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. 2020 Jun 24;58(2):632–640. doi: 10.1007/s13197-020-04577-1

Effect of succinylation on the secondary structures, surface, and thermal properties of date palm pollen protein concentrate

Haifa Sebii 1,✉, Sirine Karra 1, Brahim Bchir 1, Zeineb Nhouchi 3, Abir Mokni Ghribi 1, Romdhane Karoui 3, Christophe Blecker 2, Souhail Besbes 1
PMCID: PMC7847835  PMID: 33568857

Abstract

The present study was attempted to investigate the effect of succinylation, as chemical modification, on the functionality of male date palm pollen protein concentrate (MDPPPC). Succinylation was applied at two levels, 4 and 8 mol of succinic acid per mole of lysine. 4 M and 8 M were compared to the native MDPPPC. Findings proved that succinylation improved the surface properties of pollen protein including solubility and surfactant activity. Increased solubility of succinylated MDPPPC has been noticed especially in pH superior to pHi. The results from the differential scanning calorimetry showed a significant decrease (P < 0.05) of the denaturation temperature and the heat enthalpy for succinylated MDPPPC. β turn of succinylated MDPPPC increased significantly (P < 0.05) at the expense of β sheet indicating that the protein gained more mobility after succinylation which explains the enhancement of the functional properties and promotes the use of succinylated protein as a techno-functional ingredient.

Keywords: Succinylation, Protein concentrate, Secondary structures, Surface properties, Thermal properties

Introduction

Succinylation is one of the well-known chemical modification that has been found to influence food protein’s functionality. The succinylation of proteins involves the substitution of the free amino group, hydroxyl, or sulfhydryl groups of proteins with the succinylating agent (Kulchaiyawat et al. 2016) which replaces the positive charge with a negative charge inducing a significant modification in protein’s structure and affecting their functional properties (Shilpashree et al. 2015a).

This modification has been applied to many food proteins and all workers agreed that succinylation procedure improves solubility and some other functional properties such as foaming and emulsifying. As an example, Shilpashree et al. (2015a) reported that succinylation of milk proteins concentrate produced pronounced changes in its functional properties and solubility has been improved following succinylation which in turn enhances water-binding, emulsifying and foaming properties of milk protein concentrate. Likewise, succinylated oat protein isolate showed an increased solubility, foaming capacity and emulsifying activity (Mirmoghtadaie et al. 2009). Studies have attributed the cited improvements to structural changes of protein’s secondary and tertiary structure since succinic anhydride generates electrostatic and steric alteration of the protein (Shilpashree et al. 2015a; Achouri and Zhang 2001).

The application of succinylation to modify the structure of the protein is gaining a large interest in both food and chemistry fields. However, there is currently no published research on succinylating male date palm pollen protein concentrates (MDPPPC). Therefore, this study aimed to investigate the effect of succinylation on functional, interfacial and thermal properties of MDPPPC and to discover the relationship between structural modifications induced by succinylation and the observed changes in the studied properties.

Materials and methods

Chemical reagents

2-Octen-1-yl succinic anhydride acid and 2,4,6-trinitrobenzenesulfonic acid (TNBS) was purchased from SIGMA-ALDRICH, Co, st louis, MO, USA. All other chemicals used were of reagent grade.

Preparation of pollen protein concentrate

Date palm pollen (DPP) was mixed with distilled water at a 1:10 (w/v) ratio. pH was adjusted to 12 using 1 M NaOH. The mixture was magnetically stirred for 2 h at 30 °C then centrifuged at 10,000 g at 4 °C for 30 min. The extraction procedure was repeated twice to enhance protein extraction. Supernatants were collected prior to further precipitation. Isoelectric precipitation was carried out by adjusting the pH of supernatant to 3 with 1 M HCl and keeping it at 4 °C overnight. Finally, the protein concentrate was recovered by centrifugation 10,000 g at 4 °C for 30 min. Pollen protein concentrate was neutralized, dialyzed 5 d against ultrapure water and finally lyophilized to obtain native male date palm pollen protein concentrate (MDPPPC) (Ghribi et al. 2015).

