- Email: [email protected]
water-interface
Food Chemistry 302 (2020) 125349
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
FTIR analysis of β-lactoglobulin at the oil/water-interface a,⁎
b
Helena Schestkowa , Stephan Drusch , Anja Maria Wagemans a b
T
a
Food Colloids, Technische Universität Berlin, Königin-Luise-Straße 22, 14196 Berlin, Germany Food Technology and Food Material Science, Technische Universität Berlin, Königin-Luise-Straße 22, 14196 Berlin, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: Critical interfacial concentration Protein-stabilized emulsion FTIR subtraction method Second derivative spectra Pendant drop analysis Extrinsic fluorescence
Knowledge about the critical interfacial concentration of a protein supports our understanding of the kinetic stability of an emulsion. Its determination is currently limited to either invasive or indirect methods. The aim of our study was the determination of the critical interfacial concentration of whey protein β-lactoglobulin at oil/water-interfaces through fluorescence and pendant drop analysis and the comparison to an in situ Fouriertransform-infrared-spectroscopy (FTIR) method. Exponentially decreasing interfacial tension with increasing βlactoglobulin content (0.10–1.00 wt%) in pendant drop analysis could partly be confirmed by fluorescence spectra. A critical interfacial concentration of 0.20–0.31 wt% β-lactoglobulin (1.80–2.69 mg/m2) in oil/water (5/95)-emulsions was determined via FTIR, analyzing the Amide I/Amide II peak intensity ratio. This was confirmed by the increasing formation of intermolecular β-sheets, revealed by second derivative spectra. With this FTIR method we expand current options to investigate the interfacial behavior of food proteins by determination of secondary structure elements.
1. Introduction Oil/water-macroemulsions are known to be thermodynamically unstable (Capek, 2004; Lam & Nickerson, 2013). Thus, the kinetics of phase separation determine the macroemulsion stability which depends on the physicochemical conditions of the dispersed (oil) and the bulk (water) phase, as well as the physicochemical properties and molecular interactions of the emulsifier. In the case of protein-stabilized emulsions, kinetic stability varies with the occupation level of adsorbed protein at the oil/water-interface (Lam & Nickerson, 2013). The protein content at which the oil/water-interface is fully occupied is referred to as the critical interfacial concentration (CIC) and represents the minimum amount of protein needed to stabilize an emulsion (McClements, Decker, & Weiss, 2007; Tamm, Sauer, Scampicchio, & Drusch, 2012). At present, a non-invasive determination of the CIC in emulsions is still challenging. Current literature divides the CIC analysis into methods investigating a single drop against oil or an actual emulsion system. Pendant drop analysis can be used to determine the CIC by interfacial tension measurement of a single drop (Tamm et al., 2012). This system’s main limitation, though, is the specific interfacial area of the pendant drop that is significantly smaller than that of the multiple drops in an emulsion and therefore the amount of protein to cover the interface differs. Others described a method that measures the CIC in an
⁎
emulsion via centrifugation, followed by an analysis of the protein content in separated cream and bulk phase (Tcholakova, Denkov, Ivanov, & Campbell, 2002; Ye, 2010). However, phase separation through centrifugation alters the sample nature and may influence the sensitive equilibrium between adsorbed and bulk protein. Alternative methods for CIC determination could take advantage of changes in the secondary structure of proteins during adsorption at the oil/water-interface. A possible approach could be by an extrinsic fluorescence method that detects changes in protein polarity by emission maxima and intensity shifts of fluorescence markers like 8-anilinonaphthalene-1-sulfonic-acid (ANS) (Alizadeh-Pasdar & Li-Chan, 2000; Kato & Nakai, 1980). However, the challenge of a fluorescence measurement could be the low sensitivity as a consequence of inner filter effects of the emulsion (Alizadeh-Pasdar & Li-Chan, 2000; Kato & Nakai, 1980; Liu, Powers, Swanson, Hill, & Clark, 2005). In contrast, Fourier-transform-infrared-spectroscopy (FTIR) is a highly sensitive and non-invasive method to detect adsorption-associated structural changes in proteins (Lefèvre & Subirade, 2003). Peaks in FTIR spectra result from absorption of energy through vibration of chemical bonds such as in the peptide backbone of proteins, in particular the Amide I band (C] O stretch vibration) and Amide II band (CN stretching and in-plane NH bending vibration). These bands are sensitive to changes that can occur in hydrogen bonds of secondary structure elements such as intra- and intermolecular β-sheets (Kong & Yu, 2007).
Corresponding author. E-mail addresses: [email protected] (H. Schestkowa), [email protected] (S. Drusch), [email protected] (A.M. Wagemans).
https://doi.org/10.1016/j.foodchem.2019.125349 Received 24 April 2019; Received in revised form 1 August 2019; Accepted 8 August 2019 Available online 09 August 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
Food Chemistry 302 (2020) 125349
H. Schestkowa, et al.
Isolated protein solutions were freeze-dried. The isolated β-lg powder had an approximate β-lg content of 96 wt%, measured by HPLC according to Keppler et al. (2014).
There is controversy in current literature regarding the structure of adsorbed proteins (Zhai, Day, Aguilar, & Wooster, 2013). An increase in intermolecular and a decrease in intramolecular β-sheets has been described during adsorption at a soybean oil/water-interface (Fang & Dalgleish, 1997). Others agreed to these findings, but have found opposing trends in the formation of intermolecular β-sheets for diacyl- and triacyglycerol/water-interfaces or could only detect minor structural changes (Husband, Garrood, Mackie, Burnett, & Wilde, 2001; Sakuno, Matsumoto, Kawai, Taihei, & Matsumura, 2008). These diverse trends may result from the FTIR methods where the extraction of the protein signal was realized by subtracting plain water and oil spectra from the emulsion’s spectra. Due to a higher oil concentration, signal intensity of plain oil differs strongly from the oil signal in emulsion leading to the methodological challenge of protein signal extraction. To obtain accurate data, similar oil band shapes should be considered within the subtraction method. The aim of our study was the comparison of CIC determination of protein-stabilized oil/water-emulsions in pendant drop system with fluorescence and FTIR analysis as well as the subsequent improvement of an accurate in situ FTIR subtraction method. For this purpose, we aimed to subtract the oil signal from the protein-stabilized emulsion in form of an emulsion instead of a plain oil phase. Doing so, we analyzed the whey protein β-lactoglobulin (β-lg) as model protein for interfacial active globular proteins in water and in oil/water-emulsions with increasing protein content. In order to minimize interactions between protein and oil, we have chosen middle-chain-triacylglyceride (MCT)oil as model oil with mainly saturated fatty acids. We assumed that the adsorption-associated structural changes of β-lg would increase with increasing protein content, until the oil/water-interface is fully occupied. In detail, we hypothesized the following:
2.2. MCT-oil purification Middle-chain-triacylglyceride-oil (MCT-oil, > 99.9%, Witarix MCT 60/40, IOI Oleochemical, Hamburg, Germany) was purified with activated magnesium silicate Florisil (MgO × 3.6 SiO2 × 1.53 OH, 100%, Carl Roth GmbH, Karlsruhe, Germany), to remove interfacial active compounds, such as free fatty acids, in accordance with Schestkowa et al. (2018). 2.3. Solution and emulsion preparation Protein solutions were prepared by dissolving 0.01–1.00 wt% β-lg in distilled water and stirred for 16 h at 22 °C and 500 rpm. The pH was adjusted to pH 7.0 with 0.1 M HCl and 0.1 M NaOH. Protein solution and MCT-oil (weight ratio 95/5) were mixed for 15 s at 22 °C and 17,500 rpm using an Ultra-Turrax (IKA-Werke GmbH, Staufen, Germany). Since the proportion of the protein in relation to the oil was varied, the protein/oil ratios are additionally given below, e.g. 1.00 wt % β-lg corresponds to a protein/oil ratio of 0.95/5. In all methods, emulsions were compared to water or β-lg solutions (1.00 wt% in water). For FTIR analyses, 0.20 wt% SDS (> 99%, Serva Electrophoresis GmbH, Heidelberg, Germany) in water and 0.20 wt% SDS in emulsion (water/MCT-oil ratio 95/5) were prepared additionally. 2.4. Pendant drop analysis
• The CIC can be measured by intensity variation and blue shifts in • •
The interfacial tension analysis at the oil/water-interface and the measurement of specific interfacial area of the single drop was performed with the automated drop tensiometer OCA 20 (Dataphysics GmbH, Filderstadt, Germany) as described by Tamm et al. (2012). The protein solutions with 0.01–1.00 wt% β-lg in water were measured against MCT-oil in a quartz-glass cuvette for 1 h at 22 °C. Therefore, a 30 µL droplet was dosed by an automatic dosing system (flow rate 5 µL/ s) and the interfacial tension was extracted from droplet shape recorded with a high speed camera (approx. 2 frames per second). The curve of interfacial tension was exponentially plotted according to Schestkowa et al. (2018) and the value of the asymptote was plotted against the protein content. A significant difference from the asymptote was classified at 10% deviation.
extrinsic fluorescence emission spectra occurring through changes in molecular polarity during adsorption of increasing amounts of protein at the oil/water-interfaces. The subtraction of a surfactant-stabilized emulsion FTIR spectrum from the protein-stabilized emulsion FTIR spectrum results in an accurate protein signal, since similar oil band shapes are applied. The amount of adsorbed protein at oil/water-interfaces affects the secondary structure and therefore the location and intensity of Amide I and II bands in FTIR spectra due to secondary structure sensitive C]O and NH vibration, enabling CIC determination.
To test these hypotheses, we developed an improved FTIR method to determine changes in the structure of adsorbed β-lg, including reference and background subtraction of an SDS (sodium dodecyl sulfate) emulsion using a similar oil droplet size distribution in comparison to βlg emulsions. For CIC determination, Amide I and II band of FTIR subtraction spectra of β-lg at oil/water-interfaces and the second derivative was evaluated regarding changes in secondary structure. We compared the CIC analysis via FTIR to decreasing interfacial tension in pendant drop and emission maxima of extrinsic fluorescence spectra.
2.5. Extrinsic fluorescence measurement Fluorescence measurements were obtained with a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, Victoria, Australia) with slight modifications to Liu et al. (2005). The extrinsic fluorescence marker 8-anilinonaphthalene-1-sulfonic-acid (ANS, > 97%, Sigma Aldrich, St. Louis, USA) was used. Measurement solutions were prepared by mixing 20 µL ANS (8 mM, 50.7 mg diluted in 20 mL distilled water) and 4 mL β-lg emulsion. The spectra were recorded in quartz cells with 10 mm optical length at an excitation wavelength of 390 nm and an emission wavelength scan from 300 to 600 nm. The maximum of fluorescence intensity was plotted against the protein content in the water fraction in emulsion.
2. Materials and methods 2.1. β-lactoglobulin isolation The β-lg-rich whey protein isolate GermanProt 9000 was provided by Sachsenmilch Leppersdorf GmbH (> 90%, Leppersdorf, Germany). β-lg was isolated following the procedure of Keppler, Sönnichsen, Lorenzen, and Schwarz (2014) with slight modifications. The ultrafiltration was replaced by dialysis for 72 h with distilled water at 22 °C using BioDesignDialysis TubingTM by ThermoFisher Scientific (molecular weight cut-off 14 kDa, Waltham Massachusetts, USA). The pH was adjusted to pH 7 with 0.1 M HCl and 0.1 M NaOH, both purchased from Carl Roth GmbH (> 99.9%, analytical-grade, Karlsruhe, Germany).
