Characterization and comparative studies of galactomannans from Bauhinia vahlii, Delonix elata, and Peltophorum pterocarpum

International Journal of Biological Macromolecules 134 (2019) 498–506

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Characterization and comparative studies of galactomannans from Bauhinia vahlii, Delonix elata, and Peltophorum pterocarpum Kizukala Jamir ⁎,1, Nanibabu Badithi 1, Kusuma Venumadhav, Kottapalli Seshagirirao Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India

a r t i c l e

i n f o

Article history: Received 2 April 2019 Received in revised form 23 April 2019 Accepted 13 May 2019 Available online 14 May 2019 Keywords: Galactomannan Intrinsic viscosity Thermal property

a b s t r a c t The present investigation determines the extraction and characterization of seed galactomannans from the Leguminosae taxa of Bauhinia vahlii, Delonix elata, and Peltophorum pterocarpum. The seed galactomannans presented a total yield of 30.82, 24.01 and 25.25% with a Man/Gal ratios of 4.21:1, 2.55:1 and 3.03:1 for B. vahlii, D. elata, and P. pterocarpum, respectively exhibiting 1–4 mannose linkages with galactose side chains at C6 position. The galactomannans presented an efficient water holding capacity, solubility and emulsion properties. Intrinsic viscosity from combined Huggins and Kraemer extrapolations are 3.807, 3.424 and 3.331 dl g−1 and the viscosity average molecular weight by Mark-Houwink relationship are 5.29 × 105, 5.29 × 105 and 4.95 × 105 for B. vahlii, D. elata, and P. pterocarpum, respectively. TEM analysis of the polysaccharide showed an aggregated amorphous smooth particles, oval or tubular in shape, branched with compact surfaces. SEM micrographs of the powdered galactomannan indicate well defined amorphous material with pores and cervices on the rough surface. The thermal property studied by DSC and TG-DTA suggests good thermal stability. These findings demonstrate that the seed galactomannan of B. vahlii, D. elata, and P. pterocarpum could be explored as an effective alternative to commercial galactomannans for industrial applications. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Seed galactomannans commonly referred to as seed gums are neutral hetero-polysaccharide aplenty in nature [1]. They are hydrophilic and forms a highly stable viscous aqueous solution. They are mostly found in the endosperm of leguminous seeds as cell wall storage polysaccharide. Galactomannans have the fundamental structure consisting of the main chain of β- (1-4)-D-mannopyranose units substituted by single α-D-galactopyranose unit, branches linked α-(1-6). The significant difference between galactomannans from different plant species lies in the galactose content as well as its distribution along the mannopyranosyl backbone [2]. The structure of galactomannan, chain interaction and conformations depends on the mannose/galactose (Man/Gal) ratio which helps in determining the various industrial applications of seed galactomannans [3]. Commercial galactomannans such as locust bean gum (LBG), guar gum (GG) and tara gum (TG) are widely employed in the cosmetic, food and pharmaceutical industries. Because of the specific physicochemical properties such as high molecular weight, absence of toxicity, non-ionic character, water holding capacity, and solubility, galactomannans are used as excellent emulsifiers, binders, thickeners, excipients, stabilizing and gelling agents in ⁎ Corresponding author. E-mail address: [email protected] (K. Jamir). 1 Kizukala Jamir and Nanibabu Badithi contributed equally to this work.

https://doi.org/10.1016/j.ijbiomac.2019.05.080 0141-8130/© 2019 Elsevier B.V. All rights reserved.

food, textile, pharmaceutical, cosmetic, and paper industries [4–7]. The use of edible films or coatings is gaining importance for biodegradable packaging as well as to minimize the deleterious effects imposed by minimal processing [8]. There is a steady rise in the use of seed gums in different food products as an additive because of the increase in the consumption of ready-made meals and novelty foods such as ice cream, sauce, beverages, bakery products and for supplementation as dietary fiber [4]. Galactomannans are also used in preparing affinity and sieving matrix [9]. Thus, seed galactomannan provides an alternative to other natural polysaccharide or their synthetic counterparts. Seed galactomannan with potential industrial use is in demand worldwide, and hence the search for the newer source of these seed polysaccharides is essential. In the view of the greater potential value of galactomannans, the present investigation aimed to isolate and characterize the seed galactomannans from Bauhinia vahlii, Delonix elata, and Peltophorum pterocarpum. These plants belong to the family of Leguminosae and subfamily of Caesalpinioideae and widely distributed in the tropical and sub-tropical region. Bauhinia vahlii is a large woody creeper distributed in India. The plants are harvested from the wild as a source of food, fiber, and ethnomedicine [10]. Delonix elata or white gulmohar is a deciduous tree native to Africa. In India, the trees are found in the wild in some parts of the peninsular region. The tree is cultivated as an avenue tree and also used in agro-forestry, ethnomedicine and as a source of food [11]. Peltophorum pterocarpum commonly known as copperpod is native to south-east Asia. In India, it is

