Utilizing dry heating Maillard reaction approach to modify the functional and structural properties of sunnhemp protein isolate-dextran conjugates

doi:10.21203/rs.3.rs-4149126/v1

Sunnhemp protein isolate (SHPI) was prepared by utilizing alkaline extraction acid precipitation method. SHPI-dextran conjugates (1:1 w/w ratio) were prepared by dry heating method with Maillard reaction at 60°C for 0, 1, 3, 5, 7, and 9 days of incubation at 79% relative humidity. The functional properties of SHPI-dextran conjugates like solubility, emulsifying, foaming, water and oil binding capacities, dispersibility, and gelation were improved as compared to pure SHPI. Increment in browning index values of SHPI-dextran conjugates were observed with increase in Maillard reaction time. Conjugated SHPI reduced the percentage of α-helix and increased the content of β-sheet, β-turn and random coils content. FTIR spectroscopy confirmed the formation of covalent bonds between SHPI and dextran via Maillard reaction. XRD analysis indicated both semicrystalline and amorphous structure of SHPI-dextran conjugates as the incubation time was increased from 0 to 9 days. Decreasing trend in the values of surface hydrophobicity values were found with increase in incubation time. Free and total sulfhydryl content of SHPI was increased after conjugation with dextran up to 5 days and thereafter decreased. Incubation time of five days at 60°C and 79% RH was optimized on the basis of improvement in functional characteristics and extent of Maillard reaction time. Overall, the present study showed that conjugation of sunnhemp protein isolate with dextran successfully improved the functional characteristics of SHPI.

Sunnhemp

Dextran

Conjugate

Maillard reaction

Protein

In recent years, interest in plant-based proteins is tremendously increasing due to rising population and busy schedule of people. Animal based proteins with increasing price and limited supply are also closely related to climatic changes, biodiversity loss, freshwater bodies depletion and other health hazards (Rauw et al., 2020). The demand for plant-based proteins is on the rise throughout the world. Therefore, it is imperative to search for sustainable and ecofriendly alternatives to animal proteins (Singh et al., 2021).

Sunnhemp (Crotalaria juncea L.) is widely cultivated tropical Asian plant of the legume family (Fabaceae). Sunnhemp plant can be grown in adverse conditions like droughts, alkalinity and salinity and is mainly used for fiber production and as a fodder crop (Rawat & Saini, 2023c). Besides this, sunnhemp seeds possess excellent nutritional value. Previous studies have demonstrated that sunnhemp seeds contains high level of protein, carbohydrate, and fair level of oil (Rawat & Saini, 2022). Moreover, its seeds are also used for traditional medicinal practices to cure variety of diseases (Jagtap et al., 2006). Therefore, these underutilized sunnhemp seeds are the promising source of plant-based protein for healthy diets and sustainability.

Plant derived proteins are impeded by their inferior techno-functional characteristics including solubility, dispersibility, water and oil binding capacity, emulsifying and foaming property, and gelation as compared to animal proteins (Nasrabadi et al., 2021). Therefore, an effective and safe modification technique is required for plant proteins in order to improve their above-mentioned characteristics and broaden the utilization. Several physical treatments like high pressure processing (Zheng et al., 2020), microwave (Yan et al., 2021), pulsed electric field (Cui et al., 2020), and ultrasound (Vanga et al., 2020) methods are recently used to improve the functional traits of plant-based proteins. Among them as a green technology, ultrasonication is a significant food modification technique. It has proven to be effective in improving the functional traits of plant-based proteins (Rawat & Saini, 2023b; Zheng et al., 2019; Hadidi et al., 2020). Ultrasound is defined as sound wave having frequency higher than upper audible limit of humans (> 20 KHz) but only high intensity and low frequency ultrasound (20 to 100 KHz, 10-1000 W/cm²) is used for food protein modifications (Flores-Jiménez et al., 2019). However, from industrial application point of view, food protein lacks stability during high temperature and many other processing conditions (enzyme or solvent treatments). Therefore, they must be converted into more stable form before incorporating them into heat-treated food products (de Oliveira et al., 2016). In recent years, Maillard reaction between protein and polysaccharides has gained popularity for modifying the functional and structural characteristics of plant-based proteins in comparison to the native protein by many researchers. Maillard reaction is a green method as it demands no chemical and occur naturally under controlled conditions of moisture, temperature, relative humidity, and pH. During the Maillard reaction, a covalent bond is formed between the amino group of the protein and the reducing group of the polysaccharide (de Oliveira et al., 2016). The heating of protein and polysaccharide mixture below denaturation temperature of proteins while controlling Maillard reaction conditions produces protein-carbohydrate conjugates. Various carbohydrate sources (mono, di, oligo, and polysaccharides) have been utilized for the conjugation of food proteins to improve functional, thermal, and antioxidant properties (Nooshkam et al., 2020). Out of them, polysaccharides are safe, non-toxic, biodegradable and hydrophilic in nature having high stability, stronger molecular steric hinderance against mono and disaccharides. Moreover, polysaccharides have more reactive groups and wider molecular weight range, contributing to their structure variability. These factors are advantageous for using polysaccharides for the protein conjugation (Li et al., 2021). For protein- polysaccharide conjugation, dextran is widely accepted because of its neutral nature and superior solubility, which avoids the formation of advanced Maillard reaction products in the initial time of reaction. Dextran is a neutral polysaccharide with D-glucose units linked by α (1→6) glucosidic bonds (Liu et al., 2021). The most commonly used methods for protein-carbohydrate conjugation are either dry heating or wet heating. Dry heating method is preferred over wet heating as it gives higher degree of glycation and better handling of Maillard reaction conditions (Chen et al., 2019a). Till date, no research has been carried out on the conjugation of SHPI (ultrasound pretreated) with dextran by Maillard reaction. Therefore, the present work is undertaken to prepare sunnhemp protein isolate-dextran conjugates by using dry heating method of Maillard reaction and to analyze the influence of incubation conditions on functional and structural characteristics of conjugates. The optimum Maillard reaction conditions for obtaining SHPI-dextran conjugates were identified on the basis of functional and structural attributes.

