Emulsifying and physicochemical properties of soy hull hemicelluloses-soy protein isolate conjugates

Emulsifying and physicochemical properties of soy hull hemicelluloses-soy protein isolate conjugates

Carbohydrate Polymers 163 (2017) 181–190 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/c...

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Carbohydrate Polymers 163 (2017) 181–190

Contents lists available at ScienceDirect

Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Emulsifying and physicochemical properties of soy hull hemicelluloses-soy protein isolate conjugates Li Wang a,b,1 , Min Wu a,b,1 , Hua-Min Liu a,∗ a Province Key Laboratory of Transformation and Utilization of Cereal Resource & College of Food Science and Technology, Henan University of Technology, Zhengzhou 450001, China b Institute of Physical Science and Engineering, Zhengzhou University, Zhengzhou 450001, China

a r t i c l e

i n f o

Article history: Received 23 August 2016 Received in revised form 10 January 2017 Accepted 18 January 2017 Available online 21 January 2017 Keywords: Soy hull hemicelluloses Maillard reaction Soy protein isolate Emulsification

a b s t r a c t Protein-polysaccharide conjugates could potentially combine the excellent emulsification properties of the protein with the stabilizing effect of the polysaccharide. The investigation aimed to prepare soy hull hemicelluloses-soy protein isolate (SHH-SPI) conjugates by Maillard reaction in a controlled dry state condition and assess the suitability of the conjugates in stabilizing oil-in-water (O/W) emulsion. Results indicated that Maillard reactions occurred between amino groups and carbonyl, resulting in consumption of some functional groups and the appearance of new groups in the conjugates. The conjugates of SHHSPI obtained at the SPI content of 30% and 40% exhibited substantially improved emulsification capacity in maintaining the physical stability of O/W emulsions for a prolong storage period at heat treatment, compared with SHH and SPI alone. Overall, these results demonstrated that SHH and SPI could generate novel emulsions with improved physical and chemical stability by Mallsird reaction for application in food and pharmaceutical products. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction Many proteins are highly effective emulsifiers because they contain both charged hydrophilic regions and hydrophobic regions, which lower the surface tension and interact at the emulsion interface (Kasran, Cui, & Goff, 2013). Soy protein is an abundant byproduct of the soybean oil industry, and is commonly used as a nutritional additive in food (Su et al., 2012). Various methods based on enzymatic, chemical, physical, and genetic modifications were investigated to improve the functional properties of proteins (Seo, Karboune, Yaylayan, & L’Hocine, 2012). Among the several methods, a great deal of attention has been focused on the covalent interaction protein/polysaccharide via the Maillard reaction (Corzo-Martínez, Sánchez, Moreno, Patino, & Villamiel, 2012). Protein-polysaccharide conjugates could potentially combine the excellent emulsification properties of the protein with the stabilizing effect of the polysaccharide (Shepherd, Robertson, & Ofman, 2000). The main advantages of covalent protein/polysaccharide conjugates as compared with non-covalent complexes are the retention of molecular integrity and solubility over a wide range of

∗ Corresponding author. E-mail address: [email protected] (H.-M. Liu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.carbpol.2017.01.069 0144-8617/© 2017 Elsevier Ltd. All rights reserved.

experimental conditions (Dickinson & Galazka, 1991). In addition, the covalent conjugates have been shown to be very stable with the changes of temperature, ionic strength, and pH (Kasran, Cui, & Goff, 2013). Soy hulls, the major byproducts obtained in the soybean processing industry, and the insoluble carbohydrate fraction contains 50% hemicelluloses, 30% pectin, and 20% cellulose (Liu et al., 2013). Hemicelluloses have a very wide variety of applications and can be converted into various biopolymers by modification (Peng, Peng, Xu, & Sun, 2012). Therefore, the soy hulls were potentially inexpensive commercial sources of hemicelluloses. However, to best of our knowledge, there is little work carried out on forming SHH-SPI conjugates to improve the emulsifying properties of soy protein isolate by Maillard reaction. Maillard reaction is a reaction between the primary amine of proteins and the reducing end of carbohydrates, which generally regarded as an efficient and safe method to improve functional properties of proteins, such as emulsifying properties, heat stabil˜ ity, and antioxidant activity (Guo & Xiong, 2013; Jiménez-Castano, ˜ 2007; Sun, Hayakawa, Puangmanee, & Villamiel, & López-Fandino, Izumori, 2006). During the reaction, the conjugates of the carbohydrate and protein occur spontaneously under heating conditions without the utilization of toxic chemical products (Chevalier, Chobert, Dalgalarrondo, & Haertlé, 2001). In addition, it is well known that Maillard reaction carried out under dry state and well controlled conditions (such as reaction temperature, rela-

