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Changes of protein secondary structures of pollock surimi gels under high-temperature (100 °C and 120 °C) treatment
Accepted Manuscript Changes of protein secondary structures of pollock surimi gels under hightemperature (100 °C and 120 °C) treatment Lili Zhang, Fengxiang Zhang, Xia Wang PII:
S0260-8774(15)30026-1
DOI:
10.1016/j.jfoodeng.2015.10.025
Reference:
JFOE 8368
To appear in:
Journal of Food Engineering
Received Date: 17 November 2014 Revised Date:
24 July 2015
Accepted Date: 21 October 2015
Please cite this article as: Zhang, L., Zhang, F., Wang, X., Changes of protein secondary structures of pollock surimi gels under high-temperature (100 °C and 120 °C) treatment, Journal of Food Engineering (2015), doi: 10.1016/j.jfoodeng.2015.10.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The secondary structure of the pollock surimi gels with high-temperature (≥100 °C) treatment was mainly random coil. With treating temperature increasing, the random coil structure damaged, resulting in the disappearance of myosin heavy chain and significant decrease of actin content. In addition, the protein molecules aggregated. So the frames of the network structure became much more fragile and the holes became larger, leading to the destruction of the textural properties of the surimi gels.
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Changes of protein secondary structures of Pollock surimi gels under
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high-temperature (100 °C and 120 °C) treatment
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Lili Zhang a, Fengxiang Zhang a*, Xia Wang a
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a
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China
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*Corresponding author. Tel.: +86 0536 8462429.
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E-mail address: [email protected].
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Department of Public Health, WeiFang Medical University, Weifang, 261053, PR
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Abstract To produce instant surimi products, sterilization is essential. Previous studies found
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that the gel strength of surimi decreased significantly with treating temperature
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increasing from 100 °C to 120 °C, which affected the texture of the products. In this
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study, the changes of the secondary structures of surimi gels were studied to provide
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new train of thought for the improvement of the texture of high temperature treated
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surimi gels. Surimi gels from Alaska Pollock were obtained by heating and maintaining
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their central temperature at 100 °C and 120 °C for 10min under a constant pressure
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(0.12 MPa). Infrared (IR) spectroscopy and Raman spectroscopy showed that the
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secondary structure of the surimi gels with high temperature (≥100 °C) treatment was
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mainly random coil and the random coil structure damaged with treating temperature
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increasing, which made the frames of the network structure become much more
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fragile and the holes become larger, leading to the destruction of the textural
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properties of the surimi gels.
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Keywords: high-temperature treatment, surimi gel, Infrared spectroscopy, Raman
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spectroscopy
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1. Introduction At present, the form of surimi products in China is single, which greatly limits the edible convenience, so the development of new surimi products is very imminent.
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To produce instant surimi products, longtime shelf life is a very important goal，which
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can be realized by sterilization. Products after sterilizing especially at 121 °C can be
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stored at normal temperature and have longer shelf life. Up to now, there were very
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few reports on the effects of high temperatures (≥100 °C) on the properties of surimi
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gels. Previously it was found that high-temperature treatment (≥100 °C) significantly
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decreased the gel strength of the surimi gels, thereby affecting the texture of the
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products (Zhang et al., 2013), and adding hydrolyzed wheat gluten could significantly
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improve the textural properties of the surimi gels, and the thermal stability under high
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temperature (≥100 °C) of the gels was greatly improved (Zhang et al., 2015). The
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structure and function of the proteins are closely related, so it is very necessary to
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understand the changing mechanism of the protein spatial structures of the surimi gels
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(Nicolin et al., 1992) so as to explore other new ways to improve the texture
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characteristic of the surimi gels under high temperature treatment, providing technical
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support and theoretical basis for high-temperature treatment of instant surimi products.
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In previous study, we discussed the effect of high temperature treatment on the texture, chemical bond changes, water-binding capacity and network structure of the surimi
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gels, which primarily explained how the tertiary structure and quaternary structure
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changed with the treating temperature increasing from 100 °C to 120 °C. The primary
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structure of proteins determined the senior structure. To get stable protein senior 3
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structures, it is very necessary to clarify how the protein secondary structures change
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with high temperature treatment, which will help exploit new train of thought and
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feasible ways from different aspects to improve the texture characteristic of the surimi
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gels under high temperature treatment.
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Protein secondary structure refers to the atom arrangement (i.e. conformation) in
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the main chain of the polypeptide, which is independent of the side chain
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conformation. Secondary structural elements are in forms of α- helix, β-fold, β-corner
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and random coil, etc., which constitute the essential elements of the high protein
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structure.
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Infrared (IR) spectroscopy and Raman spectroscopy are commonly used in the
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study of protein secondary structure. Fourier transform infrared (FT-IR) spectroscopy
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has attracted considerable interest with simple operation, accurate wavelength, good
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resolution, high speed scanning and high sensitivity, which applies the earliest and
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most common way to study protein conformation (Argyri et al., 2013). Currently the
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identification of the peaks of amide I bands (1700-1600 cm-1) in IR spectrum is
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widely used, often used to analyze the secondary structure of the proteins. Raman
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spectroscopy is a branch of the vibrational spectroscopy method that is
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complementary to infrared absorbance and can be used in food analysis, since it is non-destructive, requires little pre-treatment of samples and provides information
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about different food compounds at the same time, offering quantitative analysis of
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food components with simultaneous information on molecular structure. In
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comparison to FT-IR, smaller portions of sample are required and instrumentation can 4
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be less expensive and portable. Moreover, Raman spectroscopy has the advantage
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over FT-IR that the contribution from water is very small (since H2O is a weak Raman
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scatterer) and can be used directly on foods, so it owns incomparable advantages
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when the sample is scarce or with water and is also a very useful method in the
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studies of the protein conformation and the changes of biological macromolecules.