K. Jamir et al. / International Journal of Biological Macromolecules 134 (2019) 498–506

widespread and largely planted as ornamental or shade trees [12]. Since these plants are widespread and the seeds are abundant, isolation of galactomannans from the non-conventional source would be a promising new biomaterial for various biotechnological industries. The extraction yield, physiochemical, intrinsic viscosity, structural and thermal properties of the seed polysaccharides were determined which will provide a better understanding to makes them versatile materials for their application in the food industry. 2. Materials and methods 2.1. Material and extraction of galactomannan The pods of Delonix elata (L.) Gamble (NB-2005 @ UH) and Peltophorum pterocarpum (DC.) K. Heyne (NB-2002 @ UH) were collected from University of Hyderabad campus whereas pods of Bauhinia vahlii Wight & Arn. (NB-2009 @ UH) was collected from Warangal district of Telangana, India during April–May 2014. The voucher specimens were identified by Prof. K. Seshagirirao and deposited at UH Herbarium, Department of Plant Sciences, University of Hyderabad. The seed galactomannan of B. vahlii, D. elata, and P. pterocarpum was extracted according to Cerqueira et al. [13] with minor modifications. The seeds were detached from the pods and boiled at 100 °C for 30 min to inactivate the endogenous enzymes. The seed coat, endosperm, and germ were separated from the whole seeds. The endosperm was blended in distilled water and kept for overnight stirring at 4 °C. The suspension was filtered using different grades of Buchner funnel and finally with 0.22 μM Millipore membrane filter followed by centrifugation at 10,000×g for 30 min. Furthermore, the polysaccharide solution was gradient precipitated by ethanol (10–70%). The final 70% precipitated galactomannan was lyophilized and stored dry before use. The yield of seed galactomannans is expressed as a percentage of dry mass obtained after purification in relation to the dry weight of seeds [6]. 2.2. Chemical properties The moisture and ash contents were analyzed by following the official procedures of AOAC [14]. The pH was determined by preparing a 1% (w/v) suspension of the polysaccharide, and the value was measured using a digital pH meter. The carbohydrate and protein contents of the seed galactomannan were estimated according to Dubois et al. [15] and Bradford [16], respectively. The surface charge of the polysaccharide was measured according to Varma & Jayaram Kumar [17] by determining the zeta potential value using dynamic light scattering (Horiba, SZ-100). A 0.1% (w/v) of galactomannan solution prepared in milliQ water was used for the study. Monosaccharide composition analysis of the seed gums was carried out according to Albuquerque et al. [18] with minor modifications. Briefly, 1 mg of the polysaccharide was hydrolyzed at 100 °C with 5 M trifluoroacetic acid (TFA) for 4 h followed by 1 M sodium borohydride reduction for 4 h at 25 °C and acetylation of the alditols with 1:1 (v/v) ratio of acetic anhydride and pyridine at 100 °C for 1 h. The derivatized compounds were dissolved in dichloromethane and analyzed on a gaschromatography with MS/MS (Agilent 7890A for gas chromatography system and Pegasus HT TOFMS for mass spectrometry system) equipped with an Elite-I fused silica capillary column (30 m × 0.25 mm, 1D X 1 μMdf with 100% dimethyl poly siloxane). Helium was used as carrier gas at 1 ml/min flow rate, and a 2 μl of the samples were injected manually using an injector at 10:1 split ratio. The injector temperature and the ion source temperature were maintained at 250 and 280 °C, respectively. The temperature was programmed at 110 °C consisting of an initial 2 min plateau, with a linear increase of 10 °C/ min to 200 °C, followed by 5 °C/min ramp up to 280 °C and extending for 9 min at 280 °C. The MS scans were performed at a scanning interval

