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Investigation of inhibition of lipid oxidation by L-carnosine using an oxidized-myoglobin-mediated washed fish muscle system
LWT - Food Science and Technology 97 (2018) 703–710
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Investigation of inhibition of lipid oxidation by L-carnosine using an oxidized-myoglobin-mediated washed fish muscle system
T
Shulan Xiaoa, Hong Zhuangb, Guanghong Zhoua, Jianhao Zhanga,∗ a
National Center of Meat Quality and Safety Control, Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, PR China b Quality and Safety Assessment Research Unit, 950 College Station Road, Athens, GA, 30605, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Myoglobin Antioxidant L-carnosine Lipid oxidation
The objective of the present study was to understand mechanisms of inhibitory effect of L-carnosine on lipid oxidation using an oxidized myoglobin (metMb)-mediated washed cod muscle system. Experiments were conducted to compare L-carnosine with commercial antioxidants, tert-Butylhydroquinone (TBHQ) and α-tocopherol, and metal chelating reagent EDTA for inhibition of lipid oxidation, to compare L-carnosine with TBHQ for scavenging free radicals and to evaluate the effect of L-carnosine on structure of pro-oxidant metMb with spectral methods. Results showed that the inhibitory effect of L-carnosine on lipid oxidation was comparable with TBHQ and better than α-tocopherol. However, 1,1-diphenyl-2-picrylhydrazyl assay showed that TBHQ was much more effective in scavenging free radicals than L-carnosine. Comparing with 1.5% EDTA, 1.5% L-carnosine was much more effective against lipid oxidation. Incubation of L-carnosine with metMb resulted in spectral changes of metMb in wavelengths between 370 nm and 450 nm, Soret band shifted from 409 nm to 420 nm, and reduced molar ellepticity values. These results indicate that the high inhibitory effects of L-carnosine on meat lipid oxidation may result from not only its scavenging and chelating functions but also its effect on pro-oxidant metMb structure.
1. Introduction Lipid oxidation in meat and meat products has been one of main quality concerns to meat industry and researchers because of its negative effect on quality, which includes affecting meat sensory quality, such as color, texture, and flavor, and nutritional value (Morrissey, Sheehy, Galvin, Kerry, & Buckley, 1998) then directly influencing the consumer acceptance of meat (Addis, 1986). Besides, some secondary products in lipid oxidation, like 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA), are harmful to human because of their stability, easy diffusion into cell, and reaction with critical biomolecules (Grace, Macdonald, Roberts, & Kinter, 1996). Food industry and researchers have been trying to find effective strategies to inhibit lipid oxidation in food with no harmful side effect during processing and storage. The most common method is adding antioxidants to food (Erdmann et al., 2015; Hermund et al., 2016; Shah, Bosco, & Mir, 2014). Synthetic antioxidants, such as Butyl hydroxyanisole (BHA) and tert-Butylhydroquinone (TBHQ), are common examples; however, their utilization is limited in food because of their toxic properties and unacceptable by consumer (Kansci, Genot,
∗
Meynier, & Gandemer, 1997). Thus, to find antioxidants present in natural becomes research and application interests. L-carnosine, a βalanyl-histidine dipeptide, is a natural antioxidant, which exists with high content in skeletal muscle (O'Dowd, Cairns, Trainor, Robins, & Miller, 1990) and has shown antioxidative activities in meat products (Decker & Crum, 1991; Decker & Faraji, 1990; Kohen, Yamamoto, Cundy, & Ames, 1988; Lee & Hendricks, 1997). Decker and Faraji (Decker & Faraji, 1990) demonstrated that L-carnosine effectively inhibited lipid oxidation induced by not only metal catalysts, iron and copper, but also pro-oxidant proteins, such as heme-proteins and lipoxygenase present in skeletal muscle. In addition, experiments (Liu, Xu, Dai, & Ni, 2015) have also shown that L-carnosine directly inhibited the discoloration of beef patties induced by the formation of metMb. Although the mechanism of inhibition of lipid oxidation by L-carnosine was attributed to its free radicals scavenging ability and metal chelating activity, these published data indicate that other mechanisms may also be involved in inhibition of lipid oxidation by L-carnosine. In research, demonstration of antioxidant mechanisms are much complicated in vivo because of occurrences of endogenous pro-oxidants and/or antioxidants in food. Because of it, washed fish muscle has been
Corresponding author. E-mail address: [email protected] (J. Zhang).
https://doi.org/10.1016/j.lwt.2018.08.003 Received 12 March 2018; Received in revised form 30 July 2018; Accepted 1 August 2018 Available online 02 August 2018 0023-6438/ © 2018 Published by Elsevier Ltd.
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added to WCM to the final concentration of 1.5%. L-carnosine and EDTA treated groups as well as control were added equal ethanol as THBQ and α-tocopherol treated group, and the ethanol content in all samples was less than 5%. Samples for 0 time were taken before the reaction brown bottle were placed on ice for incubation.
commonly selected for assessment of antioxidant effectiveness (Park, Undeland, Sannaveerappa, & Richards, 2013; Richards & Li, 2004) against lipid oxidation because the washing procedure not only removes the endogenous pro-oxidants and antioxidants but also maintains key meat components (myofibrillar proteins and membrane phospholipids) in the model system. Thus, the washed fish muscle provides a useful matrix to understand anti-lipid oxidation mechanism of L-carnosine in meat. And because heme-proteins, hemoglobin (Hb) and myoglobin (Mb), are considered to be main factors to accelerate lipid oxidation in muscle food (Min, Nam, & Ahn, 2010; Richards & Hultin, 2002), oxidized Mb (metmyoglobin, metMb) has been often used to stimulate lipid oxidation in vitro to avoid the interference of autoxidation of reduced Mb (oxymyoglobin, oxyMb). By using a metMb-mediated washed fish muscle system in this study, we further investigated the mechanisms and effectiveness of antioxidant L-carnosine and our data indicate that L-carnosine might inhibit lipid oxidation also through changing the structure of pro-oxidant catalyzer metMb in meat.
2.6. Analytical methods 2.6.1. Measurement of DPPH radical scavenging activity DPPH is a stable free radical in ethanol and usually used to assess the free radical scavenging activity of antioxidants. The free radical scavenging of TBHQ and L-carnosine was measured according to Saiga's (Saiga, Tanabe, & Nishimura, 2003) method with minor modifications. Four mL of antioxidant solution with different concentrations was mixed with 1 mL of 0.02% DPPH in 99.5% ethanol. Above mixture was vortex 30 s at middle speed, followed by incubating 60 min at 37 °C in the absence of light. Then, the absorbance of the mixture was measured at 517 nm. Scavenged DPPH radicals were calculated by the following equation:
2. Materials and methods 2.1. Chemicals
scavenged radicals (%) (DPPH blank + control sample − DPPH sample) = × 100 DPPH blank
Mb (from equine skeletal muscle), L-carnosine (purity: > 97%), 2Thiobarbituric acid (TBA) and Trichloroacetic acid (TCA) were obtained from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO, USA). Streptomycin sulfate, disodium ethylenediamine tetraacetate (EDTA Na2), tert-Butylhydroquinone (THBQ), α-tocopherol, 1, 1-diphenyl-2picrylhydrazyl (DPPH) bought from Shanghai Aladdin Reagent Co., Ltd. All these chemicals were reagent grade.
Where DPPH blank is the absorbance of 4 mL of 99.5% ethanol solution plus 1 mL of 0.02% DPPH solution; control sample is the absorbance of 4 mL antioxidant solution (TBHQ or L-carnosine at different concentration) plus 1 mL 99.5% ethanol; and DPPH sample is the absorbance of 4 mL of antioxidant solution (TBHQ or L-carnosine at different concentration) plus 1 mL of 0.02% DPPH solution. 2.6.2. Measurement of spectral changes of metMb after incubation with Lcarnosine The mixture of metMb and L-carnosine was incubated according to Decker (Decker, Chan, Livisay, Butterfield, & Faustman, 1995) method with minor modifications. L-carnosine and metMb were mixed in 2.5 mM PBS buffer (pH 7.4) at the ratio of 50 μM: 300 μM. The mixture was incubated at 20 °C. Then scanned the mixture using UV-2600 Spectrophotometer in wavelengths between 370 nm and 450 nm (Shimadzu Instruments, Inc, Japan) at time intervals according to the experimental design.