Succinylation procedure

The succinylation was achieved according to Shilpashree et al. (2015a). 50 mmol/L lysine dispersion was prepared by suspending the MDPPPC powder (6.48 g) in ultrapure water. The solution was magnetically stirred for 20 min at 37 °C and the pH was adjusted to 8 using 2 M NaOH. A known volume of octenyl succinic anhydride acid was added in order to get two levels of succinylation, 4 mol and 8 mol of succinic acid per mole of lysine. The mixture was magnetically stirred for 1 h at 30 °C and the pH was monitored during the reaction in order to be maintained at pH 8. Succinylated protein was recovered by precipitation at pH 3 (the isoelectric point of MDPPPC) with 1 M HCl. Four washes were carried out with ultrapure water to remove the remaining reagent. Then, the precipitate was neutralized and lyophilized to obtain two MDPPPC4M and MDPPPC8M (Fig. 1a).

Fig. 1.

Fig. 1

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a: Succinylation procedure b: Succinylation mechanism: Reaction of succinic acid anhydride with the amino group of lysine

Determination of succinylation degree

The method of trinitrobenzene sulfonic acid (TNBS) was applied to determine the number of modified amino groups (Wan et al. 2018). According to Shilpashree et al. (2015b), succinylation degree could be calculated as follows:

Succinylation degree = ((A–B) /A) × 100

A: µmol free amino-group/mg native protein.

B: µmol free amino-group/mg succcinylated protein.

Protein content

Protein content was determined using a Dumas Elementar Rapid N cube 161 15,054 (Donaustrasse, Germany) (Bchir et al. 2014). Total protein was calculated using a nitrogen conversion factor of 6.25 (Zia-Ul-Haq et al. 2007).

Nitrogen solubility index (NSI)

The solubility of nitrogen was studied in the range of pH 2–12. A 1 g/100 mL protein dispersion from samples was prepared, the pH was adjusted with 1 M NaOH or 1 M HCl. Mixtures were magnetically stirred for 1 h and finally centrifuged 10 min at 4 °C at 10,000 g. The Nitrogen content of the supernatant was determined with the Dumas method (Bchir et al. 2014). BESBES et al. (2002) reported that NSI might be calculated using the following formula:

NSI = (N in the supernatant/N in the initial dispersion) × 100

Zeta potential

30 mL of 0.1 g/100 mL protein dispersion was prepared prior to the measurement of the surface charge in the range of pH 2–12 using a Delsa Nano C Instrument (Malvern Instruments, Westborough, MA). The isoelectric point corresponds to the pH where the surface charge is null. Zeta potential was studied in a pH range 2–12. An automated titrator, linked to the Delsa Nano modulus, was used to adjust the pH of the protein dispersion using 1 M NaOH or 1 M HCl. The curve was obtained using the means of values of three replicates.

Surface tension property

Drop creation method

The surface tension was measured using an automated drop volume tensiometer TVT1 (Lauda, Germany). A 1 g/100 mL protein dispersions were used. Measurements were in dynamic mode with a syringe volume 2.5 mL, a drop creation time from 0.07 to 0.8 s/µL and at 25 ± 0.5 °C. The lifetime of the drops was measured as a function of their volume, which made it possible to calculate the surface tension (Blecker et al. 2002).

Bubble pressure method

A bubble pressure tensiometer (BP 100, Kruss GmbH, Germany) was used to determine the dynamic surface tension (mN/m) as a function of surface age (ms). The surface age is the period of time from the beginning of the production of a surface or interface to the time of the observation or measurement. A capillary was immersed in 1 g/100 mL protein dispersion to 10 mm immersion depth. An inert gas was bubbled through the capillary. Before each experiment, the capillary is rinsed with water and absolute ethanol. All analysis were done at 25 °C. A first measurement was carried out to determine the diameter of the capillary.