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tive humidity, and reaction time), which is an adequate method to improve functionality of proteins without important structural changes (Corzo-Martínez et al., 2012; Oliver, 2011). Overall, the objective of this investigation was to assess the suitability of soy hull hemicelluloses-soy protein isolate (SHH-SPI) conjugate prepared by controlled dry heating of SPI and SHH mixtures in stabilizing O/W emulsion. The effect of SPI and SHH weight ratios before conjugation on the emulsion stability were investigated by droplet size analysis and visual observation of these stabilizing O/W emulsions. Further, the conjugations were characterized by FT-IR, TGA, amino acid analysis, and color analysis to help understand the emulsification properties of the complexes from the structural perspective. 2. Materials and methods 2.1. Materials Soy hulls were obtained from Henan Sunshine Oils and Fats Group CO., Ltd. (Xingyang, China). The soy hulls were firstly ground by using a high-speed rotary cutting and then sieved though 40 mesh. The powder was dewaxed with toluene/ethanol (2:1, v/v) in a Soxhlet for 6 h. The dewaxed sample was dried at 60 ◦ C for 24 h and kept in desiccators at room temperature before used. Soy protein isolate (SPI) was purchased from Beijing Aoboxing Bio-Tech CO., Ltd. (Beijing, China). The protein, water, and ash contents of the SPI sample were 85%, 7%, and 3.2%, respectively. All chemicals and solvents used were analytical grade and used without further purification. Deionized water was used throughout. 2.2. Isolation of soy hull hemicelluloses Soy hull hemicelluloses were extracted from the soy hulls powder by hydrothermal treatment as described previously (Liu, Wang, & Liu, 2016). Briefly, a pressure glass reactor (volume 100 ml) was loaded with soy hulls and water with a solid to liquid ratio of 1:10. Agitation was set at 500 rpm and kept constant for all experiments. The reactor was heated up to the setting temperature by a magnetic heating stirrer (IKA, Germany) at a heating rate of approximately 3 ◦ C/min, and the temperature was maintained constant at the setting temperature for the desired holding time. After the extraction was complete, the reactor was cooled down to room temperature by air. When the reactor was opened, the solid and liquid mixture was removed for separation. The mixtures were separated by filtration though filter paper under vacuum, and 200 ml of deionized water was used for washing the solid products. The filtrate was concentrated to approximately 30 ml on a rotary evaporator under reduced pressure at 45 ◦ C. The water-soluble fraction was then recovered by precipitation of the concentrated water extracts in 3 vols of 95% ethanol. The precipitates formed were recovered by filtration, washed with acidified 70% ethanol, and obtained soy hull hemicelluloses. The soy hull hemicelluloses were stored in desiccators at room temperature. 2.3. Preparation of conjugates The SHH-SPI conjugates were prepared by the method of described previously (Kasran, Cui, & Goff, 2013) with some modifications. Briefly, soy protein isolate (10%, w/w) and soy hull hemicelluloses (10%, w/w) were dispersed in water with stirring for 4 h at room temperature, respectively, followed by pH adjustment to 7.0 and storage at 4 ◦ C overnight. The SPI and SHH solutions were then mixed at SPI:SHH ratios of 1:9 (SPI content of 10%), 2:8 (SPI content of 20%), 3:7 (SPI content of 30%), 4:6 (SPI content of 40%), 5:5 (SPI content of 50%), and 6:4 (SPI content of 60%), respectively (g:g, dry weight basis). The mixed solutions were freeze dried