2.2. Preparation of washed cod muscle Codfish (Gadus morhua) was obtained from a local fish market. Washed cod muscle (WCM) was prepared as described previously (Richards, He, & Grunwald, 2009). About 0.2 g g WCM were used for moisture content analysis. The rest of the samples were vacuum-packaged and stored at −40 °C for further use. 2.3. Preparation of metMb MetMb was prepared according to Lee and Richards' method (Lee, Tatiyaborworntham, Grunwald, & Richards, 2015).
2.6.3. Circular dichroism spectroscopy For evaluation of the effect of L-carnosine on structure of metMb, Circular Dichroism Spectroscopy was used to measure the incubated mixture of Mb and L-carnosine according to Richards' method (Richards et al., 2007). MetMb was firstly incubated with L-carnosine at 4 °C for 24 h. Then the mixture (containing 4.8 μM metMb) was transferred to a 2-mm cuvette containing 20 mM sodium phosphate buffers at pH 5.7. The sample was scanned from 260 to 190 nm by a Chirascan circular dichroism spectrometer (Chirascan v.4.4.2.0, Applied PhotoPhysics, Inc, UK) to determine secondary structure of metMb.
2.4. Addition of metMb to WCM MetMb was mixed with WCM in a brown bottle according to Richards and Li's method (Richards & Li, 2004) with minor modifications. In brief, the 1 mM metMb in washing buffer was added to WCM and the final metMb concentration was 40 μmol/kg WCM. Adjusted the pH of the mixture with 1 M HCl at 25 μL 1M HCl/g WCM to 5.7.2% streptomycin sulfate in milli-Q water was added to WCM mixture so that the final streptomycin sulfate was 200 ppm to inhibit growth of bacteria. Milli-Q water was added, as needed, to the brown bottle to adjust the moisture content to 88%.
2.6.4. Determination of lipid peroxides Lipid peroxides (LHP) were determined as previously described (Richards et al., 2007) with minor modifications. In brief, 0.5 g WCM in 50 mL glass tube was added 5 mL cold chloroform-methanol (1:1), mixture followed by homogenizing the mixture 30 s at 10,000 r/min. Transfer the homogenate into a new 15 mL glass tube. Then add another 5 mL cod chloroform-methanol mixture into above 50 mL sample glass tube, homogenized 30 s and transferred into above 15 mL glass tube, and added 3.00 mL cold 0.5% NaCl, vortex 30 s. Centrifuge the 15 mL glass tube 6 min at 1800 g, at 4 °C. Collected 2 mL of the lower layer from each tube to a clean glass tube, then, 1.33 mL of ice-cold chloroform/methanol (1:1) was added, followed by adding 25 μL cold ammonium thiocynate (3.94 M) and FeCl2 (18 mM, freshly prepared)
2.5. Addition of antioxidants or EDTA to WCM Prior to the addition of metMb to WCM, the antioxidants (L-carnosine, THBQ, α-tocopherol) or EDTA were added according to Richards and Li's method (Richards & Li, 2004). To be specific, 9% L-carnosine in milli-Q was added to WCM, the final concentration of L-carnosine in WCM as the experimental design (0,1%, 0.2%, 0.3%, 0.5%, 1.0%, 1.5%); the THBQ and α-tocopherol were dissolved in ethanol at the concentration of 9% first, then added to WCM to the final concentration of 1.5%; the EDTA in milli-Q water at the concentration of 3% was 704
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Fig. 1. Lipid Hydroperoxides (LHPs) values (A), thiobarbituric acid reactive substances (TBARS) (B), and hexanal content (C) in washed cod muscle treated with metMb during 2 °C storage at the present of different concentration of L-carnosine (0.1%. 0.3%, 0.5%, 1.5%) and 1.5% α-tocopherol and 1.5% TBHQ. The concentration of metMb was 40 μmol/kg washed cod, pH was 5.7. Replicates per treatment was n = 3. Means and standard deviations are shown.
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Fig. 2. Thiobarbituric acid reactive substances (TBARS) in WCM treated with 1.5% EDTA and 1.5% L-carnosine.
3. Results
accordingly, vortexed 5 s after each regent was added. Incubated above mixture at room temperature at the absence of light and read the absorbance at 500 nm immediately after incubation time was up. Standard curve was constructed using cumene hydroperoxide as standard. The lipid peroxides in WCM were expressed as μmol LHP/kg muscle.
3.1. Effect of different antioxidants (L-carnosine, α-Tocopherol, TBHQ) on lipid oxidation in WCM mediated by metMb The ability of L-carnosine at different concentrations (0.1%–1.5%) to inhibit lipid oxidation stimulated by metMb in WCM (pH = 5.7) was measured during storage at 2 °C. The formation of TBARS, hexanal, and LHPs were used as indicators of lipid oxidation. The results showed that L-carnosine in the range from 0.5% to 1.5% in WCM displayed highly inhibitory effects on lipid oxidation induced by metMb, while no inhibitory effect was noted with 0.1%–0.3% L-carnosine based on LHPs formation, TBARS values, and hexanal content (Fig. 1). To be specific, the LHPs formation, TBARS values, and hexanal content in 0.1%–0.3% L-carnosine-containing groups have no significant differences with control (WCM + metMb group). However, all above indicators in 0.5%–1.5% L-carnosine-containing groups were significantly lower than that in control and 0.1%–0.3% L-carnosine-containing groups. The LHPs formation in control group up to maximum (405 μmol LHP/kg muscle) on 1.0 days, was 7.4 times more than that in 1.5% L-carnosine treated group (55 μmol LHP/kg muscle) (Fig. 1A); TBARS values in control group up to maximum (76.43 μmol TBARS/kg muscle) on 1.5 days, was 4.6 times than that in 1.5% L-carnosine treated group (16.6 μmol TBARS/kg muscle) (Fig. 1B); and the hexanal content reached maximum on 2 days in control, 0.1% L-carnosine, 0.3% L-carnosine-containing treatments (0.54705, 0.515, 0.48145 μmol/kg muscle,
2.6.5. Determination of thiobarbituric acid reactive substances (TBARS) The value of TBARS was measured according to Richards and Dettmann's method with minor modifications (Richards, Dettmann, & Grunwald, 2005). Briefly, 0.12 g of muscle sample was added in 1.5-mL microfuge tube and mixed with 1.2-mL solution of 50% TCA with 1.3% TBA. The tubes were inverted for 10 times, heated at 65 °C in the absence of light for 1 h, and then cooled at 4 °C in the dark before centrifuged at 16,100 g and 4 °C for 5 min. Absorbance of supernatants was read at 532 nm and 600 nm. The real absorbance of supernatants was calculated as A532nm-A600nm. A standard curve was constructed using 1,1,3,3-tetraethoxypropane. Concentrations of TBARS in samples were expressed as μmol TBARS/kg muscle.
2.6.6. Determination of hexanal content Hexanal content in WCM was measured through gas chromatography (GC) method fitted with solid-phase micro-extration (SPME) technology. The samples were prepared according to Thiansilakul et al. method (2012) (Thiansilakul, Benjakul, Grunwald, & Richards, 2012). GC analysis was performed on a GC module 2010 Plus (SHIMADZU Technologies, Japan) equipped with a capillary column (DB-5, 30 m × 0.25 mm × 0.1 μm) and flame ionisation detector (FID). The method and temperature program according to Thiansilakul's method (Thiansilakul et al., 2012). The commercial hexanal was used as the standard. Identification and quantification were done by external standards and a standard curve. Samples and standard were run in triplicate.