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of 0.5 s between 45 and 450 m/z with a 70 eV. Methylation analysis of the seed polysaccharide was performed according to Ciucanu & Kerek [19] with slight modification. Methylation was carried out by dissolving 3 mg of galactomannan in 1 ml of dimethyl sulfoxide (DMSO) followed by 100 mg of dry sodium hydroxide. To the mixture, 0.5 ml of methyl iodide was added and stirred for 10 min. The reaction was terminated by adding 1 ml of distilled water, and the methylated product was extracted with dichloromethane. The methylated product was further hydrolyzed, reduced with borohydride, acetylated with acetic anhydride and pyridine. The partially methylated alditol acetates (PMAA) were analyzed by gas-chromatography with MS/MS as stated above. The result was obtained by LECO Chroma TOF optimized for Pegasus HT 4.44.0.0, and the library search was performed using NIST MS search version 11.0. 2.3. Functional properties The functional properties of the seed polysaccharide such as water absorption capacity, solubility, emulsifying capacity, and stability were determined according to Sciarini et al. [20] with some modifications. The water absorption capacity (WAC) and solubility of the galactomannan were estimated by dissolving 0.1 g of the polysaccharide in 10 ml of distilled water and centrifuged at 5000 rpm for 10 min. The supernatant was discarded, and the hydrated polymer weight determined the percentage of WAC using the following formula: %WAC ¼ Hydrated polymer weight=Dry weight  100

ð1Þ

Solubility was determined at 25 °C by dissolving 1 g of the polysaccharide in 10 ml distilled water using a magnetic stirrer for 30 min followed by centrifuging at 5000 rpm for 10 min. Aliquots of 5 ml of the supernatant were dried at 110 °C in an air convection oven until a constant weight was obtained. Solubility was calculated as: %Solubility ¼ f½ðInitial weight−Final weightÞ  10=5g  100

ð2Þ

Emulsifying capacity (EC) was studied by homogenizing 10 ml of 0.05, 0.1 and 0.5% (w/v) galactomannan gums suspension in 1 ml of rice bran oil for 1 min at 30 second pulse using a sonicator. The suspension was further centrifuged at 2500 rpm for 10 min. The EC was calculated using the following formula: %EC ¼ Emulsion volume=Total solution volume  100

ð3Þ

Emulsion stability at high temperature was also determined by heating the emulsion at 80 °C in a water bath under constant stirring for 30 min. Then, the samples were centrifuged at 2500 rpm for 10 min. Finally, the emulsion stability (ES) was calculated as: %ES ¼ Final emulsion volume=Initial emulsion volume  100

ð4Þ

The intrinsic viscosity of seed galactomannan was calculated according to Pamies et al. [21] on the principle of rolling ball time using an AMVn automated rolling ball micro-viscometer (Anton Paar, Germany) equipped with programmable tube angle. Briefly, a concentration of 5 mg/ml galactomannan solution was prepared in 5 mM NaCl and left for stirring overnight. The clear solution collected was filtered through a 0.2 μm membrane filter and stored at 4 °C before use. The intrinsic viscosity (η) was measured by determining the relative viscosity (ηrel) and converting to a specific viscosity (ηsp) by using the following equations: ηrel ¼ η=ηs ηsp ¼



ð5Þ




η−ηs =ηs ¼ ηrel −1

where ηs is the solvent viscosity.

ð6Þ

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Huggins and Kraemer's plots were obtained by extrapolating the values of ηsp/c and ln(ηrel/c) to zero concentration depending on the respective relationships given below: ηsp =c ¼ ½η þ k1 ½η2 c Huggins0




ð7Þ



ln ηrel =c ¼ ½η−k2 ½η2 c Kraemer0 s

ð8Þ

where c is the solute concentration; k1 and k2 are the Huggins and Kraemer constant, respectively. An average of four runs was performed at 25 °C and an angle of 50°. The viscosity average molecular weight (Mw) of the seed galactomannans were calculated according to Mark-Houwink relationship given for guar gum by Doublier & Launay [22] and modified by Gaisford et al. [23] given below:  0:98 ½η ¼ 11:55  10−6 ð1−rÞ  M w

ð9Þ

2.5. Thermal analysis of seed galactomannans The thermal property of the seed galactomannans were studied by differential scanning calorimetry (DSC-1, Mettler, Switzerland) and DTG-60 (Shimadzu, Japan) under a nitrogen atmosphere. Briefly, 2–3 mg of the polysaccharide powder was weighed into an aluminium pan. The pan was sealed and transferred to the heating chamber to equilibrate for 2 h at room temperature. An empty pan was used as a reference. The DSC scan was recorded between 25 and 300 °C at a rate of 10 °C/min. The onset temperature (To); peak temperature (Tp); end set (Te) and enthalpy of gelatinization (ΔH) were determined from the exotherm curves. The thermo-gravimetric analyzer (DTG-60, Shimadzu, Japan) was used to study the thermal degradation of the polysaccharide gum. The TG-DTA curves were recorded at a heating rate of 10 °C/min from 40 to 600 °C under a nitrogen atmosphere. Around 3 mg of the sample was weighted, and the mass loss percentages were determined from the curves using STARe SW 12.10 thermal analysis software.

where [η] is expressed in dl g−1 and r = 1/[(M/G) + 1].

