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Improvement in emulsifying properties of whey protein–Rhamnolipid conjugates through short-time heat treatment
Colloids and Surfaces B: Biointerfaces 181 (2019) 688–695
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Improvement in emulsifying properties of whey protein–Rhamnolipid conjugates through short-time heat treatment
T
⁎
Guorui Zhanga,b, Yuhang Lia,b, Tianwen Songa,b, Mutai Baoa,b, , Yiming Lia,b, Ximing Lic a
Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education / Institute for Advanced Ocean Study, Ocean University of China, Qingdao 266100, China b College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100, China c Petroleum Engineering Technology Research Institute, Shengli Oilfield company, Sinopec, Dongying, 257000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Whey protein Rhamnolipid Conjugate Maillard reaction Emulsion
Whey protein–rhamnolipid (WP–rhamnolipid) conjugates were prepared via Maillard reaction by heating in liquid systems. Protein-saccharide conjugates generally exhibit better technical functional properties. To optimize the reaction conditions, mixtures of whey protein and rhamnolipid at varying weight ratios, 8:1, 4:1, 2:1, 1:1, 1:2, 1:4 and 1:8, were heated at 80 ℃ for 10, 20, 40 and 60 min. Emulsifying properties, zeta potential and interfacial activity of the conjugates were evaluated, and results showed that conjugates prepared in some conditions showed significantly improved emulsifying activity, the 1:1 conjugates heated for 10 min had the highest emulsifying activity; the zeta potential decreased significantly in the early stage of heat treatment and then stabilized, indicating that the colloidal stability and electrostatic repulsion were enhanced; an increase in the rhamnolipid component could increase the interfacial activity of conjugates. The formation of conjugates and changes in reaction process were studied by browning value, free amino analysis, fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography (HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). WP–rhamnolipid conjugates formed by short-time heat treatment have great potential as highly efficient bioemulsifiers.
1. Introduction Bioemulsifiers are mainly macromolecular amphiphiles produced by microbial metabolism, mainly polysaccharides, proteins, lipoproteins, lipopolysaccharides or complex mixtures of these biopolymers [1]. Bioemulsifiers are very effective at forming stable emulsions with hydrophobic substrates, but do not have the ability to effectively reduce surface and interfacial tension compared to biosurfactant [2]. Bioemulsifiers and biosurfactants are receiving increasing attention due to their environmental friendliness and special functional properties, which can be used not only in the bioremediation processes of the petroleum industry, to enhance oil recovery and clean oil tanks and pipes, but also have potential market in the food, pharmaceutical and cosmetic industries [3,4]. Whey is a by-product of cheese production and rich in protein. Whey proteins are frequently utilized proteins used as an emulsifier, which are groups of globular proteins with rigid structures and mainly composed of β-lactoglobulin, α-lactalbumin, bovine serum albumin,
and immunoglobulins [5]. Whey proteins have high nutritional value and special functional properties and are widely used in the food industry [6]. Whey proteins are very effective as colloidal stabilizers, which are due to the combination of electrostatic and steric stabilization mechanisms that protect the emulsion droplets from coalescence [7]. In recent years, the modification of proteins through physical, chemical and enzymatic treatments have received more and more attention, especially the glycosylation modification through Maillard reaction [8,9]. The Maillard reaction, a non-enzymatic chemical reaction between protein amino groups and carbonyl compounds (usually reducing saccharides), is considered to be a potential method for protein modification [10]. The Maillard reaction occurs spontaneously, which can produce a series of complex Maillard reaction products (MRPs) [11]. The property of the protein-carbohydrate conjugates obtained by the Maillard reaction may depend on the protein conformation and the polysaccharide characteristics, For example, conjugates prepared by proteins and four different hexoses had significant differences in terms
⁎ Corresponding author at: Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education / Institute for Advanced Ocean Study, Ocean University of China, Qingdao 266100, China. E-mail address: [email protected] (M. Bao).
https://doi.org/10.1016/j.colsurfb.2019.06.015 Received 30 January 2019; Received in revised form 28 May 2019; Accepted 6 June 2019 Available online 07 June 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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of emulsifying activity, interfacial activity and emulsion dispersion stability [12]. Glycosylation could also affect the rheological properties of proteins during the heat-induced gelation [13]. The emulsifying properties of the protein-carbohydrate conjugates were improved due to conjugate formation between protein-bound lysine and carbohydrate [14]. Currently, glycosylation modification of proteins is mostly limited to saccharides. Rhamnolipid that produced by Pseudomonas aeruginosa is a kind of glycolipid biosurfactant, which consists of the hydrophilic head of one or two rhamnose molecules and the hydrophobic tail of one or two fatty acid molecules [15]. Rhamnolipid, the best studied biosurfactant, is very effective in reducing the surface tension of water [16]. Rhamnolipids from Pseudomonas aeruginosa have been currently commercialized, mainly as fungicides in agriculture or as additives to enhance bioremediation activity [17]. Thermodynamic and structural changes caused by the interaction between dirhamnolipid and bovine serum albumin have been studied by Marina Sánchez et al., which confirm the combination of dirhamnolipid and bovine serum albumin [18]. In this study, the emulsifying properties, zeta potential and interfacial activity of WP–rhamnolipid conjugates prepared under different conditions (component ratio, heat treatment time) were investigated. Emulsifying properties were investigated by emulsification index, microscopy and mean emulsion particle size. Conjugates formations were demonstrated using the degree of browning and free amino changes. The structural changes of the conjugates formed at different heat treatment times were investigated using Fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography (HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE).
Where x is the time after initial emulsification (h).
2. Materials and methods
2.7. Measurement of free amino group
2.1. Materials and chemicals
The degree of glycosylation (DG) of the conjugates was analyzed based on free amino groups determined by an o-phthaldialdehyde assay (OPA). The 200 μL solution to be tested (2 g/L) was added to a 4 mL OPA reagent, rapidly mixed with a vortex shaker, and then incubated at 25 °C for 3 min. The absorbance was measured at 340 nm using a spectrophotometer (Alpha-1860s, Lab-Spectrum Instruments Co., Ltd., Shanghai, China) [20]. The working curve was obtained using L-leucine as a standard. The DG was calculated based on the loss of free amino groups of conjugates compared to unreacted protein. Each experiment was performed in triplicate.
2.4. Emulsion particle size The emulsions after standing for 24 h were observed using an DM1000 LED optical microscope (Leica Microsystems CMS GmbH, Germany), and the mean particle size of each emulsion droplet diameter was measured by Nano Measurer software (Department of Chemistry, Surface Chemistry and Catalysis Laboratory, Fudan University, China, Xu Jie). 2.5. Zeta potential The zeta potential was measured using a Nano-ZS 90 nanoparticle size and zeta potentiometer (Malvern Instruments Ltd., UK). Each sample solution to be tested was added to the cuvette and carefully place the cuvette in the sample cell for testing. The experimental results were recorded directly, and each experiment was performed in triplicate. 2.6. Interfacial tension The 1:1 mixed solution of whey protein and rhamnolipid with different heat treatment time (0, 10, 20, 40 and 60 min, 80 ℃ water bath heating) was used as an object for subsequent experiments, because of its optimal emulsifying activity. The oil-water interfacial tension of the particles was measured at room temperature using an OCA25 contact angle measuring instrument (Dataphysics Instrument Co., Ltd. Germany). Each experiment was performed in triplicate.
