Characterization of β-lactoglobulin gels induced by high pressure processing

Innovative Food Science and Emerging Technologies 47 (2018) 335–345

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Characterization of β-lactoglobulin gels induced by high pressure processing Xiaoying Li, Like Mao, Xiaoye He, Peihua Ma, Yanxiang Gao, Fang Yuan

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Beijing Advanced Innovation Center for Food Nutrition and Human Health, Key Laboratory of Functional Dairy, Ministry of Education, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: β‑lactoglobulin Gel High pressure processing Gel strength Texture

High pressure processing (HPP) is a non-thermal technology and has been widely used in the food industry. β‑lactoglobulin (β-Lg) is a globular protein and susceptible to pressure. This study investigated the properties of β-Lg gels induced directly by HPP (0.1–600 MPa for 30 min, 25 °C) with different protein concentrations at different pH. Results showed that the lowest protein concentration required to form β-Lg gels was 20% (w/v) at 400 MPa for 30 min at pH 3.0, 5.0 and 7.0, while 14% (w/v) protein was required to form gels at 600 MPa. The gel strength and textural properties increased with the increase of protein concentration and pressure, the gel formed at pH 5.0 had the highest strength. Raman spectra suggested that the content of α-helix decreased and random coil increased of β-Lg in gels with the increase of pressure. β-Lg gels formed more regular and stable network under 600 MPa than that under 400 MPa. Industrial relevance: In recent years, high pressure processing (HPP) has been used in the food industry as an innovative technology to improve gel properties of various kinds of proteins, including β‑lactoglobulin, casein, soy protein isolate, surimi, mytolin and so on. It was found that HPP could change the structure of protein, which could cause protein denaturation and aggregation and three-dimensional network structures were formed through disulfide bonds, electrostatic interactions, hydrophobic interaction, hydrogen bonds and others. β‑lactoglobulin is the main component of whey protein, which has been widely used in the food industry as gelling agents. β‑lactoglobulin gels could be used as a wall material and applied to the delivery system of functional components such as riboflavin. The objective of the current study was to investigate the properties of β-Lg gels formed through HPP directly, focusing on the effects of pressure, protein concentration and pH on the rheological properties, texture profile, protein secondary structure and microstructure of β-Lg gels.

1. Introduction Gelation is an important characteristic of proteins, and protein gels contain three-dimensional network structures formed through protein aggregation orderly. A protein gel is also a large molecular weight complex, which is formed based on the balance between attractive and repulsive forces, including disulfide bonds, electrostatic interactions, hydrophobic interaction, hydrogen bonds and others (De Maria, Ferrari, & Maresca, 2016; Hermansson, 1979; Purwanti et al., 2011). β-lactoglobulin (β-Lg) is the main component of whey protein, which has a molecular weight of 18.4 kDa and an isoelectric point of ~5.2 (Bryant & McClements, 1998; Wu et al., 2016). β-Lg is a globular protein which consists of 162 amino acids and 5 thiol groups (Sahihi, Heidari-Koholi, & Bordbar, 2012). With a good gelation property, β-Lg is widely used in food systems. The gelation of β-Lg is largely affected by many conditions, such as protein concentration (Nicolai, Britten, & Schmitt, 2011), pH (Cao, Xia, Zhou, & Xu, 2012), temperature (Zhang,

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Yang, Tang, Chen, & You, 2015), ionic strength (Sagis et al., 2002), pressure (He, Yang, Zhu, & Mu, 2010) and enzyme (Mohtar, Perera, Quek, & Hemar, 2013). There has been extensive research regarding β-Lg gelation induced by heat, enzyme or other forces after high pressure treatment (Kavanagh, Clark, & Ross-Murphy, 2000; Line, Remondetto, & Subirade, 2005; López-Fandiño, 2006; Wu et al., 2016). For example, Ma et al. (2013) studied the effect of high pressure treatment on the properties of meat protein gel containing 0.2% CaCl2 and 0.6% κ-carrageenan induced by heating, and they found that enhanced waterbinding capacity and weakened texture profile of the gel when it was subjected to high pressure treatment. However, only little attention has been paid to β-Lg gels through high pressure processing directly. Studied have shown that hydrostatic pressure treatment (100–400 MPa, 30 min, 20 °C)of α-lactalbumin (α-La) samples (10%, w/w) did not change the rheological parameters and not formed gels (Ahmed & Ramaswamy, 2003). However, whey protein concentrate (WPC)

Corresponding author at: Box 112, No.17 Qinghua East Road, Haidian District, Beijing 100083, PR China. E-mail address: [email protected] (F. Yuan).

https://doi.org/10.1016/j.ifset.2018.03.022 Received 27 February 2017; Received in revised form 31 October 2017; Accepted 22 March 2018 Available online 23 March 2018 1466-8564/ © 2018 Published by Elsevier Ltd.

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6.5 MPa/s and released at 20 MPa/s. Compression was accompanied by increasing the temperature of about 3 °C 100 MPa−1 (Balasubramaniam, Farkas, & Turek, 2008). However, the sample temperature quickly dropped to the initial temperature due to heat transfer from the samples to the stainless steel of the vessel when the pressurization was completed (Chen & Hoover, 2003). Thus, the temperature of the samples remains 25 °C during the holding time. After the treatment, all the samples were removed from the equipment and stored at 4 °C. The properties of the samples were measured after 24 h.

