Food Chemistry 301 (2019) 125206
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The beneficial effects of rutin on myofibrillar protein gel properties and related changes in protein conformation Na Jiaa, Fengxue Zhanga,b, Qian Liub, Letian Wanga, Shiwen Lina, Dengyong Liua, a b
T
⁎
College of Food Science and Technology, Bohai University, National & Local Joint Engineering Research Center of Storage, Jinzhou, Liaoning 121013, China College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang 150030, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Rutin Myofibrillar protein Interactions Conformation Gel properties
Effects of different levels of rutin (0, 10, 50, 100 and 200 μmol/g protein) on the conformational changes and gel properties of myofibrillar protein (MP) were investigated. Rutin at 200 μmol/g caused the greatest carbonyl content. The incorporation of rutin caused the losses of thiol, free amine and α-helix contents, reduction in tryptophan intrinsic fluorescence intensity, and enhanced exposure of hydrophobic groups and protein crosslinking. When compared with control, the MP gels with 10, 50 and 100 μmol/g rutin had higher gel strength but slight lower water-holding capacity; the gels appeared to have compact microstructure with few visible pores. However, 200 μmol/g rutin was detrimental to gel properties. All the gels with rutin presented higher final storage modulus and converted to elasticity-dominant gel types. The results indicate that a slightly high concentration of rutin could improve MP gel properties which are related to the protein conformational changes induced by rutin.
1. Introduction It is well known that many plant materials are rich in phenolics that possess various beneficial bioactivities, such as anti-inflammatory, chemopreventive, antibacterial and antioxidant activities. Especially, the antioxidant activities have drawn much attention in food research. Lipid and protein oxidation are prone to occur in meat and meat products during processing, transportation, sale and storage, resulting in quality deterioration through discoloration, off-flavors, texture deterioration and loss of nutrients (Min & Ahn, 2005). Therefore, much research has been focused on the use of phenolics, derived from fruits, vegetables, herbs and spices, as natural antioxidants in meat and meat products to retard lipid and protein oxidation (Shah, Bosco, & Mir, 2014). Moreover, plant materials rich in phenolics are commonly considered to be healthier and more beneficial; thus, the application of plants or their extracts in meat processing, aiming to prevent oxidation, can improve the nutrition and health characteristics of meat products at the same time (Shah et al., 2014). While most research has concentrated on the antioxidant effects of the phenolics in meat and meat products, the covalent and non-covalent interactions between phenolics and meat protein were less studied until recent years. In fact, these interactions can modify structure, conformational and physico-chemical properties of proteins, leading to protein functional changes. For example, white grape extracts, green ⁎
tea extracts, and rosemary extracts have been proven to increase thiol loss in meat protein by forming covalent bonds between the oxidized phenolics (quinone) and thiol groups, resulting in less disulfide crosslinking, which is one of the principal forces for gelation of meat protein during heating (Jongberg, Skov, Torngren, Skibsted, & Lund, 2011; Jongberg, Torngren, Gunvig, Skibsted, & Lund, 2013; Jongberg, Terkelsen Lde, Miklos, & Lund, 2015). Similarly, high concentrations of rosmarinic acid, possibly by formation. of thiol-quinone and amine-quinone adducts, caused aggregation and decreased solubility of myofibrillar protein (MP), undermining the gelation capacity of MP, while low concentrations of rosmarinic acid improved the rheological and gelation properties of MP (Tang et al., 2017; Wang et al., 2018). Chlorogenic acid, gallic acid, and epigallocatechin-3-gallate (EGCG) have also been found to promote the loss of thiol and amine groups and initiate irreversible protein modifications, as evidenced by changes in secondary structure, tertiary structure, aggregation, and cross-linking; the gelling capacity of MP was enhanced at low concentrations, but high concentrations of phenolics were detrimental to MP gelation (Feng et al., 2017; Cao, True, Chen, & Xiong, 2016; Cao & Xiong, 2015). Accordingly, some methods have been used to retard phenolicsprotein interactions to improve the functional properties of meat protein while sustaining the antioxidant activity of the phenolics. For example, cyclodextrin derivatives were used to prevent both non-covalent
Corresponding author. E-mail addresses:
[email protected] (N. Jia),
[email protected] (D. Liu).
https://doi.org/10.1016/j.foodchem.2019.125206 Received 22 January 2019; Received in revised form 13 July 2019; Accepted 17 July 2019 Available online 18 July 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
Food Chemistry 301 (2019) 125206
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2.3. Measurement of chemical and structural changes of MP
and covalent interactions between EGCG and MP, improve the gel quality, and not markedly reduce the antioxidant ability of EGCG (Zhang et al., 2018). In addition, the overall effects of phenolics on meat protein are greatly influenced by the concentration and chemical structure of the phenolics used. According to the usually studied concentrations of phenolics ranged from 2.5 to 300 μmol/g protein, medium or high concentrations of phenolics were found to impair gel properties (Tang et al., 2017; Wang et al., 2018; Feng et al., 2017; Cao et al., 2016; Cao & Xiong, 2015). In our previous research, 10, 50, 100 and 200 μmol/g catechin were added to MP and 50, 100 and 200 μmol/ g catechin resulted in complete collapse of the gel network, and the MP suspension could not form the MP gel (Jia, Wang, Shao, Liu, & Kong, 2017). Therefore, identifying phenolics that enhance the gel properties at slightly high concentrations is another possible approach to improve the application of phenolics in meat products. Rutin, also known as rutoside or quercetin-3-O-rutinoside, is a flavonol glycoside compound widely found in plants, such as asparagus, cerrado vegetation, and citrus fruits. It was reported that rutin caused the greatest cross-linking network and increased the gel strength of gelatin gels from walleye pollock skin by mainly interacting with skeletal CeNeC and carboxyl groups of gelatin molecules (Yan, Li, Zhao, & Yi, 2011). In addition, rutin improved the film strength of soy protein isolate through covalent and non-covalent cross-linking interactions with proteins (Friesen, Chang, & Nickerson, 2015). Therefore, in the present study, rutin was selected and expected to improve the gel properties of pork MP at slightly high concentrations. The concentrations of rutin were 0, 10, 50, 100 and 200 μmol/g protein which were designed according to the usually studied concentrations of phenolics in MP systems. The MP modification and the relevant changes in gel properties induced by rutin were investigated to clarify the effects of rutin on MP structure and gel properties.