2.1. Materials and reagents

PAU 1591 variety of sunnhemp seeds were procured from Punjab Agricultural University (PAU), Ludhiana, Punjab, India. Dextran (molecular weight of 40 KDa), bovine serum albumin and O-phthalaldehyde, β-mercaptoethanol and borax were purchased from Hi-Media Laboratories Pvt. Ltd., Mumbai, India. 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) was purchased from Sigma (St. Louis, MO, USA). ANS (8-Anilino-1-naphthalenesulfonate) was purchased from Tokyo Chemical Industry (India) Pvt. Ltd. All the chemicals and solvents used throughout the present experiments were of analytical grade.

2.2. Methods

2.2.1. Preparation of SHPI

Sunnhemp protein isolate was extracted from sunnhemp flour using the alkaline solubilization acid precipitation method (Rawat & Saini, 2023a). 100 g of sunnhemp flour was dispersed in double-distilled water in the ratio of 1:10 (w/v) and stirred at 1200 rpm for 25 min in a magnetic stirrer. pH adjustment (9.5) of the dispersion was done by using 1 N HCl/ NaOH. Then, the dispersion was transferred to a continuously agitating water bath (45°C) for 2 h. Thereafter, the dispersion was centrifuged at 8000 rpm for 30 min at 5°C. After collection of the supernatant, the pH of the supernatant was adjusted to 4.5 for protein precipitation by using 1 N HCl or NaOH. The precipitated solution was left undisturbed overnight for complete precipitation of proteins followed by re-centrifugation (8000 rpm) at 4°C for 20 min. The precipitated protein was collected form the centrifuge tubes and resuspended in distilled water for pH adjustment back to 7.0 using 1 N NaOH. Then, protein solution was subjected to freeze drying and freeze dried sunnhemp protein isolate sample was collected. The obtained sunnhemp protein isolate contained 91.58 ± 0.24% protein content, which was calculated by the Folin Lowry method. BSA (Bovine serum albumin) was used as a reference protein standard. Further, the sunnhemp protein isolate was stored in airtight plastic container for further characterization.

2.2.2. Ultrasound pretreatment of SHPI

SHPI was dissolved in double distilled water (1: 20 w/v) in a beaker and pH was accurately set to 7.0 by using 1 mol/L HCl or NaOH. After that, the dispersion was magnetically stirred at 800 rpm for 20 min in a magnetic stirrer and stored overnight (5°C) for complete hydration of proteins. Next day, the protein solution was subjected to ultrasound treatment in an ultrasound processor (Snapgen, USA). The ultrasound amplitude was set at 50% for 20 min having fixed power and frequency of 500 W and 20 KHz, respectively and pulse ratio of 15 sec (on/off). During ultrasound treatment, the beaker was covered with crushed ice cubes to prevent temperature rise above 25°C. The ultrasound treated sunnhemp protein isolate was further freeze dried and stored in refrigerator. The ultrasound pretreated sunnhemp protein isolate was designated as control (no conjugation).

2.2.3. Preparation of SHPI-dextran conjugates

Sunnhemp protein isolate-dextran conjugates were prepared through dry-heating method. Briefly, SHPI and dextran were dispersed in 1:1 ratio in double distilled water (50 mL) and adjustment of pH to 7.0 was done by using 1 mol/L HCl or NaOH. Thereafter, the mixture solution was magnetically stirred at 1200 rpm for 4 h. After hydration for overnight at 4°C, the mixture solution was lyophilized followed by gentle grounding into fine powder to acquire SHPI-dextran mixture. For obtaining SHPI-dextran conjugates, the small glass petri-plates containing sample mixture was transferred into a desiccator having pre-maintained relative humidity of 79% by using saturated KBr solution and incubation temperature of 60°C. SHPI and SHPI-dextran conjugate samples were withdrawn on the 0th ,1st, 3rd, 5th ,7th and 9th days of incubation followed by lyophilization and immediately placed in a refrigerator at 4°C till characterization. The prepared SHPI-dextran conjugates for 0, 1, 3, 5, 7 and 9 days were referred as SD0, SD1, SD3, SD5, SD7 and SD9, respectively. Ultrasound treated SHPI served as control without conjugation.

2.3. Confirmation of SHPI-dextran conjugates formation

2.3.1. Browning index (BI)

BI of SHPI and conjugated SHPI samples were calculated according to the methodology followed by Chen et al. (2019a), which is used as an indicator of Maillard reaction. Briefly, SHPI-dextran conjugates (5 mg/mL) were dissolved in 0.1% w/v sodium dodecyl sulfate (SDS). The absorbance of the conjugate samples was recorded at 420 nm with the help of UV-vis-spectrophotometer.

2.3.2. Degree of glycation (DG) by OPA test

The degree of glycation of SHPI and conjugated SHPI samples were determined by following the O-phthalaldehyde (OPA) test (Yang et al., 2022). For the preparation of OPA reagent, 1 mL of OPA solution prepared in methanol (40 mg/ mL), 25 mL of borax solution (0.1 mol/L), 2.5 mL SDS solution (0.1%, w/w), and 100 µL of β-mercaptoethanol (0.4%, v/v) was diluted to 50 mL with distilled water. OPA reagent (4 mL) was added in 2 mL of protein conjugates solution (mg/ mL) prepared in sodium phosphate buffer (pH 7.0). Further incubation of conjugates solution was done in a water bath (35°C) for 20 min. The absorbance of all protein solutions was recorded in a spectrophotometer at 340 nm. For control, OPA reagent (4 mL) was mixed in distilled water (0.2 mL). A calibration curve was plotted using leucine as a reference standard. Finally, for the calculation of degree of glycation following equation was used:

$$DG \left(\%\right)=\frac{{A}_{0}-{A}_{t}}{{A}_{0}}\times 100$$

1

where,

A ₀ = Absorbance of protein sample (nm) and,

At = Absorbance of conjugated protein (nm)

2.4. Functional properties of SHPI

2.4.1. Solubility

Solubility measurement of SHPI and SHPI-dextran conjugates was done according to the previously published methodology (Chen et al., 2019a). 0.1 g of protein sample was dissolved in 20 mL of sodium phosphate buffer solution (pH 7.0, 0.1 M). The pH of the dispersion was adjusted to 7.0 by using NaOH or HCl (1 N). The protein suspensions were centrifuged at 6000 rpm for 20 min at 20°C. Folin-Lowry method was used to determine the protein concentration in the collected solution with a standard curve of BSA (Bovine serum albumin). The solubility of sunnhemp protein samples were calculated as the protein percentage in the supernatant to the protein percentage in the native SHPI.