and milled to make a powder (60 m), to yield SPI and SHH mixture. A desiccators containing saturated NaCl was placed in the oven at 60 ◦ C for 30 min to achieve an equilibrium temperature and relative humidity. And then the SPI and SHH mixture was placed in the desiccators in the presence of saturated NaCl, heated at 60 ◦ C for seven days, to induce Maillard reaction. The reaction mixture of SHH and SPI was collected and defined as SHH-SPI conjugate. The conjugates were sealed and stored at 4 ◦ C until further use. 2.4. Emulsion preparation For the preparation of O/W emulsions, samples of SPI, SHH, or SHH-SPI conjugate (0.5%, w/w) were dissolved in 1 mol/l sodium dihydrogen phosphate solution with stirring at 500 rpm for 3 h at room temperature. And then 10 ml of soy oil was added gradually to 40 ml 0.5% (g/l, w/v) amount of samples in 1 mol/l sodium dihydrogen phosphate solution and homogenized for 2 min at 10000 rpm by using a high-shear homogenizer (FA 25 model, Fluko Equipment Co., Ltd., Shanghai, China) at room temperature. 2.5. Droplet size measurement The droplet size distribution and volume-average droplet size of various O/W emulsions were determined by a laser light scattering technique, using a Zetasizer Nano (Malvern Instruments Ltd, Worcestershire, UK). The process underlying the operation of this instrument was detailed previously (Xu, Wang, Jiang, Yuan, & Gao, 2012). Distilled water was used as the dispersant and the relative refractive index of the emulsion was 1.59, i.e. the ratio of the refractive index of soy oil (1.496) to that of the aqueous medium (1.33). Emulsions were diluted to a final oil droplet concentration of 0.005% wt% with buffer solution (pH 7.0) and filtered prior to each measurement to minimize multiple scattering effects. The measurements for each emulsion were performed on at least two separately preparedly prepared samples, with each sample measured repeated at least three times. The results were described as cumulants mean diameter (size, nm) for droplet size, polydispersity index (PdI) for droplet size distribution. 2.6. Optical microscopy The microscopic observations were performed using optical microscopy (BT-1600, Dandong Bettersize Instruments Ltd., China) to compare the microstructure difference among SHH-SPI conjugates stabilized emulsions. A drop of emulsion sample was placed on a microscope slide, covered with a cover slip. Images were made immediately after preparation of emulsion. 2.7. Color analysis The method of color analysis was the same as those used by previously (Díaz, Candia, & Cobos, 2016). Briefly, these samples were conducted at 25 ◦ C using a Color Difference Meter (INESA, Shanghai, China) in the reflectance mode. Color was expressed in L∗ , a∗ and b∗ values. The L∗ value is a measurement of lightness and varies from 0 (blank) to 100 (white); the a∗ value varies from −100 (green) to +100 (red); and the b* value varies from −100 (blue) to +100 (yellow). Three measurements were performed and results were averaged. In addition, total color difference (E) was calculated using the following equation: E =



(L∗ − Lo∗ )2 + (a∗ − ao∗ )2 + (b∗ − bo∗ )

2

Where Lo ∗ , ao ∗ , and bo ∗ are the values for the SHH and SPI.

L. Wang et al. / Carbohydrate Polymers 163 (2017) 181–190

2.8. Fourier transforms infrared spectrometer analysis Fourier transform infrared spectrometer (FT-IR) spectra of the samples were recorded from an FT-IR spectrophotometer (Nicolet 510) using a KBr disk containing 1% finely ground samples. The spectra were collected in absorbance mode on sample powders obtained by grinding pellets in an agate mortar and placed directly onto the attenuated total reflectance crystal. Thirty-two scans were taken of each sample recorded from 4000 to 400 cm−1 at a resolution of 2 cm−1 in the transmission mode. All spectra were smoothed using the Savitzky-Golay function. 2.9. Thermal analysis The thermal stability of SPI, SHH, and SHH-SPI conjugates were investigated using a thermogravimetric analyzer (Shimadzu, Japan). The mass of the sample used varied from 3 to 5 mg. Thermogravimetric analysis (TGA) was carried out under the protection of nitrogen. The temperature of the sample was increased from room temperature to 600 ◦ C at a heating rate of 10 ◦ C/min. Weight loss of the sample was measured as a function of temperature. 2.10. Amino acid analysis The free amines in SHH-SPI mixture and the conjugate were quantified as described by published paper (Guo & Xiong, 2013) with slight modifications. A 1–10 mg of samples were weighed and mixed with 0.3 ml of hydrolysis acid (6 M HCl, 0.2% v/v phenol, 1nmole norvaline as internal standard). After flame-sealing the tube under vacuum, the sample was hydrolyzed at 110 ◦ C for 16 h. After cooling, the cooled and filtered hydrolyzate was dried in vacuum desiccators at 45 ◦ C and re-dissolved in citrate buffer (pH 2.2). Measurement of the amino acid content was carried out by an automatic amino acid analyzer (S-433D, Sykam CO., Fürstenfeldbruck, Germany). Identification and quantification of amino acids were achieved by comparing the retention times of the peaks with those standards. 2.11. Statistical analysis Each experiment was repeated twice or more and presented as mean ± standard deviation. Analysis was performed using the SPSS package (SPSS 11.0 for windows, SPSS Inc., Chicage, IL, USA).The statistically significant differences were determined for significance (P ≤ 0.05) according to Duncan’s multiple range test. 3. Results and discussion 3.1. Composition of hemicelluloses and its fractions The soy hull hemicelluloses were obtained from soy hull by hotcompressed water extraction at 130 ◦ C, ethanol precipitation, and lyophilized in freeze-dry apparatus. The total yield of the hemicelluloses was 10.5% (lyophilized weight, w/w) of the dried material (Liu, Wang, & Liu, 2016). To obtain high-purity hemicelluloses for raw material analysis, the hemicelluloses were firstly fractionated on Cellulose DEAE-52 column. The elution profile indicated that the hemicelluloses were mainly composed of H-1, H-2, and H-3 (Fig. S1 in Supplementary data). The protein content and monosaccharide compositions of hemicelluloses and its fractions (H-1, H-2 and H-3) were shown in Table 1. As shown, the protein content in the hemicelluloses accounted for 10.0%. In this investigation, the protein did not remove because of the protein in the hemicelluloses was used as a reactant in the technology. The monosaccharide were identified as mainly of the arabinose (32.1%), and galactose (22.6%), and galactose (22.6%) with a small amount of glucose and xylose,