2.6. FTIR analysis Infrared spectra of 0.10–1.00 wt% β-lg emulsions and 1.00 wt% β-lg water solutions as well as 0.20 wt% SDS in water and in emulsion were measured against water as background with a Bruker Tensor II spectrometer (Bruker Optic GmbH, Karlsruhe, Germany) equipped with a liquid nitrogen-cooled mercury-cadmium-telluride detector. The 2
Food Chemistry 302 (2020) 125349
H. Schestkowa, et al.
γ (c) = Ae−Bc + γ∞ = 6.3e(−6.3c) + 12.6
samples were loaded into the transmission cell AquaSpec A 741/Q (Bruker Optic GmbH, Karlsruhe, Germany) between two CaF2 windows with a path length of 7 µm. The transmission cell was cleaned with the enzyme kit ProteoClean 10 A741-C1 (Bruker Optic GmbH, Karlsruhe, Germany) and washed with distilled water. Each spectrum is an absorbance spectrum measured in transmission and a result of the average of 120 scans recorded with a resolution of 4 cm−1 at 22 °C from 1000 to 3100 cm−1. Spectra were edited with the software provided with the spectrometer (OPUS 7.5); raw spectra were offset-corrected in the region of 2000–2010 cm−1 and the second derivative of vector-normalized (1480–1780 cm−1) spectra were smoothed with the 25-point Savitsky-Golay method. FTIR subtraction method is described in section 3.3 CIC by FTIR analysis. Interfacial occupation level for CIC determined by FTIR was calculated in consideration of the specific interfacial area of the emulsions.
(1)
with lower asymptote γ∞ and the empirical variables A and B. The results showed that the higher the protein content, the lower the value of the interfacial tension approaching the asymptote γ∞ = 12.6 mN/m at protein contents from 0.3 to 1.0 wt% β-lg. No further change in interfacial tension with further increase of protein content indicates that the interface is fully occupied and no denser molecule packing at the interface is possible. There is scientific consensus that the molecular density of proteins such as β-lg varies with the preoccupation level, due to variation in the packing density of the protein film at the oil/waterinterface (Capek, 2004; Foegeding & Davis, 2011). At the CIC, the protein spreads all over the interface and, consequently, aligns more densely with increasing protein content. According to the pendant drop analysis results, it becomes clear that the CIC is a concentration region up to approx. 0.3 wt% indicated by the exponential decrease of interfacial tension. The determination of the minimum concentration of the CIC region could not be realized and needs further investigation. Tamm et al. (2012) adapted the CIC determination by calculation of two linear fits as commonly applied in the determination of the critical micellar concentration (CMC) of low molecular weight surfactants. We assume that differences result from the CIC determination method and different system parameters such as the sample composition and type of interface; they used whey protein isolate and measured at an air/water-interface (Tamm et al., 2012). The main limitation of pendant drop analysis is the differing specific interface area when measuring a 30 µL-drop against MCT-oil compared to an actual emulsion; the specific interface area of the actual β-lg emulsions amounted to 1.0754 ± 0.0793 m2/cm3 and was almost three orders of magnitude higher than the specific interface area in pendant drop analysis (0.0015 ± 0.0003 m2/cm3) whereas the protein content remained the same. With this in mind, CIC values analyzed by pendant drop should appear smaller than in emulsions due to the lower specific interface area in single drop experiments.
2.7. Droplet size measurement The oil droplet size distribution and specific interfacial area of the emulsions was measured using static laser scattering (Horiba LA-950, Retsch Technology GmbH, Haan, Germany). Doing so, the refractive index of 1.45 and a set transmission value of 98% was used. If not reported otherwise, analyses were performed at least three times (n = 3) and mean as well as standard deviation were calculated. 3. Results 3.1. Critical interfacial concentration (CIC) of proteins The adsorption of protein, such as whey protein β-lactoglobulin (βlg), at oil/water-interfaces leads to a reduction of the interfacial tension between oil and water and a fine distribution of the dispersed in continuous phase (Beverung, Radke, & Blanch, 1999). Therefore, pendant drop analysis is commonly used to observe the adsorption process of emulsifiers by means of changes in interfacial tension (Beverung et al., 1999; Rühs, Scheuble, Windhab, & Fischer, 2013; Tamm et al., 2012; Wüstneck et al., 1996). Fig. 1A shows the decrease of the interfacial tension of β-lg solution with increasing protein content against MCT-oil. The interfacial tension was plotted exponentially as a function of time similar to Eq. (1). The values of the asymptotes from this fit were plotted against the protein content (Fig. 1B). The exponential function γ© was fitted as follows Eq. (1):
3.2. CIC by fluorescence analysis For non-invasive CIC determination in actual emulsions extrinsic fluorescence was observed for ANS treated β-lg emulsions (Fig. 2A). First, intensity of emission maxima were plotted against the protein content (Fig. 2B). The extrinsic fluorescence intensity increased with increasing β-lg content in the emulsion (linear fit Imax,E(c), R2 = 0.9943) without indicating a possible shift pointing out the CIC.
Fig. 1. (A) Interfacial tension of β-lg solutions (0.01–1.00 wt% in water) in MCT-oil as function of time (n = 3, n = 1 illustrated) and values of asymptotes of interfacial tension (colored area in A) as function of protein content (B) analyzed by pendant drop and exponentially plotted function γ (c) with asymptote γ∞. 3
Food Chemistry 302 (2020) 125349
H. Schestkowa, et al.
Fig. 2. (A) Extrinsic fluorescence spectra of protein emulsions with 0.10–1.00 wt% β-lg content or rather protein/oil ratio of 0.095/5–0.95/5 (solid lines, protein content is referred to protein content in water fraction of emulsion) and 1.00 wt% β-lg in water (dashed line, n = 3, n = 1 illustrated). (B) Emission maxima (colored area in A) as function of protein content with linear fit Imax,E.
Second, a blue shift was observed compared to β-lg in water due to quenching effects (supplementary data S1 A B) (Alizadeh-Pasdar & LiChan, 2000; Kato & Nakai, 1980; Liu et al., 2005; Renard, Lefèvre, Griffin, & Griffin, 1998). The blue shift of β-lg emulsions indicates changes in molecular polarity of the protein from a less polar to a more polar environment for pressure treated proteins (Liu et al., 2005; Yang, Dunker, Powers, Clark, & Swanson, 2001). Although the peak shift is characteristic for adsorbed proteins in emulsion, the measured values did not approach the value of water and, consequently, the CIC could not be determined by extrinsic fluorescence analysis with ANS as hypothesized. Additionally, peak intensity ratio (PIR, approx. 470/ 530 nm) was calculated as shown in supplementary data S1 C D. Fluorescence results did partly indicate a similar behavior approaching an asymptote at 0.4 wt% protein (supplementary data S1) as shown for pendant drop analysis in Fig. 1B, although a higher value for a larger interface area was expected. This might be caused by the method not being sensitive enough to detect small differences in molecular polarity due to absorption and light scattering of the dispersed oil phase, summarized as inner filter effects of the emulsion (Alizadeh-Pasdar & LiChan, 2000; Kato & Nakai, 1980; Liu et al., 2005). In conclusion, accurate CIC determination should be performed by a more sensitive method in comparison to fluorescence analysis such as the secondary structure sensitive FTIR. Therefore, we developed and improved the FTIR subtraction method and further calculated the CIC based on changes in Amide I and II bands.
I and II region, taking the oil concentration in emulsion and oil droplet size distribution into account as described in depth below. Emulsions are heterogeneous systems, therefore, minor deviations in the sample characteristics (such as the measured oil droplets) alter Mie-scattering (Bassan et al., 2010), which in turn result in slightly different oil signal intensities e.g. at 1750 cm−1. To avoid theses deviations spectra with matching oil-signals were selected for subtraction assuming that the measured samples feature similar sample characteristics. Moreover, matching oil signals reduce the misinterpretation of band shapes during evaluation of difference spectra, which might occur since changes in secondary structure are easily over- or underestimated when spectra are subtracted (De Jongh, Goormaghtigh, & Ruysschaert, 1996; Dong, Huang, & Caughey, 1990). A similar approach was used by Zhai et al. (2012) to extract protein signals from synchrotron radiation circular dichroism spectra of protein-stabilized emulsions by reducing disturbing light scattering effects. In accordance with Kong and Yu (2007) we assume that water does not cause any altered vibration in an emulsion and, thus, we retained background subtraction of water. First, a background spectrum of distilled water was measured to remove the water signal from the subsequent measurements. Next, measurements of β-lg in emulsion (β-lgE), SDS in water (SDSW) and SDS in emulsion (SDSE) were performed. In order to obtain the MCT-oil signal spectrum we removed the SDS signal from SDS in emulsion by subtraction of SDSW spectra from the SDSE spectra with a subtraction factor based on the SDS band intensity at 1250 cm−1. This band was chosen due to minor overlapping with oil signals. The obtained spectrum is referred to as difference spectrum SDSE,D (Fig. 3A). Following that, the MCT-oil signal spectrum was removed from the protein emulsion by subtracting SDSE,D from the β-lgE spectrum (Fig. 3B) with an subtraction factor based on the MCT-oil band intensity at 1750 cm−1. Thus, the spectrum β-lgE,D with the Amide I and II band at approx. 1500–1700 cm−1 could be obtained. For further evaluation the β-lgE,D spectra were offset-corrected and the intensity of Amide I and II band was determined. Both Amide I and Amide II showed a protein linearity in the range from 0.10 to 1.00 wt% (protein/oil ratio of 0.095/ 5–0.95/5) with R2Amide I = 0.9980 and R2Amide II = 0.9985, respectively. The peak intensity ratio (PIR) of Amide I/Amide II was calculated (Fig. 4A) to eliminate the effect of varying repeatability precision of Amide I and Amide II absolute intensity (n = 7, 0.50 wt% β-lg in emulsion, IAmide I = 0.759 ± 0.039, IAmide II = 0.675 ± 0.040). PIR was plotted as function of protein content and resulted in a double asymptotic fit. The turning point of the fit was calculated and a
3.3. CIC by FTIR analysis An in situ FTIR subtraction method was developed to evaluate the changes in secondary structure of adsorbed proteins at oil/water-interfaces. Fig. 3B illustrates an FTIR absorbance spectrum measured in transmission of a β-lg-stabilized emulsion (β-lgE). A subtraction method to obtain the signal of the adsorbed protein by subtracting pure oil and water spectra from the emulsion spectra has already been published (Fang & Dalgleish, 1997, 1998; Fink & Gendreau, 1984; Lefèvre & Subirade, 2003; Tcholakova, Denkov, Sidzhakova, & Campbell, 2006). Following the given information in their publications, we have not been able to extract accurate Amide I and II bands due to varying oil signals (supplementary data S2). However, oil spectra shifted at approx. 1750 cm−1 showing a wider and larger band in the emulsion compared to the pure oil. Consequently, we subtracted the spectrum of an emulsion system with SDS as surfactant, which does not absorb at the Amide 4
Food Chemistry 302 (2020) 125349
H. Schestkowa, et al.
Fig. 3. FTIR subtraction method to remove (A) SDS bands at approx. 1250 cm−1 (colored area) from SDS emulsion spectra SDSE with SDS in water spectrum SDSW to obtain SDS difference spectra SDSE,D and to further remove (B) MCT-oil band at approx. 1750 cm−1 (colored area) from β-lg emulsion spectra β-lgE to obtain an β-lg spectrum β-lgE,D with Amide I and II bands at approx. 1500–1700 cm−1 (colored area).