2.4.2. Emulsifying properties (Emulsifying activity and Emulsion stability)

The emulsifying properties of all SHPI and SHPI-dextran conjugates were calculated according to the protocol described by Anzani et al. (2020). 100 mg of SHPI and SHPI-dextran conjugates powder were dispersed in distilled water (10 mL) and magnetically stirred for 10 min at 800 rpm. The solutions were mixed with 10 mL of soyabean oil and magnetically stirred for 2 min at 800 rpm. Thereafter, the protein samples were centrifuged at 3000 rpm for 5 min. The height of emulsified layer and that of total content in the centrifuge tubes were recorded. The percentage of emulsifying activity (EA) was calculated from the following formula:

$$EA \left(\%\right)=\frac{Height of emulsified layer in the tube}{Height of total content in the tube}\times 100$$

2

Emulsion stability (ES) of all the samples were determined by heating the emulsion contained in marked centrifuge tube in a water bath at 80°C for 30 min. After centrifugation at 3000 rpm for 5 min, emulsion layer was measured and ES was measured as follows:

$$ES=100-\left(\frac{R}{{R}_{I}}\times 100\right)$$

3

where, R = emulsified layer volume that was heated at 80°C for 30 min,

R _I = initial emulsified layer volume

2.4.3. Foaming properties

Foaming capacity (FC) and foam stability (FS) of SHPI and SHPI-dextran conjugates were calculated by the previously reported protocol (Zielińska et al., 2018). 0.1 g of protein sample was dispersed in 10 mL of distilled water and stirred for 30 min at 800 rpm in a magnetic stirrer. The solution was then transferred in a centrifuge tube (50 mL) and centrifuged at 8000 rpm for 2 min. The foam volume after 2 min (V₂) and 30 min (V₃₀) was recorded and foaming capacity and foam stability were calculated as follows:

$$FC \left(\%\right)=\frac{{V}_{2}}{10}\times 100$$

4

$$FS \left(\%\right)=\frac{{V}_{30}}{{V}_{2}}\times 100$$

5

2.4.4. Water and oil binding capacity

Water and oil binding capacity (WBC and OBC) of SHPI and SHPI-dextran conjugates were measured by the methodology of Shen et al. (2021). 0.5 g of protein sample was dispersed in 10 mL of distilled water or soyabean oil. The sample solution was then stirred for 5 min in a magnetic stirrer at 800 rpm. After allowing to stand for 30 min, the samples were centrifuged for 30 min at 4500 rpm. The remaining supernatant was discarded and centrifuge tubes were inverted at 45° angle for 25 min for removing excess water/oil.

The WBC/ OBC was calculated from the following formulas:

WBC/ OBC= $\frac{a-b}{c}$(6)

where, a = mass of centrifuge tube + SHPI + absorbed H₂O /soyabean oil (g)

b = mass of centrifuge tube + mass of SHPI (g)

c = mass of SHPI (g)

2.4.5. Dispersibility (D_s)

D_s profiles of SHPI and SHPI-dextran conjugates were determined by adopting the methodology of Rawat and Saini (2023b). Protein dispersion were dissolved in double distilled water (1 g/ 10 mL) and adjustment of pH was done to 7.0 by using HCl or NaOH (1 N). The dispersed solution was quickly magnetically stirred for 5 min at 1200 rpm followed by settlement of dispersion solution for 120 min. Thereafter, the volume of settled particles were recorded and the dispersibility of protein samples was determined from the following formula:

Dispersibility= $\frac{Total volume-settled volume}{Total volume }\times 100 ($7)

2.4.6. Least gelation concentration (LGC)

LGC of SHPI and SHPI-dextran conjugates were evaluated by adopting the methodology of Shen et al. (2021). Protein suspension (5 mL) was prepared in a test tube at different concentration ranging from 2 to 20% (w/v). Then, the suspensions were transferred in water bath (100°C) for 60 min. The samples were rapidly cooled for 30 min at 4°C. LGC was noted when the protein sample would not fall, when the tubes were inverted.

2.5. Structural properties

2.5.1. Circular dichroism (CD)

CD spectrophotometer (Jasco-J-815, Jasco International Co. Ltd, Japan) was used for secondary structural characterization of all SHPI and SHPI-dextran conjugates samples. All samples (1.0 mg/ 10 mL) were prepared in sodium phosphate buffer (0.05 mol/L, pH 7.0) and were scanned between 190 to 260 nm of wavelength with 1 nm of bandwidth. BeStSel (ELTE Eotvos Lorand University, Budapest, Hungary), an online tool for protein secondary structures was used to calculate the percentage of all secondary structures of all samples.

2.5.2. Fourier transform infrared spectroscopy (FTIR)

FTIR spectra of SHPI and SHPI-dextran conjugates were investigated using an FTIR spectrometer (PerkinElmer Spectrum 400, USA). The sample power was uniformly spread in to the plate at ambient condition and the wavelength range used was 400 to 4000 cm^− 1.

2.5.3. X-ray diffraction (XRD)

XRD diffractograms of all SHPI and SHPI-dextran conjugates were obtained on the X-ray diffractometer (Shimadzu XRD-7000, Japan) with a scan speed of 2°C/min and scanning range between 2θ = 5 to 45°C.