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Table 1 Monosaccharide and protein compositions of soy hull hemicelluloses and the purified fractions (H-1, H-2, and H-3). Composition (%)

Hemicelluloses

32.1 ± 0.4 22.6 ± 0.6 3.4 ± 0.3 4.6 ± 0.4 27.3 ± 0.5 10.0 ± 0.7

Arabinose Galactose Glucose Xylose Mannose Protein

Fractions H-1

H-2

H-3

4.5 ± 0.4 22.8 ± 0.4 5.0 ± 0.4 0.0 ± 0 67.7 ± 0.7 –

40.6 ± 0.5 35.4 ± 0.3 10.1 ± 0.3 13.9 ± 0.4 0.0 ± 0.5 –

40.9 ± 0.3 42.2 ± 0.6 3.1 ± 0.1 13.8 ± 0.3 0.0 ± 0 –

Table 2 Color values (L∗ , a∗ and b∗ ) and total color difference (E) of SHH, SPI, and the conjugates. Samplesa

L∗

a∗

b∗

EH

EP

SHH SPI C10 C20 C30 C40 C50 C60

72.3 ± 0.3 77.5 ± 0.3 62.7 ± 0.3 53.4 ± 0.1 50.5 ± 0.4 48.2 ± 0.3 44.4 ± 0.2 43.1 ± 0.1

0.2 ± 0.0 −1.3 ± 0.0 4.4 ± 0.1 7.0 ± 0.1 7.2 ± 0.1 7.3 ± 0.1 8.1 ± 0.1 7.6 ± 0.1

9.6 ± 0.1 18.4 ± 0.1 23.6 ± 0.1 27.2 ± 0.2 26.3 ± 0.2 25.3 ± 0.2 24.6 ± 0.1 23.3 ± 0.2

0 – 17.6 ± 0.2 26.8 ± 0.1 28.5 ± 0.2 29.7 ± 0.1 32.7 ± 0.1 33.1 ± 0.2

– 0 16.7 ± 0.1 26.9 ± 0.1 29.4 ± 0.1 31.3 ± 0.2 34.9 ± 0.2 35.8 ± 0.1

a C10, C20, C30, C40, C50 and C60 represent the conjugates obtained at the SPI content of 10%, 20%, 30%, 40%, 50%, and 60%.

which suggested that the hemicelluloses from soy hulls by hotcompressed extraction were composed mainly of arabinofuranosyl type polysaccharides (Liu, Wang, & Liu, 2016). The purified polysaccharide H-1 mainly consisted of mannose and galactose, whereas the major neutral sugar composition of H-2 and H-3 were arabinose and galactose, respectively. 3.2. Color analysis of conjugates Maillard reaction is a nonenzymatic reaction between carbonyl groups and amino groups, and it develops into a complex set of reactions that lead to significant changes in the color, flavor and structure. Color is an important characteristic of evaluation the extent of Maillard reaction and main attributes that is strongly associated with the concept of quality of a food product. Table 2 shows the CIE Lab color values (L∗ , a∗ and b* ) and total color difference (E) of SHH, SPI and their conjugates by Maillard reaction in the dry state. As shown, the color of the conjugates was significantly affected by the ratio of SHH-SPI. The SHH and SPI showed significant higher values of luminosity (L∗ ) than their conjugates by Maillard reaction. These conjugate samples were higher in a∗ and b∗ values as compared with the SHH and SPI. Significant increment of EH and EP values were observed with the increasing the ration of SHH-SPI. These results showed that an increase in reactive soy protein could accelerate the rate of Maillard reaction. This was due to that an increase in protein concentration for this investigation led to an increase of reactive intermediates which, together with other reactive precursors, condense and polymerize to form brown polymers at the final stage of Maillard reaction. 3.3. FT-IR analysis of conjugates FT-IR spectroscopy is a useful technique for studying proteincarbohydrate interactions and has been used to investigate the characteristics of Maillard reaction conjugates (Yang et al., 2015). The FT-IR spectrum of conjugates obtained at the SPI content of 30% is shown in Fig. 1 in comparison with those SPI and SHH. As shown from Fig. 1a, SPI has typical infrared absorption bands at 1630–1680 cm−1 , 1530–1560 cm−1 , and 1260–1420 cm−1 for