Thermal properties

Differential scanning calorimetry analysis

The heat flow was recorded during heating from − 50 to 250 °C at a scan rate of 5 °C/min using a Thermal Analysis Instruments Q1000 DSC (TA DSC Q1000, New Castle, DE, USA). The sample was placed in hermetic aluminum-pans and an empty one having an equal mass of 0.1 mg was used as a reference. The temperature was calibrated with two standards Indium (Tonset: 156.6 °C, DH: 28.7 J/g) and Eicosane (Tonset: 36.8 °C, DH: 247.4 J/g). Specific heat capacity (Cp) was calibrated using a Sapphire. The cell was purged with Nitrogen 50 mL/min. The analyzed sample mass was about 2 mg and the experiment was carried out in triplicate.

Thermogravimetric analysis

The thermogravimetric analyzer (Mettler Toledo DSC/TGA 1star system) was used to measure weight change during heating the sample from 25 to 250 °C with a step rate of 5 °C/min. Powders (10 mg) were placed in a ceramic pan. The Nitrogen gas flow rate was kept constant at 35 mL/min. The experiments were performed in triplicate to test the repeatability of the device. Data was collected automatically to get the weight loss rate curve.

Fourier-transform infrared (FT-IR) spectroscopy and protein secondary structure

The FT-IR measurements were performed to investigate the structural differences between native and succinylated MDPPPC. FT-IR spectrum was recorded using a Fourier transform spectrometer IRTracer-100 (Shimadzu, Duisburg, Germany) fixed with an attenuated total reflection (ATR) accessory equipped with a grip (Pike Technologies, Inc. Madison, United States). The ATR cell was made of a horizontal ZnSe crystal characterized by an incidence angle of 45° and a total reflection (n = 20). A 5 g/100 mL protein dispersion of each sample was prepared prior to the experiment. For each spectrum, 32 scans at a resolution of 4 cm−1 were obtained between 4000 and 700 cm−1. Before each measurement, the spectrum of the ZnSe crystal was recorded and used as a background spectrum. Three replicate measurements were taken (Nhouchi and Karoui 2018).

The content of each secondary structures was calculated after deconvolution and a second derivative of the spectrum. Amide I band (1600–1700 cm−1) and Amide III (1200–1300 cm−1) bands were selected. The secondary structures were assigned to wavelengths as shown in Table 3 (Kong and Yu 2007).

Statistical analysis

All measurement were performed in triplicate and the obtained results were expressed as mean values ± standard deviation. The statistical analyses were conducted using a statistical software program (SPSS for Windows version 11.0). The data were subjected to the analysis of variance using the general linear model to determine significant differences between the samples (P < 0.05).

Results and discussion

Protein content and succinylation degree

The effect of succinylation on the protein content of MDPPPC was shown in Fig. 2. From results, it was obvious that protein content decreased significantly when increasing the succinylation level reaching 60% and 46% for MDPPPC4M and MDPPPC8M, respectively, after being 70% for native MDPPPC. This could be attributed to the fact that succinylated protein became more soluble in the isoelectric point (pH 3) used for the precipitation of protein which leads to less protein recovery at the end of the succinylation procedure (Shilpashree et al. 2015b).

Fig. 2.

Fig. 2

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Protein content and succinylation degree of native and succinylated MDPPPC

Besides, Shilpashree et al. (2015a) found that maximum succinylation degree could be attained using 4 mol succinic acid per mole of lysine. In this study, doubling the molarity of succinic acid from 4 to 8 mol per mole of lysine has almost doubled the succinylation level reaching 24 and 53% for 4 M and 8 M MDPPPC, respectively, which suggests the existence of more free amino group available for succinylation.

Nitrogen solubility index and Zeta potential

Solubility is a mandatory property that affects further uses of proteins. This property is governed by the surface charge of protein which produces whether protein–protein interaction leading to aggregation and precipitation or protein-water interaction promoting solubility.