2.5.4 Surface hydrophobicity (H₀)

H₀ values of SHPI and SHPI-dextran conjugates were determined by ANS (1,8-anilinonaphthalenesulfonate) method described by Jin et al. (2021). All protein solutions (1–4 mg/ mL) were dissolved in phosphate buffer (pH 7.0, 0.1 mol/L). In 4 mL of each protein solution, 20 µL of ANS (0.01 mol/L, pH 7.0) prepared in 8.0 nmol/L in phosphate buffer was added. The samples were then immediately vortexed for 5 min and placed in dark for 20 min. The relative fluorescence intensity of all the samples were measured at 390 nm of excitation wavelength and 468 nm of emission wavelength with the slit of 2.5 nm having scanning speed of 5 nm/s. H₀ was measured by plotting the graph between initial slope of relative fluorescence intensity and protein concentration (mg/mL).

2.5.5. Free and total sulfhydryl content (FSH & TSH)

The amount of free sulfhydryl content in SHPI and SHPI-dextran conjugates were calculated by using the previously reported protocol (Jin et al., 2021). Sunnhemp protein solution (2 mg/ mL) was made in standard buffer solution (4 mM Na₂EDTA, 0.09 M Glycine, 0.086 M Tris, pH 8.0). Then, the samples were incubated in a shaking water bath at 25°C for 20 min followed by centrifugation at 6000 rpm at 5°C for 20 min. From the collected supernatant, 3 mL of protein sample were taken out and 30 µL of Ellman’s reagent (4 mg DTNB/ mL in standard buffer) was added in it, quickly vortexed and placed in dark for 30 min at room temperature. The absorbance of the protein samples was taken with the help of a spectrophotometer at 412 nm. For blank, buffer solution (3 mL) and Ellman’s reagent (30 µL) was used. For the determination of total sulfhydryl content in all protein samples, same protocol as for free sulfhydryl content was followed except that instead of using only standard buffer solution, denatured buffer solution (standard buffer + 0.5% w/v SDS solution, 8 M Urea) was used. The free and total sulfhydryl content were calculated by using the following equation:

${\mu }\text{m}\text{o}\text{l} \text{o}\text{f} \text{S}\text{H}/ \text{g} \text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n}=73.53\times {A}_{412}$ /C (8)

where,

${A}_{412}$ = absorbance of protein samples at 412 nm,

C = sunnhemp protein concentration (mg/ mL).

2.8. Statistical analysis

All the analysis were done in triplicates and the results were presented as the mean ± standard deviation using IBM SPSS statistics software. Collected data were analyzed by using One-way ANOVA (Duncan’s Multiple Range Tests). All the graphs were acquired by using OriginPro 2017 software (Origin Lab Inc.).

3.1. Confirmation of SHPI-dextran conjugates formation

3.1.1. Browning index (BI)

The extent of Maillard reaction can be monitored by measuring the browning index. The effect of glycation conditions was observed by browning index of SHPI and SHPI-dextran conjugates (Fig. 1). The BI of SHPI-dextran conjugates was increased with glycation time ranging from SD0 to SD9 samples. In the first 5 days of the Maillard reaction in samples from SD0 to SD5, the browning index was rapidly increased along with the deepening of brown color, prompting the formation of Amadori compounds (Liu et al., 2021). A slow increase in the browning index was observed as the incubation continued in SD5 to SD9 samples. When the incubation time increases, brown pigments are formed due to the polymerization in intermediate Maillard reaction products thereby increase in browning index values (Mshayisa & Van Wyk, 2021). Furthermore, during prolonged reaction time, brown color complexes were also produced called melanoidins, which may be responsible for increase in BI values. Therefore, it is crucial to optimize the conjugation time carefully (Tavasoli et al., 2022). From the results, it can be concluded that in the initial stage, conjugation was faster and gradually declined with prolonged incubation time.

3.1.2. Degree of glycation (DG)

In the Maillard reaction, a condensation reaction between an amino acid in the proteins and reducing end group of dextran initiates the covalent bond between SHPI and dextran. As a result, Schiff bases are formed, releasing one H₂O molecule and reorganization to advanced Maillard reaction products is initiated (Zhang and Wolf, 2019). DG was calculated from the reduction of available free amino groups in SHPI-dextran conjugates. A higher degree of glycation of SHPI-dextran conjugates was observed when the glycation time was increased quickly from 0 to 5 days and thereafter it was slowly increased as in SD7 and SD9 samples (Fig. 2). This may be due to the reason that further extension in incubation time may lead to fewer carbonyl groups and free amino groups availability (Chen et al., 2019a). Another possibility is because of an intermolecular reaction between proteins, which may result in aggregation of proteins during longer incubation times, potentially competing with the SHPI-dextran conjugation. During the last stage of Maillard reaction, melanoidins are formed (Xiao et al., 2021). The absorbance of protein-polysaccharide conjugates is evaluated to assess the extent of Maillard reaction at 420 nm. During later stages of Maillard reactions, accumulation of advanced end stage products (melanoidins) might be harmful to human beings. Several researchers have confirmed that early stage Maillard reaction are adequate to fabricate conjugated proteins with improved functional as well as biological characteristics. Consequently, better handling and control over the Maillard reaction conditions is required during the initial stages for food applications.

3.2. Functional properties

3.2.1. Solubility

One of the most important characteristics of proteins is their solubility, which effects their emulsifying and foaming characteristics (Dias et al., 2022). The results indicated that the solubility of SHPI-dextran conjugate samples was higher than the native SHPI without conjugation (Table 1). The reason was conjugation with dextran, which can increase the number of hydrophilic groups and enhance the steric stabilization of SHPI, thereby positive impact on solubility (Liu et al., 2021). In particular, the maximum solubility was observed in SD5 sample, thereafter solubility shows decreasing trend in SD7 and SD9 sample. Prolonged heating time promoted the development of advanced stage of Maillard reaction, which leads to lower solubility of proteins (Kutzli et al., 2020). Another reason for this is thermal denaturation of sunnhemp protein isolates resulting in a greater effect on total protein solubility than Maillard reaction under dry heating method for more than 5 days (Liu et al., 2021). Crosslinking (inter and intra molecular) between the dicarbonyl compounds (glyoxal, methylglyoxal, and 3-deoxyglucosone formed during the advanced stage of Maillard reaction also results in decrease in protein solubility (Nooshkam et al., 2020). These observations were in agreement with the results of oat protein conjugated with β-glucan (Zhong et al., 2019) and whey protein-gum acacia conjugates (Chen et al., 2019a) via Maillard reaction. Therefore, reaction process should be properly controlled for better results.