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Recently, FT-IR and Raman spectroscopy have been used to characterize the
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heat-induced denaturation of myofibrillar and connective tissue proteins of beef
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(Kirschner et al., 2004), salt- and heat-induced changes in myofibrillar protein of pork
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(Bertram et al., 2006, Böcker et al., 2006) and the structural changes in fish proteins
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(Herrero, Carmona and Careche, 2004).
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In this study, the changes of the secondary structures of the surimi gels treated with different high temperatures (100 °C and 120 °C) were studied, so as to interpret how
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high temperature treatment affected the surimi gels from a more microscopic
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perspective, further deepening the previous study and enriching the theory of high
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temperature treated surimi products, which would provide new train of thought for the
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improvement of the texture of high temperature treated surimi gels, laying foundation
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for the production of instant surimi products.
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2. Materials and methods 2.1 Materials
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Frozen Alaska Pollock (Theragra chalcogramma) surimi (grade AAA), was
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purchased from JINCAN Foods Co., Ltd., Qingdao, Shandong, China. Surimi was
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kept at -20 °C until used. 5
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All of the chemicals used were of analytical grade and were purchased from
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Sigma-Aldrich (St. Louis, Mo, USA) or Sinopharm Chemical Reagent Co., Ltd.
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(Shanghai, China).
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2.2 Preparation of the surimi gels
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To prepare surimi gels, 250 g frozen Alaska Pollock surimi was semi-thawed at
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4 °C for 4h and then cut into small pieces (about 3 cm cubes). Then the surimi was
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chopped in a Stephan vertical vacuum cutter (Model UM 5, Stephan Machinery Co.,
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Hameln, Germany). In order to keep the temperature of the sample below 4°C during
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chopping, a chilling medium (ethanol: water, 95:5) was continuously circulated in the
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double-walled chopping bowl. Firstly, the surimi was chopped for 5 min with a speed
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of 1500 rpm, then 3 % (w/w) sodium chloride was added and the mixture was
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chopped for another 5 min with a speed of 2100 rpm. In order to remove air pockets
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the chopping condition was under vacuum of 0.5 bar. The surimi sol was packed into
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plastic casings (3 cm i.d.), with both ends sealed tightly.
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2.3 High-temperature treatment
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A Steam/electric amphibious sterilization pot (JINDING Food Machinery Co., Ltd.,
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Zhucheng, Shandong, China) was used for the high-temperature treatment of the
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surimi gels. In this study, steam was used to supply the heating medium of the pot. The sterilization pot can be preset a desired temperature and the accuracy of the
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temperature in the sterilization process can be ensured by automatic temperature
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control (automatic heating when below the desired temperature and automatic stop
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when
reach
the
set
temperature). 6
In
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sterilization
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counter-pressure sterilization was used to prevent casings from bulging. Compressed
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air was passed into the pot to keep the ambient pressure and the internal pressure of
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the sample the same. After sterilization the supply of steam was stopped and the
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cooling water was supplied into the sprinkler pipe, cooling the sample. The
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compressed air can be used to compensate the pressure reduction in the pot dropped
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as caused by the decreased temperature and condensed steam. The conditions of the
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high-temperature treatment were as follows: keeping the pressure at 0.12 MPa and the
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center temperature of the samples reach 100±1 °C and 120±1 °C and maintain 10min
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respectively. the center temperature of the samples was maintain High temperature
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processing center of the sample temperature was monitored by an Ellab
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TrackSense-Pro purchased from Henglv Engineering Co., Ltd., Hubei, China. After
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high-temperature treatment the samples were cooled in ice water and then kept
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refrigerated (4 °C) until tested.
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2.4 Raman spectroscopy
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Raman spectroscopy was carried out in an inVia laser Raman spectroscopy
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(Renishaw Trading Co., Ltd., Shanghai, China). About 1 g surimi gels were placed on
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a glass sheet with a flat smooth surface. 633 argon ion laser and 1800 lines grating
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were used. The measurement conditions were as follows: the scan range was 400-4000 cm-1, the laser energy was 10 mW, the resolution was 1 cm-1, the acquisition time was 10 s and the number of the spectra collected was 40 times.
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2.5 FT-IR spectroscopy
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FT-IR spectroscopy was carried out in a 470 Fourier transform infrared 7
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spectrometer (Thermo Electron Instruments Co., Ltd., Shanghai, China).The surimi
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gels were freeze-dried and mixed with pure KBr. The mixture was grinded uniformly,
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placed in a mold and then pressed into a transparent sheet with (5-10)×107 Pa pressure
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on the hydraulic machine. The measurement conditions were as follows: the range of
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wave number was 4000-400 cm-1, the resolution was 4 cm-1 and the number of the
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spectra collected was 128 times.
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2.6 Statistical analysis
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Statistical analysis was performed by analysis of variance (ANOVA) and Duncan’s
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multiple range tests. The results were expressed as mean ± SD, and the differences
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were considered to be statistically significant at P< 0.05.
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3. Results and discussion
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3.1 Raman spectroscopy
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Fig. 1 showed the Raman spectra of the surimi gels with different high-temperature
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treatment. The Raman spectra was divided into three regions of wavenumbers and
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analyzed: 450-600 cm-1 for the recognition of the S-S stretching vibration in cystine
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or the C-C deformation vibration in aliphatic residue, 610-2000 cm-1 for the
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estimation of the secondary structure changes of the proteins and the side chains
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vibration in aromatic amino acid, and 2500-3000 cm-1 for the evaluation of the C-H stretching vibration in aliphatic residue.
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In fig. 1, the peaks between 2500-3050 cm-1 in the Raman spectra were
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corresponded to the C-H stretching vibration of a variety of aliphatic residues. As
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shown in the figure, there was a characteristic peak at 2925 cm-1, in the C-H 8
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stretching vibration region, and the peak height and peak area decreased significantly
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(P<0.05) as the temperature increased. Bouraoui have reported the same results. As
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there were very few literatures about C-H stretching vibration in amino acids, these
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phenomena could not be accurately explained (Bouraoui, Nakai and Li-Chan, 1997).