2.6. Statistical analysis

2.4. Structural properties

The data are presented as an average of triplicate observations. The statistical analysis was performed by one-way analysis of variance (ANOVA), and Turkey's multiple comparisons test at p b 0.05 using GraphPad Prism 6.

Microstructural characterization of the seed gum was investigated to determine the morphology using transmission electron microscopy and scanning electron microscopy. Transmission electron microscopy of the seed polysaccharide was investigated using JOEL JEM 1011 100 kV electron microscope. A 2 μl of the unstained polysaccharide solution (1% w/ v) was spread evenly on the carbon-coated copper electron microscopy grids and allowed to air dry for 1 h. The air-dried sample grid was incubated with 2% (w/v) aqueous uranyl acetate solution for 10 min and washed with distilled water. The microphotographs were collected at different magnifications. The surface characteristic of the powder galactomannan was studied using S-3400N, scanning electron microscope, Hitachi. The fine powder was applied on top of the aluminium stub with sticky carbon tape and gold coated for 60 s with E-1010, ion sputter, Hitachi. A 10 kV accelerating potential was used during the micrography. The attenuated transmission resonance- infrared spectroscopy (ATR-IR) spectra were recorded by PLATINUM-ATR (Bruker, Germany). Briefly, 3 mg of the seed galactomannan powder was mounted on the sample compartment base plate, and the infrared region was scanned between 4000 and 500 cm−1 with an average of 32 scans and a resolution of 4 cm−1. The spectra were obtained as percentage transmittance versus wave number using OPUS 7.0 software. The quantitative phase identification of the seed polysaccharide was carried out with X-ray diffractometer PW1830 (Philips, Netherland) with Cu irradiation at 40 kV voltage and 25 mA. The scattered radiation was measured in the angular range from 10 to 60° with a reflection angle of 2θ and at a scanning rate of 4° min−1. The data obtained were evaluated using OriginPro 8.0 software.

3. Results and discussion 3.1. Extraction of seed polysaccharide and their chemical properties The seed galactomannan was extracted by distilled water followed by subsequent ethanol gradient precipitation. The galactomannan yield from the seeds mostly depends on the extraction procedures, time of collection and plant material. The endosperm was used for polysaccharide extraction, and the total yield and chemical composition are demonstrated in Table 1. The galactomannan yield (dry weight) of D. elata (24.01%) and P. pterocarpum (25.25%) similar to galactomannan extracted from the seeds of Gleditsia triacanthos (24.73%) and Caesalpinia pulcherrima (25.70%) [13,24,25]. However, the yield of B. vahlii (30.82%) was higher than D. elata and P. pterocarpum by 5–6%. The yield of P. pterocarpum seed polysaccharide (20.4%) reported by Nwokocha & Williams [26] was lower than our report which probably due to the different extraction conditions. Minute traces of protein were reported in the samples. The moisture content of the B. vahlii, D. elata, and P. pterocarpum were 12.02, 11.13 and 11.86% while the ash content was 0.25, 0.18 and 0.24%, respectively. The total ash content in isolated polysaccharides is comparatively lower than values reported by Mirhosseini & Amid [27] in commercial gums: locust bean gum (1.2%), guar gum (1.5%), tara gum (1.5%). A 1% (w/v) of B. vahlii, D. elata and P. pterocarpum polysaccharides in water gave a pH of 6.26, 6.38 and 6.62, respectively. In particles with large positive or negative zeta potential values (+25 mV and −25 mV), the repulsive forces

Table 1 Yield and physicochemical properties of B. vahlii, D. elata and P. pterocarpum seed galactomannana. Parameters Galactomannan yield (%)⁎ Protein (%)⁎ Moisture content (%) Ash content (%)⁎ pH WAC (g/g of the sample) Solubility (%) Intrinsic viscosity (dl g−1) M w (g/mol)

B. vahlii

D. elata c

30.82 ± 1.5 2.57 ± 0.5 12.04 ± 0.7 0.25 ± 0.2 6.26 ± 0.4 18.71 ± 0.8b 73.7 ± 1.8a 3.807 5.29 × 105

P. pterocarpum c

24.01 ± 1.2 2.06 ± 0.5 11.13 ± 0.5 0.18 ± 0.8 6.38 ± 0.6 14.33 ± 0.5a 89.2 ± 0.8b 3.424 5.33 × 105

⁎ Dry weight basis; Mean values in the same column with different superscripts (a–c) are significantly different (p b 0.05).