Table 1

Effect of incubation days on the functional properties of sunnhemp protein isolate-dextran conjugates prepared by dry-heating method of Maillard reaction.
Functional property	SHPI	SD0	SD1	SD3	SD5	SD7	SD9
Solubility (%)	86.35 ± 0.37^g	87.35 ± 0.52^f	89.60 ± 0.25^e	91.52 ± 0.12^d	94.66 ± 0.27^a	93.40 ± 0.10^b	92.73 ± 0.17^c
Emulsifying activity (%)	57.03 ± 0.22^g	58.86 ± 0.36^f	60.35 ± 0.28^e	62.46 ± 0.35^d	65.68 ± 0.24^a	64.42 ± 0.21^b	63.64 ± 0.11^c
Emulsion stability (%)	65.36 ± 0.36^g	66.31 ± 0.14^f	67.70 ± 0.23^e	68.77 ± 0.21^d	71.39 ± 0.19^a	70.40 ± 0.42^b	69.56 ± 0.31^c
Foaming capacity (%)	59.88 ± 0.17^g	65.55 ± 0.36^f	69.37 ± 0.27^e	72.36 ± 0.31^d	76.80 ± 0.23^a	75.31 ± 0.18^b	74.69 ± 0.17^c
Foam stability (%)	66.26 ± 0.42^g	72.31 ± 0.17^f	74.58 ± 0.27^e	76.71 ± 0.16^d	79.68 ± 0.18^a	78.44 ± 0.36^b	77.59 ± 0.17^c
WBC (g H₂O/g protein)	4.71 ± 0.03^g	6.54 ± 0.05^f	8.13 ± 0.08^e	10.22 ± 0.04^d	13.58 ± 0.02^a	12.81 ± 0.07^b	12.33 ± 0.06^c
OBC (g oil/ g protein)	5.16 ± 0.06^g	6.86 ± 0.10^f	7.37 ± 0.09^e	8.67 ± 0.04^d	10.22 ± 0.08^a	10.08 ± 0.07^b	9.56 ± 0.05^c
Dispersibility (%)	94.34 ± 0.11^g	95.66 ± 0.06^f	96.44 ± 0.09^e	97.29 ± 0.07^d	98.54 ± 0.10^a	96.88 ± 0.04^b	95.31 ± 0.06^c
LGC (%)	12 ± 1^a	10 ± 1^b	8 ± 1^c	8 ± 1^c	6 ± 1^d	7 ± 1^d	7 ± 1^d

Mean± SD (n=7). Within the column, means having different superscripts letters are significantly different (p<0.05). SHPI: sunnhemp protein isolate without conjugation; SD0: sunnhemp protein isolate-dextran mixture at 0 day; SD1: sunnhemp protein isolate-dextran conjugates for 1 day; SD3: sunnhemp protein isolate-dextran conjugates for 3 days; SD5: sunnhemp protein isolate-dextran conjugates for 5 days; SD7: sunnhemp protein isolate-dextran conjugates for 7 days; SD9: sunnhemp protein isolate-dextran conjugates for 9 days; WBC: water binding capacity; OBC: oil binding capacity; LGC: least gelation concentration.

3.2.2. Emulsifying activity (EA) and Emulsion stability (ES)

Generally, conjugation of proteins with polysaccharides improves their interfacial functionality (Feng et al., 2023). EA and ES are the two main emulsifying characteristics of protein-based emulsifiers. EA refers to the ability of a protein to absorb to an interface, while ES represents the ability of a protein to impart strength to the emulsion to resist structural changes (Cai et al., 2023). In comparison with native SHPI sample without conjugation, EA and ES of all SHPI-dextran conjugates were remarkably improved (Table. 1). This might be due to covalent linking of dextran with sunnhemp proteins, which results in the improvement in their emulsification characteristics due to steric stabilization and development of a macromolecular stabilizing layer around oil droplets. Additionally, glycated SHPI with dextran has improved EA and ES due to their greater solubility as in line with the results. Despite this, both EA and ES of SHPI-dextran conjugates (SD0-SD9) improved up to 5 days after Maillard reaction had progressed and began to decrease thereafter in SD7 and SD9 samples (Table 1). This is due to the decreasing trend found in solubility and H₀ results of the SHPI conjugate samples after prolonged incubation time. A longer incubation time might lead to protein aggregation, which lowers the emulsifying properties of SHPI-dextran conjugates (Li et al., 2019). It is therefore important to maintain balance between hydrophobic and hydrophilic properties to achieve superior emulsifying traits of protein-polysaccharide conjugates by using Maillard reaction (Setiowati et al., 2020). Chen et al. (2019a) also reported similar results in whey protein isolate-gum acacia conjugates prepared by dry heating method for 0, 1, 3, 5, 7 days of incubation period. Likewise, Li et al. (2019) also reported reduction in emulsifying properties of soy proteins conjugated with glucose using Maillard reaction when incubation time was further extended. Overall, it can be concluded that controlled Maillard reaction conditions in the present study was successful to prevent the formation of advanced Maillard reaction products.