Succinylation can improve the functional properties of proteins including solubility and surface activity. In the matter of fact, succinylation induces great changes in protein conformation as well as in its surface charge. It replaces attractive forces by repulsive forces preventing protein aggregate formation and enhancing solubility (Shilpashree et al. 2015a).

The solubility of native and succinylated MDPPPC as a function of pH was presented in Fig. 3a. The obtained values showed that the effect of succinylation on solubility was different in each pH. The observed effects were consistent with zeta potential analysis shown in Fig. 3b. In fact, for pH from pH 2 to 5, the native MDPPPC was more soluble than succinylated MDPPPC contrarily to pH range between 5 and 12 where succinylated MDPPPC were much more soluble than native MDPPPC. Figure 3a also shows that MDPPPC8M was the least soluble up to pH 5 followed by MDPPPC4M. This observation was the inverse for pH up to 12, where MDPPPC8M was the most soluble followed by MDPPPC4M. This could be explained by the balance between repulsive and attractive forces generated by charge modification due to succinylation. Similar observations have been also reported for milk protein concentrate where higher succinylation degree leads to better solubility values and this has been explained by the existence of repulsive forces leading to the unfolding of proteins and increasing solubility (Shilpashree et al. 2015a).

Fig. 4.

Fig. 4

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Surface tension of native and succinylated MDPPPC a: Surface tension using the drop creation method; b: Surface tension using the bubble pressure method

Fig. 3.

Fig. 3

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Nitrogen solubility index (NSI) and Zeta potential of native and succinylated MDPPPC a: Nitrogen solubility index (NSI); b: Zeta potential

The Zeta potential analysis, presented in Fig. 3b, showed that the succinylated MDPPPC had more electronegative charge than the native MDPPPC. The MDPPPC8M exhibited the most electronegative charge whatever the pH when compared to MDPPPC4M. Then, the pHi of succinylated MDPPPC shifted to lower values. It becames the least for MDPPPC8M followed by MDPPPC4M and the highest pHi value was recorded for native MDPPPC. Such results proved that succinylated MDPPPC exhibited more repulsion, especially for MDPPPC8M having more linked succinic acid on the amino groups, which is the main reason for reduced solubility compared to native protein. However, in pH values superior to pHi, higher solubility was recorded for succinylated MDPPPC, the electronegativity in this pH range created intermolecular repulsion increasing protein-water interactions.

From studied properties, we may deduce that increasing the succinylation degree from 24 to 53% increases the electronegativity which is the main reason for the improved solubility especially for pH values superior to 5.

Surface properties

The curves of the surface tension of the native and succinylated MDPPPC using the drop creation method were presented in Fig. 3c. As can be shown, all samples exhibited the same shape of the curve which might be divided into two phases. The first one was a sharp decrease of the surface tension from 0 to 10 s, then, an equilibrium phase reaching 45.9; 43.5 and 41.9 mN/m for native, 4 M and 8 M succinylated MDPPPC, respectively. This significant difference (P < 0.05) was mainly attributed to the submitted modification in protein’s structure. Reported values were lower than those reported for chickpea protein concentrate which were around 52 mN/m (Ghribi et al. 2015), relatively higher than those of modified soy protein using 8 mol/L urea solution with Na2SO3 and 8 mol/L urea solution only equal to 35 and 38 mN/m, respectively, and similar to the obtained surface tension for native, modified with 4 mol/L urea solution and heat treated soy protein atteigning 45, 41 and 43 mN/m, respectively (Nir et al. 1994). Furthermore, many studies proved that succinylation improves foaming and emulsifying properties which could be strongly related to the capacity of the protein to reduce the surface tension. As an example, Shilpashree et al. (2015a) reported that the increase of the emulsifying capacity of succinylated milk protein could strongly be attributed to the increased solubility and the structural flexibility of the protein after succinylation procedure which contribute to facilitate the diffusion of the protein to the interface and help to stabilize systems. Hence and from cited examples, we may deduce that the denaturation of the protein caused whether by the thermal treatment or using chemical reagents (Urea, Na2SO3, succinic acid) is a favorable modification that leads to a better surfactant activity.