of Amide II with increasing β-lg content. As summarized by Jackson and Mantsch, Amide I and II absorption intensity is affected by the vibration of C]O and NH in the protein peptide backbone (Jackson & Mantsch, 1995). Secondary structure elements such as β-sheets are formed within this peptide backbone and are held in shape by hydrogen bonds. Vibration of electron-withdrawing groups such as C]O and NH groups is influenced by their electron density and therefore by the strength of the existing hydrogen bonds. The weaker the hydrogen bond involving the C]O group, the higher the electron density and Amide I absorption (Barth, 2007; Jackson & Mantsch, 1995). For protein adsorbed at oil/water-interfaces, the strength of hydrogen bonds inside secondary structure elements changes due to partial denaturation caused by rearrangement of hydrophobic and hydrophilic parts of the peptide backbone facing towards the oil and water phase respectively. As a result, both band intensity and frequency can vary with the protein conformation at the interface, which in turn is affected by the amount of adsorbed protein. Because of the higher secondary structure sensitivity of Amide I in
significant difference from the asymptotes was classified at 10% deviation. No correlation between PIR and the median of the oil droplet size distribution could be found (supplementary data S2). The second derivative of the Amide I region was calculated to determine the position of the maximum of broad Amide bands more precisely. Finally, the second derivative spectra of vector-normalized β-lgE,D spectra were analyzed with regards to their bands in the Amide I region (approx. 1600–1700 cm−1) as presented in Fig. 5. In order to determine the CIC of β-lg at oil/water-interfaces, the intensity of Amide I and II bands as well as the second derivative of the Amide I band as function of the β-lg content was considered. Doing so, FTIR spectra of 0.10–1.00 wt% β-lg (protein/oil ratio of 0.095/5–0.95/ 5) in emulsion and spectra of 1.00 wt% β-lg in water were recorded. The resulting FTIR difference spectra β-lgE,D of the Amide I and II region are shown in Fig. 4A. The increase of the Amide I and II intensity with increasing β-lg content was accompanied by a shift of the Amide I/Amide II PIR (Fig. 4B). Here, the intensity of Amide I increased less compared to that
Fig. 4. (A) FTIR spectra β-lgE,D of the Amide I and II region for protein emulsions with 0.10–1.00 wt% β-lg content or rather protein/oil ratio of 0.095/5–0.95/5 (solid lines) and 1.00 wt% β-lg in water (dashed line, n = 3, n = 1 illustrated). (B) Peak intensity ratio PIR of Amide I/Amide II (colored area in A) against β-lg content (protein content is referred to protein content in water fraction of emulsion) with a double asymptotic function PIR(c) and asymptotes PIR0 and PIR∞. 5
Food Chemistry 302 (2020) 125349
H. Schestkowa, et al.
Fig. 5. (A) Second derivative FTIR spectra of β-lgE,D in the Amide I region with 0.10–1.00 wt% β-lg content in emulsion or rather a protein/oil ratio of 0.095/5–0.95/ 5 (solid lines) and 1.00 wt% β-lg in water (dashed line, n = 3, n = 1 illustrated). (B) Intensity of 1630 cm−1-band of FTIR second derivative spectra (colored area in A) against protein content and exponential fit I(c) with asymptote I∞ and horizontal dashed line for 1.00 wt% β-lg in water.
in Fig. 6 (stage I). Consequently, the FTIR signal resulted mainly from protein at the oil/water-interface approaching the lower asymptote. The PIR(c) increased in the region approx. from 0.20 to 0.60 wt% protein content in the emulsion (protein/oil ratio of 0.19/5–0.57/5). Regarding changes in secondary structure through protein adsorption, we assume that the oil/water-interface is fully occupied at this region of the function (Fig. 6 stage II). The turning point at approx. 0.31 wt% of protein content could indicate a change in equilibrium between adsorbed and bulk protein towards the bulk protein side. In conclusion, we assume that the CIC region of β-lg at oil/water-interfaces ranges from 0.20 to 0.31 wt% protein content in the water phase of the emulsions (protein/oil ratio of 0.19/5–0.29/5), resulting in an interface occupation of 1.80–2.69 mg/m2. Above the CIC region, PIR(c) approached the upper asymptote PIR∞ = 1.20 at protein contents of 0.31–1.00 wt% (protein/oil ratio of 0.95/5). Here, the bulk protein signal dominates the FTIR signal of the adsorbed protein and, hence, the signal approaches the PIR of protein in water as illustrated in Fig. 6 stage III. This assumption was confirmed by the spectra of 1.0 wt% β-lg in water (without any oil) resulting in a PIR of 1.21 ± 0.14 that equals the upper asymptote PIR∞ of emulsions. This saturation of interface occupation was also reported by Taherian, Britten, Sabik, and Fustier
comparison to Amide II, the PIR of Amide I/Amide II was assumed to differ with the protein content. This is due to different secondary structure elements in adsorbed and bulk protein as well as the structural changes caused by the occupation level (Kong & Yu, 2007). In addition, variations in Amide band intensities can be explained by the displacement of water molecules through the sample; the more protein covers the oil/water-interface, the less water molecules are in contact with the protein, due to interactions with the oil phase and the lower is Amide I band intensity (Rahmelow & Hubner, 1997; Zeng, Chittur, & Lacefield, 1999). As function of the protein content, the PIR (Fig. 4B) showed a double asymptotic behavior that was empirically plotted with the correlation PIR(c), as indicated by Eq. (2):
PIR(c) = PIR ∞ +
PIR 0 − PIR ∞ 0.64 − 1.20 = 1.20 + 1 + Kcm 1 + 2.83c3.74
(2)
with upper asymptote PIR∞, lower asymptote PIR0 and the variables K and m. The PIR(c) function approached the lower asymptote PIR0 = 0.64 at 0.20 wt% of protein (protein/oil ratio of 0.19/5). At a low protein content, the equilibrium of adsorbed protein and protein in bulk phase was mostly on the side of the adsorbed protein, as illustrated
Fig. 6. Whey protein β-lg adsorption at oil/water-interfaces as a function of protein content. Equilibrium between adsorbed and bulk protein tends towards adsorbed protein at stage I and is on side of bulk protein at stage III. The critical interfacial region is in stage II; the oil/water-interface is fully occupied and the interfacial film becomes denser upon protein addition. 6
Food Chemistry 302 (2020) 125349
H. Schestkowa, et al.
spectra of adsorbed proteins at oil/water-interfaces. As hypothesized, changes in secondary structure during adsorption affected the intensity and location of hydrogen bond sensitive C]O and NH vibration in the Amide I and II band. The Amide I/Amide II peak intensity ratio of FTIR difference spectra indicated a shift of the equilibrium from adsorbed to bulk protein with increasing β-lg content. The critical interfacial concentration region could be detected via FTIR at 0.20–0.31 wt% (1.80–2.69 mg/m2) protein content in oil/water (5/95)-emulsions or rather a protein/oil ratio of 0.19/5–0.29/5. Second derivative spectra of the Amide I band supported the trends of the Amide I/Amide II peak intensity ratio. The findings extend current literature of interfacial protein film formation in respect to the film density with increasing protein content in emulsions (Capek, 2004; Foegeding & Davis, 2011). Our findings are the basis for an in situ characterization of the interfacial concentration of proteins in actual emulsions via FTIR analysis. The FTIR method can further be used to characterize concentrations relevant for analytical investigation of interfacial stabilization mechanisms of proteins. Consequently, interactions and mechanical properties of the protein film itself could be characterized by amplitude-stress tests with interfacial shear- and dilatational rheology in concentrations below the CIC to avoid the formation of interfacial multilayer and to provide defined measurement conditions. To extend the applicability of the FTIR subtraction method for food systems, the robustness should be enhanced by analyzing β-lg with structural alterations as they can be achieved via physical or biotechnological approaches. Since the FTIR method is based on relative changes in secondary structure depending on the unfolded protein at the interface, the interfacial behavior and CIC could be investigated as a function of the initial degree of denaturation and unfolding. In particular, emulsification process-related structural changes in proteins should be monitored. The sensitivity of the FTIR method could be optimized by analyzing proteins from other sources including molecules with a more or less complex secondary, tertiary and quaternary structure as well as protein mixtures. As a further step, the oil could be varied in order to investigate the influence of the interactions between protein and both saturated and unsaturated fatty acids as well as fatty acids with varying length of aliphatic chains. The latter will allow to describe the structure-function-relationships in dispersed food systems in dependence of the initial properties and interactions between its individual constituents its in.
(2011) by analyzing whey protein. To verify results from the analysis of Amide I and II band shape, band intensities of the second derivative FTIR spectra in the Amide I region (1700–1600 cm−1) were taken into account, as commonly applied for analysis of secondary structure elements of proteins (Dong et al., 1990; Kong & Yu, 2007). The change in intensity of these second derivative bands can be ascribed to changes of the secondary structure, because the spectra were vector-normalized which takes out direct effects of the protein concentration. In this case, the secondary structure varies with the occupancy at the oil/water-interface that changes with the protein concentration. The secondary structure of native β-lg consists mainly of an eight-stranded intramolecular β-sheet barrel with a short α-helix segment and additional parts with random coil structures (Dong et al., 1996). The Amide I region of the second derivative FTIR difference spectra β-lgE,D of adsorbed β-lg is shown in Fig. 5A. With increasing protein content in the emulsion, a band shift from 1641 to 1644 cm−1 occurred that indicates the formation of random coil structure elements (Jackson & Mantsch, 1995; Kong & Yu, 2007). As reported by Zhai et al. (2013), an increase of random coil structures throughout adsorption indicates unfolding of the protein at the oil/ water-interface. The observed increase of the band at 1616 cm−1 implies an adsorption-associated aggregation of β-strands accompanied by a formation of intermolecular β-sheets which is in line with Lefèvre and Subirade (2003), who emphasized the meaning of intermolecular βsheet formation during protein film formation. The intramolecular βsheet content increased with decreasing peak intensity at 1630 cm−1. Here, the intensity at 1630 cm−1 approached the minimum of the difference in the second derivative of the spectra with increasing protein concentration (Fig. 5B). The exponential approximation corresponds to Eq. (1). Furthermore, the asymptote of the peak intensity at 1630 cm−1 of adsorbed β-lg is equivalent to the value of β-lg in water (shown by the dashed line in Fig. 5B). We assume that this approximation of the peak intensity at 1630 cm−1 to the value of β-lg in water is caused by bulk protein superimposing the adsorbed protein signal with increasing protein content. These results agree with the trends from PIR in Fig. 4B, where the signal of adsorbed protein also approached the signal of water. Protein contents below 0.31 wt% β-lg (protein/oil ratio of 0.29/ 5) differed distinctively from the asymptote. Furthermore, the protein content of 0.31 wt% β-lg corresponds to the turning point of the PIR(c) curve in Fig. 4B that indicated full occupation of the oil/water-interface (Fig. 6 stage II).
Acknowledgements 4. Conclusion This research project was supported by the German Research Foundation Priority Program 1934, DispBiotech. We thank Julia Keppler from CAU Kiel for the help with the β-lg isolation and IOI Oleochemicals (Hamburg, Germany) for providing MCT-oil as well as Tobias Wollborn and Udo Fritsching (Leibniz Institute for Materials Engineering – IWT, Particles and Process Engineering, University of Bremen) for simulated protein pictures. Helena Schestkowa thanks Annette Müller and others for discussion.