2.3.1. Carbonyl content Carbonyl content was measured using the dinitrophenylhydrazine (DNPH) method as described by Cao and Xiong (2015). The absorbance was read at 370 nm and the carbonyl content was calculated using a molar extinction coefficient of 22,000 M−1 cm−1. 2.3.2. Thiol content The thiol content was determined using 5, 5′-dithio-bis (2-nitrobenzoic acid) (DTNB) (Ellman, 1959). MP samples (1 mL, 20 mg/mL) were suspended in 8 mL tris–glycine buffer (pH 8.0) containing 8 M urea. Then, the mixtures were centrifuged (10,000 g, 15 min, 4 °C). The supernatant (4.5 mL) were incubated with DTNB (0.5 mL, 10 mM) for 30 min in darkness. The absorbance was read at 412 nm. The thiol concentration was calculated using a molar extinction coefficient of 13,600 M−1 cm−1. The protein concentrations were determined using a BSA standard. 2.3.3. Free amine content Free amine content was analyzed by the method of Cao and Xiong (2015). An aliquot of 0.2 mL MP (5 mg/mL) reacted with 2 mL of 1% SDS (dissolved in PBS buffer, 0.2 M, pH 8.2) and 1 mL 2,4,6-trinitrobenzenesulfonic acid (TNBS) at 50 °C for 30 min in the dark. The absorbance was read at 420 nm and the free amines were estimated using an L-leucine standard. 2.3.4. Secondary structure Raman spectra were measured using a Raman spectrometer (LabRAM HR Evolution, Horiba Jobin Yvon, France). The MP samples (40 mg/mL) with different concentration of rutin were placed on the glass slides, respectively. The Raman spectrum was recorded in the range of 400–3600 cm−1 according to the conditions as described by Zhou et al. (2019). The proportions of secondary structures of MP were calculated using Alix’s method (Alix, Pedanou, & Berjot, 1988).
2. Materials and methods 2.1. Materials
2.3.5. Fluorescence spectroscopy Fluorescence spectroscopy was measured by a spectrofluorometer (970CRT, Jingke Co. Ltd., Shanghai, China). Aliquots of protein solution were diluted to 0.1 mg/mL with PBS (50 mM, pH 7.0) and centrifuged at 10,000 g for 30 min at 4 °C. The supernatant was added to a standard 3.5-mL quartz cell to acquire the fluorescence steady state. The emission spectra were recorded from 300 to 400 nm with the excitation wavelength set at 290 nm. The slit width was set at 10 nm, and the sensitivity of the spectrofluorometer was 2.
Fresh longissimus muscle from pork carcasses (24 h post-mortem) was obtained from the local commercial abattoir (Jinzhou, China). The meat was ground and blended homogeneously and stored at −80 °C until use. Rutin (95%) was obtained from Sigma-Aldrich (St. Louis, MO). Other chemicals were of reagent grade and obtained from Solarbio Corp. (Beijing, China).
2.2. MP preparation and treaments with rutin 2.3.6. Surface hydrophobicity Surface hydrophobicity was determined by bromophenol blue (BPB)-binding method as described by Chelh, Gatellier, and SanteLhoutellier (2006). To 1 mL of MP suspension (5 mg/mL, dissolved in 20 mM PBS, pH 7.0), 200 μL of 1 mg/mL BPB were added. The control was without MP. The mixture was reacted at room temperature for 2 h and then centrifuged at 6000 g for 15 min at 4 °C. The supernatant was diluted 1/10 with PBS (20 mM, pH 7.0). The absorbance at 595 nm was read. The amount of BPB bound (μg) was calculated as 200 μg × (Acontrol − Asample)/Acontrol, where A is the absorbance.
2.2.1. MP preparation MP was prepared as the procedure according to Park, Xiong, Alderton, and Ooixumi (2006). The protein pellet was kept at 4 °C and used within 24 h. The protein concentration was measured by the Biuret method (Gornall, Bardawill, & David, 1949).
2.2.2. MP treatments with rutin MP suspensions (40 mg/mL final protein concentration, 30 mL) were prepared by dispersion of the MP pellet into 10 mM phosphatebuffered saline solution (PBS) containing 0.6 M NaCl, pH 7.0. Various concentrations of rutin solutions were prepared by dissolving rutin in 2 mL absolute methanol and heated at 70 °C for 2 min, respectively. Rutin at four final concentrations (10, 50, 100 and 200 μmol/g protein) was added to the MP suspensions. The MP suspension added with 2 mL absolute methanol without rutin was used as a control. The MP suspensions with rutin were stored at 4 °C for 10 h to allow the MP and rutin to interact.