25.25 ± 0.8 2.12 ± 0.4 11.86 ± 0.3 0.24 ± 0.6 6.62 ± 0.2 10.23 ± 0.5b 82.4 ± 1.1c 3.331 4.95 × 105

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exceed the attractive forces leading to stable dispersion while particles with low zeta potential lack sufficient repulsive force to prevent particle aggregation [28]. The galactomannans solutions presented a negative zeta potential values for all the studied polysaccharide (−21 mV to −36.7 mV) determining their stability (Fig. 1.A). The isolated seed galactomannans are pure, stable, anhydrous with neutral pH making them suitable for their application in food industry such as in the manufacture of edible films, the use of seed galactomannans have received much attention due to their advantages over synthetic films. Gas chromatography-mass spectrometry (GC–MS/MS) analysis revealed the monosaccharide composition of the seed polysaccharide through acetylation studies (Table 2). The important monosaccharides in the seed polysaccharide were mannose and galactose. The mannose content in B. vahlii, D. elata, and P. pterocarpum were 72.5, 68.6 and 64.5% while the galactose content was 23.4, 26.9 and 30.6%, respectively. The mannose/galactose ratio of B. vahlii, D. elata, and P. pterocarpum was 4.21:1, 2.55:1 and 3.03:1, respectively. The Man/Gal ratios of the commercial galactomannans such as guar gum (GG), locust bean gum (LBG) and tara gum (TG) are 2:1, 4:1 and 3:1, respectively [29]. Nwokocha & Williams [26] reported a slightly higher Man/Gal ratio of P. pterocarpum polysaccharide (4.4:1). Besides mannose and galactose, the seed polysaccharide also contains a small amount of fucose (Fuc), arabinose (Ara) and xylose (Xyl) however rhamnose (Rha) was reported only in P. pterocarpum. The presences of these minor components in the galactomannan could be attributed to the complexity of the polysaccharide structure. The partially O-methylated alditol acetates (PMAA) of the polysaccharide samples were analyzed using GC–MS/MS. The PMAA-MS/MS patterns of the polysaccharides are shown in Fig. 1.B. Three distinct peaks were observed and using NIST MS search version 11.0, the peaks were identified as 2,3,4,6-tetra-O-methyl-α-D-galactopyranose

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Table 2 Monosaccharide composition of B. vahlii, D. elata and P. pterocarpum seed galactomannana. Sample

Monosaccharide composition (%) Man

B. vahlii

77.5 ± 0.4 D. elata 68.6 ± 0.7 P. pterocarpum 71.5 ± 0.6

a

Man/Gal

Gal

Rha

Fuc

Ara

Xyl

18.4 ± 0.5 26.9 ± 0.4 23.6 ± 0.8

–

1.3 ± 0.2 1.6 ± 0.4 0.9 ± 0.3

1.4 ± 0.1 1.3 ± 0.2 1.6 ± 0.2

1.4 ± 0.1 1.6 ± 0.2 1.5 ± 0.3

– 0.9 ± 0.5

4.21 2.55 3.03

Data are expressed as mean ± standard deviations of triplicate experiments.

(Peak 1); 2,3,6-tri-O-methyl-β-D-mannopyranose (Peak 2); and 2,3di-O-methyl-β-D-mannopyranose (Peak 3). The 2,3,4,6-tetra-Omethyl-α-D-galactopyranose indicates a non-reducing terminal branch. The 2,3,6-tri-O-methyl-β-D-mannopyranose depicts that the predominant structure is composed of 4-linked mannosyl units and the 2,3-diO-methyl-β-D-mannopyranose indicates the branching at position 6 of the major mannose backbone. Similar peak patterns were reported in all the three polysaccharide samples (Table 3). The PMAA analysis also indicated a smaller amount of galactose than that of mannose confirming the Man/Gal ratio as noted by the chemical analysis. 3.2. Functional properties Water absorption capacity (WAC) is the potential of a structure to absorb water spontaneously when submerged in water or a consistently moist environment [30]. The water absorption capacity of a polymer depends on the pore size, capillary action, surface interaction, and presences of charged protein molecules which leads to the strong

Fig. 1. (A) Zeta potential analysis of the seed galactomannans obtained by dynamic light scattering; (B) the partially O-methylated alditol acetates (PMAA) chromatogram of polysaccharides obtained by GC-MS analysis where (1) 2,3,4,6-tetra-O-methyl-α-D-galactopyranose, (2) 2,3,6-tri-O-methyl-β-D-mannopyranose, and (2) 2,3-di-O-methyl-β-Dmannopyranose.