Our study represents the first step towards an in situ measurement of the critical interfacial concentration (CIC) by FTIR in emulsion-based food systems. Therefore, we developed a FTIR subtraction method for accurate non-invasive determination of the CIC of model protein βlactoglobulin (β-lg) at MCT-oil/water-interfaces and compared the trends to both extrinsic fluorescence and pendant drop analysis. The exponential decrease of interfacial tension with increasing protein content measured by pendant drop analysis indicated a concentration region for the CIC of β-lg in oil/water (5/95)-emulsions. Due to the smaller specific interfacial area of the single drop compared to the actual emulsion system we decided to perform extrinsic fluorescence analysis of the emulsion. Peak intensity and polarity dependent blue shift of the extrinsic fluorescence emission spectra showed the low sensitivity of the fluorescence method and, therefore, could only partly confirm trends from pendant drop analysis. Consequently, for in situ CIC determination, we developed an FTIR subtraction method including the subtraction of an SDS-stabilized emulsion from protein-stabilized emulsion spectra to ensure a similarity of subtracted spectra. Doing so, we optimized previous techniques to subtract plain water and oil spectra (Fang & Dalgleish, 1997, 1998; Lee, Lefèvre, Subirade, & Paquin, 2007; Lefèvre & Subirade, 2003; Tcholakova et al., 2006). In conclusion, we recorded spectra of secondary structure sensitive FTIR
Declaration of Competing Interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125349. References Alizadeh-Pasdar, N., & Li-Chan, E. C. Y. (2000). Comparison of protein surface hydrophobicity measured at various pH values using three different fluorescent probes. Journal of Agricultural and Food Chemistry, 48(2), 328–334. https://doi.org/10.1021/
7
Food Chemistry 302 (2020) 125349
H. Schestkowa, et al.
Lefèvre, T., & Subirade, M. (2003). Formation of intermolecular β-sheet structures: A phenomenon relevant to protein film structure at oil-water interfaces of emulsions. Journal of Colloid and Interface Science, 263(1), 59–67. https://doi.org/10.1016/ S0021-9797(03)00252-2. Liu, X., Powers, J. R., Swanson, B. G., Hill, H. H., & Clark, S. (2005). Modification of whey protein concentrate hydrophobicity by high hydrostatic pressure. Innovative Food Science and Emerging Technologies, 6(3), 310–317. https://doi.org/10.1016/j.ifset. 2005.03.006. McClements, D. J., Decker, E. A., & Weiss, J. (2007). Emulsion-based delivery systems for lipophilic bioactive components. Journal of Food Science, 72(8), 109–124. https://doi. org/10.1111/j.1750-3841.2007.00507.x. Rahmelow, K., & Hubner, W. (1997). Infrared spectroscopy in aqueous solution: Difficulties and accuracy of water subtraction. Applied Spectroscopy, 51, 160–170. Renard, D., Lefèvre, J., Griffin, M. C. A., & Griffin, W. G. (1998). Effects of pH and salt environment on the association of β-lactoglobulin revealed by intrinsic fluorescence studies. International Journal of Biological Macromolecules, 22(1), 41–49. https://doi. org/10.1016/S0141-8130(97)00086-X. Rühs, P. A., Scheuble, N., Windhab, E. J., & Fischer, P. (2013). Protein adsorption and interfacial rheology interfering in dilatational experiment. European Physical Journal: Special Topics, 222(1), 47–60. https://doi.org/10.1140/epjst/e2013-01825-0. Sakuno, M. M., Matsumoto, S., Kawai, S., Taihei, K., & Matsumura, Y. (2008). Adsorption and structural change of beta-lactoglobulin at the diacylglycerol-water interface. Langmuir: The ACS Journal of Surfaces and Colloids, 24(20), 11483–11488. https://doi. org/10.1021/la8018277. Schestkowa, H., Wollborn, T., Westphal, A., Maria Wagemans, A., Fritsching, U., & Drusch, S. (2018). Conformational state and charge determine the interfacial stabilization process of beta-lactoglobulin at preoccupied interfaces. Journal of Colloid and Interface Science, 536, 300–309. https://doi.org/10.1016/j.jcis.2018.10.043. Taherian, A. R., Britten, M., Sabik, H., & Fustier, P. (2011). Ability of whey protein isolate and/or fish gelatin to inhibit physical separation and lipid oxidation in fish oil-inwater beverage emulsion. Food Hydrocolloids, 25(5), 868–878. https://doi.org/10. 1016/j.foodhyd.2010.08.007. Tamm, F., Sauer, G., Scampicchio, M., & Drusch, S. (2012). Pendant drop tensiometry for the evaluation of the foaming properties of milk-derived proteins. Food Hydrocolloids, 27(2), 371–377. https://doi.org/10.1016/j.foodhyd.2011.10.013. Tcholakova, S., Denkov, N. D., Ivanov, I. B., & Campbell, B. (2002). Coalescence in βlactoglobulin-stabilized emulsions: Effects of protein adsorption and drop size. Langmuir, 18(23), 8960–8971. https://doi.org/10.1021/la0258188. Tcholakova, S., Denkov, N. D., Sidzhakova, D., & Campbell, B. (2006). Effect of thermal treatment, ionic strength, and pH on the short-term and long-term coalescence stability of b-lactoglobulin emulsions. Langmuir: The ACS Journal of Surfaces and Colloids, 22(14), 6042–6052. https://doi.org/10.1021/la0603626. Wüstneck, R., Krägel, J., Miller, R., Fainerman, V. B., Wilde, P. J., Sarker, D. K., & Clark, D. C. (1996). Dynamic surface tension and adsorption properties of β-casein and βlactoglobulin. Food Hydrocolloids, 10(4), 395–405. https://doi.org/10.1016/S0268005X(96)80018-X. Yang, J., Dunker, A. K., Powers, J. R., Clark, S., & Swanson, B. G. (2001). Beta-lactoglobulin molten globule induced by high pressure. Journal of Agricultural and Food Chemistry, 49(7), 3236–3243. https://doi.org/10.1021/jf001226o. Ye, A. (2010). Surface protein composition and concentration of whey protein isolatestabilized oil-in-water emulsions: effect of heat treatment. Colloids and Surfaces B: Biointerfaces, 78(1), 24–29. https://doi.org/10.1016/j.colsurfb.2010.02.001. Zeng, H., Chittur, K. K., & Lacefield, W. R. (1999). Analysis of bovine serum albumin adsorption on calcium phosphate and titanium surfaces. Biomaterials, 20(4), 377–384. https://doi.org/10.1016/S0142-9612(98)00184-7. Zhai, J. L., Day, L., Aguilar, M. I., & Wooster, T. J. (2013). Protein folding at emulsion oil/ water interfaces. Current Opinion in Colloid and Interface Science, 18(4), 257–271. https://doi.org/10.1016/j.cocis.2013.03.002. Zhai, Jiali, Hoffmann, S. V., Day, L., Lee, T. H., Augustin, M. A., Aguilar, M. I., & Wooster, T. J. (2012). Conformational changes of beta-lactalbumin adsorbed at oil-water interfaces: Interplay between protein structure and emulsion stability. Langmuir, 28(5), 2357–2367. https://doi.org/10.1021/la203281c.
jf990393p. Barth, A. (2007). Infrared spectroscopy of proteins. Biochimica et Biophysica Acta – Bioenergetics, 1767(9), 1073–1101. https://doi.org/10.1016/j.bbabio.2007.06.004. Bassan, P., Kohler, A., Martens, H., Lee, J., Byrne, H. J., Dumas, P., et al. (2010). Resonant Mie Scattering (RMieS) correction of infrared spectra from highly scattering biological samples. Analyst, 135(2), 268–277. https://doi.org/10.1039/b921056c. Beverung, C. J., Radke, C. J., & Blanch, H. W. (1999). Protein adsorption at the oil/water interface: characterization of adsorption kinetics by dynamic interfacial tension measurements. Biophysical Chemistry, 81(1), 59–80. https://doi.org/10.1016/S03014622(99)00082-4. Capek, I. (2004). Degradation of kinetically-stable o/w emulsions. Advances in Colloid and Interface Science, 107(2–3), 125–155. https://doi.org/10.1016/S0001-8686(03) 00115-5. De Jongh, H. H. J., Goormaghtigh, E., & Ruysschaert, J. M. (1996). The different molar absorptivities of the secondary structure types in the amide I region: An attenuated total reflection infrared study on globular proteins. Analytical Biochemistry, 242(1), 95–103. https://doi.org/10.1006/abio.1996.0434. Dong, A., Huang, P., & Caughey, W. S. (1990). Protein secondary structures in water from second-derivative amide I infrared spectra. Biochemistry, 29(13), 3303–3308. https:// doi.org/10.1021/bi00465a022. Dong, A., Matsuura, J., Allison, S. D., Chrisman, E., Manning, M. C., & Carpenter, J. F. (1996). Infrared and circular dichroism spectroscopic characterization of structural differences between beta-lactoglobulin A and B. Biochemistry, 35, 1450–1457. https://doi.org/10.1021/bi9518104. Fang, Y., & Dalgleish, D. G. (1997). Conformation of β-lactoglobulin studied by FTIR: effect of pH, temperature, and adsorption to the oil-water interface. Journal of Colloid and Interface Science, 196(2), 292–298. https://doi.org/10.1006/jcis.1997.5191. Fang, Y., & Dalgleish, D. G. (1998). The conformation of α-lactalbumin as a function of pH, heat treatment and adsorption at hydrophobic surfaces studied by FTIR. Food Hydrocolloids, 12(2), 121–126. https://doi.org/10.1016/S0268-005X(98)00003-4. Fink, D. J., & Gendreau, R. M. (1984). Quantitative surface studies of protein adsorption by infrared spectroscopy. Analytical Biochemistry, 139(1), 140–148. https://doi.org/ 10.1016/0003-2697(84)90399-3. Foegeding, E. A., & Davis, J. P. (2011). Food protein functionality: A comprehensive approach. Food Hydrocolloids, 25(8), 1853–1864. https://doi.org/10.1016/j.foodhyd. 2011.05.008. Husband, F. A., Garrood, M. J., Mackie, A. R., Burnett, G. R., & Wilde, P. J. (2001). Adsorbed protein secondary and tertiary structures by circular dichroism and infrared spectroscopy with refractive index matched emulsions. Journal of Agricultural and Food Chemistry, 49(2), 859–866. https://doi.org/10.1021/jf000688z. Jackson, M., & Mantsch, H. H. (1995). The use and misuse of FTIR spectroscopy in the determination of protein structure. TL-30. Critical Reviews in Biochemistry and Molecular Biology, 30 VN-r(2), 95–120. https://doi.org/10.3109/ 10409239509085140. Kato, A., & Nakai, S. (1980). Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins. Biochimica et Biophysica Acta (BBA) – Protein Structure, 624(1), 13–20. https://doi.org/10.1016/0005-2795(80) 90220-2. Keppler, J. K., Sönnichsen, F. D., Lorenzen, P.-C., & Schwarz, K. (2014). Differences in heat stability and ligand binding among β-lactoglobulin genetic variants A, B and C using 1H NMR and fluorescence quenching. Biochimica et Biophysica Acta (BBA) – Proteins and Proteomics, 1844(6), 1083–1093. https://doi.org/10.1016/j.bbapap. 2014.02.007. Kong, J., & Yu, S. (2007). Fourier transform infrared spectroscopic analysis of protein secondary structures protein FTIR data analysis and band assignment. Acta Biochimica et Biophysica Sinica, 39(8), 549–559. https://doi.org/10.1186/1479-5876-10-117. Lam, R. S. H., & Nickerson, M. T. (2013). Food proteins: A review on their emulsifying properties using a structure-function approach. Food Chemistry, 141(2), 975–984. https://doi.org/10.1016/j.foodchem.2013.04.038. Lee, S. H., Lefèvre, T., Subirade, M., & Paquin, P. (2007). Changes and roles of secondary structures of whey protein for the formation of protein membrane at soy oil/water interface under high-pressure homogenization. Journal of Agricultural and Food Chemistry, 55(26), 10924–10931. https://doi.org/10.1021/jf0726076.