3.2.3. Foaming capacity and foam stability

Generally, foam represents a dispersion medium that comprises a continuous water phase as well as a gaseous discontinuous phase (Fu et al., 2021). There is a strong correlation between foam formation and protein’s solubility and hydrophobicity. Foaming capacity (FC) of proteins represents their ability to produce foam under certain conditions like their concentration, temperature and pH. A protein’s foam stability (FS) refers to how long it can maintain its foam volume (Amagliani et al., 2021). The foaming properties of all SHPI-dextran conjugates were improved in comparison to native SHPI (Table 1). This is due to the Maillard reaction that has occurred between SHPI and dextran, leading to protein structure’s unfolding. SHPI becomes more soluble, when hydrophilic groups of dextran are attached to the protein molecules, facilitating protein molecules to move quickly towards the interface, resulting in foamy liquid film formation (Yu et al., 2020). Furthermore, the effectiveness of interfacial properties (foaming) is dependent upon protein solubility. The higher the solubility of proteins, better the foaming characteristics (Hamdani et al., 2018). The maximum improvement in foaming properties was found in SD5 sample (Maillard reaction for 5 days) and thereafter decreasing trend was observed as the Maillard reaction was extended. This might to due to longer incubation time, which have promoted thermal aggregation of SHPI, reduction in solubility and surface hydrophobicity and directly affects its foaming properties. Li et al. (2019) also prepared soy proteins and glucose conjugates via Maillard reaction and noticed enhancement in foaming properties against individual soy protein isolate. According to previous researchers, it is reported that the optimum Maillard reaction condition is not necessarily obtained at higher level with superior functional properties. Thus, it is crucial to minimize thermal denaturation and cross linking of proteins during Maillard reaction (Zhang et al., 2018).

3.2.4. Water and oil binding capacity

In food products, water and oil binding capacity (WBC, OBC) affects the amount of water and oil retained by the proteins and protein water/oil interactions. Furthermore, the water binding capacity affects protein solubility, emulsification and gelation properties (Saeidy et al., 2023). WBC of all SHPI-dextran conjugates were higher than the SHPI without conjugation (Table 1). This is possibly due to the conjugation between SHPI and dextran during incubation. The strong ability of dextran to bind to water molecules makes it a highly hydrophilic polysaccharide and therefore acts as a thickening agent, and higher WBC values. The water binding capacity is directly influenced with the biopolymer solubility, consistent with the results of solubility. The further reduction in WBC in SD7 and SD9 sample is due to protein aggregation occurred during prolonged conjugation time. All SHPI-dextran conjugates showed higher OBC as compared to the pure SHPI (Table 1). This is because of Maillard reaction conditions, which have altered and unfolded protein structure, as well as exposing more hydrophobic residues, thereby possess increased WBC of SHPI-dextran conjugates (Shen & Li, 2021). Prolonged Maillard reaction time in SD7 and SD9 samples shows further decrease in WBC, which is due to formation of protein aggregates during advanced stage of Maillard reaction. Therefore, it is necessary to optimize the Maillard reaction conditions. Overall, OBC of protein conjugates are also affected by protein type, protein-polysaccharide ratio, surface hydrophobicity, and net charge on protein.

3.2.5. Dispersibility

The dispersibility of SHPI shows improvement after conjugation with dextran as compared to SHPI alone (Table 1). Compared to SHPI alone, the incorporation of dextran has increased the number of hydrophilic groups, thereby enhancing dispersibility of SHPI-dextran conjugates (Chen et al., 2019b). However, maximum enhancement was noticed in SD5 sample and thereafter decreasing trend was observed as the incubation time was extended up to 9 days. Prolonged incubation time leads to formation of protein aggregates and inter or intra molecular cross-linking was formed between some amino acids and advanced Maillard reaction products, thereby decrease in dispersibility. In this case, dispersibility trend is also in line with the solubility results. SD5 is the optimized Maillard reaction prepared SHPI-dextran conjugate, as after five days of incubation, protein aggregation and cross-linking of protein occurred as verified by emulsifying and foamability results. Improvement in dispersibility was also reported in lycopene encapsulated whey protein isolate conjugated with xylo-oligosaccharide via Maillard reaction (Jia et al., 2020).

3.2.6. Least gelation concentration (LGC)

It is important to note that lower LGC indicates a better gelling ability of proteins. Overall, SHPI-dextran conjugates showed improved gelation properties, with lower least gelation concentration compared to the native SHPI without conjugation (Table 1). This is due to the addition of dextran, which is highly hydrophilic, improving gel thickening function of SHPI (Cheng et al., 2022). Also, unfolding of proteins during conjugation resulted in more stable gel network formed by more hydrophobic protein interactions, reducing the protein concentration for gel formation (Shen et al., 2022). However, lowest LGC was observed in SD5 sample and thereafter it was increased in SD7 and SD9 sample, respectively. Prolonged conjugation time creates an additional layer of static space between polysaccharide coating and conjugated protein molecules, which inhibited the interaction of proteins with hydrophobic groups. Similar results were also reported in pea protein isolate conjugated with guar gum via Maillard reaction (Shen & Li, 2021).

3.3. Structural properties

3.3.1. Circular dichroism

Circular dichroism (CD) spectroscopy is a sensitive technique, which can be used to determine the secondary structure of proteins, especially when working with water soluble proteins (Zhao et al., 2020). Alterations in the secondary structure of glycated proteins prepared by Maillard reaction can be analyzed by CD. The SHPI-dextran conjugate samples show reduction in α-helix content with a simultaneous increase in β-sheet proportions, β-turn, and random coil content (Table 2). Protein is composed of α-helix and β-sheet proportions within their polypeptide chain. But when the Maillard reaction occurs, proteins lose their internal structure, causing them to stretch and reduction in α-helix content while increase in β-sheet content, β-turn, and random coil content (Zhang et al., 2022). The structural changes by CD analysis are also confirmed with FTIR results. Many researchers have studied that glycation between protein and polysaccharide can affect the secondary structure of protein by both Maillard reaction and partial denaturation of proteins during incubation period (Yang et al., 2023). In the present research, occurrence of conjugation between SHPI and dextran was also verified with surface hydrophobicity and sulfhydryl groups results. Therefore, the results indicated that the structural changes in SHPI-dextran conjugates is due to the modifications of SHPI by dextran and partial denaturation of SHPI during incubation period.

