The solubility of JSPI was determined according to the method used in [23] with slight modification. One (1) g of JSPI powder was diluted into 100 mL of distilled water. The suspension was mixed evenly through continuous stirring for 30 min at 500 rpm. The suspension was then kept in the refrigerator (4˚C) for overnight and centrifuged (12,600 g/20min). The soluble protein content in the supernatant was determined by the Micro-Kjeldahl method [22]. The protein solubility (PS) was estimated from the amount of protein found in the supernatant and the total sample used to prepare the solution following Equation Number 1.

The water holding ability of JSPI was determined according to the method described in [24]. One (1) g of protein sample was suspended into 10 mL of distilled water in a 15 mL graduated conical centrifuge tube. The suspension was stirred evenly at 400 rpm and allowed to stand at room temperature for 1 h. It was then centrifuged at 2217 g for 30 min, and the volume of the supernatant was measured. The water-holding capacity was expressed from the amount of water held by 1.0 g of protein sample.

The oil holding capacity of JSPI was determined following a similar method for water holding capacity, where corn oil was used as a suspension medium instead of water.

Five (5.0) g of JSPI was put into a 25 mL measuring cylinder. The initial weight and the initial volume of the sample were recorded. The JSPI powder was poured into a cylinder and tapped continuously until a constant level was obtained. Again, the weight and volume of the sample were recorded. The final weight and volume of the sample were recorded from these differences. The bulk density (g/mL) was calculated as the weight of powder (g) divided by the volume of powder (mL) according to the Equation Number 2.

The gelation capacity of JSPI was determined according to the method followed by [25] with a slight modification. A range of sample suspensions from 2% to 14% (w/v) concentrations was prepared into two different solvents; distilled water and 1.0 M NaCl solution. The test tubes containing these suspensions were then heated for 1 h in a boiling water bath and followed by a rapid cooling under cold water tap. The tubes were further cooled for 2 h at 4˚C. The least gelation concentration (LGC) was determined as the minimum concentration required to form a self-supporting gel when the sample did not fall or slip from the inverted test tube.

Foaming capacity and stability of JSPI at different pH (1.5 - 11.5) were determined according to the method described in [26]. The protein samples (2 g) were kept into 250 mL beakers and diluted with 100 mL of distilled water. The pH values were adjusted from 1.5 to 11.5 by using HCl and NaOH. The suspensions were mixed thoroughly using magnetic stirrer and finally homogenized at 5000 rpm for 5 min (WiseMix^TM, HG-15 D). The volume of the produced foam in each beaker was measured by measuring cylinder within no later than 30 s. The increment of foam volume was estimated following Equation Number 3 and expressed as percent foam capacity. The foam stability of JSPI was calculated by Equation Number 4 from the decreased foam volume after 30 min.

The emulsification capacity of JSPI was also determined at a range of pH from 1.5 to 11.5 was according to the method stated by [23] and was expressed as percent of oil emulsified per g of protein. A dispersion of 1 g isolated protein in 25 mL distilled water was prepared by continuous stirring at moderate speed for 30 min at 600 rpm. Five (5) mL refined groundnut oil was added, and after adjusting pH the mixed slurry was blended at 3000 rpm until phase separation was seen. The emulsion capacity was estimated based on separated cream following Equation Number 5.

The emulsion stability was determined according to the method used in [27] with a slight modification. The above emulsions in different pH were transferred into test tubes and held at 70˚C in a water bath for 45 min. The tubes were then allowed to stand at room temperature for 3 h. Percent stability was calculated from the height of the remaining emulsified layer and the original emulsified layer, according to Equation Number 6.

The particle size distribution of the spray dried JSPI powder was determined according to [28] with slight modification. The experiments were carried out using a Zeta Sizer (ZEN3600, Malvern Instruments Ltd., Malvern, UK). Well dispersed samples at 4 mg/100 ml (w/v) concentration were used in this test. Dispersions were prepared in distilled water with gentle stirring for 60 min (400 rpm) at ambient temperature. These dispersions were kept overnight at 4˚C for complete hydration and then diluted again with distilled water to the final concentration (4 mg/100 ml, w/v). The size distribution data were obtained and analyzed by using Malvern’s proprietary software version 7.01.