2.4. Measurement of cross-linking of MP 2.4.1. Sulfate–polyacry-lamide gel electrophoresis (SDS-PAGE) Protein cross-linking of MP with different concentrations of rutin was measured using SDS-PAGE method. The MP sample (2 mg/mL) was mixed with an equal volume of SDS–PAGE sample buffer with or without 5% β-mercaptoethanol (β-ME) and boiled for 3 min. Then, 5 μL of each sample was loaded in the wells of the 4% polyacrylamide stacking gel and separated in 12% separation gel. The gels were stained 2
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Fig. 1. Influence of rutin on the contents of carbonyl, total thiol group and free amine of MP. Error bars refer to the standard deviations obtained from triplicate sample analyses. Means with different letters (a–d) differ significantly (P < 0.05).
conducted. The results are expressed as the mean values ± standard errors. Data were analysed using the general linear model procedure of Statistix software 8.1 (Analytical Software, St. Paul, MN, USA). Significance (P < 0.05) differences between means were identified by Tukey's multiple comparison.
using 0.25% Coomassie Brilliant Blue and photographed by calibrated densitometer (GS-800, BIO-RAD, America). 2.4.2. Solubility The MP solution (10 mg/mL, dispersed in PBS buffer, 10 mM, pH7.0) was centrifuged at 10,000 g for 20 min at 4 °C. The protein concentrations of the supernatant were measured by Biuret method. The solubility (%) was expressed as the ratio of protein content in supernatant to total protein content.
3. Results and discussion 3.1. Changes in amino acid side chains
2.5. Measurement of gel properties and gel microstructure
3.1.1. Carbonyl content The carbonyl content is commonly used as a reliable indicator of protein oxidation. In meat proteins, the carbonyl compounds are formed via oxidative deamination of susceptible side chains of amino acids, such as threonine, proline, arginine, and lysine (Estévez, 2011). Therefore, rutin, as a well-known antioxidant, may affect the carbonyl content via redox-active pathways, thereby modifying the protein structure and gelation. As shown in Fig. 1A, the carbonyl content of the control was 1.65 nmol/mg protein. The addition of 10, 50 and 100 μmol/g rutin did not change the carbonyl content and showed values similar to that of the control (P > 0.05), indicating that at these concentrations, rutin could not promote or inhibit the formation of carbonyl compounds. However, 200 μmol/g rutin significantly increased the carbonyl content to 3.46 nmol/mg protein (P < 0.05). Rutin was reported to be a prooxidant at higher doses (Utrera & Estévez, 2012a). Therefore, it can be assumed that probably through its pro-oxidant activity, rutin promoted the formation of primary carbonyl compounds, such as α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS), which may be involved in the formation of final oxidant products. Moreover, it is hypothesized that a moderate extent of protein oxidation, especially in the form of protein carbonylation, may be beneficial for MP gelation (Estévez, 2011). However, it is difficult to ensure what is the moderate range of the carbonyl content, since inconsistent carbonyl content was obtained by DNPH method in different studies. Nevertheless, in the present study, the increased carbonyl content at 200 μmol/g rutin may partly contribute to the reduction of gel strength and WHC as shown in Fig. 4. The reasonable underlying links between the formation of carbonyl compounds and the loss of protein functionality have been welldescribed by Estévez and co-workers (Utrera & Estévez, 2012b; Estévez, 2011; Utrera, Parra, & Estévez, 2014). On one hand, the formation of carbonyl compounds may induce alterations in the distribution of electrical charges and overall charge arrangement of MP, further enhancing protein-protein interactions (Estévez, 2011). On the other hand, the carbonyl-amine condensations or reactions between two AAS to form Schiff bases or aldol condensation products, respectively, may result in the formation of protein cross-links and possible impairment of the gel properties of MP (Utrera & Estévez, 2012b; Liu, Xiong, & Chen, 2009). The covalent and non-covalent interactions between rutin and MP, confirmed in the subsequent sections, may offer a suitable
2.5.1. MP gel preparation MP suspensions (20 mL, 40 mg/mL) were transferred into 30 (inner diameter) × 50 (height) mm glass vials and heated at 72 °C for 10 min. Then, the formed gels were chilled and kept at 4 °C overnight. 2.5.2. Gel strength Gel strength was measured using a TA-XT Plus Texture Analyzer (Stable Micro Systems, Surry, UK) equipped with a cylinder probe (P/ 0.5). Measurements were carried out under the same controlled conditions according to our previous methods (Jia et al., 2017). 2.5.3. Water-holding capacity (WHC) Aliquots of 5.0 g MP gels were put into polypropylene tubes and then centrifuged at 4 °C (3000 g, 10 min). WHC (%) was calculated as M1/M0 × 100%, where M0 and M1 are the gel weights before and after centrifugation, respectively. 2.5.4. Microstructure The morphology of the gels was observed using a scanning electron microscopy (SEM; S-4800, Hitachi, Japan) under 10,000× magnification. Pre-treatments of the gel samples were carried out according to our previous methods (Jia et al., 2017). 2.6. Rheological behavior The rheological properties of MP suspensions (40 mg/mL in 10 mM PBS containing 0.6 M NaCl, pH 7.0) were detemined by a rheometer (Discovery DHR-1, TA, USA) according Zhao’s procedure with modifications (Zhao et al., 2013). Aliquots of 2.0 mL of samples were loaded between parallel plates (diameter of upper plate: 30-mm) and heated from 20 °C to 80 °C at 2 °C/min. The shear force was determined with a strain of 0.5% and a frequency of 0.1 Hz. Changes of storage modulus (G′) and tan δ values (the ration of G′/G″, where G″ is loss modulus) were monitored. 2.7. Statistical analysis Three
independent
experimental
trials
(replications)
were 3
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different from that of the gel without phenolics (Tang et al., 2017; Cao & Xiong, 2015).
stereochemical distance between reacting groups that may be required for the condensation reactions (Utrera & Estévez, 2012b; Estévez, 2011). In brief, the excessive intra- and intermolecular interactions and cross-links of MP may eventually impair protein hydration and swelling during gelation, leading to loss of gel strength and WHC. Previous studies also reported significant correlations between the formation of protein carbonyl compounds and loss of WHC and gelling properties of MP as well as pork and beef muscles (Utrera et al., 2014; Utrera & Estévez, 2012b; Liu, Xiong, & Chen, 2010).