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Rand and Larsson have reported that if the polarity of the surrounding environment
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increased, the intensity of the peak at 2930 cm-1 would increase (Larsson and Rand,
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1973). Therefore, the reduction of the peak intensity in this position of C-H stretching
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vibration was presumed due to the variation of the aliphatic C-H group environment.
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In the Raman spectra, -CO-NH- (peptide bond) had several characteristic vibration
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modes. The amide II bands at 1250 cm-1 and the amide I bands in the vicinity of 1650
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cm-1 were easily recognized. The precise location of the peaks in the spectrum was
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relevant to the secondary structure of the polypeptide chain, which were commonly
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used to estimate the secondary structure of the protein. Amide I bands might overlap
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with the solvent water, and the stretching vibration of aromatic ring and the C-H
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bending vibration in the region of amide III bands might mix together, therefore it
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was suggested to analyze the two areas at the same time in order to obtain more
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accurate information on the secondary structure of the protein (Bouraoui, Nakai and
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Li-Chan, 1997).
Amide I bands (1600-1700 cm-1) were mainly C=O stretching vibration, in addition
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to N-H vibration, C-C-N vibration and C-N stretching vibration (Krimms and
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Bandekar, 1986). The secondary structure of the protein could be characterized by the
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characteristic frequency of the bands in this region. In general, if the α-helix content 9
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of the protein was high, the Amide I concentrated between 1645-1657 cm-1, if the
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β-fold content of the protein was high, the intensity band would appear between
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1665-1680 cm-1, if the random coil content of the protein was high, the Amide I
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concentrated at 1660 cm-1 or so. As shown in fig. 1, the surimi gels treated with
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different high temperatures, showed the characteristic peaks at 1660 cm-1 which were
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corresponded to the random coil structure in Amide I region, and with the treating
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temperature increasing, the peak intensity declined, indicating that high-temperature
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treatment made the random coil structure of the protein subject to some degree of
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damage.
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Conformation sensitive bands appeared in the vicinity of amide III region
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(1230-1320 cm-1) reflected the secondary structure of the myosin, which came from
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the C-N stretching vibration and the N-H in-plane vibration of the peptide bond.
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Bands in this region were the characteristic bands of the α-helix moieties in myosin. If
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the α-helix content in the protein was high, a weak absorption would appear between
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1260-1320 cm-1, if the β-fold content in the protein was high, a strong absorption
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would usually appear between 1235-1245 cm-1, and if the random coil content in the
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protein was high, an absorption in 1243-1250 cm-1 would appear (Tu, 1986). Fig. 1
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illustrated that the surimi gels showed the characteristic peaks at 1249 cm-1 which were corresponded to the random coil structure, and with the treating temperature increasing, the peak intensity kept almost unchanged.
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Amide I bands and amide III bands illustrated that the increasing treating
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temperature had some impact on the random coil structure. It was probably that the 10
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secondary structure of the pollock surimi gels with high temperature (≥100 °C)
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treatment was mainly random coil, and when the treating temperature reached 120 °C,
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the structure would be affected.
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There existed two peaks at 830 cm-1 and 850 cm-1, which were mainly caused by
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the vibration of the p-substituted benzene ring of tyrosine residue. The peaks related
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to the relationship between the hydrogen bonds of the phenolic hydroxyl group and
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the environment, which could be well used as a probe for the phenolic and hydroxyl
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group and the microenvironment. If the tyrosine was a strong hydrogen bond donor or
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embedded in a hydrophobic environment, the ratio (I850/830) would be between 0.7-1.0
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(Tornberg et al., 1993). When tyrosine was both an acceptor and a moderate to weak
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hydrogen bond donor or exposed in an aqueous or polar environment, the ratio would
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be between 0.90-1.45. Fig. 1 showed that the characteristic bimodal peaks at 830 cm-1
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and 857 cm-1 which were corresponded to the tyrosine residues appeared in the surimi
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gels and in the gelling process of the surimi, I850/830 kept in 1.13-2.56. So when the
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gels were treating with 100-120 °C，the tyrosine residues mainly involved in moderate
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or weak hydrogen bonds or were exposed.
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There were several bands in the Raman spectra characterizing the aromatic amino
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acid side chains, some of which were related to the polarity of the microenvironment or the degree of participation of hydrogen bonds. Stretching vibration of the
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tryptophan ring in the gelling process of the surimi was mainly in the vicinity of 760
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cm-1. If the hydrophobic micro-environment that buried tryptophan residues changed
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into polar environment, the intensity of the peak at 760 cm-1 would drop. As shown in 11
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fig. 1, with the treating temperature increasing, the intensity of this peak decreased
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slightly. The native structure of the protein was highly folded, and the unfolding of the
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protein would lead to the exposure of the adjacent active surface and further
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interaction to form intermolecular bonds. The gradually increased intermolecular
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bonds would form hydrophobic interaction, disulfide bonds or non-covalent disulfide
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bond, thereby forming a three dimensional network (Brown et al., 2000). Heat or
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chemical treatment might change the natural structure of the protein.
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The exchange and formation of disulfide bonds played important roles in the
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process of most globulin becoming heat-induced irreversible gel. The characteristic
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frequency of the disulfide bond in the raman spectra was 500-550 cm-1. Stretching
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vibration of the disulfide bonds made the peptide or protein which contained cysteine
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residues present a peak at 510 cm-1, for which the conformation was whole twisted
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and with minimum energy. The peaks at 540 cm-1 and 525 cm-1 were identified as the
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trans-twist-trans conformation or little C-C-S-S dihedral torsion type of the disulfide
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bonds and the twist-twist-trans conformation of the disulfide bonds (Li-Chan, Nakai
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and Hirotsuka, 1994).