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a Transmittance ( %)

1448 1638 1530

b c

1388

1075

1238

889/896

1246

1739

1413

806

1310 1045

1598 1546

762

1410

1045

1638

2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 -1

Wavenumbers (cm )

900

800

700

Fig. 1. FT-IR spectra of SPI (a), SHH (b), and the conjugate (c) obtained at the SPI content of 30%.

amide I, amide II, and amide III, respectively (Qu, Huang, Wu, Sun, & Chang, 2015). The stretching vibration mode of SPI observed at 1638 cm−1 attributes to C O bands for amide I. The bending vibration mode of SPI at 1530 cm−1 belongs to the N H for amide II. The absorption band at 1240–1470 cm−1 was due to C N stretching and N H bending (amide III) vibrations. The bending vibration mode of SPI at 1388 cm−1 belongs to the C N for amide III (Qu, Huang, Wu, Sun, & Chang, 2015). The absorption band at 1238 cm−1 is attributable to C H vibration in peptide bonds (Teng, Luo, & Wang, 2012). As shown from Fig. 1b (SHH), the presence of signal at 1740 cm−1 is assigned to the acetyl and uronic ester groups or the ester linkage of the carboxylic groups in the hemicelluloses (Ma et al., 2012). The absorptions at 1413, 1310, and 1246 cm−1 are attributed to the C H and C O bending or stretching. The peak at 1045 cm−1 is assigned to the C O, C C, and glycosidic (C O C) stretching of xylans. In addition, glucan gives signal at 1081 and 1012 cm−1 corresponding to the glycosidic (C O C) stretching (Pal, Mal, & Singh, 2005). As can be seen from Fig. 1c, the conjugate reserved characteristic bands of both SPI and SHH, and showed some new features. The frequencies of the bands assigned to the amide I and II regions remained constant at 1650 cm−1 and 1540 cm−1 , respectively. However, the band assigned to carboxylate in the hemicelluloses at 1598 cm−1 was not detectable in the conjugate, indicating interactions between the protein and the hemicelluloses and highlights the lower capacity of the carbohydrate molecules to form intermolecular hydrogen bonds between themselves in presence of the protein (Carpenter & Crowe, 1989). In the amide I region, the intensity of the band at 1630–1650 cm−1 increased, which referred to typical Maillard reaction products, i.e. Schiff’s base imine group (stretching) and enaminol group (stretching), respectively (Wnorowski & Yaylayan, 2003). The result confirmed the formation of covalent bands between the amino groups and carbonyl. Interestingly, the intensity of the bands in the amide II region decreased, which is in good agreement with previously reports (Gu et al., 2010; Yang et al., 2015). It is also noteworthy that the absorptions at 1395 and 1448 cm−1 in SPI and the band at 1739 cm−1 in SHH were disappeared and became weaken in the conjugate, respectively. Maillard reactions would result in consumption of some functional groups and the appearance of other groups. For example, −NH2 groups may be decreased or lost, while the amount or groups associated with products origin from Maillard reactions

such as the C N, C O and C N may be increased by the Marillard reactions (Hui et al., 2015).