Rhamnolipid (95%) was purchased from Xi'an Boliante Chemical Industry Co., Ltd (Xi'an, China); Whey protein (total nitrogen: 13.1%, water: 5.5%) was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan); 0# diesel oil was obtained from local filling station and used directly. The reagents used in high performance liquid chromatography were chromatographic reagent grade, and all other reagents were analytical reagent grade. Reverse osmosis (RO) pure water (conductivity < 5 × 10−4 S/m) was obtained from Unique-R20 multi-purpose ultrapure water system (Research Water Purification Technology Co., Ltd., China) and used for all experiments.
2.8. Measurement of browning value Browning value can be used as a simple indication of the Maillard reaction process [21]. Solution to be tested (2 g/L) was added to the cuvette, whose absorbance was measured at 420 nm. Each experiment was performed in triplicate.
2.2. Sample preparation Whey protein (1 g/L) and rhamnolipid (1 g/L) were separately dispersed in RO pure water. The mixed solution was prepared according to different ratios (8:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:8, w/w) and mixed well. Then these solutions were heated in a water bath at 80 °C for 10, 20, 40, 60 min respectively and cooled to room temperature.
2.9. Fourier transform infrared spectroscopy The sample solution was lyophilized and ground to a fine powder, which was tableted with potassium bromide and scanned at a wavelength 4000-400 cm−1 at a resolution of 4 cm−1 using Tensor 27 Fourier transform infrared spectrum system (Bruker Co., Germany).
2.3. Emulsification index 2 mL whey protein solution (1 g/L), rhamnolipid solution (1 g/L), mixed solutions (1 g/L) with different component ratios, and conjugates (1 g/L) prepared after heating for different time were separately added to the test tube and mixed with 2 mL 0# diesel, vortexed for 2 min at 3000 rpm using a MS3 basic Vortexing machine (IKA Werke GmbH & Co. KG, Germany), then let stand. The emulsifying activity of the emulsifier was characterized by Emulsification index (EIX) [19]:
EIX =
2.10. High performance liquid chromatography To determine chromatographic conditions with better degree of separation, experiments were performed using different mobile phases, flow ratios, flow rates, detection wavelengths, and so on. The locations of pure water peaks, whey protein peaks, and rhamnolipid peaks were determined. Chromatographic conditions: Agilent 1260 high performance liquid chromatography (Agilent Technologies Inc., USA), including auto
Height of the emulsifier layer (cm) × 100% Total height of the mixture (cm) 689
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sampler, UV detector (VWD), column oven; RP-HPLC column SB-C18; column temperature: 35 °C; flow rate: 0.8 mL/min; injection amount: 20 μL; detection wavelength: 215 nm. Gradient elution method, mobile phase A: 0.1% trifluoroacetic acid (TFA) aqueous solution, mobile phase B: 0.1% TFA in acetonitrile solution, elution gradient: 0–10 min: 20%–50% B; 10–20 min: 50%–80% B; 20–24 min: 80% B; 24–24.5 min: 80%-20% B; 24.5–30 min: 20% B. 2.11. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was performed using a 12% (w/v) acrylamide separation gel and a 5% (w/v) concentrated gel, according to the method mentioned in the literature of Laemmli [22]. 20 μL sample solutions (2 g/L) were taken and added 5 μL of a buffer consisting of 0.25 M Tris-HCI (Ph 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol and 0.5 M dithiothreitol (DTT), which heated in a 100 °C water bath for 5 min. Electrophoresis was carried out in the DCode™ Universal Mutation Detection System (Bio-Rad Laboratories, USA) using Tris-Glycine running buffer containing 0.5% SDS. After the electrophoresis, the gel stained with Coomassie Brilliant Blue R250 staining solution for 1 h, then decolorized in a solution containing 10% (v/v) acetic acid and 25% (v/v) ethanol. 3. Results and discussion 3.1. Emulsifying activity
Fig. 1. The emulsification index of WP–rhamnolipid conjugates (0.1%w/v conjugates, 50% v/v diesel oil). (a) The emulsions after 24 h of storage at room temperature, (b) the emulsions after 5 days of storage at room temperature.
In the case without heat treatment, the emulsification index (day 1 and day 5) of mixtures with ratios of whey protein to rhamnolipid of 4:1, 2:1, 1:1, 1:2, 1:4 and 1:8, was higher than that of simple whey protein and rhamnolipid at the same concentration. It suggested that under this homogenization intensity of the experiment, the emulsifying activities of the mixtures mixed in these ratios are higher than whey protein and rhamnolipid. It can be seen from the EI24 that as the heat treatment time increased, the emulsification index of pure whey protein and rhamnolipid gradually decreased, the emulsification index of WP–rhamnolipid conjugates (4:1, 2:1, 1:1, 1:2, 1:4 and 1:8) showed a trend of increasing first and then decreasing, conjugates obtained after heat treatment for 10 and 20 min have higher emulsifying activity. The emulsification index of the 8:1 conjugates showed an increasing trend. These trends can still be seen in the emulsification index on day 5, In particular, emulsification index of 1:1 and 1:2 conjugates (heat treatment for 10 and 20 min) was still significantly higher than non-heat treatment mixtures. The 1:1 conjugate (heated for 10 min) has the highest EI24, while the 1:2 conjugate (heated for 10 min) has a higher emulsification index after 5 days. These trends showed that appropriate heat treatment time can surely increase the emulsifying activity of the conjugates, which was consistent with the phenomenon that the bioemulsifiers produced by microorganisms were heated at an appropriate temperature to increase the emulsifying activity [23] (Fig. 1).
uniform without oversized droplets; the emulsion stabilized by a 1:4 mixture even had a smaller mean particle size than pure rhamnolipid. These indicated that whey protein and rhamnolipid have a good synergistic effect in stabilizing the emulsion. Moreover, emulsions stabilized by the WP–rhamnolipid (including non-heat treatment and heat treatment for different times) with same ratios had similar mean particle size. These results indicated that short time heat treatment had little effect on the particle size of the emulsion. 3.3. Zeta potential Fig. 3 shows the zeta potential changes of WP–rhamnolipid conjugates as the heat treatment goes on. The zeta potential of pure whey protein and rhamnolipid was −30.1 ± 0.7 and −47.7 ± 3.1 respectively. The negative zeta potentials of WP–rhamnolipid conjugates showed a significant enhancement trend after heat treatment. In particular, the negative zeta potential enhancement was most pronounced after the shortest 10 min heat treatment, with the 1:1 conjugate enhancing the most, and the zeta potential fluctuated at a relatively consistent level during the subsequent heat treatment, which showed that the colloidal stability of WP-rhamnolipid conjugates was greatly improved after a short time heat treatment. Stronger negative zeta potential could enhance colloidal stability of protein dispersions by enhancing electrostatic repulsion between colloidal particles. Two maltose-glycated caseinate showed improved colloidal stability with increased negative zeta-potentials [25]. For glycated soy 11S glycinin using glucose, a general increase in zeta potential was observed with increasing glycation, and the trend was consistent with the improvements observed in the emulsifying activities of glycated 11S glycinin [26].