The X-ray diffraction (XRD) pattern of JSPI powder was measured to assess the crystalline/amorphous behavior of the isolated protein. This study was carried out on a Siemens (D501) X-ray diffractometer with CuKα1 radiation [29]. Diffractograms were recorded between 8˚ and 52.15˚ (2θ) at a rate of 1.20˚/min (2θ) with a step size of 0.05˚ (2θ). An anti-scatter slit of 0.15 mm and 1˚ divergence and receiving slits were used.

Particle morphology of the spray dried JSPI was observed by using a scanning electron microscope (JEOL, JSM 6300 SEM, JEOL, Tokyo, Japan). The powder sample was directly deposited on an aluminum stub using double-sided adhesive carbon conductive tape. The entire assembly was put in a desiccator containing freshly dried silica gel for 72 hours. Then the sample was sputter-coated with a thin layer of gold. The SEM micrographs were acquired at an accelerating voltage of 15 kV.

DSC tests were carried out to measure the calorimetric changes in spray dried JSPI and these changes were correlated to determine its denaturation temperature. The analysis was carried out by using DSC Q2000, (TA instruments, New Castle, USA) [30]. The JSPI powder was dispersed at 20% (w/w) concentration distilled water. About 18 ± 1 mg of sample solution was taken in the aluminum pan and sealed hermetically. The samples were scanned from 20˚C to 100˚C at a heating rate of 5˚C/min. The instrument was calibrated by using indium standard (fusion temperature = 156.61˚C and fusion enthalpy = 28.67 J/g) [31]. The software associated with the instrument (TA Universal Analysis 2000^TM) was used to analyze the DSC thermograms. The extent of denaturation was determined using a change of enthalpy (ΔH) from 50˚C to 95˚C.

The FTIR spectroscopy was used in the present study to observe the conformation and secondary structural motifs of the isolated protein. The IR spectra were acquired through Perkin Elmer FTIR (Spectrum-2) instrument operated by CPU32M software. The JSPI powder was scanned within 650 to 4000 cm⁻¹ using a triglycine sulfate (TGS) detector. A total of 8 scans at 4 cm⁻¹ resolution were accumulated at 0.2 cm/sec scanning speed. The JFSs flour and the separated JFSs starch were also scanned to observe the comparative spectra among those materials. The protein sample was diluted in deionized water at 1% concentration (w/v) for the secondary structural quantification. The spectrum of the aqueous protein solution was acquired in the same IR conditions described above. The blank spectrum of water was subtracted from the protein spectrum. The baseline subtracted protein spectra were analyzed by using Perkin Elmer’s proprietary software (Version 10.05.03), and a peak fit software Peak Fit version 4.12 (Sea Solve Software Inc. Framingham, USA). The peak Fit Software was used for quantitative analysis of the secondary structure of the protein. The original spectra (of amide region-I, 1600 - 1700 cm⁻¹) without any smoothing were fitted with Gaussian shape and were analyzed by the local least square (LLS) algorithm. Percentage of secondary structures (α-helix, β-sheets, β-turns, and random coils) was estimated using Equation Number 7 [32] [33].

where, A_ind = Sum of the area of individual secondary structure within amide I band.

A_total = Area of total amide I band.

The positions or locations of bands for each secondary structural element (β-sheets, α-helix, and β turns) of tested proteins in H₂O were considered based on the information available in the literature [12] [34]. The bands from 1620 to 1640 cm⁻¹ and 1674 cm⁻¹ to 1680 cm⁻¹ were assigned to β-sheets. The bands from 1641 to 1647 cm⁻¹ were assigned to random coil. The bands within 1648 and 1660 cm⁻¹ were assigned to α-helix. Similarly, the bands appearing at and in the vicinity of 1663 cm⁻¹, 1671 cm⁻¹, 1683 cm⁻¹, 1688 cm⁻¹, and 1694 cm⁻¹ were assigned to β turns. The peaks between 1600 cm⁻¹ - 1619 cm⁻¹ were not considered while quantifying the secondary structural features as they are known to be generated from aromatic side chains [32].