Table 1 TBARS values in WCM. L-carnosine was heated at 100 °C for 15 min, followed by cooling on top water, before it was added to WCM.
2.7. Statistical analysis Data were analyzed using one-way analysis of variance (ANOVA) with SPSS (version 12.0). The number of experimental replicates was two or three. Means were separated using the Tukey's Multiple Range Test. Significance was determined using P-value less than 0.05.
Time (days)
Control
L-carnosine (1.5%) (heat 15 min at 100 °C)
L-carnosine
0 0.5 1 1.5 2 3
6.84 ± 0.15 xa 35.24 ± 5.04 xb 59.26 ± 3.70 xc 76.43 ± 0.30 xc 72.52 ± 4.71 xc 66.26 ± 1.12 xd
5.25 6.99 3.78 4.21 4.51 4.68
3.30 ± 1.15 xa 4.57 ± 0.09 ya 4.10 ± 0.79 xa 3.94 ± 0.18 za 6.86 ± 0.28 za 10.47 ± 0.34 zb
± ± ± ± ± ±
.0.61 xa 0.84 ya 0.23 xa 0.67 za 0.79 za 0.52 za
(1.5%)
x-z – means in the same row with the same letter are not significantly different at p > 0.05. a-d – means in the same column with the same letter are not significantly different at p > 0.05. 706
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significantly higher (P < 0.05) than those with either TBHQ or L-carnosine-treated groups (Fig. 1B), which remained less than 10 μmol/kg muscle. The maximum TBARS values in 1.5% α-tocopherol treated group was 3.5, 2.9 times more than that in 1.5% TBHQ-, 1.5% L-carnosine-containing groups, respectively. The content of hexanal in all treatments showed in Fig. 1C. In 1.5% L-carnosine-, 1.5% TBHQ-containing groups, the haxanal content did not display significant differences (P > 0.05) during the whole storage period and kept at significantly lower level (< 0.05 μmol/kg muscle) compared to control. In 1.5% α-tocopherol treatment, the hexanal content was elevated greatly on 2 days and up to 0.1533 μmol/kg muscle on 3 days, which was significantly higher than that in 1.5% L-carnosine-, 1.5% TBHQ-containing groups. All above results and analysis demonstrated that L-carnosine would be as effective as TBHQ and much more effective than αtocopherol against lipid oxidation in meat. The effect of EDTA on lipid oxidation in WCM was shown in Fig. 2. The data showed that there is no significantly different between EDTA-
respectively), was about 9–14 times more than that in 1.5% L-carnosine treated group (0.0495 μmol/kg muscle) (Fig. 1C). The inhibitory percent in 1.5% L-carnosine treated group up to 87% on 0.5 days based on TBARS values. To examine the potential of L-carnosine acting as an antioxidant, its antioxidative capacity was compared with THBQ, a synthetic antioxidant broadly used in food, and α-tocopherol, another highly active and natural at the concentration of 1.5%. The results showed in Fig. 1. From the perspective of LHPs values, there was no significant difference (P > 0.05) between TBHQ and L-carnosine treatments. However, the LHPs values were significantly elevated in α-tocopherol treated group on 1.0 days and significantly higher than that in TBHQ and L-carnosine treated groups (Fig. 1A). The TBARS values were no significant differences (P > 0.05) between TBHQ and L-carnosine treatments during whole meat storage period. However, it was significantly elevated after 1.5 days of storage in the 1.5% α-tocopherol-treated group and reached maximum (30.79 μmol/kg muscle) after two days of storage. And
Fig. 3. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of TBHQ (A) and L-carnosine (B). Replicates per treatment was n = 3. Means and standard deviations are shown. 707
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Fig. 5. Circular Dichroism Spectroscopy of metMb and metMb-L-carnosine. Fig. 4. Effect of L-carnosine on the spectrum of metMb. metMb was incubated with L-carnosine at 20 °C in 2.5 mM PBS buffer (pH 7.4). The ratio of metMb and L-carnosine was 50 μM: 300 μM.
treated group and control, and the TBARS values were significantly higher than that in 1.5% L-carnosine.
that the molar ellepticity value in control metMb was much more negative than that in the metMb incubated with L-carnosine, suggesting that the number of α-helix in metMb or the secondary structure of metMb might have been greatly affected after metMb is mixed with Lcarnosine.
3.2. Thermal stability of L-carnosine as antioxidant
4. Discussion
In meat processing, the antioxidant activity of antioxidant might commonly be lowered or disappear due to heat denaturation. To measure the thermal stability of L-carnosine in antioxidant activity, Lcarnosine was heated at 100 °C for 15 min before it was added to WCM. The TBARS value in the WCM with heat-treated L-carnosine maintained a low level (< 6.99 μmol/kg muscle) and was not significantly different from non-treated L-carnosine during the whole storage period (Table 1), which indicates that the antioxidant activity of L-carnosine is not affected by heat.
Antioxidant activity of L-carnosine is concentration-dependent (Lee & Hendricks, 1997; Lee, Hendricks, & Cornforth, 1999). L-carnosine can effectively inhibit lipid oxidation induced by metMb in washed cod muscle in the concentration range from 0.5% to 1.5%, which is in accordance with previous results (Lee & Hendricks, 1997). But it did not display inhibitory effect when the concentration ranged from 0.1% to 0.3% (Fig. 1). Decker et al. (Decker & Crum, 1991) reported that, at the concentration of 0.5% and 1.5%, the L-carnosine can effectively inhibit the formation of lipid peroxides and TBARS in uncooked and salted pork during frozen storage up to 6 months. Fig. 1 shows that L-carnosine (1.5%) has stronger inhibitory effect on lipid oxidation in WCM than αtocopherol (1.5%). However, Liu et al. (Liu et al., 2015) reported that Lcarnosine and α-tocopherol showed equivalent inhibitory effect in raw beef patties, which is different from our observation. This difference may be due to the different testing systems used in experiments. In their case, some endogenous pro-oxidants and antioxidants, which were present in raw beef patties, might either act with added antioxidants or directly inhibit meat lipid oxidation, interfering the antioxidant effect of L-carnosine and α-tocopherol in experiments (Boldyrev, Dupin, Aya, Babizhaev, & Severin, 1987). Our data also show that heating L-carnosine at 100 °C for 15 min before added to WCM had no effect on its antioxidative activity (Table 1). The same result was reported by Decker and Faraji (Decker & Faraji, 1990) with a liposome model. This indicates that antioxidant capability of L-carnosine is thermally stable and can remain its antioxidative activity during thermal processing. Our results, in line with these published discoveries, indicate that L-carnosine is an effective natural antioxidant and can be added to raw meat before processing for inhibiting lipid oxidation in either raw or cooked finish products. Antioxidants are usually classified into four types based on their mechanisms against lipid oxidation, chain breaker, peroxide decomposer, metal chelator, and free radical scavenger (Yen, Chang, Lee, & Duh, 2002). Experiments have shown that different mechanisms are involved in inhibition of lipid oxidation by L-carnosine. Decker et al. (Decker, Crum, & Calvert, 1992) attributed antioxidant effect of L-carnosine to metal chelating. Chan et al. (Chan, Decker, Jin, & Butterfield, 1994) ascribed it to free radical scavenging. Decker (Decker et al., 1995) found that L-carnosine inhibited lipid oxidation through
3.3. Free radical scavenging activities Radical scavenging ability of L-carnosine was measured using DPPH and compared with TBHQ (Fig. 3). The results showed that L-carnosine hardly had any free radical scavenging ability when its concentration was < 1 mg/mL, however, it was very effective (≥71%) when its concentration was ≥5 mg/mL (Fig. 3B). On the other hand, TBHQ showed high free radical scavenging ability (≥78%) even at a much low concentration ( ∼ 5 μg/mL) (Fig. 3A). By contrast, the L-carnosine did not have any free radical scavenging ability at this concentration (data not show). This indicates that TBHQ is much more effective in scavenging free radicals compared with L-carnosine. 3.4. Effect of L-carnosine on spectrum of metMb To examine the possible effect of L-carnosine on the structure of metMb, we tested characteristic spectra of metMb in the wavelength between 370 nm and 450 nm. Results showed that Soret band of metMb was immigrated from 409 to 420 after mixing and incubating with Lcarnosine and the maximum absorbance (1.2512, 0.5887, 0.531, respectively) decreased greatly (Fig. 4). These data indicate that metMb structure or form might have been changed after incubated with Lcarnosine, such as becoming a hemichrome. 3.5. Effect of L-carnosine on molar ellepticity values of metMb Molar ellepticity values around 192 nm and 222 nm can be used as indicator for the secondary structure, α-helix, of proteins. Fig. 5 shows 708