8
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
FTIR analysis of β-lactoglobulin at the oil/water-interface a,⁎
b
Helena Schestkowa , Stephan Drusch , Anja Maria Wagemans a b
T
a
Food Colloids, Technische Universität Berlin, Königin-Luise-Straße 22, 14196 Berlin, Germany Food Technology and Food Material Science, Technische Universität Berlin, Königin-Luise-Straße 22, 14196 Berlin, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: Critical interfacial concentration Protein-stabilized emulsion FTIR subtraction method Second derivative spectra Pendant drop analysis Extrinsic fluorescence
Knowledge about the critical interfacial concentration of a protein supports our understanding of the kinetic stability of an emulsion. Its determination is currently limited to either invasive or indirect methods. The aim of our study was the determination of the critical interfacial concentration of whey protein β-lactoglobulin at oil/water-interfaces through fluorescence and pendant drop analysis and the comparison to an in situ Fouriertransform-infrared-spectroscopy (FTIR) method. Exponentially decreasing interfacial tension with increasing βlactoglobulin content (0.10–1.00 wt%) in pendant drop analysis could partly be confirmed by fluorescence spectra. A critical interfacial concentration of 0.20–0.31 wt% β-lactoglobulin (1.80–2.69 mg/m2) in oil/water (5/95)-emulsions was determined via FTIR, analyzing the Amide I/Amide II peak intensity ratio. This was confirmed by the increasing formation of intermolecular β-sheets, revealed by second derivative spectra. With this FTIR method we expand current options to investigate the interfacial behavior of food proteins by determination of secondary structure elements.
1. Introduction Oil/water-macroemulsions are known to be thermodynamically unstable (Capek, 2004; Lam & Nickerson, 2013). Thus, the kinetics of phase separation determine the macroemulsion stability which depends on the physicochemical conditions of the dispersed (oil) and the bulk (water) phase, as well as the physicochemical properties and molecular interactions of the emulsifier. In the case of protein-stabilized emulsions, kinetic stability varies with the occupation level of adsorbed protein at the oil/water-interface (Lam & Nickerson, 2013). The protein content at which the oil/water-interface is fully occupied is referred to as the critical interfacial concentration (CIC) and represents the minimum amount of protein needed to stabilize an emulsion (McClements, Decker, & Weiss, 2007; Tamm, Sauer, Scampicchio, & Drusch, 2012). At present, a non-invasive determination of the CIC in emulsions is still challenging. Current literature divides the CIC analysis into methods investigating a single drop against oil or an actual emulsion system. Pendant drop analysis can be used to determine the CIC by interfacial tension measurement of a single drop (Tamm et al., 2012). This system’s main limitation, though, is the specific interfacial area of the pendant drop that is significantly smaller than that of the multiple drops in an emulsion and therefore the amount of protein to cover the interface differs. Others described a method that measures the CIC in an
⁎
emulsion via centrifugation, followed by an analysis of the protein content in separated cream and bulk phase (Tcholakova, Denkov, Ivanov, & Campbell, 2002; Ye, 2010). However, phase separation through centrifugation alters the sample nature and may influence the sensitive equilibrium between adsorbed and bulk protein. Alternative methods for CIC determination could take advantage of changes in the secondary structure of proteins during adsorption at the oil/water-interface. A possible approach could be by an extrinsic fluorescence method that detects changes in protein polarity by emission maxima and intensity shifts of fluorescence markers like 8-anilinonaphthalene-1-sulfonic-acid (ANS) (Alizadeh-Pasdar & Li-Chan, 2000; Kato & Nakai, 1980). However, the challenge of a fluorescence measurement could be the low sensitivity as a consequence of inner filter effects of the emulsion (Alizadeh-Pasdar & Li-Chan, 2000; Kato & Nakai, 1980; Liu, Powers, Swanson, Hill, & Clark, 2005). In contrast, Fourier-transform-infrared-spectroscopy (FTIR) is a highly sensitive and non-invasive method to detect adsorption-associated structural changes in proteins (Lefèvre & Subirade, 2003). Peaks in FTIR spectra result from absorption of energy through vibration of chemical bonds such as in the peptide backbone of proteins, in particular the Amide I band (C] O stretch vibration) and Amide II band (CN stretching and in-plane NH bending vibration). These bands are sensitive to changes that can occur in hydrogen bonds of secondary structure elements such as intra- and intermolecular β-sheets (Kong & Yu, 2007).
Corresponding author. E-mail addresses: [email protected] (H. Schestkowa), [email protected] (S. Drusch), [email protected] (A.M. Wagemans).
https://doi.org/10.1016/j.foodchem.2019.125349 Received 24 April 2019; Received in revised form 1 August 2019; Accepted 8 August 2019 Available online 09 August 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
Food Chemistry 302 (2020) 125349
H. Schestkowa, et al.
Isolated protein solutions were freeze-dried. The isolated β-lg powder had an approximate β-lg content of 96 wt%, measured by HPLC according to Keppler et al. (2014).
There is controversy in current literature regarding the structure of adsorbed proteins (Zhai, Day, Aguilar, & Wooster, 2013). An increase in intermolecular and a decrease in intramolecular β-sheets has been described during adsorption at a soybean oil/water-interface (Fang & Dalgleish, 1997). Others agreed to these findings, but have found opposing trends in the formation of intermolecular β-sheets for diacyl- and triacyglycerol/water-interfaces or could only detect minor structural changes (Husband, Garrood, Mackie, Burnett, & Wilde, 2001; Sakuno, Matsumoto, Kawai, Taihei, & Matsumura, 2008). These diverse trends may result from the FTIR methods where the extraction of the protein signal was realized by subtracting plain water and oil spectra from the emulsion’s spectra. Due to a higher oil concentration, signal intensity of plain oil differs strongly from the oil signal in emulsion leading to the methodological challenge of protein signal extraction. To obtain accurate data, similar oil band shapes should be considered within the subtraction method. The aim of our study was the comparison of CIC determination of protein-stabilized oil/water-emulsions in pendant drop system with fluorescence and FTIR analysis as well as the subsequent improvement of an accurate in situ FTIR subtraction method. For this purpose, we aimed to subtract the oil signal from the protein-stabilized emulsion in form of an emulsion instead of a plain oil phase. Doing so, we analyzed the whey protein β-lactoglobulin (β-lg) as model protein for interfacial active globular proteins in water and in oil/water-emulsions with increasing protein content. In order to minimize interactions between protein and oil, we have chosen middle-chain-triacylglyceride (MCT)oil as model oil with mainly saturated fatty acids. We assumed that the adsorption-associated structural changes of β-lg would increase with increasing protein content, until the oil/water-interface is fully occupied. In detail, we hypothesized the following:
2.2. MCT-oil purification Middle-chain-triacylglyceride-oil (MCT-oil, > 99.9%, Witarix MCT 60/40, IOI Oleochemical, Hamburg, Germany) was purified with activated magnesium silicate Florisil (MgO × 3.6 SiO2 × 1.53 OH, 100%, Carl Roth GmbH, Karlsruhe, Germany), to remove interfacial active compounds, such as free fatty acids, in accordance with Schestkowa et al. (2018). 2.3. Solution and emulsion preparation Protein solutions were prepared by dissolving 0.01–1.00 wt% β-lg in distilled water and stirred for 16 h at 22 °C and 500 rpm. The pH was adjusted to pH 7.0 with 0.1 M HCl and 0.1 M NaOH. Protein solution and MCT-oil (weight ratio 95/5) were mixed for 15 s at 22 °C and 17,500 rpm using an Ultra-Turrax (IKA-Werke GmbH, Staufen, Germany). Since the proportion of the protein in relation to the oil was varied, the protein/oil ratios are additionally given below, e.g. 1.00 wt % β-lg corresponds to a protein/oil ratio of 0.95/5. In all methods, emulsions were compared to water or β-lg solutions (1.00 wt% in water). For FTIR analyses, 0.20 wt% SDS (> 99%, Serva Electrophoresis GmbH, Heidelberg, Germany) in water and 0.20 wt% SDS in emulsion (water/MCT-oil ratio 95/5) were prepared additionally. 2.4. Pendant drop analysis
• The CIC can be measured by intensity variation and blue shifts in • •
The interfacial tension analysis at the oil/water-interface and the measurement of specific interfacial area of the single drop was performed with the automated drop tensiometer OCA 20 (Dataphysics GmbH, Filderstadt, Germany) as described by Tamm et al. (2012). The protein solutions with 0.01–1.00 wt% β-lg in water were measured against MCT-oil in a quartz-glass cuvette for 1 h at 22 °C. Therefore, a 30 µL droplet was dosed by an automatic dosing system (flow rate 5 µL/ s) and the interfacial tension was extracted from droplet shape recorded with a high speed camera (approx. 2 frames per second). The curve of interfacial tension was exponentially plotted according to Schestkowa et al. (2018) and the value of the asymptote was plotted against the protein content. A significant difference from the asymptote was classified at 10% deviation.
extrinsic fluorescence emission spectra occurring through changes in molecular polarity during adsorption of increasing amounts of protein at the oil/water-interfaces. The subtraction of a surfactant-stabilized emulsion FTIR spectrum from the protein-stabilized emulsion FTIR spectrum results in an accurate protein signal, since similar oil band shapes are applied. The amount of adsorbed protein at oil/water-interfaces affects the secondary structure and therefore the location and intensity of Amide I and II bands in FTIR spectra due to secondary structure sensitive C]O and NH vibration, enabling CIC determination.
To test these hypotheses, we developed an improved FTIR method to determine changes in the structure of adsorbed β-lg, including reference and background subtraction of an SDS (sodium dodecyl sulfate) emulsion using a similar oil droplet size distribution in comparison to βlg emulsions. For CIC determination, Amide I and II band of FTIR subtraction spectra of β-lg at oil/water-interfaces and the second derivative was evaluated regarding changes in secondary structure. We compared the CIC analysis via FTIR to decreasing interfacial tension in pendant drop and emission maxima of extrinsic fluorescence spectra.
2.5. Extrinsic fluorescence measurement Fluorescence measurements were obtained with a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, Victoria, Australia) with slight modifications to Liu et al. (2005). The extrinsic fluorescence marker 8-anilinonaphthalene-1-sulfonic-acid (ANS, > 97%, Sigma Aldrich, St. Louis, USA) was used. Measurement solutions were prepared by mixing 20 µL ANS (8 mM, 50.7 mg diluted in 20 mL distilled water) and 4 mL β-lg emulsion. The spectra were recorded in quartz cells with 10 mm optical length at an excitation wavelength of 390 nm and an emission wavelength scan from 300 to 600 nm. The maximum of fluorescence intensity was plotted against the protein content in the water fraction in emulsion.
2. Materials and methods 2.1. β-lactoglobulin isolation The β-lg-rich whey protein isolate GermanProt 9000 was provided by Sachsenmilch Leppersdorf GmbH (> 90%, Leppersdorf, Germany). β-lg was isolated following the procedure of Keppler, Sönnichsen, Lorenzen, and Schwarz (2014) with slight modifications. The ultrafiltration was replaced by dialysis for 72 h with distilled water at 22 °C using BioDesignDialysis TubingTM by ThermoFisher Scientific (molecular weight cut-off 14 kDa, Waltham Massachusetts, USA). The pH was adjusted to pH 7 with 0.1 M HCl and 0.1 M NaOH, both purchased from Carl Roth GmbH (> 99.9%, analytical-grade, Karlsruhe, Germany).