3.1.3. Free amine content The effects of rutin on free amine content were also measured in the present study. As shown in Fig. 1C, the free amine content in control MP without rutin was 87.5 nmol/mg protein. The addition of 10, 50 and 100 μmol/g rutin reduced the free amine content to 84.3, 83.9, and 81.1 nmol/mg protein, respectively. These values were not significantly different from that of the control (P > 0.05). However, 200 μmol/g rutin caused significant loss of free amines in MP. The free amine content was reduced to 73.6 nmol/mg protein, 16.0% less than that of the control (P < 0.05). The formation of covalent amine-quinone adducts between the quinones of rutin and the free amines may be responsible for the loss of free amines in MP, as the free amine groups on the MP surface were inclined to be attacked by the quinones of rutin (Cao et al., 2016). Gallic acid, EGCG and rosmarinic acid were also reported to promote the loss of free amines in MP through the formation of covalent adducts between quinones and free amines (Cao et al., 2016; Feng et al., 2017; Wang et al., 2018; Zhang et al., 2018; Cao & Xiong, 2015). Moreover, based on the significant increase of the carbonyl content at 200 μmol/g rutin, the formation of carbonyl compounds through oxidative deamination process and subsequent carbonyl–amine condensations to generate Schiff bases may both be responsible for the reduction of the free amine content in MP at higher doses of rutin. Similar to the effect of thiols on gel properties of MP, the moderate MP-MP interactions or MP-rutin cross-links induced by the reduction of free amines facilitated MP gelation.
3.1.2. Total thiol content MP is rich in thiol groups, since the native myosin and actin contain 42 and 5 thiol groups, respectively (Hofmann & Hamm, 1978). The thiol groups play an important role in MP gelation because they can convert into disulfide bonds, which represent one of the main intermolecular forces in the formation of MP gels. However, it was reported that the nucleophile thiol groups in MP were prone to forming covalent bonds with phenolics, thus affecting the protein disulfide bond formation and the potential for MP gelation (Jongberg et al., 2011; Jongberg et al., 2015). Therefore, the effects of rutin on the total thiol contents were determined. As presented in Fig. 1B, 10 μmol/g rutin resulted in a slight and not significant reduction of the total thiol content compared to control (P > 0.05). Moreover, 50, 100 and 200 μmol/g rutin caused sharp and significant reduction of the total thiol content compared to control and the 10 μmol/g treatments (P < 0.05). The formation of disulfide bonds through cysteine thiol group interchanges may result in a loss of thiol content. As confirmed in subsequent section 3.3.2, the increased surface hydrophobicity will promote formation of the hydrophobic aggregates and lead to a more compact protein structure; consequently, the contact opportunities of thiol groups was enhanced and the thiol groups were prone to interact with each other to promote the formation of disulfide linking (Cavallieri & da Cunha, 2008). Similarly, it was also proved in cod protein that higher surface hydrophobicity facilitated the subsequent protein denaturation that could further cause the cross linkage of thiols (Thorarinsdottir, Arason, Bogason, & Kristbergsson, 2004). Moreover, the decrease in total thiol content may also be caused by the covalent interactions between the phenolics and thiol groups which have drawn much attention recently. In general, a preceding oxidation of the phenolics to quinone occurred, and then the thiol-quinone adducts were formed by Michael addition (Jongberg et al., 2013). For example, the rosmarinic acid-cysteine adducts formed in this way and prevented the formation of disulfide cross-link between thiols, thus the gel properties including WHC and gel strength were impaired (Tang et al., 2017). Gallic acid and EGCG were also reported to promote the loss of thiol groups by additive reactions between the quinone of phenolics and thiol groups (Feng et al., 2017; Cao et al., 2016; Cao & Xiong, 2015). The dual role of the formation of thiol-quinone adducts should be considered. On one hand, the formation of thiol-quinone adducts hindered the formation of disulfide bonds; thus, it was disadvantageous to gelation. On the other hand, low concentration of phenolics may be beneficial for the gel properties, because the non-disulfide covalent interactions occurred between quinones and nucleophile thiol groups and the phenolics acted as cross-linking agent to connect MPs and increase protein polymerization; therefore, the gel properties were
3.2. Changes in secondary structure The effects of rutin on the secondary structures of MP were measured by using Raman spectra. The proportions of α-helices, β-sheets, βturns and random coils in MP were quantified by analyzing the amide I band as shown in Table 1. The addition of rutin caused a continuous reduction in α- helices, while it increased the proportions of β-sheets, βturns and random coils in a dose-dependent manner. The α-helix was maintained by intramolecular hydrogen bonds between the carbonyl oxygen and amino hydrogen of the peptide chains. Therefore, protein unfolding, as confirmed by exposure of hydrophobic groups in section 3.3.2, may disrupt hydrogen bonding of α-helices and promote conversion of α-helices to other three secondary structures. The increase in β-sheets, maintained by intermolecular hydrogen bonds between peptide chains, was also attributed to protein aggregation caused by hydrophobic interactions. In addition, the changes in secondary structures further confirmed the unfolding of MP induced by rutin. Therefore, the exposure of active groups and further protein cross-linking contribute to protein unfolding. The appropriate extent of protein unfolding and conversion of α-helices to β-sheets were beneficial for gelation. However, excessive unfolding and aggregation of MP induced by higher doses of rutin were detrimental to gel properties, as shown by the results of gel properties and rheological properties of MP.