Table 2

Secondary structure contents of SHPI and SHPI-dextran conjugates prepared by dry-heating method of Maillard reaction at different incubation days.
Sample	α helix (%)	β-sheet (%)	β-turn (%)	Random coils (%)
SHPI	34.12 ± 0.16^a	44.33 ± 0.12^g	13.33 ± 0.10^f	8.23 ± 0.08^g
SD0	32.33 ± 0.17^b	45.33 ± 0.14^f	14.36 ± 0.22^e	9.28 ± 0.06^f
SD1	29.39 ± 0.10^c	46.82 ± 0.28^e	15.49 ± 0.36^d	10.47 ± 0.11^e
SD3	26.72 ± 0.14^d	48.37 ± 0.15^d	16.42 ± 0.32^c	11.36 ± 0.09^d
SD5	25.54 ± 0.09^e	50.18 ± 0.25^c	17.20 ± 0.50^b	12.39 ± 0.05^c
SD7	24.80 ± 0.07^f	51.29 ± 0.45^b	18.02 ± 0.23^a	13.20 ± 0.22^b
SD9	24.41 ± 0.29^g	52.13 ± 0.15^a	18.20 ± 0.21^a	13.72 ± 0.15^a
Mean ± SD (n = 7). Within the column, means having different superscripts letters are significantly different (p < 0.05). SHPI: sunnhemp protein isolate without conjugation; SD0: sunnhemp protein isolate-dextran mixture at 0 day; SD1: sunnhemp protein isolate-dextran conjugates for 1 day; SD3: sunnhemp protein isolate-dextran conjugates for 3 days; SD5: sunnhemp protein isolate-dextran conjugates for 5 days; SD7: sunnhemp protein isolate-dextran conjugates for 7 days; SD9: sunnhemp protein isolate-dextran conjugates for 9 days.

3.3.2. FTIR

FTIR analysis is widely used to study the structural changes and interaction among protein-carbohydrate complexes (Zhang et al., 2020). Interaction of functional groups at molecular level either form new bands, as well as changes in the position and intensity of absorption bands in FTIR spectrum (Ellerbrock & Gerke, 2021). Figure 3 illustrates how the Maillard reaction affects the molecular structure of SHPI-dextran conjugates by influencing the peak position or intensity in the FTIR spectrum. The peak around 1700 − 1600 cm^− 1, 1550 − 1500 cm^− 1, 1300 − 1200 cm^− 1 were attributed due to the amide I band (C = O stretching), amide II (N-H deformation) band, and amide III band (C-N stretching and N-H deformation), respectively. Sunnhemp protein samples shows amide I, amide II and amide III bands at around 1650, 1550 and 1240 cm^− 1, respectively in the FTIR spectrum. When SHPI and dextran react after conjugation, new covalent bonds are formed, which results in new bands. Furthermore, the absorption intensities of all SHPI-dextran conjugates were distinctively higher than that of SHPI without conjugation at 1040 cm^− 1, which indicated formation of new C-N covalent bond between SHPI and dextran via Maillard reaction. Compared to SHPI spectra, new peaks were observed at 1015 cm^− 1 in all the SHPI-dextran conjugates spectrum ranging from SD0 to SD9. The presence C-N glycosidic bond was attributed to this peak, which supports covalent interactions between the polysaccharide molecules and proteins. Jiang et al. (2021) also verified Maillard reaction between casein phosphopeptide and dextran with the help of FTIR spectroscopy. The results were consistent with the previously reported FTIR spectrum of whey protein conjugated with inulin using Maillard reaction in which formation of higher intensity bands were also observed (Wang et al., 2020).

3.3.3 XRD

XRD-diffraction provides direct structural information and crystalline or amorphous nature of protein molecules. The sharp peaks indicate crystalline part, while the broad peak corresponds to amorphous region (Thiangtham et al., 2019). Two major peaks at around 2θ = 11.5° and 19° were reflected in all protein and conjugate samples (Fig. 4), which are associated with the secondary structure percentage of proteins (Zhu et al., 2023). It is evident from the diffractogram that SHPI sample without conjugation shows broad semicrystalline peak with lower peak intensity. But after incorporating dextran to SHPI at zero-day, multiple characteristic peaks were observed, which shows its crystalline structure. This might be explained by the expansion of protein structure as a result of Maillard reaction, resulting in crystalline regions (Ma et al., 2023). The intensity and height of peaks were reduced in SD1 sample, indicating an amorphous structure of SHPI-dextran conjugates as the incubation time was progressed. The reduction in peak intensities and increase in amorphous structure is related to the hydrophilic nature of dextran, making conjugates more osmotic (Xue and Luo, 2023). Moreover, conjugation with polysaccharides may facilitate the mobility of proteins polymeric chains, resulting in reduced crystallinity due to the interference with the arrangements of protein chains. Similar structural changes were also reported by Ma et al. (2013) in egg white protein conjugated with maltodextrin prepared by dry heating method for different incubation days. Furthermore, there is an increase in crystallinity of SHPI-dextran conjugates samples ranging from SD7 to SD9. This is due to the rearrangement of proteins and polysaccharides to form aggregates after conjugation during prolonged incubation time. Also, extensive protein cross-linking in the advanced stage of Maillard reaction causes crystallinity. This trend was consistent with the XRD patterns of WPI- inulin Maillard reaction products incubated for 24, 48, and 72 h at 79% relative humidity and 60° C of incubation temperature (Huang et al., 2023).

3.3.4. Surface hydrophobicity (H₀)

A protein’s surface hydrophobicity is defined as the measure of number of hydrophobic groups exposed on its surface, reflects conformational changes and closely related to its other functional properties such as solubility, emulsifying and foaming properties (Luo et al., 2022). H₀ of all the conjugated SHPI samples was less as compared to unconjugated SHPI. The maximum decrease in surface hydrophobicity was found in SD9 sample as the incubation time was increased from 0 to 9 days. This trend was concurrent with the results of degree of glycation (DG). This might be due to the strong hydrophilic nature of dextran by which the surface of protein molecules is less hydrophobic for ANS (8-Anilinonaphthalene-1-sulfonic acid) to attach in comparison to unconjugated sunnhemp protein (Xue et al., 2023). Hence, SHPI-dextran conjugate samples exhibited lower H₀ and higher degree of glycation. It has been observed that surface hydrophobicity is affected by two different ways during incubation and Maillard reaction. Firstly, the attachment of hydrophilic groups of polysaccharides to the protein surface promoting hydrophilic behavior, thereby H₀ is decreased. Secondly, partial denaturation of protein during prolonged incubation due to heating, so H₀ decreased. Therefore, H₀ index is the combination of these two factors (Sun et al., 2022). Furthermore, prolonged incubation time increases the exposure of hydrophobic groups, which leads to repolymerization of hydrophobic groups and reduced hydrophobicity (Chen et al., 2023). The present findings are in line with the results as reported by Chen et al. (2019a) in whey protein-gum acacia conjugates prepared by Maillard reaction (dry-heating). In contrast, increment in H₀ of protein was observed due to unfolding of proteins molecular structure and exposure of more hydrophobic groups in hydrophilic groups after Maillard conjugation by wet- heating method (Li et al., 2019). These differences might be due to protein types, conjugation conditions, polysaccharide type, and degree of glycation (Capar & Yalcin, 2021).