2.6. FTIR analysis Infrared spectra of 0.10–1.00 wt% β-lg emulsions and 1.00 wt% β-lg water solutions as well as 0.20 wt% SDS in water and in emulsion were measured against water as background with a Bruker Tensor II spectrometer (Bruker Optic GmbH, Karlsruhe, Germany) equipped with a liquid nitrogen-cooled mercury-cadmium-telluride detector. The 2
Food Chemistry 302 (2020) 125349
H. Schestkowa, et al.
γ (c) = Ae−Bc + γ∞ = 6.3e(−6.3c) + 12.6
samples were loaded into the transmission cell AquaSpec A 741/Q (Bruker Optic GmbH, Karlsruhe, Germany) between two CaF2 windows with a path length of 7 µm. The transmission cell was cleaned with the enzyme kit ProteoClean 10 A741-C1 (Bruker Optic GmbH, Karlsruhe, Germany) and washed with distilled water. Each spectrum is an absorbance spectrum measured in transmission and a result of the average of 120 scans recorded with a resolution of 4 cm−1 at 22 °C from 1000 to 3100 cm−1. Spectra were edited with the software provided with the spectrometer (OPUS 7.5); raw spectra were offset-corrected in the region of 2000–2010 cm−1 and the second derivative of vector-normalized (1480–1780 cm−1) spectra were smoothed with the 25-point Savitsky-Golay method. FTIR subtraction method is described in section 3.3 CIC by FTIR analysis. Interfacial occupation level for CIC determined by FTIR was calculated in consideration of the specific interfacial area of the emulsions.
(1)
with lower asymptote γ∞ and the empirical variables A and B. The results showed that the higher the protein content, the lower the value of the interfacial tension approaching the asymptote γ∞ = 12.6 mN/m at protein contents from 0.3 to 1.0 wt% β-lg. No further change in interfacial tension with further increase of protein content indicates that the interface is fully occupied and no denser molecule packing at the interface is possible. There is scientific consensus that the molecular density of proteins such as β-lg varies with the preoccupation level, due to variation in the packing density of the protein film at the oil/waterinterface (Capek, 2004; Foegeding & Davis, 2011). At the CIC, the protein spreads all over the interface and, consequently, aligns more densely with increasing protein content. According to the pendant drop analysis results, it becomes clear that the CIC is a concentration region up to approx. 0.3 wt% indicated by the exponential decrease of interfacial tension. The determination of the minimum concentration of the CIC region could not be realized and needs further investigation. Tamm et al. (2012) adapted the CIC determination by calculation of two linear fits as commonly applied in the determination of the critical micellar concentration (CMC) of low molecular weight surfactants. We assume that differences result from the CIC determination method and different system parameters such as the sample composition and type of interface; they used whey protein isolate and measured at an air/water-interface (Tamm et al., 2012). The main limitation of pendant drop analysis is the differing specific interface area when measuring a 30 µL-drop against MCT-oil compared to an actual emulsion; the specific interface area of the actual β-lg emulsions amounted to 1.0754 ± 0.0793 m2/cm3 and was almost three orders of magnitude higher than the specific interface area in pendant drop analysis (0.0015 ± 0.0003 m2/cm3) whereas the protein content remained the same. With this in mind, CIC values analyzed by pendant drop should appear smaller than in emulsions due to the lower specific interface area in single drop experiments.
2.7. Droplet size measurement The oil droplet size distribution and specific interfacial area of the emulsions was measured using static laser scattering (Horiba LA-950, Retsch Technology GmbH, Haan, Germany). Doing so, the refractive index of 1.45 and a set transmission value of 98% was used. If not reported otherwise, analyses were performed at least three times (n = 3) and mean as well as standard deviation were calculated. 3. Results 3.1. Critical interfacial concentration (CIC) of proteins The adsorption of protein, such as whey protein β-lactoglobulin (βlg), at oil/water-interfaces leads to a reduction of the interfacial tension between oil and water and a fine distribution of the dispersed in continuous phase (Beverung, Radke, & Blanch, 1999). Therefore, pendant drop analysis is commonly used to observe the adsorption process of emulsifiers by means of changes in interfacial tension (Beverung et al., 1999; Rühs, Scheuble, Windhab, & Fischer, 2013; Tamm et al., 2012; Wüstneck et al., 1996). Fig. 1A shows the decrease of the interfacial tension of β-lg solution with increasing protein content against MCT-oil. The interfacial tension was plotted exponentially as a function of time similar to Eq. (1). The values of the asymptotes from this fit were plotted against the protein content (Fig. 1B). The exponential function γ© was fitted as follows Eq. (1):
3.2. CIC by fluorescence analysis For non-invasive CIC determination in actual emulsions extrinsic fluorescence was observed for ANS treated β-lg emulsions (Fig. 2A). First, intensity of emission maxima were plotted against the protein content (Fig. 2B). The extrinsic fluorescence intensity increased with increasing β-lg content in the emulsion (linear fit Imax,E(c), R2 = 0.9943) without indicating a possible shift pointing out the CIC.
Fig. 1. (A) Interfacial tension of β-lg solutions (0.01–1.00 wt% in water) in MCT-oil as function of time (n = 3, n = 1 illustrated) and values of asymptotes of interfacial tension (colored area in A) as function of protein content (B) analyzed by pendant drop and exponentially plotted function γ (c) with asymptote γ∞. 3
Food Chemistry 302 (2020) 125349
H. Schestkowa, et al.
Fig. 2. (A) Extrinsic fluorescence spectra of protein emulsions with 0.10–1.00 wt% β-lg content or rather protein/oil ratio of 0.095/5–0.95/5 (solid lines, protein content is referred to protein content in water fraction of emulsion) and 1.00 wt% β-lg in water (dashed line, n = 3, n = 1 illustrated). (B) Emission maxima (colored area in A) as function of protein content with linear fit Imax,E.
Second, a blue shift was observed compared to β-lg in water due to quenching effects (supplementary data S1 A B) (Alizadeh-Pasdar & LiChan, 2000; Kato & Nakai, 1980; Liu et al., 2005; Renard, Lefèvre, Griffin, & Griffin, 1998). The blue shift of β-lg emulsions indicates changes in molecular polarity of the protein from a less polar to a more polar environment for pressure treated proteins (Liu et al., 2005; Yang, Dunker, Powers, Clark, & Swanson, 2001). Although the peak shift is characteristic for adsorbed proteins in emulsion, the measured values did not approach the value of water and, consequently, the CIC could not be determined by extrinsic fluorescence analysis with ANS as hypothesized. Additionally, peak intensity ratio (PIR, approx. 470/ 530 nm) was calculated as shown in supplementary data S1 C D. Fluorescence results did partly indicate a similar behavior approaching an asymptote at 0.4 wt% protein (supplementary data S1) as shown for pendant drop analysis in Fig. 1B, although a higher value for a larger interface area was expected. This might be caused by the method not being sensitive enough to detect small differences in molecular polarity due to absorption and light scattering of the dispersed oil phase, summarized as inner filter effects of the emulsion (Alizadeh-Pasdar & LiChan, 2000; Kato & Nakai, 1980; Liu et al., 2005). In conclusion, accurate CIC determination should be performed by a more sensitive method in comparison to fluorescence analysis such as the secondary structure sensitive FTIR. Therefore, we developed and improved the FTIR subtraction method and further calculated the CIC based on changes in Amide I and II bands.
I and II region, taking the oil concentration in emulsion and oil droplet size distribution into account as described in depth below. Emulsions are heterogeneous systems, therefore, minor deviations in the sample characteristics (such as the measured oil droplets) alter Mie-scattering (Bassan et al., 2010), which in turn result in slightly different oil signal intensities e.g. at 1750 cm−1. To avoid theses deviations spectra with matching oil-signals were selected for subtraction assuming that the measured samples feature similar sample characteristics. Moreover, matching oil signals reduce the misinterpretation of band shapes during evaluation of difference spectra, which might occur since changes in secondary structure are easily over- or underestimated when spectra are subtracted (De Jongh, Goormaghtigh, & Ruysschaert, 1996; Dong, Huang, & Caughey, 1990). A similar approach was used by Zhai et al. (2012) to extract protein signals from synchrotron radiation circular dichroism spectra of protein-stabilized emulsions by reducing disturbing light scattering effects. In accordance with Kong and Yu (2007) we assume that water does not cause any altered vibration in an emulsion and, thus, we retained background subtraction of water. First, a background spectrum of distilled water was measured to remove the water signal from the subsequent measurements. Next, measurements of β-lg in emulsion (β-lgE), SDS in water (SDSW) and SDS in emulsion (SDSE) were performed. In order to obtain the MCT-oil signal spectrum we removed the SDS signal from SDS in emulsion by subtraction of SDSW spectra from the SDSE spectra with a subtraction factor based on the SDS band intensity at 1250 cm−1. This band was chosen due to minor overlapping with oil signals. The obtained spectrum is referred to as difference spectrum SDSE,D (Fig. 3A). Following that, the MCT-oil signal spectrum was removed from the protein emulsion by subtracting SDSE,D from the β-lgE spectrum (Fig. 3B) with an subtraction factor based on the MCT-oil band intensity at 1750 cm−1. Thus, the spectrum β-lgE,D with the Amide I and II band at approx. 1500–1700 cm−1 could be obtained. For further evaluation the β-lgE,D spectra were offset-corrected and the intensity of Amide I and II band was determined. Both Amide I and Amide II showed a protein linearity in the range from 0.10 to 1.00 wt% (protein/oil ratio of 0.095/ 5–0.95/5) with R2Amide I = 0.9980 and R2Amide II = 0.9985, respectively. The peak intensity ratio (PIR) of Amide I/Amide II was calculated (Fig. 4A) to eliminate the effect of varying repeatability precision of Amide I and Amide II absolute intensity (n = 7, 0.50 wt% β-lg in emulsion, IAmide I = 0.759 ± 0.039, IAmide II = 0.675 ± 0.040). PIR was plotted as function of protein content and resulted in a double asymptotic fit. The turning point of the fit was calculated and a
3.3. CIC by FTIR analysis An in situ FTIR subtraction method was developed to evaluate the changes in secondary structure of adsorbed proteins at oil/water-interfaces. Fig. 3B illustrates an FTIR absorbance spectrum measured in transmission of a β-lg-stabilized emulsion (β-lgE). A subtraction method to obtain the signal of the adsorbed protein by subtracting pure oil and water spectra from the emulsion spectra has already been published (Fang & Dalgleish, 1997, 1998; Fink & Gendreau, 1984; Lefèvre & Subirade, 2003; Tcholakova, Denkov, Sidzhakova, & Campbell, 2006). Following the given information in their publications, we have not been able to extract accurate Amide I and II bands due to varying oil signals (supplementary data S2). However, oil spectra shifted at approx. 1750 cm−1 showing a wider and larger band in the emulsion compared to the pure oil. Consequently, we subtracted the spectrum of an emulsion system with SDS as surfactant, which does not absorb at the Amide 4
Food Chemistry 302 (2020) 125349
H. Schestkowa, et al.
Fig. 3. FTIR subtraction method to remove (A) SDS bands at approx. 1250 cm−1 (colored area) from SDS emulsion spectra SDSE with SDS in water spectrum SDSW to obtain SDS difference spectra SDSE,D and to further remove (B) MCT-oil band at approx. 1750 cm−1 (colored area) from β-lg emulsion spectra β-lgE to obtain an β-lg spectrum β-lgE,D with Amide I and II bands at approx. 1500–1700 cm−1 (colored area).