Table 1 The secondary structure fractions of MP with different concentration of rutin. Concentration (μmol/g) 0 10 50 100 200
α-helix (%) 65.57 61.93 56.45 50.88 49.10
± ± ± ± ±
β-sheet (%) a
3.16 6.32ab 8.37ab 3.10b 3.10b
11.53 14.32 18.52 22.78 24.15
Means with different letters (a–b) in each column differ significantly (P < 0.05). 4
± ± ± ± ±
β-turn (%) b
2.42 4.84ab 6.42ab 2.37a 2.37a
13.57 14.15 15.00 15.87 16.15
± ± ± ± ±
Random coil (%) b
0.49 0.99ab 1.31ab 0.48a 0.48a
9.64 ± 0.19b 9.86 ± 0.39ab 10.20 ± 0.51ab 10.54 ± 0.19a 10.65 ± 0.19a
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Fig. 2. Tryptophan fluorescence of MP suspensions with or without rutin (A); influence of rutin on the surface hydrophobicity of MP (B). Error bars refer to the standard deviations obtained from triplicate sample analyses. Means with different letters (a–b) differ significantly (P < 0.05).
around the tryptophan residues in proteins, also leading to exposure of tryptophan residues to a more polar microenvironment and thus the reduction of FI, due to the water quenching effect (Acero-Lopez, Ullah, Offengenden, Jung, & Wu, 2012). Therefore, the decrease in FI might primarily be caused by blocking of tryptophan residues in a polar environment, penetration of water molecules, and interactions between rutin and tryptophan residues, rather than the exposure of the tryptophan at the protein surface. In addition, it should be noted that when investigating protein oxidation by assessing tryptophan oxidation, the decreased tryptophan FI may not be completely caused by the prooxidation activity of the antioxidants but can also be explained by the interactions between tryptophan and antioxidants.
3.3. Changes of tertiary structure 3.3.1. Intrinsic tryptophan fluorescence Tryptophan, as an essential amino acid, is commonly contained in many protein-based foods and dietary proteins. Due to the presence of the aromatic ring, tryptophan is able to emit fluorescence at 330–370 nm when excited at 290 nm. The tryptophan content in pork protein is less than that of other amino acids, but tryptophan is the only fluorescence-emitting amino acid naturally present in meat (Estévez, Kylli, Puolanne, Kivikari, & Heinonen, 2008). In many reports on meat protein oxidation, tryptophan loss is often characterized as an oxidative change because tryptophan contains nucleophilic side chains and may be oxidized by reactive oxygen species. In addition, when other molecules interact with proteins, the protein conformation may also be affected, changing tryptophan fluorescence; thus, tryptophan fluorescence was determined in the present study. As presented in Fig. 2A, the tryptophan maximum fluorescence emission wavelength (λmax) of MP was near 335 nm when excited at 290 nm. The fluorescence intensity (FI) and λmax of tryptophan are sensitive to the polarity of its microenvironment and could be used to monitor conformational changes of proteins. As reviewed by Cao and Xiong (2015) and Cao et al. (2016), in the folded state, tryptophan residues were situated in the core of the protein molecule, thus having a high FI and a short λmax, whereas in a partially or completely unfolded state, tryptophan residues were exposed to the hydrophilic solvent environment and were located on the surface of the protein, thus having a reduced FI and a higher λmax (red shift). In the present study, the addition of rutin decreased the FI of tryptophan in a dose-dependent manner, indicating that rutin induced the MP conformational changes and the tryptophan residues were exposed to a more hydrophilic environment. Moreover, the decreased FI may also be related to the possible interactions between rutin and tryptophan residues. However, the λmax did not increased as expected, but showed a slight blue shift from 335 to 330 nm with the increasing rutin concentrations. Similar changes in FI and λmax were also reported by Cao et al. (2016), who found that the addition of gallic acid promoted the loss of intrinsic fluorescence and a slight blue shift of λmax. A possible explanation is that protein unfolding indeed occurred, as confirmed by the increase in surface hydrophobicity described in Section 3.2.2, but it may be mainly caused by the exposure of other hydrophobic groups rather than tryptophan; in contrast, part of the tryptophan residues may be occluded and still be buried within the protein structure (Sponton, Perez, Carrara, & Santiago, 2015; Jiang, Chen, & Xiong, 2009); thus, the λmax became shorter. The occluded tryptophan that did not participate in protein hydrophobic interactions would be in a more polar environment and these tryptophan residues would not contribute to the fluorescence emission (Sponton et al., 2015). Meanwhile, partial protein unfolding potentially enhances penetration of water molecules
3.3.2. Surface hydrophobicity Surface hydrophobicity plays an important role in determining protein functional properties. In the present study, the changes in MP surface hydrophobicity induced by rutin were investigated to further illustrate the effects of these changes on thermal gelation of MP. As shown in Fig. 2B, the addition of 10 μmol/g rutin increased the surface hydrophobicity of MP, but no significant differences were observed when compared with the control (P > 0.05). Moreover, 50 and 100 μmol/g rutin significantly increased the surface hydrophobicity (P < 0.05). Native myosin has hydrophobic residues strongly concentrated in the core of the helix (Maclachlan & Karn, 1982); therefore, the increased surface hydrophobicity indicated that treatments with rutin induced exposure of the hydrophobic domains originally buried in the native proteins, reflecting the unfolding of protein conformation and allowing more functional groups to participate in the aggregation and gelation process (Cao & Xiong, 2015). However, 200 μmol/g rutin resulted in a slight and not significant reduction of the surface hydrophobicity compared to the 50 and 100 μmol/g treatments (P > 0.05), and it was still higher than the control and 10 μmol/g treatments. The decreases in surface hydrophobicity at 200 μmol/g rutin might be attributed to the formation of hydrophobic aggregates, because the exposure of hydrophobic groups facilitated subsequent hydrophobic interactions between MPs and made the hydrophobic groups unavailable for bromophenol blue. Besides, large entanglements may be formed through the cross-linking between MPs induced by rutin and will bury some hydrophobic residues inside. Furthermore, the non-covalent hydrophobic interactions between rutin and the exposed hydrophobic groups may also result in shielding of the hydrophobic amino acid residues. In brief, when compared with the control, the incorporation of rutin increased the surface hydrophobicity of MP; consequently, the moderate increased surface hydrophobicity potentially promoted the hydrophobic interactions between MPs during thermal gelation and contributed to the gel properties.