(15–20%, w/w) formed gels on pressurization (300 MPa, 30 min), which was assigned that β-Lg appeared to be sensitive to pressure and the interaction between β-Lg and α-La in the network (Ipsen, Olsen, Skibsted, & Qvist, 2002). In addition, Olsen, Ipsen, Otte, and Skibsted (1999) monitored the state of aggregation and thermal gelation properties of pressure-treated β-Lg immediately after depressurization and after storage for 24 h at 5 °C. β-Lg formed soluble aggregates when it was subjected to the pressure of 150 MPa or higher than 300 MPa for 30 min, but continued for 30 min, a pressure of 450 MPa caused gelation of the 5% β-Lg solution. Generally, the gelation process includes protein denaturation and aggregation (Ramaswamy, Singh, & Sharma, 2015). The premise condition for the formation of protein gel is protein denaturation (Chawla, Patil, & Singh, 2011), and non-covalent bonds (hydrogen, ionic, hydrophobic bonds) between globular proteins can be affected by high pressure processing (HPP) and thus protein structure is modified (Singh, Sharma, & Ramaswamy, 2015). For example, Molina, Defaye, and Ledward (2002) found that soy protein isolate (12%, w/v) formed a gel after being pressurized at 500 MPa and 25 °C for 30 min, and the gelation process might involve hydrophobic bonds and disulfide bonds. In addition, HPP is especially beneficial for heat-sensitive components from preventing thermal destruction (Acero-Lopez, Ullah, Offengenden, Jung, & Wu, 2012). Compared with the formation of gel induced by acid, enzyme or ion, HPP is of great interest because this process does not need to add other chemical substances and it can be performed at room temperature and is energy-effective (Qin et al., 2017; Yang, Wang, & Chen, 2017; Zhang, Yang, Tang, Chen, & You, 2016). Moreover, it is possible to keep the original color, flavor and freshness of protein gels after treatment (Mensi et al., 2013). In addition, the stability of the gels is an important factor during its application. Le and Turgeon (2015) found that the higher availability of proteins favored the formation of β-Lg and xanthan gum gels, which could weaken gel stability and limit its development in new functional semi-solid products. However, Mensi et al. (2013) prepared β-Lg aggregates by HPP, which entrapped β-carotene and improved its storage stability. The objective of the current study was to investigate the properties of β-Lg gels formed through HPP directly, focusing on the effects of pressure, protein concentration and pH on the rheological properties, texture profile, protein secondary structure and microstructure of β-Lg gels. The results obtained from this study would be meaningful to develop functional foods through non-thermal processing.

2.3. Rheological measurements Dynamic oscillatory shear tests were performed using a controlled stress rheometer AR-1500ex (TA Instruments, Delaware, USA) fitted with a parallel plate geometry (40 mm diameter, 5 mm gap). The temperature was controlled at 25 °C using a precise circulating water system. The dynamic oscillation analysis determined the storage modulus (G') and the loss modulus (G″) of gels. G′ and G″ were obtained directly from TA software. Each measurement was repeated for three times. The linear viscoelastic region (LVR) was determined by applying a strain sweep test from 0.01 to 100%, at a constant temperature of 25 °C and a constant frequency of 1 Hz (Liu, Wang, Sun, & Gao, 2016). Samples were equilibrated for 2 min before test to remove residual stress. The frequency sweep test was performed from 0.1 rad/s to 100 rad/s under a shear strain of 1%. The strain was selected based on linear part of the linear viscoelastic range. New sample was used for each measurement. Power Law models based on Eqs. (1) and (2) were also used to evaluate the frequency dependence of G′ and G″ by non-linear regression feature of SPSS 18.0 software (SPSS Inc., Chicago, USA). ′

G′ = K′⋅ωn

(1) ″

G″ = K″⋅ωn

(2)

where K′ and K″ were constants, n′ and n″ referred to the frequency exponent, ω was the angular frequency. The values of n′ and n″ were characteristic index for the gel, which reflected the dependence of the viscoelastic modulus on frequency (Özkan, Xin, & Chen, 2002).

2. Materials & methods

2.4. Texture profile analysis (TPA)

2.1. Preparation of β-Lg solutions

Texture profile analysis was performed using TA-XT Plus Texture Analyzer (Stable Micro Systems, Godalming, UK) at room temperature. Before the test, each sample was cut into required size (height: 1.0 cm, diameter: 2.5 cm), and then was equilibrated for 2 h at room temperature. The gel was subjected to a compression test using a cylindrical probe (P/0.25) at a 60 mm/min test speed with a trigger force of 0.5 N and deformation of 30%. Textural properties of the gels including hardness (N), cohesiveness, springiness (mm) and chewiness (mJ) were obtained from the software (Chen et al., 2010). Each measurement was repeated for six times.

The β-Lg (97.7%, protein) used in this study was purchased from Davisco Food International (Le Sueur, MN, USA). β-Lg (14, 16, 18, 20, 22%, w/v) was dissolved in different buffer solutions at pH 3.0 (10 mM glycine-HCl buffer), pH 5.0 (10 mM acetic acid buffer), pH 7.0 (10 mM phosphate buffer) and the samples were stirred at room temperature for fully dissolution. The stock solutions were then stored at 4 °C overnight for further use. 2.2. High pressure treatment β-Lg solutions were transferred to centrifuge tubes and vacuum sealed in polyethylene bags. The bags were then put into HPP L2-700/1 ultra-high pressure equipment (Tianjin Huatai Senmiao Biotechnology and Technique Co. Ltd., Tianjin, China), and the samples were highpressure treated at 200 MPa, 400 MPa and 600 MPa for 30 min at 25 °C with the water as medium. Untreated samples (0.1 MPa) were used as controls. In other words, this experimental procedure was that the pressure increased from 0.1 MPa to certain pressure that we had set and then held for 30 min, finally the equipment pressure released to 0.1 MPa. Each time pressure was raised at an approximate rate of

2.5. Raman spectroscopy Raman spectra of the gels were recorded using a high resolution Raman spectrometer (LabRAM HR Evolution, HORIBA Jobin Yvon S.A.S, Japan) at an excitation wavelength of 532 nm at room temperature. β-Lg (pH 5.0, 22% w/v) treated under different pressures were measured in the wavelength range of 100–4000 cm−1, scanning time of 20 s, accumulation time of 3 times, power of 20 mW, resolution of 0.7 cm−1. The data was processed by LabSpec 5 (HORIBA Jobin Yvon S.A.S, Japan) (Ikeda & Li-Chan, 2004). 336

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2.6. Scanning electron microscopy (SEM) analysis Microstructure of freeze-dried β-Lg gels samples was observed by SEM (JEOL, JSM-6701F, Japan) at an accelerating voltage of 5.0 kV. The surfaces of the samples were sprayed with gold before the observation to avoid charging under the electron beam. The samples were observed in 5 to 10 regions. 2.7. Statistical analysis The experiment was repeated at least three times, and the results were expressed by M (mean) ± SD (standard deviation). Analysis of variances was obtained by SPSS 18.0 (SPSS Inc., Chicago, USA), significant level (p < 0.05) of Duncan multiple range test was used to determine differences among treatments.

Fig. 1. Appearance of β-Lg gels with different pressures and protein concentrations. A: pH 5.0, 20% (w/v), 400 MPa; B: pH 5.0, 20% (w/v), 600 MPa; C: pH 5.0, 22% (w/v), 400 MPa; D: pH 5.0, 22% (w/v), 600 MPa.

and treated under different pressures were shown in Fig. 1. Visually, the gel texture was more compact with the increase of pressure and it was easier to form a smoother gel. From these images (Fig. 1), it can be seen that the samples treated under 400 MPa was not able to form a selfsupporting structure, and the samples treated under 600 MPa formed soft-solid-like structure.