of Amide II with increasing β-lg content. As summarized by Jackson and Mantsch, Amide I and II absorption intensity is affected by the vibration of C]O and NH in the protein peptide backbone (Jackson & Mantsch, 1995). Secondary structure elements such as β-sheets are formed within this peptide backbone and are held in shape by hydrogen bonds. Vibration of electron-withdrawing groups such as C]O and NH groups is influenced by their electron density and therefore by the strength of the existing hydrogen bonds. The weaker the hydrogen bond involving the C]O group, the higher the electron density and Amide I absorption (Barth, 2007; Jackson & Mantsch, 1995). For protein adsorbed at oil/water-interfaces, the strength of hydrogen bonds inside secondary structure elements changes due to partial denaturation caused by rearrangement of hydrophobic and hydrophilic parts of the peptide backbone facing towards the oil and water phase respectively. As a result, both band intensity and frequency can vary with the protein conformation at the interface, which in turn is affected by the amount of adsorbed protein. Because of the higher secondary structure sensitivity of Amide I in
significant difference from the asymptotes was classified at 10% deviation. No correlation between PIR and the median of the oil droplet size distribution could be found (supplementary data S2). The second derivative of the Amide I region was calculated to determine the position of the maximum of broad Amide bands more precisely. Finally, the second derivative spectra of vector-normalized β-lgE,D spectra were analyzed with regards to their bands in the Amide I region (approx. 1600–1700 cm−1) as presented in Fig. 5. In order to determine the CIC of β-lg at oil/water-interfaces, the intensity of Amide I and II bands as well as the second derivative of the Amide I band as function of the β-lg content was considered. Doing so, FTIR spectra of 0.10–1.00 wt% β-lg (protein/oil ratio of 0.095/5–0.95/ 5) in emulsion and spectra of 1.00 wt% β-lg in water were recorded. The resulting FTIR difference spectra β-lgE,D of the Amide I and II region are shown in Fig. 4A. The increase of the Amide I and II intensity with increasing β-lg content was accompanied by a shift of the Amide I/Amide II PIR (Fig. 4B). Here, the intensity of Amide I increased less compared to that
Fig. 4. (A) FTIR spectra β-lgE,D of the Amide I and II region for protein emulsions with 0.10–1.00 wt% β-lg content or rather protein/oil ratio of 0.095/5–0.95/5 (solid lines) and 1.00 wt% β-lg in water (dashed line, n = 3, n = 1 illustrated). (B) Peak intensity ratio PIR of Amide I/Amide II (colored area in A) against β-lg content (protein content is referred to protein content in water fraction of emulsion) with a double asymptotic function PIR(c) and asymptotes PIR0 and PIR∞. 5
Food Chemistry 302 (2020) 125349
H. Schestkowa, et al.
Fig. 5. (A) Second derivative FTIR spectra of β-lgE,D in the Amide I region with 0.10–1.00 wt% β-lg content in emulsion or rather a protein/oil ratio of 0.095/5–0.95/ 5 (solid lines) and 1.00 wt% β-lg in water (dashed line, n = 3, n = 1 illustrated). (B) Intensity of 1630 cm−1-band of FTIR second derivative spectra (colored area in A) against protein content and exponential fit I(c) with asymptote I∞ and horizontal dashed line for 1.00 wt% β-lg in water.
in Fig. 6 (stage I). Consequently, the FTIR signal resulted mainly from protein at the oil/water-interface approaching the lower asymptote. The PIR(c) increased in the region approx. from 0.20 to 0.60 wt% protein content in the emulsion (protein/oil ratio of 0.19/5–0.57/5). Regarding changes in secondary structure through protein adsorption, we assume that the oil/water-interface is fully occupied at this region of the function (Fig. 6 stage II). The turning point at approx. 0.31 wt% of protein content could indicate a change in equilibrium between adsorbed and bulk protein towards the bulk protein side. In conclusion, we assume that the CIC region of β-lg at oil/water-interfaces ranges from 0.20 to 0.31 wt% protein content in the water phase of the emulsions (protein/oil ratio of 0.19/5–0.29/5), resulting in an interface occupation of 1.80–2.69 mg/m2. Above the CIC region, PIR(c) approached the upper asymptote PIR∞ = 1.20 at protein contents of 0.31–1.00 wt% (protein/oil ratio of 0.95/5). Here, the bulk protein signal dominates the FTIR signal of the adsorbed protein and, hence, the signal approaches the PIR of protein in water as illustrated in Fig. 6 stage III. This assumption was confirmed by the spectra of 1.0 wt% β-lg in water (without any oil) resulting in a PIR of 1.21 ± 0.14 that equals the upper asymptote PIR∞ of emulsions. This saturation of interface occupation was also reported by Taherian, Britten, Sabik, and Fustier
comparison to Amide II, the PIR of Amide I/Amide II was assumed to differ with the protein content. This is due to different secondary structure elements in adsorbed and bulk protein as well as the structural changes caused by the occupation level (Kong & Yu, 2007). In addition, variations in Amide band intensities can be explained by the displacement of water molecules through the sample; the more protein covers the oil/water-interface, the less water molecules are in contact with the protein, due to interactions with the oil phase and the lower is Amide I band intensity (Rahmelow & Hubner, 1997; Zeng, Chittur, & Lacefield, 1999). As function of the protein content, the PIR (Fig. 4B) showed a double asymptotic behavior that was empirically plotted with the correlation PIR(c), as indicated by Eq. (2):
PIR(c) = PIR ∞ +
PIR 0 − PIR ∞ 0.64 − 1.20 = 1.20 + 1 + Kcm 1 + 2.83c3.74
(2)
with upper asymptote PIR∞, lower asymptote PIR0 and the variables K and m. The PIR(c) function approached the lower asymptote PIR0 = 0.64 at 0.20 wt% of protein (protein/oil ratio of 0.19/5). At a low protein content, the equilibrium of adsorbed protein and protein in bulk phase was mostly on the side of the adsorbed protein, as illustrated
Fig. 6. Whey protein β-lg adsorption at oil/water-interfaces as a function of protein content. Equilibrium between adsorbed and bulk protein tends towards adsorbed protein at stage I and is on side of bulk protein at stage III. The critical interfacial region is in stage II; the oil/water-interface is fully occupied and the interfacial film becomes denser upon protein addition. 6
Food Chemistry 302 (2020) 125349
H. Schestkowa, et al.
spectra of adsorbed proteins at oil/water-interfaces. As hypothesized, changes in secondary structure during adsorption affected the intensity and location of hydrogen bond sensitive C]O and NH vibration in the Amide I and II band. The Amide I/Amide II peak intensity ratio of FTIR difference spectra indicated a shift of the equilibrium from adsorbed to bulk protein with increasing β-lg content. The critical interfacial concentration region could be detected via FTIR at 0.20–0.31 wt% (1.80–2.69 mg/m2) protein content in oil/water (5/95)-emulsions or rather a protein/oil ratio of 0.19/5–0.29/5. Second derivative spectra of the Amide I band supported the trends of the Amide I/Amide II peak intensity ratio. The findings extend current literature of interfacial protein film formation in respect to the film density with increasing protein content in emulsions (Capek, 2004; Foegeding & Davis, 2011). Our findings are the basis for an in situ characterization of the interfacial concentration of proteins in actual emulsions via FTIR analysis. The FTIR method can further be used to characterize concentrations relevant for analytical investigation of interfacial stabilization mechanisms of proteins. Consequently, interactions and mechanical properties of the protein film itself could be characterized by amplitude-stress tests with interfacial shear- and dilatational rheology in concentrations below the CIC to avoid the formation of interfacial multilayer and to provide defined measurement conditions. To extend the applicability of the FTIR subtraction method for food systems, the robustness should be enhanced by analyzing β-lg with structural alterations as they can be achieved via physical or biotechnological approaches. Since the FTIR method is based on relative changes in secondary structure depending on the unfolded protein at the interface, the interfacial behavior and CIC could be investigated as a function of the initial degree of denaturation and unfolding. In particular, emulsification process-related structural changes in proteins should be monitored. The sensitivity of the FTIR method could be optimized by analyzing proteins from other sources including molecules with a more or less complex secondary, tertiary and quaternary structure as well as protein mixtures. As a further step, the oil could be varied in order to investigate the influence of the interactions between protein and both saturated and unsaturated fatty acids as well as fatty acids with varying length of aliphatic chains. The latter will allow to describe the structure-function-relationships in dispersed food systems in dependence of the initial properties and interactions between its individual constituents its in.
(2011) by analyzing whey protein. To verify results from the analysis of Amide I and II band shape, band intensities of the second derivative FTIR spectra in the Amide I region (1700–1600 cm−1) were taken into account, as commonly applied for analysis of secondary structure elements of proteins (Dong et al., 1990; Kong & Yu, 2007). The change in intensity of these second derivative bands can be ascribed to changes of the secondary structure, because the spectra were vector-normalized which takes out direct effects of the protein concentration. In this case, the secondary structure varies with the occupancy at the oil/water-interface that changes with the protein concentration. The secondary structure of native β-lg consists mainly of an eight-stranded intramolecular β-sheet barrel with a short α-helix segment and additional parts with random coil structures (Dong et al., 1996). The Amide I region of the second derivative FTIR difference spectra β-lgE,D of adsorbed β-lg is shown in Fig. 5A. With increasing protein content in the emulsion, a band shift from 1641 to 1644 cm−1 occurred that indicates the formation of random coil structure elements (Jackson & Mantsch, 1995; Kong & Yu, 2007). As reported by Zhai et al. (2013), an increase of random coil structures throughout adsorption indicates unfolding of the protein at the oil/ water-interface. The observed increase of the band at 1616 cm−1 implies an adsorption-associated aggregation of β-strands accompanied by a formation of intermolecular β-sheets which is in line with Lefèvre and Subirade (2003), who emphasized the meaning of intermolecular βsheet formation during protein film formation. The intramolecular βsheet content increased with decreasing peak intensity at 1630 cm−1. Here, the intensity at 1630 cm−1 approached the minimum of the difference in the second derivative of the spectra with increasing protein concentration (Fig. 5B). The exponential approximation corresponds to Eq. (1). Furthermore, the asymptote of the peak intensity at 1630 cm−1 of adsorbed β-lg is equivalent to the value of β-lg in water (shown by the dashed line in Fig. 5B). We assume that this approximation of the peak intensity at 1630 cm−1 to the value of β-lg in water is caused by bulk protein superimposing the adsorbed protein signal with increasing protein content. These results agree with the trends from PIR in Fig. 4B, where the signal of adsorbed protein also approached the signal of water. Protein contents below 0.31 wt% β-lg (protein/oil ratio of 0.29/ 5) differed distinctively from the asymptote. Furthermore, the protein content of 0.31 wt% β-lg corresponds to the turning point of the PIR(c) curve in Fig. 4B that indicated full occupation of the oil/water-interface (Fig. 6 stage II).
Acknowledgements 4. Conclusion This research project was supported by the German Research Foundation Priority Program 1934, DispBiotech. We thank Julia Keppler from CAU Kiel for the help with the β-lg isolation and IOI Oleochemicals (Hamburg, Germany) for providing MCT-oil as well as Tobias Wollborn and Udo Fritsching (Leibniz Institute for Materials Engineering – IWT, Particles and Process Engineering, University of Bremen) for simulated protein pictures. Helena Schestkowa thanks Annette Müller and others for discussion.