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The surimi gels showed a weak peak at 528 cm-1 in this region of the Raman
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spectrum (Fig. 1), which was identified as a twist-twist-trans conformation of the disulfide bonds in peptide. It was probably that the reason why the strength raised
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with the treating temperature increasing was due to such kind of conformation
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converted by other disulfide bonds after high-temperature treatment. Bouraoui, Nakai
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and Li-Chan (1997) have reported that the peak intensity at 530 cm-1 nearby would 12
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increase after high-temperature treatment, and inferred the formation of twist-
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twist-trans conformation disulfide bond. 3.2 FT-IR spectroscopy
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IR spectroscopy is very useful in the research of the secondary structure and kinetic
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properties of the protein, which has decades of history. IR spectroscopy reflects
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changes in the protein amide bands (mainly the amide I and amide II bands). The
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vibration frequency of the amide I, II and III bands are very sensitive to the secondary
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structure of the protein and the information is more subtle than UV and fluorescence
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spectroscopy. IR bands can be divided into three categories: 1. the characteristic
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absorption peaks of hydrogen bonding carboxyl or amine groups exist in all the of
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protein spectra; 2. the amino acids constituting proteins will cause partial absorption;
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3. the amide bands at 1700-1500 cm-1. The peaks between 1700-1600 cm-1 are mainly
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C=O stretching vibration, corresponding to the amide I bands, and the peaks between
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1600-1500 cm-1 are mainly C-N stretching vibration and N-H bending vibration,
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corresponding to the amide II bands. The amide I and amide II bands can be used for
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the ownership of the protein secondary structure and to explore the internal hydrogen
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bonding condition. There were lots of studies on amide I and amide II bands.
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The absorption of the characteristic peak of amide I bands is the strongest and is
sensitive to regional changes in protein secondary structure, often used to analyze the
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secondary structure of proteins. The characteristic absorption peak was caused by the
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C-N stretching vibration of the protein polypeptide backbone. The amide I bands
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consist of
three carbonyl stretching bands at 1640-1630 cm-1,1660-1680 cm-1 and 13
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1700-1680 cm-1 respectively. At present the peaks of amide I have been vested
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maturely, The relationship between the protein secondary structure and the peaks is as
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follows: 1650-1660 cm-1 for the α-helix; 1600-1640 cm-1 for β-fold, 1660-1695 cm-1
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for β-corner, and 1640-1650 cm-1 for the random coil. As shown in Fig. 2, the
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characteristic peak appeared in the vicinity of 1645 cm-1, corresponding to the amide I
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bands. The peak was judged as random coil, indicating that the protein secondary
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structure of the surimi gels treated with high temperature was mainly random coil.
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Moreover, the figure also showed that with the treating temperature increasing, both
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the area and intensity of the peak declined, indicating that high temperature damaged
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the random coil structure.
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In addition, the IR spectroscopy of the surimi gels showed characteristic peaks at 2930 cm-1, corresponding to the C-H stretching vibration and displaying the
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characteristics of the saturated aliphatic. It has also been shown in the Raman
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spectrum.
In previous study (Zhang et al., 2013), according to the sodium dodecyl
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sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron
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microscopy (SEM) results, we hypothesized that high temperature treatment led to the
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formation of a protein polymer interconnected by covalent bonds and high-temperature treatment damaged the gel network structure in some degree, the
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network frames becoming more fragile and the holes in the network becoming bigger.
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Previously study with transmission electron microscope also found that under high
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temperature conditions, myofibrillar protein molecules clumped together chaotically, 14
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reducing the crosslinking degree of proteins, and thereby destroying the network
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structure. Because of high temperature, the gel network structure became uneven and
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rough with big holes, affecting the gel strength of surimi products.
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Consolidated the above results, we hypothesized that increasing high-temperature
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treatment damaged the secondary structure, leading to the protein aggregation,
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thereby reducing the hydration between proteins and water, and further affecting the
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gel properties of the surimi gels.
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4. Conclusion
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The secondary structure of the pollock surimi gels with high temperature (≥100 °C)
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treatment was mainly random coil. With treating temperature increasing, the random
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coil structure damaged, leading to the protein aggregation, which made the frames of
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the network structure become much more fragile and the holes become larger, and
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thereby the hydration between proteins and water reduced, resulting in the destruction
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of the textural properties of the surimi gels.
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Acknowledgments
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This study was supported by Shandong Provincial Natural Science Foundation,
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China (Project No. ZR2010HQ044) and the science and technology planning project
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of WeiFang Medical University (Complex properties of food gums and their improve mechanisms on the surimi with low gel strength).
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polypeptides and proteins. Adv. Protein Chem. 38, 181-364.
Tornberg, E., Andersson, A., Göransson, A., von Seth, G., 1993. Water and fat
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distribution in pork in relation to sensory properties. In: Pork Quality, Genetic and
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Metabolic Factors, Oxon, pp. 239-258.
344 345
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Tu, A.T., 1986. Proteins. In: Raman spectroscopy in biology: Principles and applications, New York, pp. 65-116.
Zhang, L.L., Xue, Y., Xu, J., Li, Z.J., Xue, C.H., 2013. Effects of high-temperature
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treatment (≥100 °C) on Alaska Pollock (Theragra chalcogramma) surimi gels. J.
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Food. Eng. 115 (1), 115-120.
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Zhang, L.L., Zhang, F.X., Wang X., 2015. Effects of hydrolyzed wheat gluten on the properties of high-temperature (≥100 °C) treated surimi gels. Food Hydrocolloids 45, 196-202.
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Wavenumber (cm )
Fig. 1 Raman spectropy of the surimi gels with different high-temperature treatment
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Fig.2 FT-IR spectropy of the surimi gels with different high-temperature treatment A: 100 °C; B: 120 °C
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·The changes of the protein secondary structures of the surimi gels were studied. ·The secondary structure of the surimi gels treated with 100-120 °C was mainly random coil. ·The random coil structure damaged as treating temperature increased.
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·High-temperature treatment resulted in serious damage to the network structure of the surimi gels.