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Fig. 3. Native (-βME) and reducing (+βME) SDS-PAGE gels of MP with different concentrations of rutin (0, 10, 50, 100 and 200 μmol/g) (A); and influence of rutin on the solubility of MP (B). Error bars refer to the standard deviations obtained from triplicate sample analyses. Means with different letters (a–c) differ significantly (P < 0.05). MHC: myosin heavy chain.
3.4.2. Solubility The addition of rutin induced modification of protein structure and thus, has a major impact on protein solubility. As shown in Fig. 3B, compared to the control, the solubility decreased significantly by 10.0%, 11.0%, 16.1% and 27.1% in MP containing 10, 50, 100 and 200 μmol/g rutin, respectively (P < 0.05). Protein solubility is mainly determined by electrostatic repulsions and hydrophobic interactions between protein molecules; protein solubility is higher when electrostatic repulsions are greater than hydrophobic interactions (Mohan, Ramachandran, Sankar, & Anandan, 2007). Therefore, the decrease in solubility may be ascribed to the formation of protein aggregates via hydrophobic interactions. In addition, the cross-linking of proteinprotein or protein-rutin via covalent interactions may also reduce the solubility of proteins. It is commonly accepted that low protein solubility is associated with a high degree of protein denaturation. In addition, high protein solubility is beneficial for protein functionality. However, in the present study, the addition of 10, 50 and 100 μmol/g rutin reduced the solubility. In contrast, the gel properties improved with increase in concentration of rutin. The lower solubility can be attributed to moderate polymerization of proteins and MP-rutin crosslinking rather than denaturation and precipitation of proteins. Addition of 200 μmol/g rutin reduced the solubility together with the gel properties, since high concentration of rutin resulted in excessive MP-MP and MP-rutin interactions, leading to aggregates and precipitation of
3.4. Cross-linking and solubility of MP 3.4.1. Cross-linking SDS-PAGE was used to observe the cross-linking of MP with or without rutin. Fig. 3A depicts the non-reduced samples without β-ME (–β-ME) and reduced samples with β-ME (+β-ME). As shown in Fig. 3A (–β-ME), compared to the control, the addition of 10, 50 and 100 μmol/ g rutin showed reduction in myosin heavy chain (MHC) band intensity and more polymers at the top of the stacking gel, especially at 100 μmol/g rutin, indicating that the MHC participated in the polymerization of MP. At 200 μmol/g rutin, the intensity of the MHC band was significantly reduced. Intensity of the actin band was also reduced, but no more polymers were observed due to the insolubility of some of the polymers induced by rutin (Cao et al., 2016). After being reduced by β-ME (Fig. 3A, +β-ME), most MHC and actin molecules were recovered and no significant changes in band intensity were observed, suggesting that disulfide linkages mainly contributed to protein polymerization from MHC. However, there were still few residual polymers, suggesting that these polymers were formed via non-disulfide bonds, probably via formation of the carbonyl-amine, thiol-quinone, and amine-quinone adducts, as verified in Section 3.1. As mentioned above, the oxidized rutin could also act as a cross-linking agent to facilitate protein polymerization.
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Fig. 4. Influence of rutin on the gel strength (A) and water-holding capacity (B) of MP; SEM photographs (10,000 × ) (C) of MP gels with or without rutin: (a) control; (b) 10 μmol/g rutin; (c) 50 μmol/g rutin; (d) 100 μmol/g rutin; and (e) 200 μmol/g rutin. Error bars refer to the standard deviations obtained from triplicate sample analyses. Means with different letters (a–b) differ significantly (P < 0.05).