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Acknowledgement
quenching the ferryl-myoglobin radicals formed by hydrogen peroxideactivated metMb. The same author also reported that L-carnosine could quench unsaturated aldehydic lipid oxidation products, 4-hydroxy-2trans-nonenal (HNE). Grunwald (Grunwald, Tatiyaborworntham, Faustman, & Richards, 2017) reported that HNE can covalently bound to sperm whale Mb, and the adducted Mb promoted lipid oxidation in washed muscle more effectively than non-adducted Mb. Thus, quenching could be another mechanism for L-carnosine to reduce lipid oxidation induced by Mb in meat. In addition, Nagasawa et al. (Nagasawa, Yonekura, Nishizawa, & Kitts, 2001) found that either Lhistidine or β-alanine by itself did not affect lipid oxidation and they concluded that the dipeptide linkage in β-alanyl-histidine was essential for inhibitory effect of L-carnosine on lipid oxidation. In the present study, we hypothesized that in addition to metal chelating and free radical scavenging, the effect of L-carnosine on prooxidant metMb may also be involved in its inhibition of lipid oxidation. To determine the inhibitory effect of L-carnosine act as a metal chelator in lipid oxidation mediated by metMb, a comparison of L-carnosine to EDTA in inhibition of lipid oxidation was made using TBARS measurement results. The samples treated with EDTA (1.5%) showed no differences (P > 0.05) from the control group during the whole storage based on their TBARS values (Fig. 2). This is in agreement with Richards and Li's results (Richards & Li, 2004), indicating that the chelating activity of EDTA has no effect on lipid oxidation mediated with metMb in WCM. This is also supported by Grunwald and Richards' (Grunwald & Richards, 2006) findings that hemin released from metMb was the primary promoter of lipid oxidation in washed fish muscle other than free iron. On the other hand, a great inhibition of lipid oxidation by 1.5% L-carnosine demonstrates that the other mechanisms may be involved rather than chelation. The DPPH free radical scavenging experiment showed that TBHQ can scavenge almost 100% DPPH free radicals at 100 μg/mL concentration. By contrast, L-carnosine showed almost no scavenging ability at the same concentration (data not show). However, the TBARS values between TBHQ (1.5%) and L-carnosine (1.5%) treatments were similar to each other. These results indicate that the mechanism of Lcarnosine in inhibiting lipid oxidation may not be solely due to its free radical scavenging activity. The differences in visible spectroscopy and circular dichroism spectroscopy measurements between metMb and metMb-carnosine mixture suggest the structural change in metMb. In the spectrum of metMb-carnosine, the immigration of Soret band and the decrease of maximum absorbance could be due to the formation of hemichrome or other forms of metMb. The results of circular dichroism spectroscopy indicate that L-carnosine may affect the secondary structure of metMb. These data also suggest that L-carnosine may inhibit lipid oxidation in meat through changing metMb structures (eg. Unfolded the metMb) and subsequently affecting metMb pro-oxidant activity (Olcott & Lukton, 1961).
This work was supported by the National major Science and Technology project [2016YFD0401502]. The author would like to thank Dr. Haizhou Wu of Chalmers University of Technology for his kind advice on the revision of this paper. References Addis, P. B. (1986). Occurrence of lipid oxidation products in foods. Food and Chemical Toxicology. An International Journal Published for the British Industrial Biological Research Association, 24(10–11), 1021. Boldyrev, A. A., Dupin, A. M., Aya, B., Babizhaev, M. A., & Severin, S. E. (1987). The antioxidative properties of carnosine, a natural histidine containing dipeptide. Biochemistry International, 15(6), 1105. Chan, W. K. M., Decker, E. A., Jin, B. L., & Butterfield, D. A. (1994). EPR Spin-trapping studies of the hydroxyl radical scavenging activity of carnosine and related dipeptides. Journal of Agricultural and Food Chemistry, 42(7), 162–169. Decker, E. A., Chan, W. K. M., Livisay, S. A., Butterfield, D. A., & Faustman, C. (1995). 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Determination of sitespecific modifications of glucose-6-phosphate dehydrogenase by 4-hydroxy-2nonenal using matrix assisted laser desorption time-of-flight mass spectrometry. Free Radical Research Communications, 25(1), 23–29. Grunwald, E. W., & Richards, M. P. (2006). Studies with myoglobin variants indicate that released hemin is the primary promoter of lipid oxidation in washed fish muscle. Journal of Agricultural and Food Chemistry, 54(12), 4452. Grunwald, E. W., Tatiyaborworntham, N., Faustman, C., & Richards, M. P. (2017). Effect of 4-hydroxy-2-nonenal on myoglobin-mediated lipid oxidation when varying histidine content and hemin affinity. Food Chemistry, 227, 289–297. Hermund, D. B., Karadag, A., Andersen, U., Jonsdottir, R., Kristinsson, H. G., Alasalvar, C., et al. (2016). Oxidative stability of granola bars enriched with multilayered fish oil emulsion in the presence of novel brown seaweed based antioxidants. Journal of Agricultural and Food Chemistry, 64(44), 8359–8368. Kansci, G., Genot, C., Meynier, A., & Gandemer, G. (1997). The antioxidant activity of carnosine and its consequences on the volatile profiles of liposomes during iron/ ascorbate induced phospholipid oxidation. Food Chemistry, 60(2), 165–175. Kohen, R., Yamamoto, Y., Cundy, K. C., & Ames, B. N. (1988). Antioxidant activity of carnosine, homocarnosine, and anserine present in muscle and brain. Proceedings of the National Academy of Sciences of the United States of America, 85(9), 3175–3179. Lee, B. J., & Hendricks, D. G. (1997). Antioxidant effects of L-carnosine on liposomes and beef homogenates. Journal of Food Science, 62(5), 931–1000. Lee, B. J., Hendricks, D. G., & Cornforth, D. P. (1999). A comparison of carnosine and ascorbic acid on color and lipid stability in a ground beef pattie model system. Meat Science, 51(3), 245–253. Lee, S. K., Tatiyaborworntham, N., Grunwald, E. W., & Richards, M. P. (2015). Myoglobin and haemoglobin-mediated lipid oxidation in washed muscle: Observations on crosslinking, ferryl formation, porphyrin degradation, and haemin loss rate. Food Chemistry, 167, 258–263. Liu, F., Xu, Q., Dai, R., & Ni, Y. (2015). Effects of natural antioxidants on colour stability, lipid oxidation and metmyoglobin reducing activity in raw beef patties. Acta Scientiarum Polonorum Technologia Alimentaria, 14(1), 37. Min, B., Nam, K. C., & Ahn, D. U. (2010). Catalytic mechanisms of metmyoglobin on the oxidation of lipids in phospholipid liposome model system. Food Chemistry, 123(2), 231–236. Morrissey, P. A., Sheehy, P. J. A., Galvin, K., Kerry, J. P., & Buckley, D. J. (1998). Lipid stability in meat and meat products. Meat Science, 49(5), S73. Nagasawa, T., Yonekura, T., Nishizawa, N., & Kitts, D. D. (2001). In vitro and in vivo inhibition of muscle lipid and protein oxidation by carnosine. Molecular and Cellular Biochemistry, 225(1–2), 29–34. O'Dowd, J. J., Cairns, M. T., Trainor, M., Robins, D. J., & Miller, D. J. (1990). Analysis of carnosine, homocarnosine, and other histidyl derivatives in rat brain. Journal of Neurochemistry, 55(2), 446–452. Olcott, H. S., & Lukton, A. (1961). Hemichrome and hemochrome formation with anserine, carnosine and related compounds. Archives of Biochemistry and Biophysics, 93(3), 666. Park, S. Y., Undeland, I., Sannaveerappa, T., & Richards, M. P. (2013). Novel interactions of caffeic acid with different hemoglobins: Effects on discoloration and lipid oxidation in different washed muscles. Meat Science, 95(1), 110–117. Richards, M. P., Dettmann, M. A., & Grunwald, E. W. (2005). Pro-oxidative characteristics of trout hemoglobin and myoglobin: A role for released heme in oxidation of lipids. Journal of Agricultural and Food Chemistry, 53(26), 10231–10238.