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Table 3 Identification of peaks by GC–MS/MS through methylation analysis of B. vahlii, D. elata and P. pterocarpum seed galactomannan. B. vahlii PMAA peak 2,3,4,6-Tetra-O-methyl-α-D-galactopyranose 2,3,6-Tri-O-methyl-β-D-mannopyranose 2,3-Di-O-methyl-β-D-mannopyranose

D. elata

P. pterocarpum

Linkage type

RA (%)

RT (min)

RA (%)

RT (min)

RA (%)

RT (min)

Terminal 1,4 1,4,6

22 25 46

10.05 10.23 10.33

24 21 49

10.02 10.20 10.29

18 20 46

10.31 10.48 10.58

RA: relative abundance; RT: retention time.

correlation of protein hydration with the polar constituents along with the hydrophilic interaction through hydrogen bonding [31]. The WAC of the B. vahlii, D. elata, and P. pterocarpum seed polysaccharide were 18.71, 14.33 and 10.23 g/g of the sample, respectively (Table 1). The high WAC of the seed gums is likely due to the presence of protein residues and surface interaction between the water molecules and particles, making them ideal for their applications in the food industry. Solubility and emulsifying properties are an essential functional property of a polysaccharide to determine their possible industrial applications. At 25 °C, D. elata (89.2%) presented the highest solubility followed by P. pterocarpum (82.4%) and B. vahlii (73.7%) (Table 1). With increasing temperature (50 to 80 °C), the solubility of the polysaccharide increased however beyond 80 °C; the solubility reduced likely due to the gelling effect (Data not showed). The principle of polymer emulsifying capacity is mostly associated with a high surface active which controls the surface tension. Fig. 2.A and B represent the emulsifying capacity and stability of the seed polysaccharides. The emulsion capacity values increased with increasing polysaccharide concentrations, and the order is D. elata b P. pterocarpum b B. vahlii. Molecules

with larger Man/Gal ratio can develop intermolecular associations between the unsubstituted regions of mannan backbone while smaller Man/Gal ratio molecules may develop more gel-like layer around the oil droplets enhancing the emulsion capacity and stability [32]. At 0.5% of galactomannan, D. elata with least Man/Gal ratio exhibited the highest the emulsifying capacity (100%) followed by P. pterocarpum (98.71%) and B. vahlii (93.33%) (Fig. 2.A). The emulsion capacities of the seed galactomannans were in agreement with the galactomannans extracted from the seeds of Trigonella foenum-graecum, Prosopis glandulosa and Gleditsia triacanthos [20,31,33] however the values of emulsion capacity reported by Wu et al. [32] were comparatively lower (b 70). The emulsion stability of the gums studied at 80 °C is shown in Fig. 2. B. The stability of the emulsions at 0.5% gum suspension was 93.76, 92.05 and 90.91% for P. pterocarpum, D. elata and B. vahlii, respectively with no significant differences. The findings were consistent with the results observed by Martinez-Avila et al. [33] and Sciarini et al. [20]. At high temperature, emulsions are usually thermodynamically unstable; however, our study showed excellent emulsion stabilization effect

Fig. 2. (A) Emulsion capacity and stability as a function of galactomannan concentration; (B) Analysis of intrinsic viscosity potential of the seed galactomannans determined by Huggins (●) and Kraemer (●) equations.

K. Jamir et al. / International Journal of Biological Macromolecules 134 (2019) 498–506

even at 80 °C. The study also observed that emulsion stability is independent of polysaccharide concentration. A polymer with higher surface active reduces the surface tension forming a steric layer around the droplet thereby stabilizing the emulsion [34]. Besides surface active, the emulsion properties of the polymer also depend on the molecular weight, Man/Gal ratio, interfacial property, and interactions of protein residue [33,34]. The intrinsic viscosity of a polymer is the hydrodynamic volume of an individual molecule in solution. The intrinsic viscosity of the seed galactomannan determined graphically by Huggins and Kraemer equations are presented in Fig. 2.C. The intrinsic viscosity polysaccharide obtained by combined Huggins and Kraemer extrapolation are 3.807, 3.424 and 3.331 dl g−1 for B. vahlii, D. elata, and P. pterocarpum, respectively. The intrinsic viscosity values were higher than the value for P. pterocarpum (3.14 dl g−1) reported by Nwokocha & Williams [26]. The intrinsic viscosity values are however lower than Delonix regia (9.9 dl g−1), guar gum (10.3 dl g−1), Prosopis juliflora (9.4 dl g−1), Detarium senegalense (8.9 dl g−1) polysaccharide [35–37]. The Huggins constant (k1) obtained from the slope were 0.280, 0.339 and 0.261 while the Kraemer constant (k2) calculated from the slope were −0.022, −0.017 and −0.046 for B. vahlii, D. elata, and P. pterocarpum, respectively. Huggins and Kraemer coefficient constants values depend on the solvent quality. The values of k1 in a flexible polymer chain lie between 0.2 and 0.8 depending on the polymer-solvent interactions and k1 b 0.5 is considered as a good solvent condition [21]. With increasing affinity between the polymer and solvent, the value of k1 decreases. This result is supported by Kraemer coefficient constant where negative k2 values depict good polymer solvation [38]. The viscosity average molecular weight of the seed galactomannans are 5.29 × 105, 5.29 × 105 and 4.95 × 105 for B. vahlii, D. elata, and P. pterocarpum, respectively which are lower than those reported in C. pulcherrima (2.79 × 106 Da), G. triacanthos (1.62 × 106 Da), A. pavonina (1.81 × 106 Da), S. japonica (1.34 × 106 Da) [13,39]. The differences probably depend on the