3. Results and discussion 3.1. The appearance of β-Lg gels The physical state of β-Lg gels under different pressures, concentrations and pH values were shown in Table 1. Gel formation can be induced at a higher pressure with a certain protein concentration. The solutions containing 20 and 22% (w/v) β-Lg can form a gel after high pressure treatment at 400 MPa. At the pressure of 600 MPa, the gel can be formed when the concentration was over 14% (w/v). In addition, we also observed a little exudation of non-incorporated liquid, which could be related to the water holding capacity of β-Lg gels induced by HPP. The precondition of gel formation is protein denaturation (Molina et al., 2002). Dumay, Kalichevsky, and Cheftel (1994) found that high pressure treatment at > 400 MPa could induce β-Lg denaturation, which was in accordance with the present study. In addition, the strength of protein gel was different with different pH values, because pH can change the surface charge of protein and thus it affected intermolecular protein interactions and the protein aggregation (Baneful & Bhattacharya, 2012; Grinberg & Tolstoguzov, 1997). Table 1 indicates that the gel formed at pH 5.0 showed a much smoother and more compact appearance than those formed at pH 3.0 and 7.0. Although it has been shown that pressurized weak acid buffer has a huge effect on the pH value due to the effect on the ionization equilibrium, and the pH of acetic acid buffer could decrease one pH unit over a pressure range of 500 MPa (Orlien, Olsen, & Skibsted, 2007), in our study, at pH around 5.0, intermolecular electrostatic interactions between the proteins was weaker, and it was easier to form gels with greater hardness under the same pressure. The appearances of β-Lg gels with different protein concentrations

3.2. Rheological properties of β-Lg gels 3.2.1. Strain sweep Strain sweep curves of β-Lg gels under different conditions were shown in Fig. 2, which were representative curves at pH 3.0, 5.0 and 7.0. It can be seen that the curves could be divided into two regions: in one region, G' remained constant in a certain strain range (LVE); in the other region, G′ decreased dramatically when the strain increased to a certain value, which was in the nonlinear viscoelastic region. The strain where G′ reduced to 95% of G0 (the initial value of G') was defined as the critical strain value γc (Wang, Li, Wang, Wu, & Özkan, 2011). The LVE was generally ≥1% for biological macromolecular gels (Clark & Ross-Murphy, 1987). β-Lg gels induced by ultra-high pressure treatments belong to macromolecular gels, and the γc was around 10% in the present study (Fig. 2). However, this value was higher than that of the gel used in this study (1%), confirming that the present experiment worked well within the linear region. Hyun et al. (2011) observed that the evolution of the viscoelastic properties of the complex fluid could be divided into four types according to the changes of G′ and G″ with the change of strain: strain thinning (type I: G′ and G″ decreasing); strain hardening (type II: G' and G" increasing); weak strain overshoot (type III: G′ decreasing, G″ increasing followed by decreasing); strong strain overshoot (type IV: G′ and G″ increasing followed by decreasing). In the present study (Fig. 2), it can be observed that the transition of viscoelastic properties of β-Lg gels had the characteristic of type I, because G′ and G″ were following monotone decreasing with the change of the strain. The strength of the gel induced by high pressure was related to pressure intensity and protein concentration. From Fig. 2, it was observed that the G' increased with the increase of protein concentration under the same pH and pressure, which suggested that the gel strength was significantly improved. The G' increased with the increase of pressure under the same pH and protein concentration. For example, the G0 of 22% (w/v) β-Lg gels at pH 5.0 increased from 1537 to 7778 Pa when the pressure increased from 400 to 600 MPa, which indicated that higher pressure can result in enhanced strength of β-Lg gels. It also can be seen from Fig. 2 that the viscoelastic properties were divided into three G′ levels at the same pH value: low level (~200 Pa) for 14% (w/v) β-Lg gels formed at 600 MPa; intermediate level (~500–3000 Pa) for 20% (w/v), 22% (w/v) β-Lg gels formed at 400 MPa and 16% (w/v) βLg gels formed at 600 MPa; high level (> 3000 Pa) for 18% (w/v), 20% (w/v) and 22% (w/v) β-Lg gels formed at 600 MPa. These results

Table 1 The states of β-Lg gels under different pressures, concentrations and pH values. Conditions

14%(w/v)

16%(w/v)

18%(w/v)

20%(w/v)

22%(w/v)

pH 3.0

− − − + − − − + − − − +

− − − + − − − + − − − +

− − − ++ − − − +++ − − − ++

− − + ++ − − + +++ − − + ++

− − + ++ − − ++ +++ − − + ++

pH 5.0

pH 7.0

0.1 MPa 200 MPa 400 MPa 600 MPa 0.1 MPa 200 MPa 400 MPa 600 MPa 0.1 MPa 200 MPa 400 MPa 600 MPa

“—” means that the sample did not form a gel; “+” means that the sample formed a gel induced by high pressure processing but it was coarser and lesscompacted; “++” means that the gel was glossier and smoother; “+++” means that the gel showed a much smoother and more compact appearance. 337

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Fig. 2. Strain sweep curves of β-Lg gels under different conditions. (A1): the change of G' at pH 3.0; (A2): the change of G″ at pH 3.0; (B1): the change of G′ at pH 5.0; (B2): the change of G″ at pH 5.0; (C1): the change of G' at pH 7.0; (C2): the change of G″ at pH 7.0.

results were accordance with the states that we observed in Table 1. In addition, the γc of the gel became smaller with the increase of the concentration of β-Lg. The G′ of 14% (w/v) β-Lg gels formed at 600 MPa at pH 5.0 remained essentially unchanged in the strain range from 0.01 to 100%. And the γc of 22% (w/v) β-Lg gels formed at 600 MPa was 10.1%, which was because of the larger concentration of

suggested that both the pressure and protein concentration played a crucial role in the gel strength of β-Lg gels. The higher the pressure and the lower the protein concentration, the lower the gel strength; otherwise, the lower the pressure and the higher the protein concentration, the gel strength was of intermediate level; the higher the protein concentration and the pressure, the gel strength was the highest. These

338

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Fig. 3. Frequency sweep curves of β-Lg gels under different conditions. (A1): the change of G′ at pH 3.0; (A2): the change of G″ at pH 3.0; (B1): the change of G′ at pH 5.0; (B2): the change of G″ at pH 5.0; (C1): the change of G′ at pH 7.0; (C2): the change of G″ at pH 7.0.