3.3.5. Free and total sulfhydryl content

The free and total sulfhydryl groups are reactive chemistry groups with a reducing capability, which plays a significant role in maintaining the structural and functional characteristics of proteins (Li et al., 2021). The free and total sulfhydryl content of all SHPI-dextran conjugates were increased as compared to SHPI. The free and total sulfhydryl content shows increasing trend with increase in incubation (SD0-SD5), thereafter opposite trend was observed in SD7 and SD9, respectively (Table 3). The increment in sulfhydryl groups could be explained mainly by following three reasons: i) the extended spatial structure of SHPI during Maillard conjugation exposed buried sulfhydryl groups to the protein surface; ii) conjugation caused disulfide bonds to be cleaved, exposing new free sulfhydryl groups; iii) the intermediate Maillard reaction products could influence the SH groups/disulfide bonds exchange reaction and the exposure of free sulfhydryl groups (Li et al., 2019; Ai et al., 2021). The reduction in sulfhydryl groups as in SD7 and SD9 samples is probably owing to two main reasons: Firstly, incubation conditions for the Maillard reaction can create oxidized states other than disulfides as sulfhydryl groups are more prone to oxidation during heat treatment. Secondly, formation of inter and intra molecular disulfide bonds during incubation. Maillard reaction occurred when amino group of protein and carbonyl groups of reducing sugars, formed Schiff base and rearrangement to advanced stage Maillard reaction products (Jia et al., 2022). As a consequence of modification or fragmentation, these molecules become more reactive and further react with guanidines, amines, and sulfhydryl groups. In conclusion, due to the oxidation of SH groups, protein aggregates were formed during prolonged incubation, thereby decrease in free and total sulfhydryl content (Li et al., 2019). Similar sulfhydryl content trend was also observed in soy protein isolate conjugated with glucose via Maillard reaction (Li et al., 2019). These differences in the results can be attributed by the Maillard reaction conditions like incubation time and temperature, biopolymer ratio, relative humidity, protein source, and carbohydrate type (Shen & Li, 2021).

Table 3

Surface hydrophobicity, free and total sulfhydryl content of SHPI and SHPI-dextran conjugates prepared by dry- heating method of Maillard reaction at different incubation days.
Sample code	Surface hydrophobicity (H₀)	Free sulfhydryl content (µmol/g)	Total sulfhydryl content (µmol/g)
SHPI	198.4 ± 0.24^a	14.15 ± 0.04	87.13 ± 0.08
SD0	192.4 ± 0.15^b	15.32 ± 0.06	89.35 ± 0.10
SD1	181.6 ± 0.21^c	17.34 ± 0.07	92.44 ± 0.03
SD3	177.5 ± 0.83^d	18.96 ± 0.09	95.13 ± 0.06
SD5	171.6 ± 0.15^e	20.41 ± 0.05	97.22 ± 0.07
SD7	169.6 ± 0.19^f	19.23 ± 0.08	96.58 ± 0.09
SD9	168.7 ± 0.09^g	18.35 ± 0.04	95.87 ± 0.03
Mean ± SD (n = 7). Within the column, means having different superscripts letters are significantly different (p < 0.05). SHPI: sunnhemp protein isolate without conjugation; SD0: sunnhemp protein isolate-dextran conjugates at 0 day; SD1: sunnhemp protein isolate-dextran conjugates for 1 day; SD3: sunnhemp protein isolate-dextran conjugates for 3 days; SD5: sunnhemp protein isolate-dextran conjugates for 5 days; SD7: sunnhemp protein isolate-dextran conjugates for 7 days; SD9: sunnhemp protein isolate-dextran conjugates for 9 days.

In the present study, SHPI-dextran conjugates were successfully fabricated through controlled dry heating method via Maillard reaction for different incubation times (0, 1, 3, 5, 7, and 9 days) and their functional and structural properties were investigated. The results shows that the browning index and degree of glycation were increased with incubation time. Continuously increasing trend in browning index shows a progressing Maillard reaction. Functional characteristics such as emulsifying property, solubility, foaming property, WBC, OBC, dispersibility, and LGC of SHPI were improved after conjugation with dextran. FTIR, circular dichroism and free sulfhydryl content results confirmed the occurrence of conjugation between SHPI and dextran. Sunnhemp protein-dextran conjugate prepared with five days of incubation time at 60°C of incubation temperature and 79% relative humidity shows highest improvement in all the functional properties against SHPI alone and other conjugate samples. Surface hydrophobicity of SHPI-dextran conjugates was initially increased up to first five days by increasing incubation time and thereafter got decreased. In conclusion, this study provides a useful knowledge to expand the application of Maillard reaction prepared plant-based protein conjugates as functional ingredient in food applications.

Funding

The author (s) received no financial support for the research, authorship, and/or publication of this article.

CRediT authorship contribution statement

Rashmi Rawat: Formal analysis, Investigation, Methodology, Writing – original draft.

Charanjiv Singh Saini: Conceptualization, Resources, Writing – review & editing.

Declaration of Competing Interest

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

Acknowledgment

The authors are thankful to the Central Research Laboratory, Sant Longowal Institute of Engineering and Technology, Longowal, Sangrur, Punjab, for providing the necessary research facilities.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflict of interest

The authors declare that they have no conflict of interest.

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No competing interests reported.