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extraction and purification processes which influence the intrinsic viscosity and, thereby, the viscosity average molecular weight. 3.3. Structural properties Fig. 3 presented the TEM and SEM analysis of the seed galactomannans. Transmission electron microscopy was carried out at 500 nm to study the microstructure morphology of the seed gums. Transmission electron microscopic studies of the seed polysaccharide suggested an aggregated amorphous, smooth particles which differ in shapes and size. The particles were oval or tubular shape, branched with compact surfaces. The surface topography of the powdered seed polysaccharides was also investigated by scanning electron microscopy. Scanning electron micrograph of the powered polysaccharide indicates well defined amorphous material with pores and cervices on the rough surface. The presence of pores and cervices on the particle surface can help in the entrapment of drug which is an important feature for drug release study [40]. The particles are seen as fibrous aggregates of nonuniform size and distribution. Due to the hydrophilicity property of the galactomannans, the increase in size and surface area of a particle increases the hydration capacity which in turn influences the intrinsic viscosity and molecular weight of the polymer. The size, shape and surface architecture of the galactomannan may be affected by the method of extraction and purification or preparation of the product [41]. The typical polysaccharide nature of B. vahlii, D. elata, and P. pterocarpum was also demonstrated by ATR-IR spectroscopy (Fig. 4. A). The spectra consist of broadband between 3600 and 3000 cm−1 with a significant peak at 3295 cm−1 which arises due to the O\\H stretch vibration of polymer and water involved in hydrogen bonding [42]. The absorption peaks observed between 3000 and 2800 cm−1 attributes to the different stretching vibration mode of C\\H bonds [43]. An absorption band around 2890 cm−1 occurs due to the symmetric C\\H stretch while the peak at 2980 cm−1 which is highly

Fig. 3. The micrographs of the galactomannan gums studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

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Fig. 4. (A) The structural analysis of the seed galactomannans was also evaluated using attenuated transmission resonance- infrared spectroscopy (ATR-IR) in the spectral region between 4000 to 500 cm−1; (B) X-ray diffraction (XRD) spectrum of the galactomannans isolates recorded between 10–60° at 2θ; (C) differential scanning calorimetry (DSC) scans of the seed galactomannans recorded between 25–300 °C at a rate of 10 °C min−1.

pronounced in B. vahlii arises due to the asymmetric C\\H stretch. A sharp band at 1640 cm−1 is due to the absorption of the amino group. Bands at 1417 and 1376 cm−1 are attributed to deformation in the C\\H stretching [43]. Coupling mode of C\\O in C–O–C (glycosidic) stretching and C–O–H (pyranose) bending vibration gives rise to bands at 1246, 1149 and 1075 cm−1. In the anomeric region (950–700 cm−1), the seed galactomannans exhibited characteristic absorption at 871 and 812 cm−1 due to the skeletal mode vibrations elucidating the presence of α-linked D-galactopyranose units and βlinked D-mannopyranose units, respectively [9] where the intensity difference between the two absorption bands is an indication of mannose and galactose content. The X-ray diffraction patterns of the seed galactomannan of B. vahlii, D. elata and P. pterocarpum confirm the amorphous structure by presenting a broad peak around diffraction angle 2θ = 20° (Fig. 4.B). The seed galactomannans also presented a scattering peak at around 44°. Similar patterns were observed in Gleditsia species by Jiang et al. [44]. When polysaccharides naturally interact with water, results in amorphous-crystalline transitions which have a significant impact the molecular mobility and functional properties [45]. The crystallinity index value of the seed galactomannan denoted by the alignment of molecules in a particle structure [43] is 0.288, 0.291 and 0.307 for B. vahlii, P. pterocarpum, and D. elata, respectively. The degree of crystallinity of the seed galactomannans was higher than native guar gum