S0260-8774(15)30026-1
DOI:
10.1016/j.jfoodeng.2015.10.025
Reference:
JFOE 8368
To appear in:
Journal of Food Engineering
Received Date: 17 November 2014 Revised Date:
24 July 2015
Accepted Date: 21 October 2015
Please cite this article as: Zhang, L., Zhang, F., Wang, X., Changes of protein secondary structures of pollock surimi gels under high-temperature (100 °C and 120 °C) treatment, Journal of Food Engineering (2015), doi: 10.1016/j.jfoodeng.2015.10.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The secondary structure of the pollock surimi gels with high-temperature (≥100 °C) treatment was mainly random coil. With treating temperature increasing, the random coil structure damaged, resulting in the disappearance of myosin heavy chain and significant decrease of actin content. In addition, the protein molecules aggregated. So the frames of the network structure became much more fragile and the holes became larger, leading to the destruction of the textural properties of the surimi gels.
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Changes of protein secondary structures of Pollock surimi gels under
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high-temperature (100 °C and 120 °C) treatment
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Lili Zhang a, Fengxiang Zhang a*, Xia Wang a
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a
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China
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*Corresponding author. Tel.: +86 0536 8462429.
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E-mail address: [email protected].
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Department of Public Health, WeiFang Medical University, Weifang, 261053, PR
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Abstract To produce instant surimi products, sterilization is essential. Previous studies found
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that the gel strength of surimi decreased significantly with treating temperature
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increasing from 100 °C to 120 °C, which affected the texture of the products. In this
12
study, the changes of the secondary structures of surimi gels were studied to provide
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new train of thought for the improvement of the texture of high temperature treated
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surimi gels. Surimi gels from Alaska Pollock were obtained by heating and maintaining
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their central temperature at 100 °C and 120 °C for 10min under a constant pressure
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(0.12 MPa). Infrared (IR) spectroscopy and Raman spectroscopy showed that the
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secondary structure of the surimi gels with high temperature (≥100 °C) treatment was
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mainly random coil and the random coil structure damaged with treating temperature
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increasing, which made the frames of the network structure become much more
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fragile and the holes become larger, leading to the destruction of the textural
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properties of the surimi gels.
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Keywords: high-temperature treatment, surimi gel, Infrared spectroscopy, Raman
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spectroscopy
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1. Introduction At present, the form of surimi products in China is single, which greatly limits the edible convenience, so the development of new surimi products is very imminent.
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To produce instant surimi products, longtime shelf life is a very important goal，which
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can be realized by sterilization. Products after sterilizing especially at 121 °C can be
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stored at normal temperature and have longer shelf life. Up to now, there were very
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few reports on the effects of high temperatures (≥100 °C) on the properties of surimi
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gels. Previously it was found that high-temperature treatment (≥100 °C) significantly
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decreased the gel strength of the surimi gels, thereby affecting the texture of the
33
products (Zhang et al., 2013), and adding hydrolyzed wheat gluten could significantly
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improve the textural properties of the surimi gels, and the thermal stability under high
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temperature (≥100 °C) of the gels was greatly improved (Zhang et al., 2015). The
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structure and function of the proteins are closely related, so it is very necessary to
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understand the changing mechanism of the protein spatial structures of the surimi gels
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(Nicolin et al., 1992) so as to explore other new ways to improve the texture
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characteristic of the surimi gels under high temperature treatment, providing technical
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support and theoretical basis for high-temperature treatment of instant surimi products.
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In previous study, we discussed the effect of high temperature treatment on the texture, chemical bond changes, water-binding capacity and network structure of the surimi
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gels, which primarily explained how the tertiary structure and quaternary structure
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changed with the treating temperature increasing from 100 °C to 120 °C. The primary
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structure of proteins determined the senior structure. To get stable protein senior 3
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structures, it is very necessary to clarify how the protein secondary structures change
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with high temperature treatment, which will help exploit new train of thought and
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feasible ways from different aspects to improve the texture characteristic of the surimi
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gels under high temperature treatment.
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Protein secondary structure refers to the atom arrangement (i.e. conformation) in
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the main chain of the polypeptide, which is independent of the side chain
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conformation. Secondary structural elements are in forms of α- helix, β-fold, β-corner
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and random coil, etc., which constitute the essential elements of the high protein
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structure.
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Infrared (IR) spectroscopy and Raman spectroscopy are commonly used in the
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study of protein secondary structure. Fourier transform infrared (FT-IR) spectroscopy
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has attracted considerable interest with simple operation, accurate wavelength, good
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resolution, high speed scanning and high sensitivity, which applies the earliest and
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most common way to study protein conformation (Argyri et al., 2013). Currently the
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identification of the peaks of amide I bands (1700-1600 cm-1) in IR spectrum is
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widely used, often used to analyze the secondary structure of the proteins. Raman
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spectroscopy is a branch of the vibrational spectroscopy method that is
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complementary to infrared absorbance and can be used in food analysis, since it is non-destructive, requires little pre-treatment of samples and provides information
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about different food compounds at the same time, offering quantitative analysis of
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food components with simultaneous information on molecular structure. In
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comparison to FT-IR, smaller portions of sample are required and instrumentation can 4
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be less expensive and portable. Moreover, Raman spectroscopy has the advantage
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over FT-IR that the contribution from water is very small (since H2O is a weak Raman
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scatterer) and can be used directly on foods, so it owns incomparable advantages
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when the sample is scarce or with water and is also a very useful method in the
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studies of the protein conformation and the changes of biological macromolecules.
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Recently, FT-IR and Raman spectroscopy have been used to characterize the
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heat-induced denaturation of myofibrillar and connective tissue proteins of beef
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(Kirschner et al., 2004), salt- and heat-induced changes in myofibrillar protein of pork
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(Bertram et al., 2006, Böcker et al., 2006) and the structural changes in fish proteins
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(Herrero, Carmona and Careche, 2004).