3.4. Thermal analysis of conjugates Thermal gravimetric analysis (TG) can be employed to determine temperatures and rates of pyrolysis, while differential thermal analysis (DTG) curves show the exothermic or endothermic nature of the reactions that accompanied with the pyrolysis and combustion. Such information is very valuable in assessing the chemistry of the thermal decomposition of different macromolecule. The thermal properties of the SPI, SHH, and the conjugates, were investigated by TG and DTG, and their curves are shown in Fig. 2. As can be seen from the figure, the weight loss of all samples occurred at the beginning is attributed to the evaporation of water. The results indicated the water probably adsorbed on the surface of the materials. As can be observed from Fig. 2A, the SPI began to decompose at 202 ◦ C, and the maximum rate of weight loss was observed at 298.5 ◦ C and 315.4 ◦ C. During the degradation, non-covalent bonds, including intermolecular and intramolecular hydrogen bonds and electrostatic bonds, and hydrophobic interaction decomposed, then covalent bonds between C N, C (O) NH, C (O) NH2 of amino acid residues were broken as temperature increased (Liu, Li, & Sun, 2015). The protein backbone was completely decomposed and released various gases, such as CO, CO2 , and NH3 (Das, Routray, & Nayak, 2008; Schmidt, Giacomelli, & Soldi, 2005). From Fig. 2B, the hemicellulosic fraction began to decompose at 198 ◦ C. Beyond the temperatures, the thermal degradation takes place and the major decomposition temperatures of the sample occurred between 198 and 350 ◦ C. In this stage, the DTG showed two degradation peaks: the first is at lower temperature (at 260.4 ◦ C), related to initial hemicelluloses loss, and the second (323.3 ◦ C) related the organic material oxidation (Zafeiropoulos, Baillie, & Matthews, 2001). When the temperature is above 350 ◦ C, the slope in the TGA cure changes and weight loss slows down, which might be due to the further breakage of the hemicelluloses and the small amount flammable gas (Soliman, El-Shinnawy, & Mobarak, 1997). While at 50% weight loss, the decomposition temperature was occurred at 351.9 and 280.9 ◦ C for SPI and SHH, respectively. These results indicated that the thermal stability of SPI is higher than the SHH.

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Fig. 2. Thermograms (TG and DTG) of soy hull hemicelluloses, soy protein, and their conjugates (A-SPI, B-SHH, C-conjugate obtained at the SPI content of 10%, D-conjugate obtained at the SPI content of 20%, E-conjugate obtained at the SPI content of 30%, F-conjugate obtained at the SPI content of 40%, G-conjugate obtained at the SPI content of 50%, and H-conjugate obtained at the SPI content of 60%).

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Table 3 Amino acid composition of SHH-SPI mixture (SPI content of 30%) and the conjugate. Amino acid (%, dry weight basis)

SPI-SHH mixture

Conjugates

Asparaginic acid Threonine Serine Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Proline Total amino acid

3.4 ± 0.04 0.4 ± 0.03 1.0 ± 0.01 3.6 ± 0.09 0.8 ± 0.02 1.3 ± 0.03 0.8 ± 0.01 0.1 ± 0.01 1.0 ± 0.02 1.2 ± 0.04 0.6 ± 0.02 0.9 ± 0.01 0.7 ± 0.01 1.1 ± 0.03 1.4 ± 0.02 1.7 ± 0.05 20.0 ± 0.43

2.9 ± 0.06 0.3 ± 0.00 0.8 ± 0.02 3.1 ± 0.05 0.8 ± 0.02 0.7 ± 0.01 0.6 ± 0.01 0.1 ± 0.00 0.8 ± 0.01 1.0 ± 0.02 0.5 ± 0.01 0.9 ± 0.02 0.6 ± 0.01 0.8 ± 0.01 1.2 ± 0.03 1.3 ± 0.02 16.4 ± 0.33

The total loss rate of amino acid in this investigate was much lower as compared with the protein-small carbohydrate molecule systems (Rufian-Henares, Guerra-Hernández, & Garcı´ıA-Villanova, 2002). However, the conjugate of proteins with small carbohydrate molecules such as glucose or lactose is liable to react with most lysyl residues exposed outside and to result in insoluble aggregates due to the progressive side reaction (Muppalla, Sonavale, Chawla, & Sharma, 1997). To improve the functional properties of the conjugate, the conjugate with polysaccharides was desirable for industrial applications (Muppalla, Sonavale, Chawla, & Sharma, 1997). Maillard-induced glycosylation occurs primarily at the ␧amino group of the lysine residues, but can also occur to a lesser extent at the imidazole group of histidine, the indole group of tryptophan, the guanidine group of arginine residues, and at the N-terminus of the protein (Ames, 1992). As shown from Table 3, the asparaginic acid, glutamic acid, alanine, and proline were the dominating amino acid resides reacting with SHH. 3.6. Emulsifying properties of conjugates