3.2. Emulsion structure and particle size distribution As can be seen from Fig. 2, the mean particle size of the emulsion stabilized by rhamnolipid was small and dense, the mean particle size of the emulsion stabilized by whey protein was the largest. The mean particle sizes of emulsions stabilized by pure whey protein and rhamnolipid were 11.25 μm and 5.70 μm respectively, mean particle sizes of emulsions stabilized by the mixtures (4:1, 2:1, 1:1, 1:2 and 1:4) were 9.53 μm, 7.39 μm, 6.88 μm, 6.42 μm and 5.03 μm, respectively. It can be seen that a small amount of rhamnolipid could significantly reduce the emulsion droplet size, which might be due to the fact that rhamnolipid can effectively reduce the interfacial tension [24]. Interestingly, the emulsion particle size distribution stabilized by a 1:1 mixture was more
3.4. Interfacial tension It can be seen from Fig. 4(a) that the whey protein solution had the highest interfacial tension, while the rhamnolipid solution had the 690
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Fig. 2. Size distribution and emulsion optical micrograph of whey protein, rhamnolipid and mixtures with different component ratios. All calibration bars were 100 μm.
of brown matter and the decrease in the amount of free amino groups in the protein. Therefore, measurements of browning value and free amino group are common indicators of the Maillard reaction process. The degree of browning was measured by the absorbance at 420 nm of conjugate solutions at different heat treatment times. There was no significant change in the degree of browning, in the first 20 min of heat treatment, the browning deepened but not much, browning began to weaken as the heat treatment continued, and weakened to near the initial level after 60 min of heat treatment. This trend also occurd during the glycation of the soy protein isolate–lactose blended at 75 ℃ [30].The Maillard reaction occurs mainly between the ε-lysyl amino groups exposed on the surface of the protein and the reducing end carbonyl group of the polysaccharide molecules. According to the Fig. 5, as the heat treatment time increased, the concentrations of free amino groups presented in the mixture solutions were continuously reduced. In the first 10 min of heat treatment, the rate of free amino groups reduction in the solution was the highest, and after 60 min of heat treatment, the concentrations of free amino groups were reduced
lowest interfacial tension, and the addition of rhamnolipid component significantly reduced interfacial tension. The reason is that whey protein is a bioemulsifier, which can form a stable emulsion with hydrocarbons, but cannot effectively reduce the interfacial tension [27], rhamnolipid is an excellent biosurfactant, which can strongly reduce the interfacial tension [28,29]. As can be seen from Fig. 4(b), the WP–rhamnolipid conjugates formed by the short-time heat treatment showed no significant difference from the mixtures of whey protein and rhamnolipid in reducing the interfacial tension, which indicated that short-time heating had little effect on the interfacial activity of conjugates.
3.5. Measurement of degree of glycosylation and browning value The 1:1 conjugate was selected for subsequent experiments due to its optimal emulsifying activity, to investigate the progress and structural changes of the reaction during heating. The Maillard reaction is generally accompanied by the production 691
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Fig. 5. Free amino groups (left axis) and absorbance at 420 nm (right axis) of WP–rhamnolipid conjugates (heat treatment for 0, 10, 20, 40 and 60 min). Fig. 3. Zeta potential of WP–rhamnolipid conjugates (heat treatment for 0, 10, 20, 40 and 60 min): ▪, whey protein : Rhamnolipid = 4:1; •, whey protein : Rhamnolipid = 2:1; ▲, whey protein : Rhamnolipid = 1:1; ▼, whey protein : Rhamnolipid = 1:2; ◆, whey protein : Rhamnolipid = 1:4.
degree of graft reactions by short-term hydrothermal treatment.
3.6. Fourier transform infrared spectroscopy (FTIR)
by about 14.4%.The concentrations of free amino groups were not significantly changed when pure whey protein solution was heated in the absence of rhamnolipid. All these indicated that the reaction between the relevant group of whey protein and rhamnolipid can form conjugates, the reaction rate was faster at the beginning of the heat treatment, and as the heat treatment continued, the reaction rate slowed down. According to reports, the concentrations of free amino groups of αlactalbumin–acacia gum (α-la : AG) conjugates that prepared by Maillard reaction using the dry heating method, were reduced by about 12% [21], the concentrations of free amino groups of Soy protein isolate–maltodextrin (SPI-MD) conjugates that synthesized via the Maillard reaction under high-temperature (90, 115 and 140 °C) and shorttime (2 h) dry-heating conditions, were reduced by about 6.86％ ± 0.17％ (90 °C), 14.05％ ± 1.55％ (115 °C) and 26.46％ ± 3.17％ (140 °C) [31], the concentrations of free amino groups of peanut protein isolate–galactomannan conjugates through classical heating for 8 h, were reduced by about 11.98% [32]. It can be seen that whey protein and rhamnolipid can achieve a relatively high
The Maillard reaction is a reaction between a carbonyl compound and an amino compound, which is also referred to as a non-enzymatic browning reaction. The heat treatment leads to the Maillard reaction. In the initial stage, the amino groups are condensed with the carbonyl groups to form Schiff bases; as the reaction goes further, the reducing ketones, aldehydes and unsaturated carbonyl compounds are formed; finally, after complicated reaction processes, some brown or black macromolecular substances are formed. It can be expected that the chemical changes of whey protein and rhamnolipid during the Maillard reaction would result in some variations in the FTIR spectrum due to the utilization of several functional groups such as amino groups [33]. The fingerprint region (the region from 1800 to 800 cm−1) of the IR spectrum is a very useful part for analysis of proteinaceous material, because bonds forming amide groups (C]O, NeH, and CeN) absorb is formed within this range [34]. It can be seen from Fig. 6 that as the heat treatment progressed, the infrared peak at the 1651 cm−1 of the conjugate gradually showed a sharp small peak at 1653 cm−1, and the infrared peak shape at 1600-1500 cm−1 became sharp, indicating that the secondary structure of the protein might have changed which may
Fig. 4. Interfacial tension of WP–rhamnolipid conjugates: (a) whey protein, rhamnolipid and mixtures with different component ratios; (b) WP–rhamnolipid conjugates (heat treatment for 0, 10, 20, 40 and 60 min). 692
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Fig. 6. Infrared spectrogram of WP–rhamnolipid conjugates (heat treatment for 0, 10, 20, 40 and 60 min) and several enlarged views.
mixture, liquid chromatogram of the conjugates heated for 10 min and 20 min (line C and line D) did not change significantly, indicating that no reaction occurred or the reaction product was too low to be detected by liquid chromatography. According to liquid chromatogram of conjugates heating for 40 min and 60 min (Fig. 7, line E and line F), as the heat treatment time increased, it was apparent that two new peaks appeared at 17.8 and 19.4 min, as well as peak area gradually increased. The conjugates were eluted after whey protein and precedes rhamnolipid. These changes confirmed that the WP–rhamnolipid conjugates were formed in this process and the polarity of conjugates was between the whey protein and the rhamnolipid.