protein in the gels. As the gel strength was increasing, the strain and γc were decreasing when the same level of stress was applied. This finding was in agreement with Rafe's study where an extension of the mixed gels' LVE toward higher strain values was observed with the increase of

protein concentration (Rafe, Razavi, & Farhoosh, 2013). 3.2.2. Frequency sweep Fig. 3 shows the evolution of G′ and G″ as affected by protein 339

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Table 2 Power Law parameters for β-Lg gels under different conditions. pH

3.0

5.0

7.0

Conditions

20%-400 MPa 22%-400 MPa 14%-600 MPa 16%-600 MPa 18%-600 MPa 20%-600 MPa 22%-600 MPa 20%-400 MPa 22%-400 MPa 14%-600 MPa 16%-600 MPa 18%-600 MPa 20%-600 MPa 22%-600 MPa 20%-400 MPa 22%-400 MPa 14%-600 MPa 16%-600 MPa 18%-600 MPa 20%-600 MPa 22%-600 MPa

G' = K′·ωn′

G″ = K″·ωn″

K′

n′

405.011 ± 5.790a 1048.580 ± 4.791b 85.593 ± 3.258c 575.727 ± 6.633d 3408.751 ± 16.586e 3599.866 ± 17.403f 4409.423 ± 49.333g 539.782 ± 4.526a 1301.061 ± 17.150b 117.159 ± 1.717c 702.199 ± 5.738d 3677.570 ± 13.018e 4663.524 ± 24.300f 6386.610 ± 36.323g 475.707 ± 5.810a 1263.402 ± 18.087b 95.646 ± 1.032c 601.240 ± 14.659d 3535.672 ± 16.015e 3793.686 ± 44.772f 5472.794 ± 34.543g

0.089 0.090 0.146 0.103 0.088 0.078 0.129 0.094 0.090 0.158 0.089 0.083 0.082 0.085 0.107 0.083 0.135 0.093 0.082 0.133 0.085

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.005a 0.002a 0.008b 0.004c 0.002a 0.001d 0.004e 0.003a 0.004ad 0.004b 0.003ad 0.002c 0.002c 0.002cd 0.004a 0.003b 0.004c 0.006d 0.001b 0.004c 0.002b

R2

K″

n″

0.914 0.985 0.941 0.958 0.986 0.989 0.977 0.971 0.926 0.976 0.970 0.989 0.985 0.983 0.957 0.954 0.969 0.901 0.989 0.976 0.979

83.332 ± 0.848a 191.549 ± 1.865b 23.963 ± 0.421c 133.254 ± 1.090d 483.467 ± 6.638e 581.931 ± 8.560f 897.277 ± 18.642g 104.276 ± 0.730a 239.601 ± 2.653b 34.021 ± 0.548c 142.153 ± 1.322d 508.792 ± 2.966e 659.283 ± 6.392f 1134.811 ± 6.503g 105.582 ± 0.549a 205.221 ± 2.330b 25.382 ± 0.381c 133.768 ± 1.870d 483.741 ± 5.472e 590.092 ± 7.450f 914.894 ± 6.530g

0.054 0.012 0.120 0.039 0.017 0.016 0.018 0.027 0.022 0.143 0.023 0.024 0.024 0.027 0.019 0.016 0.112 0.015 0.012 0.018 0.015

R2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.004a 0.004b 0.003c 0.003d 0.005b 0.006b 0.007b 0.003a 0.004a 0.005b 0.003a 0.003a 0.004a 0.002a 0.002a 0.003a 0.005b 0.004a 0.004a 0.005a 0.005a

0.891 0.960 0.976 0.927 0.960 0.986 0.942 0.903 0.960 0.963 0.944 0.940 0.938 0.970 0.925 0.894 0.937 0.952 0.941 0.950 0.887

Different superscript letters in the same pH column indicate significant differences (p < 0.05).

with Chang, Li, Wang, Bi, and Adhikari (2014) and Nicole, Caimeng, Eric, and Yufei (2014). However, no consistent changes in n′ and n″ were found in the present study. The protein concentration has a crucial effect on gel formation, and gelation occurs when protein concentration is higher than a critical value. When there is insufficient protein, it only forms suspension of aggregates (Nicolai et al., 2011). The current study found that the lowest protein concentration required forming β-Lg gels under 400 MPa was 20% (w/v) and 14% (w/v) under 600 MPa at different pH. Over 400 MPa, β-Lg was denatured (Dumay et al., 1994; Galazka, Sumner, & Ledward, 1996) and they aggregated and cross-linked to form gels as a result of intermolecular interactions. Mu et al. (Kanno & Mu, 2002) also found that 14% (w/v) β-Lg can form a gel under 800 MPa for 5 min at 30 °C. The β-Lg cannot form a gel when the concentration was lower than 14% (w/v) under 600 MPa, which was because that the intermolecular interaction was not strong enough to form a stable network. It can be seen from Fig. 3 that with higher protein concentration, G′ and G″ were greater under the same pH and pressure. For example, for β-Lg gel with 14% (w/v) protein formed under 600 MPa and pH 5.0, the G′ and G″ were 238 Pa and 73 Pa. However, for β-Lg gel with 22% (w/v) protein the two values were 9107 Pa and 1173 Pa, respectively, which indicated that gel strength changed with protein concentration. Protein denaturation can be induced through different approaches. Under high pressure, the protein structure expanded, and the hydrophobic groups were exposed (Loupiac, Bonetti, Pin, & Calmettes, 2006). Therefore, the adjacent molecules formed a three-dimensional network structure through hydrogen bonding, disulfide bonds, Van der Waals force and hydrophobic interactions to maintain a stable system. Water and other components in the system were trapped in the gel network (He et al., 2014). The strength of the protein gels can be enhanced by high pressure treatment. For example, G' of β-Lg gels with 20% (w/v) protein processed under 400 MPa was 567 Pa, while it was 5781 Pa when processed under 600 MPa at pH 5.0. This difference in G' was attributed to the fact that the pressure could change protein conformation and enhance the interactions between protein and water molecules. The interactions could affect the processes of protein denaturation, aggregation and gelation, and thus the gel network became more compact and gel strength was increased significantly (Abd ElSalam, El-Shibiny, & Salem, 2009; Patel, Singh, Havea, Considine, & Creamer, 2005).