The dynamic surface tension measured by bubble pressure or maximum bubble pressure technique reflects the presence of surface active species that can migrate and adsorb to the surface of the newly formed bubble. It’s the only way that allows measurements of dynamic surface tensions of surfactant solutions in the short time range down to milliseconds and even below (Fainerman and Miller 2004). Fainerman and Miller (2004) reported that the surface tension γ can be calculated from the measured maximum capillary pressure P and the radius of the capillary r using the Laplace equation:

γ=f(r.P/2)

In our case: The radius of the capillary r, determined in the first measurement, was equal to 0.229 mm. The correction factory f, needed for capillaries with the radius r > 0.1 mm, was equal to 0.45.

The obtained data from bubble pressure tensiometer was presented in Fig. 3d. From the result, it was clear that succinylation affects the surface tension of the pollen protein concentrate. During the injection of the gas, a consecutive bubbling procedure took place at the tip of the capillary and the surface age increased leading to the migration of surfactant molecules to the surface of the bubble which reduces the surface tension. Increasing the succinylation degree improved the ability of MDPPPC to reduce the surface tension. In fact, at a surface age of 10,000 ms, MDPPPC8M was the most surface active since it reduced the surface tension to 46 mN/m, followed by MDPPPC4M reducing the surface tension to 48 mN/m and the native protein was the least surface active reaching only 50 mN/m. This result suggested that although the reduced protein content of the succinylated MDPPPC, proteins were more flexible and capable to reach the interface at milliseconds scale, leading to the rapid reduction of the interfacial tension. Findings from surface tension measurements using the two previous methods showed that succinylation contributes to improving the interfacial properties of MDPPPC leading to lowing the surface tension with increased succinylation degree.

Thermal properties

DSC was carried out to discuss the thermal stability of protein on the basis of their peak temperature (endothermic or exothermic). This thermal analysis gave also information about protein denaturation and aggregation through the heat enthalpy value (Pugliese et al. 2016).

The thermodynamic properties of native and succinylated MDPPPC were presented in Table 1.A. The peak temperature value shifted significantly (P < 0.5) from 103.2 °C for the native MDPPPC to 85.8 °C and 83.3 °C for 4 M and 8 M succinylated MDPPPC, respectively. Besides, it should be noticed that the heat enthalpy dropped from 347.9 J/g for native MDPPPC to 248.3 J/g and 236.9 J/g for 4 M and 8 M succinylated MDPPPC, respectively. Similar decreases were observed for wheat gluten protein after chemical modifications (deamidation and succinylation) (Liu et al. 2018) and for ovalbumin with increased succinylation degree (Broersen et al. 2007). According to Liu et al. (2018), the observed decrease indicated a protein denaturation caused by succinylation. The latter might cause modifications in protein’s conformation by breaking bonds and creating new ones which could create a smaller protein aggregate less stable against heating treatment. Furthermore, the same study suggested that succinylated samples contain more hydrophilic amino acids than the native protein since hydrophobic amino acids induce high thermal stability. All findings reflected a partial denaturation of the MDPPPC leading to the formation of fewer protein aggregates contained in the succinylated MDPPPC which increased the mobility of the protein.

Table 1.

Thermal properties of native and succinylated pollen protein concentrates

Sample Onset (°C) Endset (°C) Peak (°C) Enthalpy (J/g)
A: The thermodynamic properties determined by differential scanning calorimetry (DSC)
Native 23.7 ± 0.2c 191.4 ± 1.6b 103.2 ± 0.4c 347.9 ± 0.9c
4 M 11.37 ± 0.9b 169.9 ± 1.2a 85.8 ± 0.6b 248.3 ± 1.7b
8 M 0.09 ± 0.8a 168.8 ± 1.6a 83.3 ± 0.3a 236.9 ± 0.2a
B: Weight loss values obtained by thermogravimetric analysis (TGA)
Sample Step 1 Step 2
T °C Weight loss (%) T °C Weight loss (%)
Native 0_160  − 9.04 ± 0.09c 160_250  − 36.83 ± 0.34b
4 M 0_160  − 4.34 ± 0.02b 160_250  − 42.26 ± 0.08c
8 M 0_160  − 3.07 ± 0.01a 160_250  − 34.13 ± 0.23a
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All the data are expressed as mean ± SD and are the mean of three replicates