be related to the process of Maillard reaction [35]. At the same time, the conjugates showed three distinct new infrared peaks at the infrared wavelengths of 1052 cm−1, 669 cm−1 and 420 cm−1, respectively. The peaks that appearing at around 669 cm−1 and 420 cm−1 may be formed by the bending vibration of aldehydes and amines produced by the Maillard reaction; the infrared peak that appearing around 1052 cm−1 may be caused by the CeO stretching vibration in the Maillard reaction products, which proved the progress of the Maillard reaction. 3.7. High performance liquid chromatography (HPLC) HPLC can be used to separate components with different polarities in the mixture through a stationary phase column, and then the separated components are tested by detectors [36]. Fig. 7 shows the liquid chromatogram of whey protein and WP–rhamnolipid conjugates, the liquid chromatogram of pure water was uniformly subtracted as the background. It can be seen from the liquid chromatogram of whey protein (Fig. 7, line A) and non-heat treatment WP–rhamnolipid mixture (Fig. 7, line B) that whey protein peak appeared mainly at 6–16 min, and the peak shape was more complicated; the rhamnolipid peak appeared after 21 min, and there was a clear rhamnolipid characteristic peak at 21.2 min. Compared to the non-heat treatment
3.8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) can separate different proteins depending on the molecular weight, which was used to confirm the covalent linkages between whey protein and rhamnolipid during relatively short heating treatment time. As can be seen from Fig. 8, there was no significant change in the band pattern of the WP-rammnolipid mixture sample (lane 3) compared to the pattern of whey protein, indicating that no conjugate was formed between whey protein and rhamnolipid, or the yield of these 693
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4. Conclusion WP–rhamnolipid conjugates were prepared under different conditions in this study. Contrast with the same mass concentration of pure whey protein and rhamnolipid, conjugates formed by appropriate heating time showed improved emulsifying activity, wherein the conjugates with a ratio of 1:1 (heated for 10 min) had the best emulsifying activity. In particular, the emulsion droplets stabilized by a 1:1 ratio of the mixture are more uniform without oversized droplets. Short-time heating had little effect on the interfacial activity of conjugates, while it could enhance the negative zeta potential. The occurrence of the reaction was confirmed using browning analysis and free amino analysis. The formation of conjugates and the structural change during the heat treatment were indicated by change of the fingerprint region of the infrared spectrum and the appearance of new infrared peaks, which were further confirmed through the formation of new bands of gel electrophoresis and new liquid chromatogram peaks. WP–rhamnolipid conjugates formed by short-time heating Maillard reaction can offer substantial potential for producing effective emulsifiers. Declaration of competing interest Fig. 7. Liquid chromatogram of whey protein and WP–rhamnolipid conjugates (heat treatment for 0, 10, 20, 40 and 60 min).
We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgments This research was supported by the Fundamental Research Funds for the Central Universities (201822009); the Shandong Provincial Natural Science Foundation, China (ZR2018MD017); the National Key Research and Development Program (2016YFC1402301); the Open Foundation of Key Laboratory of Marine Spill Oil Identification and Damage Assessment Technology of SOA (201702); the Program for Innovative Research Team in University (IRT1289). This is MCTL Contribution No. 195. References [1] E. Rosenberg, E.Z. Ron, High- and low-molecular-mass microbial surfactants, Appl. Microbiol. Biotechnol. 52 (1999) 154–162. [2] S.K. Satpute, A.G. Banpurkar, P.K. Dhakephalkar, I.M. Banat, B.A. Chopade, Methods for investigating biosurfactants and bioemulsifiers: a review, Crit. Rev. Biotechnol. 30 (2010) 127–144. [3] I.M. Banat, Biosurfactants production and possible uses in microbial enhanced oilrecovery and oil pollution remediation - a review, Bioresour. Technol. 51 (1995) 1–12. [4] F. Zhao, J.D. Zhou, F. Ma, R.J. Shi, S.Q. Han, J. Zhang, Y. Zhang, Simultaneous inhibition of sulfate-reducing bacteria, removal of H2S and production of rhamnolipid by recombinant Pseudomonas stutzeri Rhl: applications for microbial enhanced oil recovery, Bioresour. Technol. 207 (2016) 24–30. [5] J.E. Kinsella, D.M. Whitehead, Proteins in whey: chemical, physical, and functional properties, Adv. Food Nutr. Res. 33 (1989) 343–438. [6] G. Panaras, G. Moatsou, S. Yanniotis, I. Mandala, The influence of functional properties of different whey protein concentrates on the rheological and emulsification capacity of blends with xanthan gum, Carbohydr. Polym. 86 (2011) 433–440. [7] E. Dickinson, Colloids in food: ingredients, structure, and stability, Annu. Rev. Food Sci. Technol. 6 (6) (2015) 211–233. [8] H.R. Sharif, P.A. Williams, M.K. Sharif, S. Abbas, H. Majeed, K.G. Masamba, W. Safdar, F. Zhong, Current progress in the utilization of native and modified legume proteins as emulsifiers and encapsulants - a review, Food Hydrocolloid 76 (2018) 2–16. [9] X.L. Zhang, H. Gao, C.Y. Wang, A. Qayum, Z.S. Mu, Z.L. Gao, Z.M. Jiang, Characterization and comparison of the structure and antioxidant activity of glycosylated whey peptides from two pathways, Food Chem. 257 (2018) 279–288. [10] S. Shrestha, M.B. Sadiq, A.K. Anal, Culled banana resistant starch-soy protein isolate conjugate based emulsion enriched with astaxanthin to enhance its stability, Int. J. Biol. Macromol. 120 (2018) 449–459. [11] H. Yu, Y.X. Seow, P.K.C. Ong, W.B. Zhou, Kinetic study of high-intensity ultrasoundassisted Maillard reaction in a model system of D-glucose and glycine, Food Chem. 269 (2018) 628–637. [12] N. Cheetangdee, K. Fukada, Emulsifying activity of bovine beta-lactoglobulin conjugated with hexoses through the Maillard reaction, Colloids Surf. A 450 (2014)
Fig. 8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns for whey protein and rhamnolipid conjugates: Lane 1, molecular weight markers (11–180 kDa); lane2, whey protein; lanes 3, WP–rhamnolipid mixture sample; lanes 4–7, WP–rhamnolipid conjugates heated at 80 °C for 10, 20, 40, and 60 min.