Fig. 4. Frequency sweep curves of 20% (w/v) β-Lg gels under 600 MPa and different pH values (f = 1 Hz and strain 1%).

concentration, pressure and pH at a frequency of 1 Hz, which were presenting a representative curve in Fig. 3. Morris and Ross-Murphy (1981) gave a definition of a gel that the elasticity modulus (G′) of the typical gel should be much larger than the viscous modulus (G″), both G′ and G″ were almost independent of frequency in the range of 10−2–102 rad/s, and G′ was paralleled with G″. Winter and Chambon (1986) also found that G′ and G″ were paralleled and G' > G″ for a typical gel system. It was seen that the gels after all treatments showed G′ > G″ and they were independent of frequency as indicated in Fig. 3. The Power Law model parameters for β-Lg gels under different conditions were listed in Table 2, which shows that the result obtained from frequency sweep test was well consistent with this model (R2 > 0.887). At K′ > K″, the viscous components dominated β-Lg gels in the range of 0.1–100 rad/s and the changes of protein concentration and pressure also had significant influence on the structure of the gels (p < 0.05). In addition, at n′ > n″, the dependence of G′ and G″ on frequency was different, and this finding was in accordance 340

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Fig. 5. Textural property of β-Lg gels as function of protein concentration, pH value and pressure (mean ± SD, n = 6). Different superscript letters in the same pH column indicate significant differences (p < 0.05). (A): Hardness (N); (B): Cohesiveness; (C): Springiness (mm); (D): Chewiness (mJ).

linking had a large influence on whey protein isolate (WPI) gelation induced by pressure and the degree of cross-linking under alkaline conditions was higher than that under acidic conditions. When the pH was over 6.8, the degree of ionization and the activity of sulfhydryl groups were increased, which promoted the formation of intermolecular disulfide bonds, and the gel strength was generally higher. The strength of the gel at pH 7.0 was higher than that at pH 3.0, which was presumably due to the different level of the disulfide cross-linking.

pH has an important effect on protein gelation and the properties of the gels formed. In the current study, it was found that β-Lg gels induced by high pressure were pH-sensitive, because of the effect of pH on the kinetics of β-Lg denaturation and aggregation (Ikeuchi et al., 2001). Changes of G′ of β-Lg gels with 20% (w/v) protein as a function of frequency were shown in Fig. 4. The gel formed at pH 5.0 had the highest gel strength (9107 Pa), followed by those formed at pH 7.0 (7754 Pa) and 3.0 (7690 Pa). At pH 5.0, which was closer to the isoelectric point (pI ≈ 5.2) of β-Lg after pressure-treated, the electrostatic repulsion force between protein molecules was rather small and the aggregation was relatively fast, which contributed to the highest gel strength. He, Azuma and Yang, (2010) found that the disulfide cross-

3.3. Textural property of β-Lg gels Protein concentration, pH and pressure had large effects on the 341

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Fig. 5. (continued)

with the increases in both G' and protein concentration during frequency sweep test. Fig. 5 also presents that the hardness, cohesiveness, springiness and chewiness of β-Lg gels increased significantly with the increase of pressure (p < 0.05). It was proposed that during high pressure processing the volume of the protein solution decreased and the amino acid residues around the water molecules were rearranged, resulting in the change in protein structure. High pressure processing can speed up the aggregation of β-Lg, which could contribute to the exposure of sulfydryl groups of β-Lg and the formation of intermolecular disulfide bonds. Therefore, the hardness, cohesiveness, springiness and chewiness of the gel were improved (Funtenberger, Dumay, & Cheftel, 1997; Kanno & Mu, 2002).

textural properties of protein gels. Kanno, Mu, Hagiwara, Ametani, and Azuma (1998) found that the hardness of pressure-induced gels from WPI increased with increasing WPI concentration (12–18%) and hydrostatic pressure, and they thought that β-Lg predominantly participated in pressure-induced aggregation and gelation, in which intermolecular SeS bonding was likely to play a critical role. Fig. 5 shows that the increase in the protein concentration led to higher hardness, springiness and chewiness of the gels (p < 0.05) under the same pressure and pH. However, the cohesiveness did not change significantly when protein concentration was over 18% (w/v). This finding was attributed to the denser network of the gels with higher protein concentration. Hardness, springiness and chewiness were increased (Le & Turgeon, 2015), which were consistent 342

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decreased with the increase of pressure, which was in agreement with previous findings (Ngarize, Herman, Adams, & Howell, 2004). The region between 1200 and 1300 cm−1 was an amide III region, resulted mainly from CeN stretching and NeH in-plane bending vibration (Sun et al., 2011). A peak at 1450 cm−1 was the characteristic of the deformation of CH2 and CH3. 1600–1700 cm−1 were C]O stretching, NeH in-plane bending and CeN stretching vibration, which were among amide I (Rygula et al., 2013). 1663–1675 cm−1 was the random coil structure of β-Lg, and the random coil structure appeared at 1663 cm−1 and 1670 cm−1 in the current study. In addition, the peak area was larger with the increase of pressure, and the random coil content increased by HPP. The peaks at 1858 cm−1, 2012 cm−1, 2166 cm−1 and 2418 cm−1 disappeared with the increase of pressure, which may be due to the formation of a more compact three-dimensional network by pressurizing. The bands at 2571 cm−1 and 2574 cm−1 were attributed to stretching vibration of -SH in cysteine residues. It can be seen from Fig. 6 that the peak intensity decreased or was even not detected as the pressure increased, which may be related to the denaturation of β-Lg induced by pressure that promoted the conversion between -SH and SeS, and it was conducive to the formation of stable network structure (Ngarize, Adams, & Howell, 2005). The bands between 2800 cm−1 to 3000 cm−1 were characteristics of the stretching vibration of CH3. The two peaks were broaden in this range, which may be caused by the changes in the side chains of amino acids after protein aggregation by pressurizing (Rafe & Razavi, 2015).

Fig. 6. Raman spectra of 22% (w/v) β-Lg at pH 5.0 after different pressure treatments: (A) 0.1 MPa; (B) 200 MPa; (C) 600 MPa.

