPLC-based kinetic monitoring approach, the formation of crosslinked products, including dimers, trimers, and polydisperse aggregates, was meticulously analyzed. This method also facilitated the tracking of the kinetics of the residual amount of native RA, for process productivity quantification. The tracing of the kinetics indicated that the enzymatic reaction was not complete in the applied conditions, and that the final mixture consisted of 60 % native RA and 40 % crosslinking products (including 20 % RA dimers, 6 % RA trimers, and 14 % RA polydisperse products). This suggests that the influence of operating conditions on reaction kinetics should be further studied. The structural changes were associated with a slight decrease in denaturation temperature and an increase in surface hydrophobicity, confirming the modification of the RA conformation.

Functionally, CRA exhibited highly remarkable gelling properties, forming stable gels at protein concentrations as low as 3 %, in stark contrast to native RA. Furthermore, CRA significantly improved emulsion stability in a mayonnaise-type model, reducing droplet size, enhancing long-term physical stability, and thickening emulsion texture. These functional improvements can be attributed to the modified molecular structure and surface properties induced by enzymatic crosslinking.

This study hence demonstrated the potential of transglutaminase crosslinking as a targeted and food-compatible method to valorize RA proteins as a functional texturizing ingredient in plant-based emulsions and gels. Future research should focus on identifying the specific crosslinked species responsible for functional enhancements, to favor their formation, as well as the optimization of the process conditions for industrial applications.

### CRediT authorship contribution statement

**Romain Faure:** Writing – review & editing, Writing – original draft, Validation, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.
**Romain Kapel:** Writing – review & editing, Visualization, Validation, Supervision, Resources, Project

administration, Methodology, Investigation, Funding acquisition, Conceptualization.
**Philippe Marchal:** Writing – review & editing, Visualization, Validation, Resources, Methodology, Formal analysis, Data curation.
**Ghislain Génin:** Writing – review & editing, Visualization, Software, Methodology, Formal analysis, Data curation.
**Sophie Beaubier:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

### Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors used ChatGPT/OpenAI in order to rephrase in the writing process. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

### Funding

This work was funded by the French National Research Agency (ANR) as part of the JCJC project "ANR-23-CE51-0003-01".

### Declaration of Competing interest

None.

### Acknowledgments

We thank Alexander Kriznik from the Biophysics and Structural Biology Core Facility of UMS2008 IBSLor (Université de Lorraine-CNRS-INSERM – http://umsibslor.univ-lorraine.fr) for its help in physicochemical properties measurements. We also thank Philippe Arnoux and Maud Lebrun from SAMPL – LRGP (Université de Lorraine-CNRS – https://lrgp-nancy.cnrs.fr/), for the formations of specific apparatus.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodhyd.2025.112100.

### Data availability

Data will be made available on request.

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