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In this study, the changes of the secondary structures of the surimi gels treated with different high temperatures (100 °C and 120 °C) were studied, so as to interpret how
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high temperature treatment affected the surimi gels from a more microscopic
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perspective, further deepening the previous study and enriching the theory of high
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temperature treated surimi products, which would provide new train of thought for the
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improvement of the texture of high temperature treated surimi gels, laying foundation
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for the production of instant surimi products.
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2. Materials and methods 2.1 Materials
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Frozen Alaska Pollock (Theragra chalcogramma) surimi (grade AAA), was
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purchased from JINCAN Foods Co., Ltd., Qingdao, Shandong, China. Surimi was
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kept at -20 °C until used. 5
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All of the chemicals used were of analytical grade and were purchased from
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Sigma-Aldrich (St. Louis, Mo, USA) or Sinopharm Chemical Reagent Co., Ltd.
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(Shanghai, China).
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2.2 Preparation of the surimi gels
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To prepare surimi gels, 250 g frozen Alaska Pollock surimi was semi-thawed at
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4 °C for 4h and then cut into small pieces (about 3 cm cubes). Then the surimi was
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chopped in a Stephan vertical vacuum cutter (Model UM 5, Stephan Machinery Co.,
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Hameln, Germany). In order to keep the temperature of the sample below 4°C during
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chopping, a chilling medium (ethanol: water, 95:5) was continuously circulated in the
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double-walled chopping bowl. Firstly, the surimi was chopped for 5 min with a speed
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of 1500 rpm, then 3 % (w/w) sodium chloride was added and the mixture was
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chopped for another 5 min with a speed of 2100 rpm. In order to remove air pockets
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the chopping condition was under vacuum of 0.5 bar. The surimi sol was packed into
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plastic casings (3 cm i.d.), with both ends sealed tightly.
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2.3 High-temperature treatment
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A Steam/electric amphibious sterilization pot (JINDING Food Machinery Co., Ltd.,
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Zhucheng, Shandong, China) was used for the high-temperature treatment of the
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surimi gels. In this study, steam was used to supply the heating medium of the pot. The sterilization pot can be preset a desired temperature and the accuracy of the
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temperature in the sterilization process can be ensured by automatic temperature
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control (automatic heating when below the desired temperature and automatic stop
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when
reach
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counter-pressure sterilization was used to prevent casings from bulging. Compressed
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air was passed into the pot to keep the ambient pressure and the internal pressure of
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the sample the same. After sterilization the supply of steam was stopped and the
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cooling water was supplied into the sprinkler pipe, cooling the sample. The
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compressed air can be used to compensate the pressure reduction in the pot dropped
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as caused by the decreased temperature and condensed steam. The conditions of the
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high-temperature treatment were as follows: keeping the pressure at 0.12 MPa and the
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center temperature of the samples reach 100±1 °C and 120±1 °C and maintain 10min
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respectively. the center temperature of the samples was maintain High temperature
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processing center of the sample temperature was monitored by an Ellab
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TrackSense-Pro purchased from Henglv Engineering Co., Ltd., Hubei, China. After
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high-temperature treatment the samples were cooled in ice water and then kept
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refrigerated (4 °C) until tested.
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2.4 Raman spectroscopy
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Raman spectroscopy was carried out in an inVia laser Raman spectroscopy
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(Renishaw Trading Co., Ltd., Shanghai, China). About 1 g surimi gels were placed on
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a glass sheet with a flat smooth surface. 633 argon ion laser and 1800 lines grating
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were used. The measurement conditions were as follows: the scan range was 400-4000 cm-1, the laser energy was 10 mW, the resolution was 1 cm-1, the acquisition time was 10 s and the number of the spectra collected was 40 times.
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2.5 FT-IR spectroscopy
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FT-IR spectroscopy was carried out in a 470 Fourier transform infrared 7
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spectrometer (Thermo Electron Instruments Co., Ltd., Shanghai, China).The surimi
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gels were freeze-dried and mixed with pure KBr. The mixture was grinded uniformly,
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placed in a mold and then pressed into a transparent sheet with (5-10)×107 Pa pressure
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on the hydraulic machine. The measurement conditions were as follows: the range of
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wave number was 4000-400 cm-1, the resolution was 4 cm-1 and the number of the
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spectra collected was 128 times.
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2.6 Statistical analysis
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Statistical analysis was performed by analysis of variance (ANOVA) and Duncan’s
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multiple range tests. The results were expressed as mean ± SD, and the differences
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were considered to be statistically significant at P< 0.05.
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3. Results and discussion
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3.1 Raman spectroscopy
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Fig. 1 showed the Raman spectra of the surimi gels with different high-temperature
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treatment. The Raman spectra was divided into three regions of wavenumbers and
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analyzed: 450-600 cm-1 for the recognition of the S-S stretching vibration in cystine
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or the C-C deformation vibration in aliphatic residue, 610-2000 cm-1 for the
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estimation of the secondary structure changes of the proteins and the side chains
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vibration in aromatic amino acid, and 2500-3000 cm-1 for the evaluation of the C-H stretching vibration in aliphatic residue.
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In fig. 1, the peaks between 2500-3050 cm-1 in the Raman spectra were
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corresponded to the C-H stretching vibration of a variety of aliphatic residues. As
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shown in the figure, there was a characteristic peak at 2925 cm-1, in the C-H 8
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stretching vibration region, and the peak height and peak area decreased significantly
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(P<0.05) as the temperature increased. Bouraoui have reported the same results. As
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there were very few literatures about C-H stretching vibration in amino acids, these
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phenomena could not be accurately explained (Bouraoui, Nakai and Li-Chan, 1997).
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Rand and Larsson have reported that if the polarity of the surrounding environment
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increased, the intensity of the peak at 2930 cm-1 would increase (Larsson and Rand,
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1973). Therefore, the reduction of the peak intensity in this position of C-H stretching
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vibration was presumed due to the variation of the aliphatic C-H group environment.