Table 3 Assignment of major bands in the Raman spectra of β-Lg gels induced by high pressure processing. Wavenumber (cm−1)

Assignment

830, 855 940 1004 1255, 1262, 1266 1450 1600–1700

Tyr CeC stretching(α-helix) Phe Amide III (CeN stretching, NeH bending vibration) CH2 deformation, CH3 deformation Amide I (C]O stretching, NeH bending vibration, CeN stretching) -SH stretching CH3 deformation

2571, 2574 2800–3000

3.5. SEM images SEM was applied to observe the microstructure of β-Lg gels (Fig. 7). The images clearly showed the difference in the structures of β-Lg induced by different pressures. The native β-Lg solutions had spherical structures (Fig. 7A). Fig. 7B shows a number of fragments of the β-Lg solution after high pressure treatment at 200 MPa, which suggested that the failure of the development of a gel network. However, the protein formed a relatively thick microstructure with rough surface after the treatment at 400 MPa (Fig. 7C) and a network structure with a certain pore and smooth surface after the treatment at 600 MPa (Fig. 7D) was observed. These findings also suggested that the lowest pressure required to induce β-Lg gelation was 400 MPa and the pressure lower than 400 MPa would not result in a gel. In addition, the structure of βLg gels was more regular when treated under 600 MPa than that of the gel treated at 400 MPa, which was consistent with the findings regarding gel hardness, springiness and chewiness, as they all increased with the increase of pressure. These findings may be caused by intermolecular and intramolecular forces of β-Lg, which increased with the increase of pressure and the network structure was more stable (Mensi et al., 2013).

pH of the systems could modify the net charge of the molecules and thus affect intermolecular attraction and repulsion between protein molecules and the surrounding solvent molecules (Baneful & Bhattacharya, 2012). Fig. 5 shows that the gel at pH 5.0 exhibited the biggest hardness, followed by the gels at pH 7.0 and pH 3.0 under the same protein concentration and pressure. 3.4. Raman spectra tests High pressure treatment led to detectable changes in Raman bands of protein structures (Fig. 6), assignments of which were summarized in Table 3. Doublet bands at 830 cm−1 and 850 cm−1 arose from Fermi resonance of the benzene skeleton vibration and out-of-plane ringbending vibration of para-substituted benzene bands, and the relative intensity suggested the nature of hydrogen bonds (strong or weak) and the ionization state of the phenolic hydroxyl group in the tyrosine side chain (Bellocq, Lord, & Mendelsohn, 1972; Ogawa et al., 1999; Van Dael, Lafaut, & Van Cauwelaert, 1987). If the relative intensity at 855 cm−1 to 830 cm−1 was higher, the hydrogen bonds of the tyrosine side were in polar or hydrophobic environment. Instead, the exposed tyrosine residues were buried in the molecular interior due to the aggregation of the protein. In the current study, there was an obvious peak at 830 cm−1. Moreover, the peak intensity and the relative intensity (I855/I830) decreased with the increase of pressure, presumably because that the exposed tyrosine residues were embedded in molecular interior in the protein aggregation due to HPP, which led to the decrease of Raman intensity. The peak at 940 cm−1 was resulted from CeC stretching vibrations of β-Lg and its intensity was proportional to the fraction of α-helix. I940/ I1004 reflected the changes of the content of α-helix caused by different treatment conditions (Ikeda & Li-Chan, 2004). The peak at 1004 cm−1 was often used for normalizing the intensity of other bands. As seen from Fig. 6, the value of I940/I1004 decreased with the increase of pressure (from 0.99 to 0.91), showing that the α-helix content of β-Lg

4. Conclusions This work systematically studied the effects of different pH and protein concentrations on the gelation of β-Lg gels induced by HPP and gel rheological properties. In the current study, it was found that the lowest protein concentration required to form β-Lg gels was 20% (w/v) under 400 MPa and 14% (w/v) under 600 MPa. β-Lg formed more compact soft-solid-like gel structure by increasing pressure. The G′ and G″, textural properties of β-Lg gels increased with the increase of protein concentration and processing pressures, and the gel strength reached the highest level at pH 5.0, followed by pH 7.0 and finally pH 3.0. Based on the structural investigation, some Raman peaks of βLg gels decreased or even disappeared by high pressure treatment, which might be caused by the changes of protein structure. The current study revealed that high pressure treatment could induce the formation of β-Lg gels, which could be used to develop novel functional foods. 343

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Fig. 7. SEM images of 22% (w/v) β-Lg at pH 5.0. (A) 0.1 MPa; (B) 200 MPa; (C) 400 MPa; (D) 600 MPa.

Therefore, further studies to evaluate the physicochemical stability of the β-Lg gels induced by HPP and how to improve the stability will be necessary, which are very important on the practical application. In addition, the formation of HPP-induced gels is difficult to be detected in real time, so the mechanism of the gels is difficult to explore and still have to be clarified on HPP-induced gels behavior.

Bellocq, A. M., Lord, R. C., & Mendelsohn, R. (1972). Laser-excited Raman spectroscopy of biomolecules. III. Native bovine serum albumin and β-lactoglobulin. Biochimica et Biophysica Acta (BBA)-Protein Structure, 257, 280–287. Bryant, C. M., & McClements, D. J. (1998). Molecular basis of protein functionality with special consideration of cold-set gels derived from heat-denatured whey. Trends in Food Science & Technology, 9, 143–151. Cao, Y., Xia, T., Zhou, G., & Xu, X. (2012). The mechanism of high pressure-induced gels of rabbit myosin. Innovative Food Science & Emerging Technologies, 16, 41–46. Chang, Y. Y., Li, D., Wang, L. J., Bi, C. H., & Adhikari, B. (2014). Effect of gums on the rheological characteristics and microstructure of acid-induced SPI-gum mixed gels. Carbohydrate Polymers, 108, 183–191. Chawla, R., Patil, G. R., & Singh, A. K. (2011). High hydrostatic pressure technology in dairy processing: A review. Journal of Food Science and Technology, 48, 260–268. Chen, H., & Hoover, D. G. (2003). Pressure inactivation kinetics of Yersinia enterocolitica ATCC 35669. International Journal of Food Microbiology, 87, 161–171. Chen, C., Wang, R., Sun, G., Fang, H., Ma, D., & Yi, S. (2010). Effects of high pressure level and holding time on properties of duck muscle gels containing 1% curdlan. Innovative Food Science & Emerging Technologies, 11, 538–542. Clark, A. H., & Ross-Murphy, S. B. (1987). Structural and mechanical properties of biopolymer gels. Biopolymers (pp. 57–192). . De Maria, S., Ferrari, G., & Maresca, P. (2016). Effects of high hydrostatic pressure on the conformational structure and the functional properties of bovine serum albumin. Innovative Food Science & Emerging Technologies, 33, 67–75. Dumay, E. M., Kalichevsky, M. T., & Cheftel, J. C. (1994). High-pressure unfolding and aggregation of beta-lactoglobulin and the baroprotective effects of sucrose. Journal of Agricultural and Food Chemistry, 42, 1861–1868. Funtenberger, S., Dumay, E., & Cheftel, J. C. (1997). High pressure promotes β-lactoglobulin aggregation through SH/SS interchange reactions. Journal of Agricultural and Food Chemistry, 45, 912–921. Galazka, V. B., Sumner, I. G., & Ledward, D. A. (1996). Changes in protein-protein and protein-polysaccharide interactions induced by high pressure. Food Chemistry, 57,