combinations was too low to detect by SDS-PAGE. It can be seen from the mixture samples after heat treatment for 10, 20, 40, 60 min (lane 4, lane 5, lane 6 and lane 7) that as the heat treatment time increased, the two bands below the electropherogram were obviously shifted upwards, which might be caused by the combination of whey protein and rhamnolipid to produce a larger molecular weight product. Due to the small molecular weight of rhamnolipids, the shift in protein bands at large molecular weights was not significant. In the lanes of the conjugates treated with heat treatment for 40 min and 60 min, it can be seen that a new band was produced at the 46 kDa position, and the new band produced by the heat treatment for 60 min was clearer, indicating the formation of new reaction products (WP–rhamnolipid conjugates). SDS-PAGE was also reported to demonstrate the formation ofα-Lactalbumin: acacia gum (α-la : AG) conjugate [21], soy β-conglycinin–dextran conjugate [37], and whey protein isolate–sugar beet pectin (WPI–SBP) [38] treated by heating.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Improvement in emulsifying properties of whey protein–Rhamnolipid conjugates through short-time heat treatment
T
⁎
Guorui Zhanga,b, Yuhang Lia,b, Tianwen Songa,b, Mutai Baoa,b, , Yiming Lia,b, Ximing Lic a
Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education / Institute for Advanced Ocean Study, Ocean University of China, Qingdao 266100, China b College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100, China c Petroleum Engineering Technology Research Institute, Shengli Oilfield company, Sinopec, Dongying, 257000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Whey protein Rhamnolipid Conjugate Maillard reaction Emulsion
Whey protein–rhamnolipid (WP–rhamnolipid) conjugates were prepared via Maillard reaction by heating in liquid systems. Protein-saccharide conjugates generally exhibit better technical functional properties. To optimize the reaction conditions, mixtures of whey protein and rhamnolipid at varying weight ratios, 8:1, 4:1, 2:1, 1:1, 1:2, 1:4 and 1:8, were heated at 80 ℃ for 10, 20, 40 and 60 min. Emulsifying properties, zeta potential and interfacial activity of the conjugates were evaluated, and results showed that conjugates prepared in some conditions showed significantly improved emulsifying activity, the 1:1 conjugates heated for 10 min had the highest emulsifying activity; the zeta potential decreased significantly in the early stage of heat treatment and then stabilized, indicating that the colloidal stability and electrostatic repulsion were enhanced; an increase in the rhamnolipid component could increase the interfacial activity of conjugates. The formation of conjugates and changes in reaction process were studied by browning value, free amino analysis, fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography (HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). WP–rhamnolipid conjugates formed by short-time heat treatment have great potential as highly efficient bioemulsifiers.
1. Introduction Bioemulsifiers are mainly macromolecular amphiphiles produced by microbial metabolism, mainly polysaccharides, proteins, lipoproteins, lipopolysaccharides or complex mixtures of these biopolymers [1]. Bioemulsifiers are very effective at forming stable emulsions with hydrophobic substrates, but do not have the ability to effectively reduce surface and interfacial tension compared to biosurfactant [2]. Bioemulsifiers and biosurfactants are receiving increasing attention due to their environmental friendliness and special functional properties, which can be used not only in the bioremediation processes of the petroleum industry, to enhance oil recovery and clean oil tanks and pipes, but also have potential market in the food, pharmaceutical and cosmetic industries [3,4]. Whey is a by-product of cheese production and rich in protein. Whey proteins are frequently utilized proteins used as an emulsifier, which are groups of globular proteins with rigid structures and mainly composed of β-lactoglobulin, α-lactalbumin, bovine serum albumin,
and immunoglobulins [5]. Whey proteins have high nutritional value and special functional properties and are widely used in the food industry [6]. Whey proteins are very effective as colloidal stabilizers, which are due to the combination of electrostatic and steric stabilization mechanisms that protect the emulsion droplets from coalescence [7]. In recent years, the modification of proteins through physical, chemical and enzymatic treatments have received more and more attention, especially the glycosylation modification through Maillard reaction [8,9]. The Maillard reaction, a non-enzymatic chemical reaction between protein amino groups and carbonyl compounds (usually reducing saccharides), is considered to be a potential method for protein modification [10]. The Maillard reaction occurs spontaneously, which can produce a series of complex Maillard reaction products (MRPs) [11]. The property of the protein-carbohydrate conjugates obtained by the Maillard reaction may depend on the protein conformation and the polysaccharide characteristics, For example, conjugates prepared by proteins and four different hexoses had significant differences in terms
⁎ Corresponding author at: Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education / Institute for Advanced Ocean Study, Ocean University of China, Qingdao 266100, China. E-mail address: [email protected] (M. Bao).
https://doi.org/10.1016/j.colsurfb.2019.06.015 Received 30 January 2019; Received in revised form 28 May 2019; Accepted 6 June 2019 Available online 07 June 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
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of emulsifying activity, interfacial activity and emulsion dispersion stability [12]. Glycosylation could also affect the rheological properties of proteins during the heat-induced gelation [13]. The emulsifying properties of the protein-carbohydrate conjugates were improved due to conjugate formation between protein-bound lysine and carbohydrate [14]. Currently, glycosylation modification of proteins is mostly limited to saccharides. Rhamnolipid that produced by Pseudomonas aeruginosa is a kind of glycolipid biosurfactant, which consists of the hydrophilic head of one or two rhamnose molecules and the hydrophobic tail of one or two fatty acid molecules [15]. Rhamnolipid, the best studied biosurfactant, is very effective in reducing the surface tension of water [16]. Rhamnolipids from Pseudomonas aeruginosa have been currently commercialized, mainly as fungicides in agriculture or as additives to enhance bioremediation activity [17]. Thermodynamic and structural changes caused by the interaction between dirhamnolipid and bovine serum albumin have been studied by Marina Sánchez et al., which confirm the combination of dirhamnolipid and bovine serum albumin [18]. In this study, the emulsifying properties, zeta potential and interfacial activity of WP–rhamnolipid conjugates prepared under different conditions (component ratio, heat treatment time) were investigated. Emulsifying properties were investigated by emulsification index, microscopy and mean emulsion particle size. Conjugates formations were demonstrated using the degree of browning and free amino changes. The structural changes of the conjugates formed at different heat treatment times were investigated using Fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography (HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE).
Where x is the time after initial emulsification (h).
2. Materials and methods
2.7. Measurement of free amino group
2.1. Materials and chemicals
The degree of glycosylation (DG) of the conjugates was analyzed based on free amino groups determined by an o-phthaldialdehyde assay (OPA). The 200 μL solution to be tested (2 g/L) was added to a 4 mL OPA reagent, rapidly mixed with a vortex shaker, and then incubated at 25 °C for 3 min. The absorbance was measured at 340 nm using a spectrophotometer (Alpha-1860s, Lab-Spectrum Instruments Co., Ltd., Shanghai, China) [20]. The working curve was obtained using L-leucine as a standard. The DG was calculated based on the loss of free amino groups of conjugates compared to unreacted protein. Each experiment was performed in triplicate.
2.4. Emulsion particle size The emulsions after standing for 24 h were observed using an DM1000 LED optical microscope (Leica Microsystems CMS GmbH, Germany), and the mean particle size of each emulsion droplet diameter was measured by Nano Measurer software (Department of Chemistry, Surface Chemistry and Catalysis Laboratory, Fudan University, China, Xu Jie). 2.5. Zeta potential The zeta potential was measured using a Nano-ZS 90 nanoparticle size and zeta potentiometer (Malvern Instruments Ltd., UK). Each sample solution to be tested was added to the cuvette and carefully place the cuvette in the sample cell for testing. The experimental results were recorded directly, and each experiment was performed in triplicate. 2.6. Interfacial tension The 1:1 mixed solution of whey protein and rhamnolipid with different heat treatment time (0, 10, 20, 40 and 60 min, 80 ℃ water bath heating) was used as an object for subsequent experiments, because of its optimal emulsifying activity. The oil-water interfacial tension of the particles was measured at room temperature using an OCA25 contact angle measuring instrument (Dataphysics Instrument Co., Ltd. Germany). Each experiment was performed in triplicate.