hydrogen bonding between the hydroxyl groups of rutin and the –NH + 3 groups of MP amino acids was strong due to large molecular size and the sugar moieties of rutin, and thus promote an increased amount of hydrogen bonding within the heat-induced MP gels (Friesen et al., 2015). Moreover, the rutin-protein cross-linking may also be enhanced by the formation of thiol-quinone and amine-quinone adducts, since rutin may be oxidized to form quinone that have higher cross-linking capacity (Cao & Xiong, 2015) . Similarly, rutin was reported to significantly increase puncture strength of soy protein isolate films (Friesen et al., 2015). It was also reported that rutin was a highly effective cross-linking agent for pollock skin gelatin and increased the gel strength of gelatin gels, because rutin could reinforce the intermolecular interaction by entering the spacing of polypeptide chains of gelatin, mainly interacting with the skeletal CeNeC group and carboxyl group in the formation of gels and there were more binding sites of rutin-modified xerogel (Yan et al., 2011). Therefore, in the present study, rutin may enhance the gel strength according to similar principles. However, 200 μmol/g rutin resulted in significant lower gel strength than other rutin treatment groups (P < 0.05), but it still showed no significant reduction compared to control (P > 0.05). The decrease in the gel strength of MP with high concentration of rutin might be due to the coverage of protein surfaces by excess rutin molecules or sufficient covalent and non-covalent interactions between rutin and MP or MP and MP, thus decreasing the opportunity for protein-protein interactions during thermal gelation (Yan et al., 2011). In addition, protein precipitation can occur at high concentration of rutin and insufficient
proteins. Similarly, high concentration of gallic acid and rosmarinic acid decreased the solubility and gel properties of MP (Cao & Xiong, 2015; Wang et al., 2018).
3.5. Gel properties and microstructure 3.5.1. Gel strength and WHC Gel strength is one of the crucial functional properties of heat-induced MP gel and protein gelation is the most important textureforming property in processed meat products. As presented in Fig. 4A, the addition of 10, 50 and 100 μmol/g rutin increased the gel strength by 6.39%, 8.84% or 19.1% compared to control, indicating the formation of a stronger gel network, although no significant differences were observed (P > 0.05). In contrast, in our previous research, the catechin was found to reduce the gel strength and destroyed the gel network at the same determined concentrations (Jia et al., 2017). Moreover, rutin was also better than rosmarinic acid and EGCG, since 1.25 mM (62.5 μmol/g) rosmarinic acid or 1000 ppm (2.2 μmol/g) EGCG were detrimental to gel properties (Tang et al., 2017; Feng et al., 2017). Therefore, rutin was relatively beneficial for improving MP gel properties, as the rutin-modified gels had a higher degree of crosslinking. On one hand, the protein–protein interactions, including hydrophobic interactions and formation of disulfide bonds, as important intermolecular forces in the formation of the heat-induced MP gels, were reinforced by addition of rutin as mentioned above. On the other hand, the rutin-protein cross-linking via hydrogen bonding may also be beneficial to the formation of MP gel network. The formation of 7
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formed between some proteins (Tang et al., 2017; Cao & Xiong, 2015). Finally, from 50 °C to 80 °C, the G′ showed a continuous increase because of the intensification of both myosin head-head and tail–tail interactions, and ultimately formed a permanent and irreversible gel network structure. The addition of rutin caused a decrease in G′ below 50 °C, especially at a concentration of 200 μmol/g rutin, suggesting that rutin weakened the interactions between MPs. In the initial stage of gel formation, the covalent-linking between rutin and MP, as proved by the loss of thiol and free amine content, will block the proteins’ intermolecular interactions. As a result, the formation of an elastic protein network was hindered. The two transition peak temperatures of MP with 200 μmol/g rutin shifted to 44 °C and 46 °C, respectively, indicating the severely reduced protein stability at high concentration of rutin, since excessive rutin coating the protein surface may further reduce the opportunity of MP interactions. However, further heating process (ranged from 50 °C to 80 °C) caused higher G′ value of MP with rutin than control, particularly the final G′. In the final stage of gel formation, higher heating temperature resulted in sufficient unfolding of protein and exposure of more functional groups, which facilitated gelation; consequently, the rutin-MP interactions could not hinder the intermolecular interactions of MPs any more. In addition, the interactions between rutin and MP might increase the molecular size of MP, thus possibly increasing entanglements between protein chains and contribute to the increase of G′ (Wang et al., 2018; Feng et al., 2017). As aforementioned, rutin promoted the exposure of hydrophobic groups and thus the hydrophobic interaction force was enhanced during the thermal gelation process, resulting in shrinking of the MP gel with higher G′. The G′ of gel with 200 μmol/g rutin was slight lower than that of the gel with 100 μmol/g rutin, but the MP still formed an integrated three-dimensional gel network with relatively higher G′ than that of the MP with 0, 10 or 50 μmol/g rutin. Therefore, the rutin was beneficial to the formation of MP gel at relatively high concentrations. These results are consistent with those of gel strength. The G″ describes the viscosity properties of the MP suspensions during the gelation process. The ratio of the G″ to the G′ is expressed as tan δ, which reflects whether the elasticity-dominant or the viscositydominant is the main type of the formed gel. As shown in Fig. 5B, when the temperature was below 50 °C, the MP with 200 µmol/g rutin had a high tan δ value, which indicated that the viscosity played a leading role in the MP system. This can be explained by the weaken interactions of MPs, thus the gel network had not begun to form and the MP systems were prone to be in the solution state. The results were consistent with the low G′ of the MP with 200 µmol/g rutin (Fig. 5A). In the final stage of the thermal gelation, all the MP with or without rutin could form good gel structures and all the MP gels converted to the elasticitydominant types, as evidenced by a sharp drop in the tan δ.