Values with different letters in the same column differ significantly (P < 0.05)

The denaturation process of proteins is an intramolecular change involving the destruction of internal order, and in some cases, the complete unfolding of peptide chains (Guerrero and De la Caba 2010). Anhydrides such as acetic anhydride (neutral) and succinic anhydride (negatively charged) interact with the positively charged N terminal amino group of the protein which leads to the change in its net charge and its denaturation (Yada 2017).

Usually, protein denaturation leads to the alteration of some functional properties including the loss of the solubility as a result of the denaturation at low pH (Yada 2017). However, several studies reported a positive effect of partial protein denaturation in food applications. For example, Kato et al. (1981) stated an improvement in protein’s functionality involving a great increase in surface hydrophobicity and a remarkable amelioration in foaming and emulsifying properties for heat denaturated lysozyme despite the small change in the conformation. In addition, succinylation has been found to improve the solubility, the water, and fat binding capacity, the foaming property of sesame protein (Zaghloul and Prakash 2002) and the emulsifying properties of arabinoxylan-protein gum (Xiang and Runge 2016). Similarly, the partial denaturation of MDPPPC, caused by the succinylation, had also induced an enhancement in functional properties such as solubility and surfactant activity, discussed in previous sections, which promote the use of succinylated MDPPPC as a technofunctional ingredient.

The thermogravimetric analysis (TGA) gives an indication about material’s changes under heating treatment. The water evaporation, the lost of crystalliferous water, the release of low molecular volatile matter, and the decomposition of material are the main event that could occur during TGA with the rise of temperature (Yu et al. 2015).

Table 1.B showed the weight loss as a function of temperature for native and succinylated MDPPPC. As can be seen from results, the weight loss was relatively small until 160 °C and became more important above this temperature with a noticed significant differences (P < 0.05) between samples. However, increasing succinylation degree provoked less weight loss for MDPPPC8M (37%) when compared with MDPPPC4M (46%). The succinylation had led to the formation of protein aggregates of different size having different thermal stability.

FT-IR and secondary structures

FT-IR is one of the oldest and reliable techniques that allows discovering polypeptides and proteins secondary structures. The secondary structures of a protein are the β sheet, α helix, Random coil, and β turn. These structures could be deduced from the correlation with the frequencies of amide I band. This latter, which occurs between 1600–1700 cm−1, is the most sensitive spectral region to the protein’s secondary structures (Hesso et al. 2015). The amide I band is mostly due to C = O stretching (80%) and N–H bending (20%) (Kong and Yu 2007). Although the amide III that occurs band between 1229–1301 cm−1, does not mainly correspond to one vibration mode like amide I, it can be also used to determine the secondary structures of protein (Nawrocka 2014).

As far as we know, there is no relevant information available about MDPPPC secondary structure. Then, in order to gain basic knowledge about the composition of the protein secondary structure, FT-IR was conducted firstly on native concentrate. Afterward, the effect of succinylation, as chemical modification, on protein secondary structures was depicted after comparing native protein with succinylated samples.

Results presented in Table 2 showed that, from the amide I band, the amounts of secondary structures for native PPC followed the order of 50% β sheet > 31% α helix > 11% Random coil > 7% β turn. For 4 M, the unordered structures (16%) and β turn (20%) increase significantly (P < 0.05) at the expense of β sheet (38%) and α helix (25%). Whereas, in the case of 8 M α helix (37%), Random coil (13%) and β turn (12%) increase significantly (P < 0.05) while β sheet (35%) declined. Equally from amide III, the same order of secondary structures was noticed for native MDPPPC: 35% β sheet > 28% α helix > 21% Random coil > 14% β turn. The same changes were recorded for succinylated 4 M and 8 M MDPPPC where random coils (22%) and β turn (22%) increased for 4 M and α helix (31%), Random coil (21%) and β turn (24%) increased for 8 M. Collected values also showed that increasing succinylation degree contributed to losing more of the β sheet structure.