5. Conclusions The present study demonstrated that L-carnosine can inhibit lipid oxidation mediated by metMb in a WCM system as effectively as TBHQ. As a natural antioxidant, L-carnosine showed much stronger antioxidant capability than α-tocopherol in the WCM system. By comparison with metal chelating reagent EDTA and effective free radical scavenging chemical TBHQ, our results suggest that the inhibition of lipid oxidation in meat by L-carnosine may not be due to only the chelation of prooxidant metals and free radical scavenging. Indirect measurements of structure of metMb with spectroscopic methods indicate that the inhibitory effect of L-carnosine may be involved in inducing structural change of pro-oxidant metMb, which further inactivates the pro-oxidant property of metMb. However, further study is needed to provide direct evidence that L-carnosine can cause structure changes in pro-oxidant metMb and thereafter inhibit lipid oxidation in meat products. 709
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55(9), 3643–3654. Saiga, A., Tanabe, S., & Nishimura, T. (2003). Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment. Journal of Agricultural and Food Chemistry, 51(12), 3661–3667. Shah, M. A., Bosco, S. J., & Mir, S. A. (2014). Plant extracts as natural antioxidants in meat and meat products. Meat Science, 98(1), 21–33. Thiansilakul, Y., Benjakul, S., Grunwald, E. W., & Richards, M. P. (2012). Retardation of myoglobin and haemoglobin-mediated lipid oxidation in washed bighead carp by phenolic compounds. Food Chemistry, 134(2), 789–796. Yen, W. J., Chang, L. W., Lee, C. P., & Duh, P. D. (2002). Inhibition of lipid peroxidation and nonlipid oxidative damage by carnosine. Journal of the American Oil Chemists’ Society, 79(4), 329–333.
Richards, M. P., He, C., & Grunwald, E. W. (2009). Phenylalanine substitution at site B10 (L29F) inhibits metmyoglobin formation and myoglobin-mediated lipid oxidation in washed fish muscle: Mechanistic implications. Journal of Agricultural and Food Chemistry, 57(17), 7997. Richards, M. P., & Hultin, H. O. (2002). Contributions of blood and blood components to lipid oxidation in fish muscle. Journal of Agricultural and Food Chemistry, 50(3), 555. Richards, M. P., & Li, R. (2004). Effects of released iron, lipid peroxides, and ascorbate in trout hemoglobin-mediated lipid oxidation of washed cod muscle. Journal of Agricultural and Food Chemistry, 52(13), 4323–4329. Richards, M. P., Nelson, N. M., Kristinsson, H. G., Mony, S. S., Petty, H. T., & Oliveira, A. C. (2007). Effects of fish heme protein structure and lipid substrate composition on hemoglobin-mediated lipid oxidation. Journal of Agricultural and Food Chemistry,
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Investigation of inhibition of lipid oxidation by L-carnosine using an oxidized-myoglobin-mediated washed fish muscle system
T
Shulan Xiaoa, Hong Zhuangb, Guanghong Zhoua, Jianhao Zhanga,∗ a
National Center of Meat Quality and Safety Control, Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, PR China b Quality and Safety Assessment Research Unit, 950 College Station Road, Athens, GA, 30605, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Myoglobin Antioxidant L-carnosine Lipid oxidation
The objective of the present study was to understand mechanisms of inhibitory effect of L-carnosine on lipid oxidation using an oxidized myoglobin (metMb)-mediated washed cod muscle system. Experiments were conducted to compare L-carnosine with commercial antioxidants, tert-Butylhydroquinone (TBHQ) and α-tocopherol, and metal chelating reagent EDTA for inhibition of lipid oxidation, to compare L-carnosine with TBHQ for scavenging free radicals and to evaluate the effect of L-carnosine on structure of pro-oxidant metMb with spectral methods. Results showed that the inhibitory effect of L-carnosine on lipid oxidation was comparable with TBHQ and better than α-tocopherol. However, 1,1-diphenyl-2-picrylhydrazyl assay showed that TBHQ was much more effective in scavenging free radicals than L-carnosine. Comparing with 1.5% EDTA, 1.5% L-carnosine was much more effective against lipid oxidation. Incubation of L-carnosine with metMb resulted in spectral changes of metMb in wavelengths between 370 nm and 450 nm, Soret band shifted from 409 nm to 420 nm, and reduced molar ellepticity values. These results indicate that the high inhibitory effects of L-carnosine on meat lipid oxidation may result from not only its scavenging and chelating functions but also its effect on pro-oxidant metMb structure.
1. Introduction Lipid oxidation in meat and meat products has been one of main quality concerns to meat industry and researchers because of its negative effect on quality, which includes affecting meat sensory quality, such as color, texture, and flavor, and nutritional value (Morrissey, Sheehy, Galvin, Kerry, & Buckley, 1998) then directly influencing the consumer acceptance of meat (Addis, 1986). Besides, some secondary products in lipid oxidation, like 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA), are harmful to human because of their stability, easy diffusion into cell, and reaction with critical biomolecules (Grace, Macdonald, Roberts, & Kinter, 1996). Food industry and researchers have been trying to find effective strategies to inhibit lipid oxidation in food with no harmful side effect during processing and storage. The most common method is adding antioxidants to food (Erdmann et al., 2015; Hermund et al., 2016; Shah, Bosco, & Mir, 2014). Synthetic antioxidants, such as Butyl hydroxyanisole (BHA) and tert-Butylhydroquinone (TBHQ), are common examples; however, their utilization is limited in food because of their toxic properties and unacceptable by consumer (Kansci, Genot,
∗
Meynier, & Gandemer, 1997). Thus, to find antioxidants present in natural becomes research and application interests. L-carnosine, a βalanyl-histidine dipeptide, is a natural antioxidant, which exists with high content in skeletal muscle (O'Dowd, Cairns, Trainor, Robins, & Miller, 1990) and has shown antioxidative activities in meat products (Decker & Crum, 1991; Decker & Faraji, 1990; Kohen, Yamamoto, Cundy, & Ames, 1988; Lee & Hendricks, 1997). Decker and Faraji (Decker & Faraji, 1990) demonstrated that L-carnosine effectively inhibited lipid oxidation induced by not only metal catalysts, iron and copper, but also pro-oxidant proteins, such as heme-proteins and lipoxygenase present in skeletal muscle. In addition, experiments (Liu, Xu, Dai, & Ni, 2015) have also shown that L-carnosine directly inhibited the discoloration of beef patties induced by the formation of metMb. Although the mechanism of inhibition of lipid oxidation by L-carnosine was attributed to its free radicals scavenging ability and metal chelating activity, these published data indicate that other mechanisms may also be involved in inhibition of lipid oxidation by L-carnosine. In research, demonstration of antioxidant mechanisms are much complicated in vivo because of occurrences of endogenous pro-oxidants and/or antioxidants in food. Because of it, washed fish muscle has been
Corresponding author. E-mail address: [email protected] (J. Zhang).
https://doi.org/10.1016/j.lwt.2018.08.003 Received 12 March 2018; Received in revised form 30 July 2018; Accepted 1 August 2018 Available online 02 August 2018 0023-6438/ © 2018 Published by Elsevier Ltd.