swell of protein are adverse to gelation (Silber, Davitt, Khairutdinov, & Hurst, 1998). The WHC reflects the amount of water trapped in the formed 3dimensional network of MP gels, and is associated with spatial structure of protein gel. As presented in Fig. 4B, the MP gels with 10, 50 and 100 μmol/g rutin showed slight lower WHC compared to that of the control, but no significant differences were observed (P > 0.05); furthermore, 200 μmol/g rutin resulted in the lowest WHC. The addition of 10, 50 and 100 μmol/g rutin had a slight negative effect on WHC, in contrast to its positive effect on gel strength. The reduction in WHC may be caused by the formation of a more compact structure of MP gels, because the enhanced hydrophobic force may cause shrinkage of the MP gel during heating. In addition, less water would be entrapped in gels due to the reduced inter-chain space of MP gels brought about by hydrogen bonding between the sugar moieties on the rutin structure and water within the gels or the proteins (Friesen et al., 2015). 3.5.2. Microstructure of MP gels The microstructures of the MP gels with or without rutin are shown in Fig. 4C. The control MP gel had an intact, continuous, homogeneous, and porous microstructure. After addition of rutin, obvious morphological changes were observed compared to that of the control. When 10, 50, and 100 μmol/g rutin was added, the MP gels displayed similar surface microstructures with slight agglomerate, but still flat and uniform surface structures. Moreover, these MP gels also displayed a compact and layered structure with few visible pores, because the unfolding and cross-linking of proteins promoted aggregation of MP, leading to the formation of larger micelles in the matrix and a more compact network structure. Moreover, the MP gels with 200 μmol/g rutin presented an overlapped, obvious agglomerate and rough surface structure consisting of discontinuous protein aggregates. The more compact and shrunken structure resulted in the formation of some small apertures between the micelles in MP gels, thus leading to lower WHC. The above results indicated that only the 200 μmol/g rutin exhibited the unfavorable effect on MP gel. 3.6. Dynamic rheological properties The rheological properties of MP samples during gelation process are evaluated by G′ that describe the gel elasticity. The G′ of MP samples in the present or absence of rutin is shown in Fig. 5A. The control MP without rutin exhibited a typical G′ curve with two transition peaks at 46 °C and 50 °C. The increase in G′ from 40 °C to 46 °C (first peak) was mainly caused by the head-head interactions of the unfolded myosin and indicated the formation of elastic gel networks. Then, the G′ decreased and showed a low value at 50 °C (second peak), indicating weakening of the gel elasticity due to the subsequent tail–tail interactions of myosin and the disturbance of the cross-linking previously
Fig. 5. Storage modulus (A) and tan δ value (B) of MP during thermal gelation with or without rutin. 8
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4. Conclusions
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Rutin has a significant impact on MP structure and MP gel properties. The amino residue side-chain group was modified as confirmed by continuous reduction of thiol and free amine contents and the greatest carbonyl content at 200 μmol/g rutin. The incorporation of rutin caused conversions of α-helix to β-sheet, β-turn and random coil. The tertiary structure was also changed as the FI of intrinsic tryptophan decreased, the λmax showed a slight blue shift and the surface hydrophobicity increased. Accordingly, with the addition of 10, 50 and 100 μmol/g rutin, the MP gel strength increased and the WHC showed a slight decrease; with the addition of 200 μmol/g rutin, the lowest gel strength and WHC were observed. The SEM and dynamic rheological results proved the beneficial effects of 10, 50 and 100 μmol/g rutin on the MP gel properties and the negative effect of 200 μmol/g rutin. A slightly high concentration of rutin could improve the gel properties of MP due to moderate MP-MP interactions induced by rutin and the cross-linking capacity of rutin. However, 200 μmol/g rutin destroyed the gel properties owing to the excessive MP-MP or MP-rutin interactions. Declaration of Competing Interest None. Acknowledgment This study was funded by the National Natural Science Foundation of China (Grant No: 31301509). References Acero-Lopez, A., Ullah, A., Offengenden, M., Jung, S., & Wu, J. (2012). Effect of high pressure treatment on ovotransferrin. Food Chemistry, 135, 2245–2252. Alix, A. J. P., Pedanou, G., & Berjot, M. (1988). Fast determination of the quantitative secondary structure of proteins by using some parameters of the raman amide I band. Journal of Molecular Structure, 174, 159–164. Cao, Y., & Xiong, Y. L. (2015). Chlorogenic acid-mediated gel formation of oxidatively stressed myofibrillar protein. Food Chemistry, 180, 235–243. Cao, Y., True, A. D., Chen, J., & Xiong, Y. L. (2016). Dual role (anti- and pro-oxidant) of gallic acid in mediating myofibrillar protein gelation and gel in vitro digestion. Journal of Agricultural and Food Chemistry, 15, 3054–3061. Cavallieri, A. L. F., & da Cunha, R. L. (2008). The effects of acidification rate, pH and ageing time on the acidic cold set gelation of whey proteins. Food Hydrocolloids, 22, 439–448. Chelh, I., Gatellier, P., & Sante-Lhoutellier, V. (2006). Technical note: A simplified procedure for myofibril hydrophobicity determination. Meat Science, 74, 681–683. Ellman, G. L. (1959). Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics, 82, 70–77. Estévez, M. (2011). Protein carbonyls in meat system: A review. Meat Science, 89(3), 259–279. Estévez, M., Kylli, P., Puolanne, E., Kivikari, R., & Heinonen, M. (2008). Fluorescence spectroscopy as a novel approach for the assessment of myofibrillar protein oxidation in oil-in-water emulsions. Meat Science, 80, 1290–1296. Feng, X., Chen, L., Lei, N., Wang, S., Xu, X., Zhou, G., & Li, Z. (2017). Emulsifying properties of oxidatively stressed myofibrillar protein emulsion gels prepared with (−)-epigallocatechin-3-gallate and NaCl. Journal of Agricultural and Food Chemistry, 65, 2816–2826. Friesen, K., Chang, C., & Nickerson, M. (2015). Incorporation of phenolic compounds, rutin and epicatechin, into soy protein isolate films: Mechanical, barrier and crosslinking properties. Food Chemistry, 172, 18–23.
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