Rhamnolipid (95%) was purchased from Xi'an Boliante Chemical Industry Co., Ltd (Xi'an, China); Whey protein (total nitrogen: 13.1%, water: 5.5%) was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan); 0# diesel oil was obtained from local filling station and used directly. The reagents used in high performance liquid chromatography were chromatographic reagent grade, and all other reagents were analytical reagent grade. Reverse osmosis (RO) pure water (conductivity < 5 × 10−4 S/m) was obtained from Unique-R20 multi-purpose ultrapure water system (Research Water Purification Technology Co., Ltd., China) and used for all experiments.
2.8. Measurement of browning value Browning value can be used as a simple indication of the Maillard reaction process [21]. Solution to be tested (2 g/L) was added to the cuvette, whose absorbance was measured at 420 nm. Each experiment was performed in triplicate.
2.2. Sample preparation Whey protein (1 g/L) and rhamnolipid (1 g/L) were separately dispersed in RO pure water. The mixed solution was prepared according to different ratios (8:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:8, w/w) and mixed well. Then these solutions were heated in a water bath at 80 °C for 10, 20, 40, 60 min respectively and cooled to room temperature.
2.9. Fourier transform infrared spectroscopy The sample solution was lyophilized and ground to a fine powder, which was tableted with potassium bromide and scanned at a wavelength 4000-400 cm−1 at a resolution of 4 cm−1 using Tensor 27 Fourier transform infrared spectrum system (Bruker Co., Germany).
2.3. Emulsification index 2 mL whey protein solution (1 g/L), rhamnolipid solution (1 g/L), mixed solutions (1 g/L) with different component ratios, and conjugates (1 g/L) prepared after heating for different time were separately added to the test tube and mixed with 2 mL 0# diesel, vortexed for 2 min at 3000 rpm using a MS3 basic Vortexing machine (IKA Werke GmbH & Co. KG, Germany), then let stand. The emulsifying activity of the emulsifier was characterized by Emulsification index (EIX) [19]:
EIX =
2.10. High performance liquid chromatography To determine chromatographic conditions with better degree of separation, experiments were performed using different mobile phases, flow ratios, flow rates, detection wavelengths, and so on. The locations of pure water peaks, whey protein peaks, and rhamnolipid peaks were determined. Chromatographic conditions: Agilent 1260 high performance liquid chromatography (Agilent Technologies Inc., USA), including auto
Height of the emulsifier layer (cm) × 100% Total height of the mixture (cm) 689
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sampler, UV detector (VWD), column oven; RP-HPLC column SB-C18; column temperature: 35 °C; flow rate: 0.8 mL/min; injection amount: 20 μL; detection wavelength: 215 nm. Gradient elution method, mobile phase A: 0.1% trifluoroacetic acid (TFA) aqueous solution, mobile phase B: 0.1% TFA in acetonitrile solution, elution gradient: 0–10 min: 20%–50% B; 10–20 min: 50%–80% B; 20–24 min: 80% B; 24–24.5 min: 80%-20% B; 24.5–30 min: 20% B. 2.11. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was performed using a 12% (w/v) acrylamide separation gel and a 5% (w/v) concentrated gel, according to the method mentioned in the literature of Laemmli [22]. 20 μL sample solutions (2 g/L) were taken and added 5 μL of a buffer consisting of 0.25 M Tris-HCI (Ph 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol and 0.5 M dithiothreitol (DTT), which heated in a 100 °C water bath for 5 min. Electrophoresis was carried out in the DCode™ Universal Mutation Detection System (Bio-Rad Laboratories, USA) using Tris-Glycine running buffer containing 0.5% SDS. After the electrophoresis, the gel stained with Coomassie Brilliant Blue R250 staining solution for 1 h, then decolorized in a solution containing 10% (v/v) acetic acid and 25% (v/v) ethanol. 3. Results and discussion 3.1. Emulsifying activity
Fig. 1. The emulsification index of WP–rhamnolipid conjugates (0.1%w/v conjugates, 50% v/v diesel oil). (a) The emulsions after 24 h of storage at room temperature, (b) the emulsions after 5 days of storage at room temperature.
In the case without heat treatment, the emulsification index (day 1 and day 5) of mixtures with ratios of whey protein to rhamnolipid of 4:1, 2:1, 1:1, 1:2, 1:4 and 1:8, was higher than that of simple whey protein and rhamnolipid at the same concentration. It suggested that under this homogenization intensity of the experiment, the emulsifying activities of the mixtures mixed in these ratios are higher than whey protein and rhamnolipid. It can be seen from the EI24 that as the heat treatment time increased, the emulsification index of pure whey protein and rhamnolipid gradually decreased, the emulsification index of WP–rhamnolipid conjugates (4:1, 2:1, 1:1, 1:2, 1:4 and 1:8) showed a trend of increasing first and then decreasing, conjugates obtained after heat treatment for 10 and 20 min have higher emulsifying activity. The emulsification index of the 8:1 conjugates showed an increasing trend. These trends can still be seen in the emulsification index on day 5, In particular, emulsification index of 1:1 and 1:2 conjugates (heat treatment for 10 and 20 min) was still significantly higher than non-heat treatment mixtures. The 1:1 conjugate (heated for 10 min) has the highest EI24, while the 1:2 conjugate (heated for 10 min) has a higher emulsification index after 5 days. These trends showed that appropriate heat treatment time can surely increase the emulsifying activity of the conjugates, which was consistent with the phenomenon that the bioemulsifiers produced by microorganisms were heated at an appropriate temperature to increase the emulsifying activity [23] (Fig. 1).
uniform without oversized droplets; the emulsion stabilized by a 1:4 mixture even had a smaller mean particle size than pure rhamnolipid. These indicated that whey protein and rhamnolipid have a good synergistic effect in stabilizing the emulsion. Moreover, emulsions stabilized by the WP–rhamnolipid (including non-heat treatment and heat treatment for different times) with same ratios had similar mean particle size. These results indicated that short time heat treatment had little effect on the particle size of the emulsion. 3.3. Zeta potential Fig. 3 shows the zeta potential changes of WP–rhamnolipid conjugates as the heat treatment goes on. The zeta potential of pure whey protein and rhamnolipid was −30.1 ± 0.7 and −47.7 ± 3.1 respectively. The negative zeta potentials of WP–rhamnolipid conjugates showed a significant enhancement trend after heat treatment. In particular, the negative zeta potential enhancement was most pronounced after the shortest 10 min heat treatment, with the 1:1 conjugate enhancing the most, and the zeta potential fluctuated at a relatively consistent level during the subsequent heat treatment, which showed that the colloidal stability of WP-rhamnolipid conjugates was greatly improved after a short time heat treatment. Stronger negative zeta potential could enhance colloidal stability of protein dispersions by enhancing electrostatic repulsion between colloidal particles. Two maltose-glycated caseinate showed improved colloidal stability with increased negative zeta-potentials [25]. For glycated soy 11S glycinin using glucose, a general increase in zeta potential was observed with increasing glycation, and the trend was consistent with the improvements observed in the emulsifying activities of glycated 11S glycinin [26].