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added to WCM to the final concentration of 1.5%. L-carnosine and EDTA treated groups as well as control were added equal ethanol as THBQ and α-tocopherol treated group, and the ethanol content in all samples was less than 5%. Samples for 0 time were taken before the reaction brown bottle were placed on ice for incubation.
commonly selected for assessment of antioxidant effectiveness (Park, Undeland, Sannaveerappa, & Richards, 2013; Richards & Li, 2004) against lipid oxidation because the washing procedure not only removes the endogenous pro-oxidants and antioxidants but also maintains key meat components (myofibrillar proteins and membrane phospholipids) in the model system. Thus, the washed fish muscle provides a useful matrix to understand anti-lipid oxidation mechanism of L-carnosine in meat. And because heme-proteins, hemoglobin (Hb) and myoglobin (Mb), are considered to be main factors to accelerate lipid oxidation in muscle food (Min, Nam, & Ahn, 2010; Richards & Hultin, 2002), oxidized Mb (metmyoglobin, metMb) has been often used to stimulate lipid oxidation in vitro to avoid the interference of autoxidation of reduced Mb (oxymyoglobin, oxyMb). By using a metMb-mediated washed fish muscle system in this study, we further investigated the mechanisms and effectiveness of antioxidant L-carnosine and our data indicate that L-carnosine might inhibit lipid oxidation also through changing the structure of pro-oxidant catalyzer metMb in meat.
2.6. Analytical methods 2.6.1. Measurement of DPPH radical scavenging activity DPPH is a stable free radical in ethanol and usually used to assess the free radical scavenging activity of antioxidants. The free radical scavenging of TBHQ and L-carnosine was measured according to Saiga's (Saiga, Tanabe, & Nishimura, 2003) method with minor modifications. Four mL of antioxidant solution with different concentrations was mixed with 1 mL of 0.02% DPPH in 99.5% ethanol. Above mixture was vortex 30 s at middle speed, followed by incubating 60 min at 37 °C in the absence of light. Then, the absorbance of the mixture was measured at 517 nm. Scavenged DPPH radicals were calculated by the following equation:
2. Materials and methods 2.1. Chemicals
scavenged radicals (%) (DPPH blank + control sample − DPPH sample) = × 100 DPPH blank
Mb (from equine skeletal muscle), L-carnosine (purity: > 97%), 2Thiobarbituric acid (TBA) and Trichloroacetic acid (TCA) were obtained from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO, USA). Streptomycin sulfate, disodium ethylenediamine tetraacetate (EDTA Na2), tert-Butylhydroquinone (THBQ), α-tocopherol, 1, 1-diphenyl-2picrylhydrazyl (DPPH) bought from Shanghai Aladdin Reagent Co., Ltd. All these chemicals were reagent grade.
Where DPPH blank is the absorbance of 4 mL of 99.5% ethanol solution plus 1 mL of 0.02% DPPH solution; control sample is the absorbance of 4 mL antioxidant solution (TBHQ or L-carnosine at different concentration) plus 1 mL 99.5% ethanol; and DPPH sample is the absorbance of 4 mL of antioxidant solution (TBHQ or L-carnosine at different concentration) plus 1 mL of 0.02% DPPH solution. 2.6.2. Measurement of spectral changes of metMb after incubation with Lcarnosine The mixture of metMb and L-carnosine was incubated according to Decker (Decker, Chan, Livisay, Butterfield, & Faustman, 1995) method with minor modifications. L-carnosine and metMb were mixed in 2.5 mM PBS buffer (pH 7.4) at the ratio of 50 μM: 300 μM. The mixture was incubated at 20 °C. Then scanned the mixture using UV-2600 Spectrophotometer in wavelengths between 370 nm and 450 nm (Shimadzu Instruments, Inc, Japan) at time intervals according to the experimental design.
2.2. Preparation of washed cod muscle Codfish (Gadus morhua) was obtained from a local fish market. Washed cod muscle (WCM) was prepared as described previously (Richards, He, & Grunwald, 2009). About 0.2 g g WCM were used for moisture content analysis. The rest of the samples were vacuum-packaged and stored at −40 °C for further use. 2.3. Preparation of metMb MetMb was prepared according to Lee and Richards' method (Lee, Tatiyaborworntham, Grunwald, & Richards, 2015).
2.6.3. Circular dichroism spectroscopy For evaluation of the effect of L-carnosine on structure of metMb, Circular Dichroism Spectroscopy was used to measure the incubated mixture of Mb and L-carnosine according to Richards' method (Richards et al., 2007). MetMb was firstly incubated with L-carnosine at 4 °C for 24 h. Then the mixture (containing 4.8 μM metMb) was transferred to a 2-mm cuvette containing 20 mM sodium phosphate buffers at pH 5.7. The sample was scanned from 260 to 190 nm by a Chirascan circular dichroism spectrometer (Chirascan v.4.4.2.0, Applied PhotoPhysics, Inc, UK) to determine secondary structure of metMb.
2.4. Addition of metMb to WCM MetMb was mixed with WCM in a brown bottle according to Richards and Li's method (Richards & Li, 2004) with minor modifications. In brief, the 1 mM metMb in washing buffer was added to WCM and the final metMb concentration was 40 μmol/kg WCM. Adjusted the pH of the mixture with 1 M HCl at 25 μL 1M HCl/g WCM to 5.7.2% streptomycin sulfate in milli-Q water was added to WCM mixture so that the final streptomycin sulfate was 200 ppm to inhibit growth of bacteria. Milli-Q water was added, as needed, to the brown bottle to adjust the moisture content to 88%.
2.6.4. Determination of lipid peroxides Lipid peroxides (LHP) were determined as previously described (Richards et al., 2007) with minor modifications. In brief, 0.5 g WCM in 50 mL glass tube was added 5 mL cold chloroform-methanol (1:1), mixture followed by homogenizing the mixture 30 s at 10,000 r/min. Transfer the homogenate into a new 15 mL glass tube. Then add another 5 mL cod chloroform-methanol mixture into above 50 mL sample glass tube, homogenized 30 s and transferred into above 15 mL glass tube, and added 3.00 mL cold 0.5% NaCl, vortex 30 s. Centrifuge the 15 mL glass tube 6 min at 1800 g, at 4 °C. Collected 2 mL of the lower layer from each tube to a clean glass tube, then, 1.33 mL of ice-cold chloroform/methanol (1:1) was added, followed by adding 25 μL cold ammonium thiocynate (3.94 M) and FeCl2 (18 mM, freshly prepared)
2.5. Addition of antioxidants or EDTA to WCM Prior to the addition of metMb to WCM, the antioxidants (L-carnosine, THBQ, α-tocopherol) or EDTA were added according to Richards and Li's method (Richards & Li, 2004). To be specific, 9% L-carnosine in milli-Q was added to WCM, the final concentration of L-carnosine in WCM as the experimental design (0,1%, 0.2%, 0.3%, 0.5%, 1.0%, 1.5%); the THBQ and α-tocopherol were dissolved in ethanol at the concentration of 9% first, then added to WCM to the final concentration of 1.5%; the EDTA in milli-Q water at the concentration of 3% was 704
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Fig. 1. Lipid Hydroperoxides (LHPs) values (A), thiobarbituric acid reactive substances (TBARS) (B), and hexanal content (C) in washed cod muscle treated with metMb during 2 °C storage at the present of different concentration of L-carnosine (0.1%. 0.3%, 0.5%, 1.5%) and 1.5% α-tocopherol and 1.5% TBHQ. The concentration of metMb was 40 μmol/kg washed cod, pH was 5.7. Replicates per treatment was n = 3. Means and standard deviations are shown.
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Fig. 2. Thiobarbituric acid reactive substances (TBARS) in WCM treated with 1.5% EDTA and 1.5% L-carnosine.