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In the Raman spectra, -CO-NH- (peptide bond) had several characteristic vibration
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modes. The amide II bands at 1250 cm-1 and the amide I bands in the vicinity of 1650
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cm-1 were easily recognized. The precise location of the peaks in the spectrum was
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relevant to the secondary structure of the polypeptide chain, which were commonly
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used to estimate the secondary structure of the protein. Amide I bands might overlap
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with the solvent water, and the stretching vibration of aromatic ring and the C-H
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bending vibration in the region of amide III bands might mix together, therefore it
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was suggested to analyze the two areas at the same time in order to obtain more
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accurate information on the secondary structure of the protein (Bouraoui, Nakai and
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Li-Chan, 1997).
Amide I bands (1600-1700 cm-1) were mainly C=O stretching vibration, in addition
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to N-H vibration, C-C-N vibration and C-N stretching vibration (Krimms and
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Bandekar, 1986). The secondary structure of the protein could be characterized by the
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characteristic frequency of the bands in this region. In general, if the α-helix content 9
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of the protein was high, the Amide I concentrated between 1645-1657 cm-1, if the
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β-fold content of the protein was high, the intensity band would appear between
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1665-1680 cm-1, if the random coil content of the protein was high, the Amide I
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concentrated at 1660 cm-1 or so. As shown in fig. 1, the surimi gels treated with
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different high temperatures, showed the characteristic peaks at 1660 cm-1 which were
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corresponded to the random coil structure in Amide I region, and with the treating
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temperature increasing, the peak intensity declined, indicating that high-temperature
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treatment made the random coil structure of the protein subject to some degree of
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damage.
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Conformation sensitive bands appeared in the vicinity of amide III region
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(1230-1320 cm-1) reflected the secondary structure of the myosin, which came from
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the C-N stretching vibration and the N-H in-plane vibration of the peptide bond.
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Bands in this region were the characteristic bands of the α-helix moieties in myosin. If
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the α-helix content in the protein was high, a weak absorption would appear between
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1260-1320 cm-1, if the β-fold content in the protein was high, a strong absorption
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would usually appear between 1235-1245 cm-1, and if the random coil content in the
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protein was high, an absorption in 1243-1250 cm-1 would appear (Tu, 1986). Fig. 1
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illustrated that the surimi gels showed the characteristic peaks at 1249 cm-1 which were corresponded to the random coil structure, and with the treating temperature increasing, the peak intensity kept almost unchanged.
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Amide I bands and amide III bands illustrated that the increasing treating
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temperature had some impact on the random coil structure. It was probably that the 10
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secondary structure of the pollock surimi gels with high temperature (≥100 °C)
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treatment was mainly random coil, and when the treating temperature reached 120 °C,
202
the structure would be affected.
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There existed two peaks at 830 cm-1 and 850 cm-1, which were mainly caused by
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the vibration of the p-substituted benzene ring of tyrosine residue. The peaks related
205
to the relationship between the hydrogen bonds of the phenolic hydroxyl group and
206
the environment, which could be well used as a probe for the phenolic and hydroxyl
207
group and the microenvironment. If the tyrosine was a strong hydrogen bond donor or
208
embedded in a hydrophobic environment, the ratio (I850/830) would be between 0.7-1.0
209
(Tornberg et al., 1993). When tyrosine was both an acceptor and a moderate to weak
210
hydrogen bond donor or exposed in an aqueous or polar environment, the ratio would
211
be between 0.90-1.45. Fig. 1 showed that the characteristic bimodal peaks at 830 cm-1
212
and 857 cm-1 which were corresponded to the tyrosine residues appeared in the surimi
213
gels and in the gelling process of the surimi, I850/830 kept in 1.13-2.56. So when the
214
gels were treating with 100-120 °C，the tyrosine residues mainly involved in moderate
215
or weak hydrogen bonds or were exposed.
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There were several bands in the Raman spectra characterizing the aromatic amino
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acid side chains, some of which were related to the polarity of the microenvironment or the degree of participation of hydrogen bonds. Stretching vibration of the
219
tryptophan ring in the gelling process of the surimi was mainly in the vicinity of 760
220
cm-1. If the hydrophobic micro-environment that buried tryptophan residues changed
221
into polar environment, the intensity of the peak at 760 cm-1 would drop. As shown in 11
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fig. 1, with the treating temperature increasing, the intensity of this peak decreased
223
slightly. The native structure of the protein was highly folded, and the unfolding of the
224
protein would lead to the exposure of the adjacent active surface and further
225
interaction to form intermolecular bonds. The gradually increased intermolecular
226
bonds would form hydrophobic interaction, disulfide bonds or non-covalent disulfide
227
bond, thereby forming a three dimensional network (Brown et al., 2000). Heat or
228
chemical treatment might change the natural structure of the protein.
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The exchange and formation of disulfide bonds played important roles in the
230
process of most globulin becoming heat-induced irreversible gel. The characteristic
231
frequency of the disulfide bond in the raman spectra was 500-550 cm-1. Stretching
232
vibration of the disulfide bonds made the peptide or protein which contained cysteine
233
residues present a peak at 510 cm-1, for which the conformation was whole twisted
234
and with minimum energy. The peaks at 540 cm-1 and 525 cm-1 were identified as the
235
trans-twist-trans conformation or little C-C-S-S dihedral torsion type of the disulfide
236
bonds and the twist-twist-trans conformation of the disulfide bonds (Li-Chan, Nakai
237
and Hirotsuka, 1994).