3.2. Emulsion structure and particle size distribution As can be seen from Fig. 2, the mean particle size of the emulsion stabilized by rhamnolipid was small and dense, the mean particle size of the emulsion stabilized by whey protein was the largest. The mean particle sizes of emulsions stabilized by pure whey protein and rhamnolipid were 11.25 μm and 5.70 μm respectively, mean particle sizes of emulsions stabilized by the mixtures (4:1, 2:1, 1:1, 1:2 and 1:4) were 9.53 μm, 7.39 μm, 6.88 μm, 6.42 μm and 5.03 μm, respectively. It can be seen that a small amount of rhamnolipid could significantly reduce the emulsion droplet size, which might be due to the fact that rhamnolipid can effectively reduce the interfacial tension [24]. Interestingly, the emulsion particle size distribution stabilized by a 1:1 mixture was more
3.4. Interfacial tension It can be seen from Fig. 4(a) that the whey protein solution had the highest interfacial tension, while the rhamnolipid solution had the 690
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Fig. 2. Size distribution and emulsion optical micrograph of whey protein, rhamnolipid and mixtures with different component ratios. All calibration bars were 100 μm.
of brown matter and the decrease in the amount of free amino groups in the protein. Therefore, measurements of browning value and free amino group are common indicators of the Maillard reaction process. The degree of browning was measured by the absorbance at 420 nm of conjugate solutions at different heat treatment times. There was no significant change in the degree of browning, in the first 20 min of heat treatment, the browning deepened but not much, browning began to weaken as the heat treatment continued, and weakened to near the initial level after 60 min of heat treatment. This trend also occurd during the glycation of the soy protein isolate–lactose blended at 75 ℃ [30].The Maillard reaction occurs mainly between the ε-lysyl amino groups exposed on the surface of the protein and the reducing end carbonyl group of the polysaccharide molecules. According to the Fig. 5, as the heat treatment time increased, the concentrations of free amino groups presented in the mixture solutions were continuously reduced. In the first 10 min of heat treatment, the rate of free amino groups reduction in the solution was the highest, and after 60 min of heat treatment, the concentrations of free amino groups were reduced
lowest interfacial tension, and the addition of rhamnolipid component significantly reduced interfacial tension. The reason is that whey protein is a bioemulsifier, which can form a stable emulsion with hydrocarbons, but cannot effectively reduce the interfacial tension [27], rhamnolipid is an excellent biosurfactant, which can strongly reduce the interfacial tension [28,29]. As can be seen from Fig. 4(b), the WP–rhamnolipid conjugates formed by the short-time heat treatment showed no significant difference from the mixtures of whey protein and rhamnolipid in reducing the interfacial tension, which indicated that short-time heating had little effect on the interfacial activity of conjugates.
3.5. Measurement of degree of glycosylation and browning value The 1:1 conjugate was selected for subsequent experiments due to its optimal emulsifying activity, to investigate the progress and structural changes of the reaction during heating. The Maillard reaction is generally accompanied by the production 691
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Fig. 5. Free amino groups (left axis) and absorbance at 420 nm (right axis) of WP–rhamnolipid conjugates (heat treatment for 0, 10, 20, 40 and 60 min). Fig. 3. Zeta potential of WP–rhamnolipid conjugates (heat treatment for 0, 10, 20, 40 and 60 min): ▪, whey protein : Rhamnolipid = 4:1; •, whey protein : Rhamnolipid = 2:1; ▲, whey protein : Rhamnolipid = 1:1; ▼, whey protein : Rhamnolipid = 1:2; ◆, whey protein : Rhamnolipid = 1:4.
degree of graft reactions by short-term hydrothermal treatment.
3.6. Fourier transform infrared spectroscopy (FTIR)
by about 14.4%.The concentrations of free amino groups were not significantly changed when pure whey protein solution was heated in the absence of rhamnolipid. All these indicated that the reaction between the relevant group of whey protein and rhamnolipid can form conjugates, the reaction rate was faster at the beginning of the heat treatment, and as the heat treatment continued, the reaction rate slowed down. According to reports, the concentrations of free amino groups of αlactalbumin–acacia gum (α-la : AG) conjugates that prepared by Maillard reaction using the dry heating method, were reduced by about 12% [21], the concentrations of free amino groups of Soy protein isolate–maltodextrin (SPI-MD) conjugates that synthesized via the Maillard reaction under high-temperature (90, 115 and 140 °C) and shorttime (2 h) dry-heating conditions, were reduced by about 6.86％ ± 0.17％ (90 °C), 14.05％ ± 1.55％ (115 °C) and 26.46％ ± 3.17％ (140 °C) [31], the concentrations of free amino groups of peanut protein isolate–galactomannan conjugates through classical heating for 8 h, were reduced by about 11.98% [32]. It can be seen that whey protein and rhamnolipid can achieve a relatively high
The Maillard reaction is a reaction between a carbonyl compound and an amino compound, which is also referred to as a non-enzymatic browning reaction. The heat treatment leads to the Maillard reaction. In the initial stage, the amino groups are condensed with the carbonyl groups to form Schiff bases; as the reaction goes further, the reducing ketones, aldehydes and unsaturated carbonyl compounds are formed; finally, after complicated reaction processes, some brown or black macromolecular substances are formed. It can be expected that the chemical changes of whey protein and rhamnolipid during the Maillard reaction would result in some variations in the FTIR spectrum due to the utilization of several functional groups such as amino groups [33]. The fingerprint region (the region from 1800 to 800 cm−1) of the IR spectrum is a very useful part for analysis of proteinaceous material, because bonds forming amide groups (C]O, NeH, and CeN) absorb is formed within this range [34]. It can be seen from Fig. 6 that as the heat treatment progressed, the infrared peak at the 1651 cm−1 of the conjugate gradually showed a sharp small peak at 1653 cm−1, and the infrared peak shape at 1600-1500 cm−1 became sharp, indicating that the secondary structure of the protein might have changed which may
Fig. 4. Interfacial tension of WP–rhamnolipid conjugates: (a) whey protein, rhamnolipid and mixtures with different component ratios; (b) WP–rhamnolipid conjugates (heat treatment for 0, 10, 20, 40 and 60 min). 692
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Fig. 6. Infrared spectrogram of WP–rhamnolipid conjugates (heat treatment for 0, 10, 20, 40 and 60 min) and several enlarged views.
mixture, liquid chromatogram of the conjugates heated for 10 min and 20 min (line C and line D) did not change significantly, indicating that no reaction occurred or the reaction product was too low to be detected by liquid chromatography. According to liquid chromatogram of conjugates heating for 40 min and 60 min (Fig. 7, line E and line F), as the heat treatment time increased, it was apparent that two new peaks appeared at 17.8 and 19.4 min, as well as peak area gradually increased. The conjugates were eluted after whey protein and precedes rhamnolipid. These changes confirmed that the WP–rhamnolipid conjugates were formed in this process and the polarity of conjugates was between the whey protein and the rhamnolipid.