3. Results
accordingly, vortexed 5 s after each regent was added. Incubated above mixture at room temperature at the absence of light and read the absorbance at 500 nm immediately after incubation time was up. Standard curve was constructed using cumene hydroperoxide as standard. The lipid peroxides in WCM were expressed as μmol LHP/kg muscle.
3.1. Effect of different antioxidants (L-carnosine, α-Tocopherol, TBHQ) on lipid oxidation in WCM mediated by metMb The ability of L-carnosine at different concentrations (0.1%–1.5%) to inhibit lipid oxidation stimulated by metMb in WCM (pH = 5.7) was measured during storage at 2 °C. The formation of TBARS, hexanal, and LHPs were used as indicators of lipid oxidation. The results showed that L-carnosine in the range from 0.5% to 1.5% in WCM displayed highly inhibitory effects on lipid oxidation induced by metMb, while no inhibitory effect was noted with 0.1%–0.3% L-carnosine based on LHPs formation, TBARS values, and hexanal content (Fig. 1). To be specific, the LHPs formation, TBARS values, and hexanal content in 0.1%–0.3% L-carnosine-containing groups have no significant differences with control (WCM + metMb group). However, all above indicators in 0.5%–1.5% L-carnosine-containing groups were significantly lower than that in control and 0.1%–0.3% L-carnosine-containing groups. The LHPs formation in control group up to maximum (405 μmol LHP/kg muscle) on 1.0 days, was 7.4 times more than that in 1.5% L-carnosine treated group (55 μmol LHP/kg muscle) (Fig. 1A); TBARS values in control group up to maximum (76.43 μmol TBARS/kg muscle) on 1.5 days, was 4.6 times than that in 1.5% L-carnosine treated group (16.6 μmol TBARS/kg muscle) (Fig. 1B); and the hexanal content reached maximum on 2 days in control, 0.1% L-carnosine, 0.3% L-carnosine-containing treatments (0.54705, 0.515, 0.48145 μmol/kg muscle,
2.6.5. Determination of thiobarbituric acid reactive substances (TBARS) The value of TBARS was measured according to Richards and Dettmann's method with minor modifications (Richards, Dettmann, & Grunwald, 2005). Briefly, 0.12 g of muscle sample was added in 1.5-mL microfuge tube and mixed with 1.2-mL solution of 50% TCA with 1.3% TBA. The tubes were inverted for 10 times, heated at 65 °C in the absence of light for 1 h, and then cooled at 4 °C in the dark before centrifuged at 16,100 g and 4 °C for 5 min. Absorbance of supernatants was read at 532 nm and 600 nm. The real absorbance of supernatants was calculated as A532nm-A600nm. A standard curve was constructed using 1,1,3,3-tetraethoxypropane. Concentrations of TBARS in samples were expressed as μmol TBARS/kg muscle.
2.6.6. Determination of hexanal content Hexanal content in WCM was measured through gas chromatography (GC) method fitted with solid-phase micro-extration (SPME) technology. The samples were prepared according to Thiansilakul et al. method (2012) (Thiansilakul, Benjakul, Grunwald, & Richards, 2012). GC analysis was performed on a GC module 2010 Plus (SHIMADZU Technologies, Japan) equipped with a capillary column (DB-5, 30 m × 0.25 mm × 0.1 μm) and flame ionisation detector (FID). The method and temperature program according to Thiansilakul's method (Thiansilakul et al., 2012). The commercial hexanal was used as the standard. Identification and quantification were done by external standards and a standard curve. Samples and standard were run in triplicate.
Table 1 TBARS values in WCM. L-carnosine was heated at 100 °C for 15 min, followed by cooling on top water, before it was added to WCM.
2.7. Statistical analysis Data were analyzed using one-way analysis of variance (ANOVA) with SPSS (version 12.0). The number of experimental replicates was two or three. Means were separated using the Tukey's Multiple Range Test. Significance was determined using P-value less than 0.05.
Time (days)
Control
L-carnosine (1.5%) (heat 15 min at 100 °C)
L-carnosine
0 0.5 1 1.5 2 3
6.84 ± 0.15 xa 35.24 ± 5.04 xb 59.26 ± 3.70 xc 76.43 ± 0.30 xc 72.52 ± 4.71 xc 66.26 ± 1.12 xd
5.25 6.99 3.78 4.21 4.51 4.68
3.30 ± 1.15 xa 4.57 ± 0.09 ya 4.10 ± 0.79 xa 3.94 ± 0.18 za 6.86 ± 0.28 za 10.47 ± 0.34 zb
± ± ± ± ± ±
.0.61 xa 0.84 ya 0.23 xa 0.67 za 0.79 za 0.52 za
(1.5%)
x-z – means in the same row with the same letter are not significantly different at p > 0.05. a-d – means in the same column with the same letter are not significantly different at p > 0.05. 706
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significantly higher (P < 0.05) than those with either TBHQ or L-carnosine-treated groups (Fig. 1B), which remained less than 10 μmol/kg muscle. The maximum TBARS values in 1.5% α-tocopherol treated group was 3.5, 2.9 times more than that in 1.5% TBHQ-, 1.5% L-carnosine-containing groups, respectively. The content of hexanal in all treatments showed in Fig. 1C. In 1.5% L-carnosine-, 1.5% TBHQ-containing groups, the haxanal content did not display significant differences (P > 0.05) during the whole storage period and kept at significantly lower level (< 0.05 μmol/kg muscle) compared to control. In 1.5% α-tocopherol treatment, the hexanal content was elevated greatly on 2 days and up to 0.1533 μmol/kg muscle on 3 days, which was significantly higher than that in 1.5% L-carnosine-, 1.5% TBHQ-containing groups. All above results and analysis demonstrated that L-carnosine would be as effective as TBHQ and much more effective than αtocopherol against lipid oxidation in meat. The effect of EDTA on lipid oxidation in WCM was shown in Fig. 2. The data showed that there is no significantly different between EDTA-
respectively), was about 9–14 times more than that in 1.5% L-carnosine treated group (0.0495 μmol/kg muscle) (Fig. 1C). The inhibitory percent in 1.5% L-carnosine treated group up to 87% on 0.5 days based on TBARS values. To examine the potential of L-carnosine acting as an antioxidant, its antioxidative capacity was compared with THBQ, a synthetic antioxidant broadly used in food, and α-tocopherol, another highly active and natural at the concentration of 1.5%. The results showed in Fig. 1. From the perspective of LHPs values, there was no significant difference (P > 0.05) between TBHQ and L-carnosine treatments. However, the LHPs values were significantly elevated in α-tocopherol treated group on 1.0 days and significantly higher than that in TBHQ and L-carnosine treated groups (Fig. 1A). The TBARS values were no significant differences (P > 0.05) between TBHQ and L-carnosine treatments during whole meat storage period. However, it was significantly elevated after 1.5 days of storage in the 1.5% α-tocopherol-treated group and reached maximum (30.79 μmol/kg muscle) after two days of storage. And
Fig. 3. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of TBHQ (A) and L-carnosine (B). Replicates per treatment was n = 3. Means and standard deviations are shown. 707
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Fig. 5. Circular Dichroism Spectroscopy of metMb and metMb-L-carnosine. Fig. 4. Effect of L-carnosine on the spectrum of metMb. metMb was incubated with L-carnosine at 20 °C in 2.5 mM PBS buffer (pH 7.4). The ratio of metMb and L-carnosine was 50 μM: 300 μM.
treated group and control, and the TBARS values were significantly higher than that in 1.5% L-carnosine.
that the molar ellepticity value in control metMb was much more negative than that in the metMb incubated with L-carnosine, suggesting that the number of α-helix in metMb or the secondary structure of metMb might have been greatly affected after metMb is mixed with Lcarnosine.