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The surimi gels showed a weak peak at 528 cm-1 in this region of the Raman
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spectrum (Fig. 1), which was identified as a twist-twist-trans conformation of the disulfide bonds in peptide. It was probably that the reason why the strength raised
241
with the treating temperature increasing was due to such kind of conformation
242
converted by other disulfide bonds after high-temperature treatment. Bouraoui, Nakai
243
and Li-Chan (1997) have reported that the peak intensity at 530 cm-1 nearby would 12
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increase after high-temperature treatment, and inferred the formation of twist-
245
twist-trans conformation disulfide bond. 3.2 FT-IR spectroscopy
247
IR spectroscopy is very useful in the research of the secondary structure and kinetic
248
properties of the protein, which has decades of history. IR spectroscopy reflects
249
changes in the protein amide bands (mainly the amide I and amide II bands). The
250
vibration frequency of the amide I, II and III bands are very sensitive to the secondary
251
structure of the protein and the information is more subtle than UV and fluorescence
252
spectroscopy. IR bands can be divided into three categories: 1. the characteristic
253
absorption peaks of hydrogen bonding carboxyl or amine groups exist in all the of
254
protein spectra; 2. the amino acids constituting proteins will cause partial absorption;
255
3. the amide bands at 1700-1500 cm-1. The peaks between 1700-1600 cm-1 are mainly
256
C=O stretching vibration, corresponding to the amide I bands, and the peaks between
257
1600-1500 cm-1 are mainly C-N stretching vibration and N-H bending vibration,
258
corresponding to the amide II bands. The amide I and amide II bands can be used for
259
the ownership of the protein secondary structure and to explore the internal hydrogen
260
bonding condition. There were lots of studies on amide I and amide II bands.
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The absorption of the characteristic peak of amide I bands is the strongest and is
sensitive to regional changes in protein secondary structure, often used to analyze the
263
secondary structure of proteins. The characteristic absorption peak was caused by the
264
C-N stretching vibration of the protein polypeptide backbone. The amide I bands
265
consist of
three carbonyl stretching bands at 1640-1630 cm-1,1660-1680 cm-1 and 13
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1700-1680 cm-1 respectively. At present the peaks of amide I have been vested
267
maturely, The relationship between the protein secondary structure and the peaks is as
268
follows: 1650-1660 cm-1 for the α-helix; 1600-1640 cm-1 for β-fold, 1660-1695 cm-1
269
for β-corner, and 1640-1650 cm-1 for the random coil. As shown in Fig. 2, the
270
characteristic peak appeared in the vicinity of 1645 cm-1, corresponding to the amide I
271
bands. The peak was judged as random coil, indicating that the protein secondary
272
structure of the surimi gels treated with high temperature was mainly random coil.
273
Moreover, the figure also showed that with the treating temperature increasing, both
274
the area and intensity of the peak declined, indicating that high temperature damaged
275
the random coil structure.
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In addition, the IR spectroscopy of the surimi gels showed characteristic peaks at 2930 cm-1, corresponding to the C-H stretching vibration and displaying the
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characteristics of the saturated aliphatic. It has also been shown in the Raman
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spectrum.
In previous study (Zhang et al., 2013), according to the sodium dodecyl
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sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron
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microscopy (SEM) results, we hypothesized that high temperature treatment led to the
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formation of a protein polymer interconnected by covalent bonds and high-temperature treatment damaged the gel network structure in some degree, the
285
network frames becoming more fragile and the holes in the network becoming bigger.
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Previously study with transmission electron microscope also found that under high
287
temperature conditions, myofibrillar protein molecules clumped together chaotically, 14
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reducing the crosslinking degree of proteins, and thereby destroying the network
289
structure. Because of high temperature, the gel network structure became uneven and
290
rough with big holes, affecting the gel strength of surimi products.
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Consolidated the above results, we hypothesized that increasing high-temperature
292
treatment damaged the secondary structure, leading to the protein aggregation,
293
thereby reducing the hydration between proteins and water, and further affecting the
294
gel properties of the surimi gels.
295
4. Conclusion
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The secondary structure of the pollock surimi gels with high temperature (≥100 °C)
297
treatment was mainly random coil. With treating temperature increasing, the random
298
coil structure damaged, leading to the protein aggregation, which made the frames of
299
the network structure become much more fragile and the holes become larger, and
300
thereby the hydration between proteins and water reduced, resulting in the destruction
301
of the textural properties of the surimi gels.
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Acknowledgments
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This study was supported by Shandong Provincial Natural Science Foundation,
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China (Project No. ZR2010HQ044) and the science and technology planning project
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of WeiFang Medical University (Complex properties of food gums and their improve mechanisms on the surimi with low gel strength).
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References
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Argyri, A.A., Jarvis, R.M., Wedge, D., Xu, Y., Panagou, E.Z., Goodacre, R., Nychas,
309
G.E., 2013. A comparison of Raman and FT-IR spectroscopy for the prediction of 15
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meat spoilage. Food Control 29, 461-470. Bertram, H.C., Kohler, A., Böcker, U., Ofstad, R., Andersen, H.J., 2006. Heat-induced
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changes in myofibrillar protein structures and myowater of two pork qualities. A
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combined FT-IR spectroscopy and low-field NMR relaxometry study. J. Agric.
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Food Chem. 54, 1740-1747.
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Böcker, U., Ofstad, R., Bertram, H.C., Egelandsdal, B., Kohler, A., 2006. Salt-induced
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changes in pork myofibrillar tissue investigated by FT-IR microspectroscopy and
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light microscopy. J. Agric. Food Chem. 54, 6733-6740.
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Bouraoui, M., Nakai, S., Li-Chan, E., 1997. In situ investigation of protein structure
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Brown, R.J.S., Capozzi, F., Cavani, C., Cremonini, M.A., Petracci, M., Placucci, G.,
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2000. Relationships between 1H NMR relaxation data and some technological
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parameters of meat: A chemometric approach. J. Magn. Reson. 147 (1), 89-94.
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Wavenumber (cm )
Fig. 1 Raman spectropy of the surimi gels with different high-temperature treatment
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Fig.2 FT-IR spectropy of the surimi gels with different high-temperature treatment A: 100 °C; B: 120 °C
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·The changes of the protein secondary structures of the surimi gels were studied. ·The secondary structure of the surimi gels treated with 100-120 °C was mainly random coil. ·The random coil structure damaged as treating temperature increased.
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·High-temperature treatment resulted in serious damage to the network structure of the surimi gels.