Our study represents the first step towards an in situ measurement of the critical interfacial concentration (CIC) by FTIR in emulsion-based food systems. Therefore, we developed a FTIR subtraction method for accurate non-invasive determination of the CIC of model protein βlactoglobulin (β-lg) at MCT-oil/water-interfaces and compared the trends to both extrinsic fluorescence and pendant drop analysis. The exponential decrease of interfacial tension with increasing protein content measured by pendant drop analysis indicated a concentration region for the CIC of β-lg in oil/water (5/95)-emulsions. Due to the smaller specific interfacial area of the single drop compared to the actual emulsion system we decided to perform extrinsic fluorescence analysis of the emulsion. Peak intensity and polarity dependent blue shift of the extrinsic fluorescence emission spectra showed the low sensitivity of the fluorescence method and, therefore, could only partly confirm trends from pendant drop analysis. Consequently, for in situ CIC determination, we developed an FTIR subtraction method including the subtraction of an SDS-stabilized emulsion from protein-stabilized emulsion spectra to ensure a similarity of subtracted spectra. Doing so, we optimized previous techniques to subtract plain water and oil spectra (Fang & Dalgleish, 1997, 1998; Lee, Lefèvre, Subirade, & Paquin, 2007; Lefèvre & Subirade, 2003; Tcholakova et al., 2006). In conclusion, we recorded spectra of secondary structure sensitive FTIR
Declaration of Competing Interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125349. References Alizadeh-Pasdar, N., & Li-Chan, E. C. Y. (2000). Comparison of protein surface hydrophobicity measured at various pH values using three different fluorescent probes. Journal of Agricultural and Food Chemistry, 48(2), 328–334. https://doi.org/10.1021/
7
Food Chemistry 302 (2020) 125349
H. Schestkowa, et al.
Lefèvre, T., & Subirade, M. (2003). Formation of intermolecular β-sheet structures: A phenomenon relevant to protein film structure at oil-water interfaces of emulsions. Journal of Colloid and Interface Science, 263(1), 59–67. https://doi.org/10.1016/ S0021-9797(03)00252-2. Liu, X., Powers, J. R., Swanson, B. G., Hill, H. H., & Clark, S. (2005). Modification of whey protein concentrate hydrophobicity by high hydrostatic pressure. Innovative Food Science and Emerging Technologies, 6(3), 310–317. https://doi.org/10.1016/j.ifset. 2005.03.006. McClements, D. J., Decker, E. A., & Weiss, J. (2007). Emulsion-based delivery systems for lipophilic bioactive components. Journal of Food Science, 72(8), 109–124. https://doi. org/10.1111/j.1750-3841.2007.00507.x. Rahmelow, K., & Hubner, W. (1997). Infrared spectroscopy in aqueous solution: Difficulties and accuracy of water subtraction. Applied Spectroscopy, 51, 160–170. Renard, D., Lefèvre, J., Griffin, M. C. A., & Griffin, W. G. (1998). Effects of pH and salt environment on the association of β-lactoglobulin revealed by intrinsic fluorescence studies. International Journal of Biological Macromolecules, 22(1), 41–49. https://doi. org/10.1016/S0141-8130(97)00086-X. Rühs, P. A., Scheuble, N., Windhab, E. J., & Fischer, P. (2013). Protein adsorption and interfacial rheology interfering in dilatational experiment. European Physical Journal: Special Topics, 222(1), 47–60. https://doi.org/10.1140/epjst/e2013-01825-0. Sakuno, M. M., Matsumoto, S., Kawai, S., Taihei, K., & Matsumura, Y. (2008). Adsorption and structural change of beta-lactoglobulin at the diacylglycerol-water interface. Langmuir: The ACS Journal of Surfaces and Colloids, 24(20), 11483–11488. https://doi. org/10.1021/la8018277. Schestkowa, H., Wollborn, T., Westphal, A., Maria Wagemans, A., Fritsching, U., & Drusch, S. (2018). Conformational state and charge determine the interfacial stabilization process of beta-lactoglobulin at preoccupied interfaces. Journal of Colloid and Interface Science, 536, 300–309. https://doi.org/10.1016/j.jcis.2018.10.043. Taherian, A. R., Britten, M., Sabik, H., & Fustier, P. (2011). Ability of whey protein isolate and/or fish gelatin to inhibit physical separation and lipid oxidation in fish oil-inwater beverage emulsion. Food Hydrocolloids, 25(5), 868–878. https://doi.org/10. 1016/j.foodhyd.2010.08.007. Tamm, F., Sauer, G., Scampicchio, M., & Drusch, S. (2012). Pendant drop tensiometry for the evaluation of the foaming properties of milk-derived proteins. Food Hydrocolloids, 27(2), 371–377. https://doi.org/10.1016/j.foodhyd.2011.10.013. Tcholakova, S., Denkov, N. D., Ivanov, I. B., & Campbell, B. (2002). Coalescence in βlactoglobulin-stabilized emulsions: Effects of protein adsorption and drop size. Langmuir, 18(23), 8960–8971. https://doi.org/10.1021/la0258188. Tcholakova, S., Denkov, N. D., Sidzhakova, D., & Campbell, B. (2006). Effect of thermal treatment, ionic strength, and pH on the short-term and long-term coalescence stability of b-lactoglobulin emulsions. Langmuir: The ACS Journal of Surfaces and Colloids, 22(14), 6042–6052. https://doi.org/10.1021/la0603626. Wüstneck, R., Krägel, J., Miller, R., Fainerman, V. B., Wilde, P. J., Sarker, D. K., & Clark, D. C. (1996). Dynamic surface tension and adsorption properties of β-casein and βlactoglobulin. Food Hydrocolloids, 10(4), 395–405. https://doi.org/10.1016/S0268005X(96)80018-X. Yang, J., Dunker, A. K., Powers, J. R., Clark, S., & Swanson, B. G. (2001). Beta-lactoglobulin molten globule induced by high pressure. Journal of Agricultural and Food Chemistry, 49(7), 3236–3243. https://doi.org/10.1021/jf001226o. Ye, A. (2010). Surface protein composition and concentration of whey protein isolatestabilized oil-in-water emulsions: effect of heat treatment. Colloids and Surfaces B: Biointerfaces, 78(1), 24–29. https://doi.org/10.1016/j.colsurfb.2010.02.001. Zeng, H., Chittur, K. K., & Lacefield, W. R. (1999). Analysis of bovine serum albumin adsorption on calcium phosphate and titanium surfaces. Biomaterials, 20(4), 377–384. https://doi.org/10.1016/S0142-9612(98)00184-7. Zhai, J. L., Day, L., Aguilar, M. I., & Wooster, T. J. (2013). Protein folding at emulsion oil/ water interfaces. Current Opinion in Colloid and Interface Science, 18(4), 257–271. https://doi.org/10.1016/j.cocis.2013.03.002. Zhai, Jiali, Hoffmann, S. V., Day, L., Lee, T. H., Augustin, M. A., Aguilar, M. I., & Wooster, T. J. (2012). Conformational changes of beta-lactalbumin adsorbed at oil-water interfaces: Interplay between protein structure and emulsion stability. Langmuir, 28(5), 2357–2367. https://doi.org/10.1021/la203281c.
jf990393p. Barth, A. (2007). Infrared spectroscopy of proteins. Biochimica et Biophysica Acta – Bioenergetics, 1767(9), 1073–1101. https://doi.org/10.1016/j.bbabio.2007.06.004. Bassan, P., Kohler, A., Martens, H., Lee, J., Byrne, H. J., Dumas, P., et al. (2010). Resonant Mie Scattering (RMieS) correction of infrared spectra from highly scattering biological samples. Analyst, 135(2), 268–277. https://doi.org/10.1039/b921056c. Beverung, C. J., Radke, C. J., & Blanch, H. W. (1999). Protein adsorption at the oil/water interface: characterization of adsorption kinetics by dynamic interfacial tension measurements. Biophysical Chemistry, 81(1), 59–80. https://doi.org/10.1016/S03014622(99)00082-4. Capek, I. (2004). Degradation of kinetically-stable o/w emulsions. Advances in Colloid and Interface Science, 107(2–3), 125–155. https://doi.org/10.1016/S0001-8686(03) 00115-5. De Jongh, H. H. J., Goormaghtigh, E., & Ruysschaert, J. M. (1996). The different molar absorptivities of the secondary structure types in the amide I region: An attenuated total reflection infrared study on globular proteins. Analytical Biochemistry, 242(1), 95–103. https://doi.org/10.1006/abio.1996.0434. Dong, A., Huang, P., & Caughey, W. S. (1990). Protein secondary structures in water from second-derivative amide I infrared spectra. Biochemistry, 29(13), 3303–3308. https:// doi.org/10.1021/bi00465a022. Dong, A., Matsuura, J., Allison, S. D., Chrisman, E., Manning, M. C., & Carpenter, J. F. (1996). Infrared and circular dichroism spectroscopic characterization of structural differences between beta-lactoglobulin A and B. Biochemistry, 35, 1450–1457. https://doi.org/10.1021/bi9518104. Fang, Y., & Dalgleish, D. G. (1997). Conformation of β-lactoglobulin studied by FTIR: effect of pH, temperature, and adsorption to the oil-water interface. Journal of Colloid and Interface Science, 196(2), 292–298. https://doi.org/10.1006/jcis.1997.5191. Fang, Y., & Dalgleish, D. G. (1998). The conformation of α-lactalbumin as a function of pH, heat treatment and adsorption at hydrophobic surfaces studied by FTIR. Food Hydrocolloids, 12(2), 121–126. https://doi.org/10.1016/S0268-005X(98)00003-4. Fink, D. J., & Gendreau, R. M. (1984). Quantitative surface studies of protein adsorption by infrared spectroscopy. Analytical Biochemistry, 139(1), 140–148. https://doi.org/ 10.1016/0003-2697(84)90399-3. Foegeding, E. A., & Davis, J. P. (2011). Food protein functionality: A comprehensive approach. Food Hydrocolloids, 25(8), 1853–1864. https://doi.org/10.1016/j.foodhyd. 2011.05.008. Husband, F. A., Garrood, M. J., Mackie, A. R., Burnett, G. R., & Wilde, P. J. (2001). Adsorbed protein secondary and tertiary structures by circular dichroism and infrared spectroscopy with refractive index matched emulsions. Journal of Agricultural and Food Chemistry, 49(2), 859–866. https://doi.org/10.1021/jf000688z. Jackson, M., & Mantsch, H. H. (1995). The use and misuse of FTIR spectroscopy in the determination of protein structure. TL-30. Critical Reviews in Biochemistry and Molecular Biology, 30 VN-r(2), 95–120. https://doi.org/10.3109/ 10409239509085140. Kato, A., & Nakai, S. (1980). Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins. Biochimica et Biophysica Acta (BBA) – Protein Structure, 624(1), 13–20. https://doi.org/10.1016/0005-2795(80) 90220-2. Keppler, J. K., Sönnichsen, F. D., Lorenzen, P.-C., & Schwarz, K. (2014). Differences in heat stability and ligand binding among β-lactoglobulin genetic variants A, B and C using 1H NMR and fluorescence quenching. Biochimica et Biophysica Acta (BBA) – Proteins and Proteomics, 1844(6), 1083–1093. https://doi.org/10.1016/j.bbapap. 2014.02.007. Kong, J., & Yu, S. (2007). Fourier transform infrared spectroscopic analysis of protein secondary structures protein FTIR data analysis and band assignment. Acta Biochimica et Biophysica Sinica, 39(8), 549–559. https://doi.org/10.1186/1479-5876-10-117. Lam, R. S. H., & Nickerson, M. T. (2013). Food proteins: A review on their emulsifying properties using a structure-function approach. Food Chemistry, 141(2), 975–984. https://doi.org/10.1016/j.foodchem.2013.04.038. Lee, S. H., Lefèvre, T., Subirade, M., & Paquin, P. (2007). Changes and roles of secondary structures of whey protein for the formation of protein membrane at soy oil/water interface under high-pressure homogenization. Journal of Agricultural and Food Chemistry, 55(26), 10924–10931. https://doi.org/10.1021/jf0726076.
8