Table 2.

Assignment of Amide I and amide III frequencies to protein’s secondary structures and the secondary structures of native and succinylated pollen protein concentrates

Secondary structure assignment Mean wavenumber (cm−1)
Amide I Amide III
β sheet 1622–1641 1220–1245
Random coil 1641–1651 1245–1270
α helix 1650–1660 1270–1295
β turn 1660–1680 1295–1330
β sheet (%) Random coil (%) β turn (%) α helix (%)
Amide I
 Native 50.00 ± 0.45c 11.20 ± 0.13a 7.51 ± 0.84a 31.29 ± 2.32b
 4 M 38.07 ± 0.05b 16.52 ± 0.01c 20.06 ± 0.29c 25.35 ± 0.10a
 8 M 35.73 ± 0.11a 13.73 ± 0.76b 12.89 ± 0.56b 37.64 ± 0.69c
Amide III
 Native 35.79 ± 0.30c 21.29 ± 0.06a 14.17 ± 0.30a 28.74 ± 0.13a
 4 M 26.44 ± 0.31b 22.38 ± 0.02b 22.56 ± 0.16b 28.63 ± 0.01a
 8 M 22.54 ± 0.05a 21.59 ± 0.08a 24.74 ± 0.29c 31.13 ± 0.49b
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All the data are expressed as mean ± SD and are the mean of three replicates

Values with different letters in the same column differ significantly (P < 0.05)

Several studies discussed the effect of succinylation on protein’s secondary structures. Indeed, Achouri and Zhang (2001) reported that succinylation of soy protein isolate caused a significant loss in the content of β sheet with an increase in the amounts of β turn and random coil with increasing modification’s degree. Furthermore, Liu et al. (2018) showed that after succinylation of wheat gluten, a significant increase in the β sheet and random coil contents with a decrease of β turn and no change in the α helix content. Hence, a variety of changes in secondary structure could be observed depending on the method as well as the used reagent during the modification procedure.

In this work, the presence of the succinic acid has certainly induced changes in protein’s structure. It had probably lead to the destruction of some secondary structures (β sheet) to form new aggregates. On one hand, the significant increase of β turn for succinylated DPPPC at the expense of β sheet indicated that the protein gained more mobility after succinylation. On the other hand, the succinylation procedure contributed to the reorganization of hydrogen bonding which resulted in a more flexible protein (Wang et al. 2017) enhancing other studied properties such as solubility and surface tension property.

Conclusion

In the present investigation, applying succinylation to MDPPPC had been found to be a successful method to improve protein’s functionality. Although the partial denaturation induced by succinylation, mainly determined with the thermodynamic properties, the study revealed an improved solubility, especially in acidic pH, for succinylated MDPPPC against the native protein.

Although reduced protein content of MDPPPC4M and MDPPPC8M compared with native protein, they exhibited a better capacity of reducing the surface tension reaching lower values compared to native protein. After modification, β turn content increased which made the protein more flexible and capable of rapidly reaching the interface and lowering the surface tension at the milliseconds scale. Hence, succinylated MDPPPC prepared by this method could replace native MDPPPC partially or completely in some food products in which the solubility and the interfacial properties are the main criteria. However, several additional studies are needed to demonstate the improvement of the emulsifying and the foaming properties of MDPPPC after the succinylation procedure.

Acknowledgment

This work was funded by the Ministry of Higher Education and Scientific Research–Tunisia and Wallonie Bruxelles International (Axis 2, project 9).

Compliance with ethical standards

Conflict of interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Footnotes

Publisher's Note

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