The solubility of JSPI was found as 78.44%. This value of solubility is comparable with the percent solubility of other proteins isolated from different sources. Reference [4] found variation in solubility of the protein isolates prepared by different drying methods, such as vacuum dried, spray dried and freeze dried lentil protein isolate showed 51%, 81% and 78% solubility respectively. The literature suggests that the spray dried powder possesses more amorphous particles, which provides increased solubility during rehydration [15] [41]. The percent solubility of rice bran protein concentrate ranged from 47.69% to 73% based on varieties of rice [36]. Protein isolate with higher solubility is preferred for direct consumption as well as utilization as a food ingredient. Therefore, using a spray dryer for commercial production of JSPI is recommended to produce the concentrated protein with higher solubility.

The water holding capacity of the JSPI was 2.89 mL H₂O/g protein, which is higher than the water holding capacity of cowpea protein, 2.20 mL H₂0/g [42], but lower than that of the wheat bran protein, 4.20 mL H₂0/g [2]. High water holding capacity of proteins helps to reduce moisture loss from bakery goods and maintains freshness and moist mouth feel of the products. The obtained values indicate that the JSPI powder needs to be mixed with a portion of wheat protein to have adequate swelling and water retention during food preparation.

The oil-holding capacity of JSPI was 1.57 mL oil/g protein, which is slightly lower compared to wheat bran protein 1.70 mL/g [2]. Higher oil holding capacity is advantageous for oily food like sausages, mayonnaise, and salad dressing. Therefore, utilization of JSPI combined with a protein of higher oil holding capacity is preferred for such a food.

This is an important parameter of a powder or a mix of powders to determine the packaging requirement of the product. The bulk density of JSPI was 0.67 g/mL, which is less compared to the bulk density of casein 0.89 g/mL [35]. Low bulk density is advantageous for the formulation of weaning foods regarding packaging and transportation [43].

In the process of gel formation, about 6% JSPI in 1.0 M NaCl solution initiated forming a gel; however, this gel was not consistent enough to withstand the gravity force at the inverted position of the container. At least 12% (w/v) JSPI concentration in that solution was required to make a firm gel (Table 1). The protein did not form a gel in pure water. Reference [4] found the least gelling concentration of lentil protein isolate was 14% in a phosphate buffer of pH 7.0. The lower least gelation concentration implies the greater gelling capacity of the protein [44].

The foaming capacity (FC) of isolated jackfruit seeds protein was pH-dependent. As shown in Figure 2, the alkaline solution resulted in better foam formation. The FC increased from the lowest value of 6% at pH 5.5 to 42%, 25%, 46%, 58%, and 74% at pH 1.5, 3.5, 7.5, 9.5, and 11.5, respectively. The findings comply with the previous observation that the alkaline solution is favorable for the protein solubility and foaming capacity [45]. This trend could be due to the fact that the variation of pH affects the net charge and electrostatic balance of the protein solution. The solubility of protein tends to be minimum near the isoelectric point

Note: Symbols: − no gel; + weak gel; ++ strong gel; +++ very strong gel.

(IP) because of negligible or zero net charge. The IPs of the most plant proteins are reportedly fallen between 4 and 5 [21]. The higher FC at pH 11.5 was likely due to the increased net charges on the protein molecules, which weakened the hydrophobic interactions and increased the flexibility of the protein [2]. These phenomena allowed the protein to diffuse more rapidly to the air-water interface to encapsulate air particles and then enhanced the foam formation.