For the conjugates of SPI and SHH (As can be seen from Fig. 2C–H), there are 3 steps of thermal degradation of the conjugates in the temperature range of 30–600 ◦ C. In the first stage in TG curves, observed at room temperature to 140 ◦ C, is related to the loss of adsorbed and bounder water. It is possible to observe some differences when compared with TG results for the conjugates and their individual components. Moisture seems to be lost at higher temperatures (at peaks of 55–90 ◦ C) for the conjugates than for SPI (at peak of 55 ◦ C) and SHH (at peak of 55 ◦ C) samples, which may be explained on basis of a greater amount of water binding taking place in the conjugates through hydrogen bonding with protein and hemicelluloses. The temperature range for the second step of thermal degradation is 150–500 ◦ C. This corresponds to the decomposition of SPI and SHH from the conjugates. The third step of thermal degradation in the temperature range of 500–600 ◦ C might be due to oxidation of partially decomposed SPI under air flow. During the whole temperature range, the maximum mass loss rates of the conjugate samples were lower than that of SPI and SHH. With the increase of SPI content, the maximum mass loss rate decreased, and the temperature at the maximum weight loss rate on the DTG curve shifted from 264.1 ◦ C to 308.5 ◦ C, suggesting the stability of the conjugates increased. Addition, it can be clearly noted that the conjugates at the SPI contents from 20 to 60% have higher content of residues (31.3–45.6%) than that of SPI and SHH, which presumed due to the higher thermal stability of the conjugates. The possible reason for the thermal stability increased was that SHH reacted with those groups on an unfolded protein surface by Maillard reaction, so cross-linkages due to protein–protein interaction increased. These results support a conclusion that increase the content of protein in the conjugates was beneficed to improvements thermal stability. 3.5. Analysis of free amino acid groups Glycosylation occurred by the covalent attachment of carbonyl groups in reducing sugars with free amino groups in proteins to form Schiff base. Therefore, the loss of available primary amino group is an indicator used to compare the sugar reactivity in the Maillard reaction. The amino acid compositions of SHH-SPI mixture and the conjugate obtained at protein content of 30% are presented in Table 3. The decrease in free amino group content and the browing (From Table 1) indicated that SPI was successfully linked with SHH under the conditions investigated. As shown from Table 3, the conjugate lost 3.6% of the available amino groups, confirming a higher degree of Maillard conjugation as compared with previously reported free amine loss value (1.4%) during whey protein isolate-dextran conjugates process (Wang & Ismail, 2012).

Fig. 3 shows the z-average droplet size, distribution, and microphotographs of fresh emulsions stabilized by SHH-SPI Maillard reaction products, SPI, and SHH. In general, the fine emulsion is relatively more uniform in droplet size as compared with the coarse emulsion. As shown, all SPI/SHH conjugates could form emulsions with monomodal droplet distributions and with average droplet size below 400 nm (d3,4 ). In terms of average droplet size and polydispersity, it was found that emulsions stabilized by the conjugates obtained at the SPI content of 30% and 40% showed much better emulsion abilities when compared with other conjugate samples, SPI, and SHH stabilized emulsions. One noteworthy point was that the emulsion stabilized by conjugate obtained at the SPI content of 60% had the larger mean droplet size of 383 nm in comparison to the SPI and SHH stabilized emulsions which had mean droplet size of 262 nm and 305 nm, respectively. It was mentioned that average droplet size (d4,3 ) represents the particle diameter which is responsible for the volume mean of the system. In fact, the influence of particles with larger diameter on the total volume of the system is more pronounced even when they constitute fewer numbers (Farzi, Emam-Djomeh, & Mohammadifar, 2013). Therefore, this parameter (d4,3 ) is more influenced by the largest particles in the system. According to the results by color and thermal analysis of conjugates, the higher ratio of protein in conjugates may accelerate the rate of Maillard reaction and made protein–protein interaction increase. Therefore, the unabsorbed composition in the conjugates maybe increase and these unadsorbed compositions could create large sizes (Farzi, Saffari, Emam-Djomeh, & Mohammadifar, 2011; Mohammadifar, Musavi, Kiumarsi, & Williams, 2006). Aggregations and large flocs in the emulsions stabilized by conjugates obtained at the SPI content of 50% and 60% were also observed by the microphotographs (Fig. 3). The optimum ration for better emulsion abilities also depends on the types of protein and polysaccharide employed. Akhtar and Dickinson (Akhtar & Dickinson, 2007) reported that the optimum ratio of whey protein/maltodextrin conjugates was 1:1 and 1:2. In other investigations with Maillard-type conjugates of bovine serum albumin/dextran (Dickinson & Semenova, 1992) and whey protein/dextran (Akhtar & Dickinson, 2003), the optimum protein/polysaccharide ratio was around 1:3. Based on the above results and supported by other work, 30% and 40% protein contents in conjugates were therefore chosen for the further test of emulsion stability. 3.7. Stability of conjugates stabilized emulsions Emulsions are inclined to break down over time through a variety of polysicochemical mechanisms, including floccula-

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Fig. 3. Z-average droplet size, distribution, and microstructure of various fresh emulsions stabilized by SHH-SPI Maillard reaction products (A-conjugate obtained at the SPI content of 10%, B-conjugate obtained at the SPI content of 20%, C-conjugate obtained at the SPI content of 30%, D-conjugate obtained at the SPI content of 40%, E-conjugate obtained at the SPI content of 50%, and F-conjugate obtained at the SPI content of 60%), SPI (G), and SHH (H).