Acknowledgements We thank the financial support from the National Natural Science Foundation of China (No. 31371836 and No. 31701648) and National Key R&D Program of China (No. 2016YFD0400804). References Abd El-Salam, M. H., El-Shibiny, S., & Salem, A. (2009). Factors affecting the functional properties of whey protein products: A review. Food Reviews International, 25, 251–270. Acero-Lopez, A., Ullah, A., Offengenden, M., Jung, S., & Wu, J. (2012). Effect of high pressure treatment on ovotransferrin. Food Chemistry, 135, 2245–2252. Ahmed, J., & Ramaswamy, H. S. (2003). Effect of hydrostatic pressure and temperature on rheological characteristics of α‑lactalbumin. Australian Journal of Dairy Technology, 58, 233–237. Balasubramaniam, V. M., Farkas, D., & Turek, E. J. (2008). Preserving foods through highpressure processing. Food Technology, 62(11), 32–38. Baneful, S., & Bhattacharya, S. (2012). Food gels: Gelling process and new applications. Critical Reviews in Food Science and Nutrition, 52, 334–346.

344

Innovative Food Science and Emerging Technologies 47 (2018) 335–345

X. Li et al.

Formation, structure and applications. Food Hydrocolloids, 25, 1945–1962. Nicole, M., Caimeng, Z., Eric, K., & Yufei, H. (2014). Salt and acid-induced soft tofu-type gels: Rheology, structure and fractal analysis of viscoelastic properties as a function of coagulant concentration. International Journal of Food Engineering, 10, 595–611. Ogawa, M., Nakamura, S., Horimoto, Y., An, H., Tsuchiya, T., & Nakai, S. (1999). Raman spectroscopic study of changes in fish actomyosin during setting. Journal of Agricultural and Food Chemistry, 47, 3309–3318. Olsen, K., Ipsen, R., Otte, J., & Skibsted, L. H. (1999). Effect of high pressure on aggregation and thermal gelation of β‑lactoglobulin. Milchwissenschaft, 54(10), 543–546. Orlien, V., Olsen, K., & Skibsted, L. H. (2007). In situ measurements of pH changes in βlactoglobulin solutions under high hydrostatic pressure. Journal of Agricultural and Food Chemistry, 55, 4422–4428. Özkan, N., Xin, H., & Chen, X. D. (2002). Application of a depth sensing indentation hardness test to evaluate the mechanical properties of food materials. Journal of Food Science, 67, 1814–1820. Patel, H. A., Singh, H., Havea, P., Considine, T., & Creamer, L. K. (2005). Pressure-induced unfolding and aggregation of the proteins in whey protein concentrate solutions. Journal of Agricultural and Food Chemistry, 53, 9590–9601. Purwanti, N., Smiddy, M., Van der Goot, A. J., De Vries, R., Alting, A., & Boom, R. (2011). Modulation of rheological properties by heat-induced aggregation of whey protein solution. Food Hydrocolloids, 25, 1482–1489. Qin, X. S., Chen, S. S., Li, X. J., Luo, S. Z., Zhong, X. Y., Jiang, S. T., ... Zheng, Z. (2017). Gelation properties of transglutaminase-induced soy protein isolate and wheat gluten mixture with ultrahigh pressure pretreatment. Food and Bioprocess Technology, 1–9. Rafe, A., & Razavi, S. M. (2015). Effect of thermal treatment on chemical structure of βlactoglobulin and basil seed gum mixture at different states by ATR-FTIR spectroscopy. International Journal of Food Properties, 18, 2652–2664. Rafe, A., Razavi, S. M., & Farhoosh, R. (2013). Rheology and microstructure of basil seed gum and β‑lactoglobulin mixed gels. Food Hydrocolloids, 30, 134–142. Ramaswamy, H. S., Singh, A., & Sharma, M. (2015). Back extrusion rheology for evaluating the transitional effects of high pressure processing of egg components. Journal of Texture Studies, 46, 34–45. Rygula, A., Majzner, K., Marzec, K. M., Kaczor, A., Pilarczyk, M., & Baranska, M. (2013). Raman spectroscopy of proteins: A review. Journal of Raman Spectroscopy, 44, 1061–1076. Sagis, L. M., Veerman, C., Ganzevles, R., Ramaekers, M., Bolder, S. G., & van der Linden, E. (2002). Mesoscopic structure and viscoelastic properties of β-lactoglobulin gels at low pH and low ionic strength. Food Hydrocolloids, 16, 207–213. Sahihi, M., Heidari-Koholi, Z., & Bordbar, A. K. (2012). The interaction of polyphenol flavonoids with β-lactoglobulin: Molecular docking and molecular dynamics simulation studies. Journal of Macromolecular Science, 51, 2311–2323. Singh, A., Sharma, M., & Ramaswamy, H. S. (2015). Effect of high pressure treatment on rheological characteristics of egg components. International Journal of Food Properties, 18, 558–571. Sun, W., Zhao, Q., Zhao, M., Yang, B., Cui, C., & Ren, J. (2011). Structural evaluation of myofibrillar proteins during processing of Cantonese sausage by Raman spectroscopy. Journal of Agricultural and Food Chemistry, 59, 11070–11077. Van Dael, H., Lafaut, J. P., & Van Cauwelaert, F. (1987). Tyrosine group behaviour in bovine α‑lactalbumin as revealed by its Raman effect. European Biophysics Journal, 14, 409–414. Wang, Y., Li, D., Wang, L. J., Wu, M., & Özkan, N. (2011). Rheological study and fractal analysis of flaxseed gum gels. Carbohydrate Polymers, 86, 594–599. Winter, H. H., & Chambon, F. (1986). Analysis of linear viscoelasticity of a crosslinking polymer at the gel point. Journal of Rheology, 30, 367–382. Wu, X., Nishinari, K., Gao, Z., Zhao, M., Zhang, K., Fang, Y., ... Jiang, F. (2016). Gelation of β‑lactoglobulin and its fibrils in the presence of transglutaminase. Food Hydrocolloids, 52, 942–951. Yang, C., Wang, Y., & Chen, L. (2017). Fabrication, characterization and controlled release properties of oat protein gels with percolating structure induced by cold gelation. Food Hydrocolloids, 62, 21–34. Zhang, Z., Yang, Y., Tang, X., Chen, Y., & You, Y. (2015). Chemical forces and water holding capacity study of heat-induced myofibrillar protein gel as affected by high pressure. Food Chemistry, 188, 111–118. Zhang, Z., Yang, Y., Tang, X., Chen, Y., & You, Y. (2016). Chemical forces study of heatinduced myofibrillar protein gel as affected by partial substitution of NaCl with KCl, MgCl2 and CaCl2. CyTA-Journal of Food, 14, 239–247.