be related to the process of Maillard reaction [35]. At the same time, the conjugates showed three distinct new infrared peaks at the infrared wavelengths of 1052 cm−1, 669 cm−1 and 420 cm−1, respectively. The peaks that appearing at around 669 cm−1 and 420 cm−1 may be formed by the bending vibration of aldehydes and amines produced by the Maillard reaction; the infrared peak that appearing around 1052 cm−1 may be caused by the CeO stretching vibration in the Maillard reaction products, which proved the progress of the Maillard reaction. 3.7. High performance liquid chromatography (HPLC) HPLC can be used to separate components with different polarities in the mixture through a stationary phase column, and then the separated components are tested by detectors [36]. Fig. 7 shows the liquid chromatogram of whey protein and WP–rhamnolipid conjugates, the liquid chromatogram of pure water was uniformly subtracted as the background. It can be seen from the liquid chromatogram of whey protein (Fig. 7, line A) and non-heat treatment WP–rhamnolipid mixture (Fig. 7, line B) that whey protein peak appeared mainly at 6–16 min, and the peak shape was more complicated; the rhamnolipid peak appeared after 21 min, and there was a clear rhamnolipid characteristic peak at 21.2 min. Compared to the non-heat treatment
3.8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) can separate different proteins depending on the molecular weight, which was used to confirm the covalent linkages between whey protein and rhamnolipid during relatively short heating treatment time. As can be seen from Fig. 8, there was no significant change in the band pattern of the WP-rammnolipid mixture sample (lane 3) compared to the pattern of whey protein, indicating that no conjugate was formed between whey protein and rhamnolipid, or the yield of these 693
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4. Conclusion WP–rhamnolipid conjugates were prepared under different conditions in this study. Contrast with the same mass concentration of pure whey protein and rhamnolipid, conjugates formed by appropriate heating time showed improved emulsifying activity, wherein the conjugates with a ratio of 1:1 (heated for 10 min) had the best emulsifying activity. In particular, the emulsion droplets stabilized by a 1:1 ratio of the mixture are more uniform without oversized droplets. Short-time heating had little effect on the interfacial activity of conjugates, while it could enhance the negative zeta potential. The occurrence of the reaction was confirmed using browning analysis and free amino analysis. The formation of conjugates and the structural change during the heat treatment were indicated by change of the fingerprint region of the infrared spectrum and the appearance of new infrared peaks, which were further confirmed through the formation of new bands of gel electrophoresis and new liquid chromatogram peaks. WP–rhamnolipid conjugates formed by short-time heating Maillard reaction can offer substantial potential for producing effective emulsifiers. Declaration of competing interest Fig. 7. Liquid chromatogram of whey protein and WP–rhamnolipid conjugates (heat treatment for 0, 10, 20, 40 and 60 min).
We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgments This research was supported by the Fundamental Research Funds for the Central Universities (201822009); the Shandong Provincial Natural Science Foundation, China (ZR2018MD017); the National Key Research and Development Program (2016YFC1402301); the Open Foundation of Key Laboratory of Marine Spill Oil Identification and Damage Assessment Technology of SOA (201702); the Program for Innovative Research Team in University (IRT1289). This is MCTL Contribution No. 195. References [1] E. Rosenberg, E.Z. Ron, High- and low-molecular-mass microbial surfactants, Appl. Microbiol. Biotechnol. 52 (1999) 154–162. [2] S.K. Satpute, A.G. Banpurkar, P.K. Dhakephalkar, I.M. Banat, B.A. Chopade, Methods for investigating biosurfactants and bioemulsifiers: a review, Crit. Rev. Biotechnol. 30 (2010) 127–144. [3] I.M. Banat, Biosurfactants production and possible uses in microbial enhanced oilrecovery and oil pollution remediation - a review, Bioresour. Technol. 51 (1995) 1–12. [4] F. Zhao, J.D. Zhou, F. Ma, R.J. Shi, S.Q. Han, J. Zhang, Y. Zhang, Simultaneous inhibition of sulfate-reducing bacteria, removal of H2S and production of rhamnolipid by recombinant Pseudomonas stutzeri Rhl: applications for microbial enhanced oil recovery, Bioresour. Technol. 207 (2016) 24–30. [5] J.E. Kinsella, D.M. Whitehead, Proteins in whey: chemical, physical, and functional properties, Adv. Food Nutr. Res. 33 (1989) 343–438. [6] G. Panaras, G. Moatsou, S. Yanniotis, I. Mandala, The influence of functional properties of different whey protein concentrates on the rheological and emulsification capacity of blends with xanthan gum, Carbohydr. Polym. 86 (2011) 433–440. [7] E. Dickinson, Colloids in food: ingredients, structure, and stability, Annu. Rev. Food Sci. Technol. 6 (6) (2015) 211–233. [8] H.R. Sharif, P.A. Williams, M.K. Sharif, S. Abbas, H. Majeed, K.G. Masamba, W. Safdar, F. Zhong, Current progress in the utilization of native and modified legume proteins as emulsifiers and encapsulants - a review, Food Hydrocolloid 76 (2018) 2–16. [9] X.L. Zhang, H. Gao, C.Y. Wang, A. Qayum, Z.S. Mu, Z.L. Gao, Z.M. Jiang, Characterization and comparison of the structure and antioxidant activity of glycosylated whey peptides from two pathways, Food Chem. 257 (2018) 279–288. [10] S. Shrestha, M.B. Sadiq, A.K. Anal, Culled banana resistant starch-soy protein isolate conjugate based emulsion enriched with astaxanthin to enhance its stability, Int. J. Biol. Macromol. 120 (2018) 449–459. [11] H. Yu, Y.X. Seow, P.K.C. Ong, W.B. Zhou, Kinetic study of high-intensity ultrasoundassisted Maillard reaction in a model system of D-glucose and glycine, Food Chem. 269 (2018) 628–637. [12] N. Cheetangdee, K. Fukada, Emulsifying activity of bovine beta-lactoglobulin conjugated with hexoses through the Maillard reaction, Colloids Surf. A 450 (2014)
Fig. 8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns for whey protein and rhamnolipid conjugates: Lane 1, molecular weight markers (11–180 kDa); lane2, whey protein; lanes 3, WP–rhamnolipid mixture sample; lanes 4–7, WP–rhamnolipid conjugates heated at 80 °C for 10, 20, 40, and 60 min.
combinations was too low to detect by SDS-PAGE. It can be seen from the mixture samples after heat treatment for 10, 20, 40, 60 min (lane 4, lane 5, lane 6 and lane 7) that as the heat treatment time increased, the two bands below the electropherogram were obviously shifted upwards, which might be caused by the combination of whey protein and rhamnolipid to produce a larger molecular weight product. Due to the small molecular weight of rhamnolipids, the shift in protein bands at large molecular weights was not significant. In the lanes of the conjugates treated with heat treatment for 40 min and 60 min, it can be seen that a new band was produced at the 46 kDa position, and the new band produced by the heat treatment for 60 min was clearer, indicating the formation of new reaction products (WP–rhamnolipid conjugates). SDS-PAGE was also reported to demonstrate the formation ofα-Lactalbumin: acacia gum (α-la : AG) conjugate [21], soy β-conglycinin–dextran conjugate [37], and whey protein isolate–sugar beet pectin (WPI–SBP) [38] treated by heating.
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