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Fig. 4. Z-average droplet size and distribution in emulsions stabilized by SPI, SHH, and the conjugates obtained at the SPI content of 30% (C30) and 40% (C40) during storage at 60 ◦ C.

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Fig. 5. Microstructure of emulsions stabilized by SPI (III), SHH (IV), and their conjugates obtained at SPI contents of 30% (I) and 40% (II) after various days of storage at 60 ◦ C.

tion, gravitational separation, coalescence and Ostwald ripening (Ettoumi, Chibane, & Romero, 2016). Their will develop into two layers, cream (consisting of oil droplets) and serum. The creaming rate is affected by many variasles, e.g., droplet size, flocculated state of oil droplets, interaction between interfacial emulsifier and water phase, and even inter-droplet interactions. Fig. 4A–H shows the changes of droplet size in oil/water emulsions stabilized by SPI, SHH, and their conjugates during storage at pH 7.0 and 60 ◦ C. Initially, all the particle size distributions (except SPI) were unimodal, and the diameter of droplets were 139, 158, 262, and 305 nm for conjugates obtained at the SPI content of 30% and 40%, SPI, and SHH stabilized emulsions, respectively. Comparing the average droplet size after storage various days, it was observed that as the storage time increased, the average droplet size of all studied emulsions decreased. For SPI- and SHH-stabilized emulsions, the average droplet sizes only increased slightly after 4 days of storage. However, sharp increase in droplet sizes were observed on storage for up to 5 days, and bi-modal distribution were observed after 4 days. In contrast, the particle size distribution of SHH-SPI conjugatestabilized emulsions remained mono-modal distribution for up to 6 days, and the average droplet size of conjugate-stabilized emulsions remained smaller as compared with SPI- and SHH-stabilized emulsions during the whole storage period. The excellent emulsification capacity exhibited by their conjugates from Maillard reaction of SPI and SHH was resulted from the combination of the steric stabilizing effect of attached SHH and the good emulsifying property of SPI, which could effectively prevent re-coalescence of oil droplets (Dickinson & Galazka, 1991; Chiu, Chen, & Chang, 2009). Diftis and Kiosseoglou (Diftis & Kiosseoglou, 2003, 2006) reported that soy protein/dextran or soy protein/carboxymethyl cellulose conjugates could reduce the droplet-to-droplet interactions in oil/water emulsions, which prevented droplet coalescence and creaming.

3.8. Microstructure To further investigate the role of conjugates of SPI and SHH in the stability of oil/water emulsion, the microstructure of oil/water emulsions, prepared with the conjugates obtained at the SPI 30% and 40%, SPI, and SHH after 0, 2, 4, and 6 days storage at 60 ◦ C were shown in Fig. 5. Microstructure exhibited some difference among SPI, SHH, and conjugates stabilized emulsions after storage. As shown, the emulsions stabilized by SPI and SHH appeared flocculation and coalescence after 4 or 6 days, which appeared empty spaces between the droplets which were in accordance with the bimodal droplet size distribution (as shown in Fig. 4). By comparison, the conjugate-stabilized emulsions showed no flocculation and homogeneous droplet which was in accordance with the bimodal droplet distribution. The results also indicated that the conjugates have much more effective emulsion stabilities as compared with untreated SPI and SHH. 4. Conclusion The current investigation prepared SHH-SPI conjugates by controlled dry heating of various ratios of SHH and SPI mixtures and assess the suitability of the conjugates in stabilizing O/W emulsion. The effect of SPI and SHH weight ratios before conjugation on the emulsion stability were investigated and the properties of conjugates were confirmed by FT-IR, thermal, color, and amino acid analysis. The optimized SHH-SPI conjugates (obtained at SPI content of 30% and 40%) exhibited substantially improved emulsification capacity in maintaining the physical stability of O/W emulsions for a prolong storage period at heat treatment, compared with SHH and SPI alone. Maillard reactions occurred between amino groups and carbonyl in SHH and SPI mixtures, resulting in consumption of some functional groups and the appearance of new groups in the conjugates. Thermogravimetric analysis showed that

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