3.2. Thermal stability of L-carnosine as antioxidant
4. Discussion
In meat processing, the antioxidant activity of antioxidant might commonly be lowered or disappear due to heat denaturation. To measure the thermal stability of L-carnosine in antioxidant activity, Lcarnosine was heated at 100 °C for 15 min before it was added to WCM. The TBARS value in the WCM with heat-treated L-carnosine maintained a low level (< 6.99 μmol/kg muscle) and was not significantly different from non-treated L-carnosine during the whole storage period (Table 1), which indicates that the antioxidant activity of L-carnosine is not affected by heat.
Antioxidant activity of L-carnosine is concentration-dependent (Lee & Hendricks, 1997; Lee, Hendricks, & Cornforth, 1999). L-carnosine can effectively inhibit lipid oxidation induced by metMb in washed cod muscle in the concentration range from 0.5% to 1.5%, which is in accordance with previous results (Lee & Hendricks, 1997). But it did not display inhibitory effect when the concentration ranged from 0.1% to 0.3% (Fig. 1). Decker et al. (Decker & Crum, 1991) reported that, at the concentration of 0.5% and 1.5%, the L-carnosine can effectively inhibit the formation of lipid peroxides and TBARS in uncooked and salted pork during frozen storage up to 6 months. Fig. 1 shows that L-carnosine (1.5%) has stronger inhibitory effect on lipid oxidation in WCM than αtocopherol (1.5%). However, Liu et al. (Liu et al., 2015) reported that Lcarnosine and α-tocopherol showed equivalent inhibitory effect in raw beef patties, which is different from our observation. This difference may be due to the different testing systems used in experiments. In their case, some endogenous pro-oxidants and antioxidants, which were present in raw beef patties, might either act with added antioxidants or directly inhibit meat lipid oxidation, interfering the antioxidant effect of L-carnosine and α-tocopherol in experiments (Boldyrev, Dupin, Aya, Babizhaev, & Severin, 1987). Our data also show that heating L-carnosine at 100 °C for 15 min before added to WCM had no effect on its antioxidative activity (Table 1). The same result was reported by Decker and Faraji (Decker & Faraji, 1990) with a liposome model. This indicates that antioxidant capability of L-carnosine is thermally stable and can remain its antioxidative activity during thermal processing. Our results, in line with these published discoveries, indicate that L-carnosine is an effective natural antioxidant and can be added to raw meat before processing for inhibiting lipid oxidation in either raw or cooked finish products. Antioxidants are usually classified into four types based on their mechanisms against lipid oxidation, chain breaker, peroxide decomposer, metal chelator, and free radical scavenger (Yen, Chang, Lee, & Duh, 2002). Experiments have shown that different mechanisms are involved in inhibition of lipid oxidation by L-carnosine. Decker et al. (Decker, Crum, & Calvert, 1992) attributed antioxidant effect of L-carnosine to metal chelating. Chan et al. (Chan, Decker, Jin, & Butterfield, 1994) ascribed it to free radical scavenging. Decker (Decker et al., 1995) found that L-carnosine inhibited lipid oxidation through
3.3. Free radical scavenging activities Radical scavenging ability of L-carnosine was measured using DPPH and compared with TBHQ (Fig. 3). The results showed that L-carnosine hardly had any free radical scavenging ability when its concentration was < 1 mg/mL, however, it was very effective (≥71%) when its concentration was ≥5 mg/mL (Fig. 3B). On the other hand, TBHQ showed high free radical scavenging ability (≥78%) even at a much low concentration ( ∼ 5 μg/mL) (Fig. 3A). By contrast, the L-carnosine did not have any free radical scavenging ability at this concentration (data not show). This indicates that TBHQ is much more effective in scavenging free radicals compared with L-carnosine. 3.4. Effect of L-carnosine on spectrum of metMb To examine the possible effect of L-carnosine on the structure of metMb, we tested characteristic spectra of metMb in the wavelength between 370 nm and 450 nm. Results showed that Soret band of metMb was immigrated from 409 to 420 after mixing and incubating with Lcarnosine and the maximum absorbance (1.2512, 0.5887, 0.531, respectively) decreased greatly (Fig. 4). These data indicate that metMb structure or form might have been changed after incubated with Lcarnosine, such as becoming a hemichrome. 3.5. Effect of L-carnosine on molar ellepticity values of metMb Molar ellepticity values around 192 nm and 222 nm can be used as indicator for the secondary structure, α-helix, of proteins. Fig. 5 shows 708
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Acknowledgement
quenching the ferryl-myoglobin radicals formed by hydrogen peroxideactivated metMb. The same author also reported that L-carnosine could quench unsaturated aldehydic lipid oxidation products, 4-hydroxy-2trans-nonenal (HNE). Grunwald (Grunwald, Tatiyaborworntham, Faustman, & Richards, 2017) reported that HNE can covalently bound to sperm whale Mb, and the adducted Mb promoted lipid oxidation in washed muscle more effectively than non-adducted Mb. Thus, quenching could be another mechanism for L-carnosine to reduce lipid oxidation induced by Mb in meat. In addition, Nagasawa et al. (Nagasawa, Yonekura, Nishizawa, & Kitts, 2001) found that either Lhistidine or β-alanine by itself did not affect lipid oxidation and they concluded that the dipeptide linkage in β-alanyl-histidine was essential for inhibitory effect of L-carnosine on lipid oxidation. In the present study, we hypothesized that in addition to metal chelating and free radical scavenging, the effect of L-carnosine on prooxidant metMb may also be involved in its inhibition of lipid oxidation. To determine the inhibitory effect of L-carnosine act as a metal chelator in lipid oxidation mediated by metMb, a comparison of L-carnosine to EDTA in inhibition of lipid oxidation was made using TBARS measurement results. The samples treated with EDTA (1.5%) showed no differences (P > 0.05) from the control group during the whole storage based on their TBARS values (Fig. 2). This is in agreement with Richards and Li's results (Richards & Li, 2004), indicating that the chelating activity of EDTA has no effect on lipid oxidation mediated with metMb in WCM. This is also supported by Grunwald and Richards' (Grunwald & Richards, 2006) findings that hemin released from metMb was the primary promoter of lipid oxidation in washed fish muscle other than free iron. On the other hand, a great inhibition of lipid oxidation by 1.5% L-carnosine demonstrates that the other mechanisms may be involved rather than chelation. The DPPH free radical scavenging experiment showed that TBHQ can scavenge almost 100% DPPH free radicals at 100 μg/mL concentration. By contrast, L-carnosine showed almost no scavenging ability at the same concentration (data not show). However, the TBARS values between TBHQ (1.5%) and L-carnosine (1.5%) treatments were similar to each other. These results indicate that the mechanism of Lcarnosine in inhibiting lipid oxidation may not be solely due to its free radical scavenging activity. The differences in visible spectroscopy and circular dichroism spectroscopy measurements between metMb and metMb-carnosine mixture suggest the structural change in metMb. In the spectrum of metMb-carnosine, the immigration of Soret band and the decrease of maximum absorbance could be due to the formation of hemichrome or other forms of metMb. The results of circular dichroism spectroscopy indicate that L-carnosine may affect the secondary structure of metMb. These data also suggest that L-carnosine may inhibit lipid oxidation in meat through changing metMb structures (eg. Unfolded the metMb) and subsequently affecting metMb pro-oxidant activity (Olcott & Lukton, 1961).
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5. Conclusions The present study demonstrated that L-carnosine can inhibit lipid oxidation mediated by metMb in a WCM system as effectively as TBHQ. As a natural antioxidant, L-carnosine showed much stronger antioxidant capability than α-tocopherol in the WCM system. By comparison with metal chelating reagent EDTA and effective free radical scavenging chemical TBHQ, our results suggest that the inhibition of lipid oxidation in meat by L-carnosine may not be due to only the chelation of prooxidant metals and free radical scavenging. Indirect measurements of structure of metMb with spectroscopic methods indicate that the inhibitory effect of L-carnosine may be involved in inducing structural change of pro-oxidant metMb, which further inactivates the pro-oxidant property of metMb. However, further study is needed to provide direct evidence that L-carnosine can cause structure changes in pro-oxidant metMb and thereafter inhibit lipid oxidation in meat products. 709
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