393–398. Grinberg, V. Y., & Tolstoguzov, V. B. (1997). Thermodynamic incompatibility of proteins and polysaccharides in solutions. Food Hydrocolloids, 11, 145–158. He, J. S., Azuma, N., & Yang, H. (2010). Effects of pH and ionic strength on the rheology and microstructure of a pressure-induced whey protein gel. International Dairy Journal, 20, 89–95. He, X. H., Liu, H. Z., Liu, L., Zhao, G. L., Wang, Q., & Chen, Q. L. (2014). Effects of high pressure on the physicochemical and functional properties of peanut protein isolates. Food Hydrocolloids, 36, 123–129. He, J. S., Yang, H., Zhu, W., & Mu, T. H. (2010). Kinetics of microstructure formation of highpressure induced gel from a whey protein isolatevol. 215, no. 1. Kinetics of microstructure formation of high-pressure induced gel from a whey protein isolate (pp. 012169–). IOP Publishing. Hermansson, A. M. (1979). Aggregation and denaturation involved in gel formation—Chapter 5. Functionality and protein structure (pp. 81–103). . Hyun, K., Wilhelm, M., Klein, C. O., Cho, K. S., Nam, J. G., Ahn, K. H., ... McKinley, G. H. (2011). A review of nonlinear oscillatory shear tests: Analysis and application of large amplitude oscillatory shear (LAOS). Progress in Polymer Science, 36, 1697–1753. Ikeda, S., & Li-Chan, E. C. (2004). Raman spectroscopy of heat-induced fine-stranded and particulate β‑lactoglobulin gels. Food Hydrocolloids, 18, 489–498. Ikeuchi, Y., Nakagawa, K., Endo, T., Suzuki, A., Hayashi, T., & Ito, T. (2001). Pressureinduced denaturation of monomer β-lactoglobulin is partially irreversible: Comparison of monomer form (highly acidic pH) with dimer form (neutral pH). Journal of Agricultural and Food Chemistry, 49, 4052–4059. Ipsen, R., Olsen, K., Skibsted, L. H., & Qvist, K. B. (2002). Gelation of whey protein induced by high pressure. Milchwissenschaft, 57(11−12), 650–653. Kanno, C., & Mu, T. H. (2002). Gel formation of individual milk whey proteins under hydrostatic pressure. Progress in Biotechnology, 19, 453–460. Kanno, C., Mu, T. H., Hagiwara, T., Ametani, M., & Azuma, N. (1998). Gel formation from industrial milk whey proteins under hydrostatic pressure: Effect of hydrostatic pressure and protein concentration. Journal of Agricultural and Food Chemistry, 46, 417–424. Kavanagh, G. M., Clark, A. H., & Ross-Murphy, S. B. (2000). Heat-induced gelation of globular proteins: Part 3. Molecular studies on low pH β-lactoglobulin gels. International Journal of Biological Macromolecules, 28, 41–50. Le, X. T., & Turgeon, S. L. (2015). Textural and waterbinding behaviors of β-lactoglobulin-xanthan gum electrostatic hydrogels in relation to their microstructure. Food Hydrocolloids, 49, 216–223. Line, V. L. S., Remondetto, G. E., & Subirade, M. (2005). Cold gelation of β‑lactoglobulin oil-in-water emulsions. Food Hydrocolloids, 19, 269–278. Liu, F., Wang, D., Sun, C., & Gao, Y. (2016). Influence of polysaccharides on the physicochemical properties of lactoferrin–polyphenol conjugates coated β‑carotene emulsions. Food Hydrocolloids, 52, 661–669. López-Fandiño, R. (2006). High pressure-induced changes in milk proteins and possible applications in dairy technology. International Dairy Journal, 16, 1119–1131. Loupiac, C., Bonetti, M., Pin, S., & Calmettes, P. (2006). β‑lactoglobulin under high pressure studied by small-angle neutron scattering. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1764, 211–216. Ma, F., Chen, C., Zheng, L., Zhou, C., Cai, K., & Han, Z. (2013). Effect of high pressure processing on the gel properties of salt-soluble meat protein containing CaCl2 and κcarrageenan. Meat Science, 95, 22–26. Mensi, A., Choiset, Y., Haertlé, T., Reboul, E., Borel, P., Guyon, C., ... Chobert, J. M. (2013). Interlocking of β‑carotene in β‑lactoglobulin aggregates produced under high pressure. Food Chemistry, 139, 253–260. Mohtar, N. F., Perera, C. O., Quek, S. Y., & Hemar, Y. (2013). Optimization of gelatine gel preparation from New Zealand hoki (Macruronus novaezelandiae) skins and the effect of transglutaminase enzyme on the gel properties. Food Hydrocolloids, 31, 204–209. Molina, E., Defaye, A. B., & Ledward, D. A. (2002). Soy protein pressure-induced gels. Food Hydrocolloids, 16, 625–632. Morris, E. R., & Ross-Murphy, S. B. (1981). Techniques in the life science. B 310, 1. Ngarize, S., Adams, A., & Howell, N. (2005). A comparative study of heat and high pressure induced gels of whey and egg albumen proteins and their binary mixtures. Food Hydrocolloids, 19, 984–996. Ngarize, S., Herman, H., Adams, A., & Howell, N. (2004). Comparison of changes in the secondary structure of unheated, heated, and high-pressure-treated β‑lactoglobulin and ovalbumin proteins using Fourier transform Raman spectroscopy and self-deconvolution. Journal of Agricultural and Food Chemistry, 52, 6470–6477. Nicolai, T., Britten, M., & Schmitt, C. (2011). β‑Lactoglobulin and WPI aggregates:

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