The foaming stability (FS) requires the formation of a thick, cohesive, and viscoelastic film around each gas bubble [46]. Isolated jackfruit seeds protein showed poor foam stability at pH 5.5 (0.83%). On both sides of pH 5.5, the foaming stability gradually increased and reached its maximum value of 47% at pH 11.5. The solutions with pH 7.5, 9.5, and 1.5 showed about 26%, 39 %, and 21% FSs, respectively (Figure 2). The previous researches also observed a considerable effect of pH on the stability of foams [35] [47]. The improvement of FS in the alkaline pH is likely due to increased solubility and surface activity of the soluble protein.

The pH of solution has a significant effect on emulsion properties also. As shown in Figure 3, JSPI had a minimum emulsion capacity (EC) (2%) at pH 5.5, which increased on either side of this pH. The pH 11.5 of the solution showed the highest EC of 63%, which was followed by the pH of 9.5 and 7.5, with EC values of 56% and 25%, respectively. The observed results suggest that the alkaline pH improved the emulsion capacity from the acidic one. Dependence of emulsion capacity on pH was expected, as the emulsion capacity of a protein is known to depend on the hydrophilic-lipophilic balance and electrostatic repulsion at the isoelectric point. The JSPI had minimum emulsion stability at pH 5.5 (0.43% after 3 h) and reached the maximum value of 52% at pH 11.5 (Figure 3).

It is reported that various factors including pH, net charge, interfacial tension, viscosity, and protein conformation could affect the values of ES [48].

The particle size and size distribution curve of the spray dried JSPI are presented in Figure 4. The curve suggests that about 75.0% (by volume) particles ranged from 100 to 1000 nm and around 23.0% of the particles were smaller than 100 nm. About 2% particles’ diameter was larger than 1000 nm. The variation of size found in this curve reflects the disparity of morphological image (Figure 6). This observation consents to the previous finding that the micro particulation process of bio particles delivers finer to comparatively larger particles [49].

The XRD diffractogram is presented in Figure 5. Absence of any sharp peak

suggests that the spray-dried JSPI particles were amorphous in nature. However, two broad peaks were found at 9.0 and 19.0 degrees 2θ on the JSPI diffractogram. Reference [50] observed three peaks (at 8.5, 19.5 and 24.5 degrees 2θ) on the soy protein isolate diffractogram and reference [4] reported two broad peaks (at 8.5 and 24.5 degrees 2θ) on the lentil protein isolate diffractogram. It is reported that the powder form of protein normally possesses the amorphous behavior [29] [51].

The SEM micrographs of JSPI powder presented in two images with two different magnifications appear varying sizes of particles (Figure 6). The surface of the spray-dried protein isolate produced more or less spherical appearance with dimples and surface folds. Higher magnification micrograph shows the smooth-surfaced hollow particles, although they possess indented surface. The low diffusion rate of the moisture from the aqueous droplets containing skin-forming solids is the primary reason for the formation of hollow particles in spray dried particles [52].

According to the DSC thermogram (Figure 7), about 1.810 J/g energy required to denature the jackfruit protein isolate. The endothermic peak produced by the thermal scanning from 20˚C to 100˚C suggests that the denaturation temperature (Td) of JSPI was about 77.83˚C. The denaturation temperature of the spray-dried lentil protein isolate was reported as 123.6˚C [4]. They concluded that the Td value can be changed with the variation of crops or with the verities of same crop. Reference [14] suggested that the variation of Td can be due to the variation of concentration of protein in the solution. They demonstrated that the Td values vary from 75˚C to 80˚C with the variation of concentration of whey protein isolate from 20% to 40%.

A cut off portion of the amide region-I (1600 - 1700 cm⁻¹) from the original absorption spectrum and its fitted spectra are presented in Figure 8. The amide-I region was selected for secondary structural quantification because of its very high signal to noise ratio [53] [54]. As seen in the figure, there were nine bands (excluding side chain) produced for the best fitting of the amide-I region with an r² value of 0.97. The quantification tools of the assigned bands estimated that the JFSs protein contained 50.28% β-sheet, 21.71% α-helix, 8.86% β-turn, and 19.15% unordered structure. The result advised that the β-sheet is the dominant secondary structure of JFSs protein.