(0.240), Gleditsia melanacantha (0.278), G. microphylla (0.285) and G. sinensis (0.297) but lower than Cassia grandis (0.334) [18,44,46]. The crystallinity of the material is attributed to the extraction and drying process which might have improved the chain organization within the molecules. 3.4. Thermal properties Thermal stability of the polysaccharide is a valuable property to determine the material suitable for food applications where thermal processing is required [43]. The physical and chemical changes during thermal processing of a polysaccharide as a function of temperature were assessed by differential scanning calorimetry (DSC) and thermogravimetric-differential thermal analysis (TG-DTA). The relative purity of a polysaccharide is depicted through a sharp symmetric melting endotherm whereas the broad asymmetric curve indicates impurities or presences of more than one thermal process. DSC has been instrumental in studying the phase transitions of polymers because of its accuracy and sensitivity. The thermal properties of the seed gums determined through DSC heating curves were presented in Fig. 4.C. The seed polysaccharides revealed a glass transition temperature (Tg) at around 90 °C for B. vahlii, D. elata, and P. pterocarpum, respectively which occurred due to loss of free or trapped moisture in the sample. The Man/Gal ratio and molecular weights significantly influence the

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polysaccharide. The second weight loss event marks with an onset weight loss at 285, 293 and 297 °C and maximum oxidation exhibiting at 321, 316 and 319 °C for B. vahlii, D. elata, and P. pterocarpum, respectively. These values are in agreement with Cerqueira et al. [7] for G. triacanthos, C. pulcherrima and A. pavonina seed galactomannan with the values at 309.81, 321.73 and 320.62 °C, respectively. The thermal decomposition pattern of the seed galactomannans exhibits good thermal stability which can tolerate most temperatures applied in the thermal treatment of food products such as sterilization and pasteurization. The differential thermal analysis (DTA) curves for the seed gums reveals the transition temperature at about 90 °C. The dehydration, depolymerization and decomposition steps in DTA conform with the DSC curves of the galactomannans. Since polysaccharides majorly have carboxylate or carboxylic acid functional groups, the evolution of CO2 from the corresponding carbohydrate backbone as a result of thermal scission of the carboxylate groups may be a probable mechanism for the thermal transitions [47]. 4. Conclusion The present investigation evaluates the extraction and characterization of water-soluble seed galactomannans from the Leguminosae taxa of Bauhinia vahlii, Delonix elata, and Peltophorum pterocarpum. The galactomannans were extracted from the endosperm with distilled water followed by subsequent ethanol gradient precipitation. The yield was 30.82, 24.01 and 25.25% with a Man/Gal ratios of 4.21:1, 2.55:1 and 3.03:1 for B. vahlii, D. elata, and P. pterocarpum, respectively. The seed galactomannan exhibited a β-1-4-linked mannose linked with D-galactose units where the galactose side chains are attached at the C6 position. The functional properties of the seed galactomannans demonstrated adequate characteristics which can be explored in the food industry. The thermal decomposition pattern of the seed galactomannans exhibited a good thermal stability. Thus, based on the yield, physicochemical, structural and functional properties, this study demonstrates that the non-conventional sources of galactomannans from B. vahlii, D. elata, and P. pterocarpum could be an ideal candidate for industrial applications. Declaration of Competing Interest Authors have no conflict of interest to declare. Acknowledgment Fig. 5. The thermogravimetric-differential thermal analysis (TG-DTA) curves of the seed galactomannans recorded at a heating rate of 10°C min−1 from 40–600°C under a nitrogen atmosphere.

thermal behavior of the galactomannan [7]. High mannose content in the polysaccharide sample indicates a lower branching and increased binding energy between the backbones of the monosaccharides. The second peak was observed at around 260 °C due to the cleavage of the glycosidic linkage. Fig. 5 represents the TGA and DTA pattern of B. vahlii, D. elata, and P. pterocarpum seed galactomannans, respectively. TGA helps in analyzing the thermal stability and decomposition pattern of the polysaccharide. The result of TGA curves of the galactomannan isolates revealed three events loss of mass as earlier reports [47,48]. The first step of mass loss takes place at 121, 127 and 131 °C for B. vahlii, D. elata, and P. pterocarpum, respectively which occurs due to desorption or loss of moisture bound to the sample associated to the hydrophilic nature of the polysaccharide functional groups. The first step of mass loss are 11.56, 11.08, and 10.97% for B. vahlii, D. elata, and P. pterocarpum, respectively which are in good agreement with the moisture content of the polysaccharide. Post-hydration, the second and the significant loss of weight event is attributed to the thermal degradation of the

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