Conjugation with alginate oligosaccharide via the controlled Maillard reaction in a dry state is an effective method for the preparation of salmon myofibrillar protein with excellent anti-inflammatory activity
The efficacy of the controlled Maillard reaction with alginate oligosaccharide (AO) in a dry state was examined for development of an anti-inflammatory compound from fish myofibrillar protein (Mf). Lyophilized Mf from spawned-out chum salmon was mixed with AO (half of the total protein weight) and incubated at 60 °C and 35 % relative humidity for 0–6 h, followed by digestion with pepsin and trypsin. The anti-inflammatory activity of the digested peptide was improved with the progress of the AO conjugation, and dMSA4 (prepared from Mf–AO conjugate by reaction for 4 h, with 49.6 μg/mg protein of AO attached) was most effective in inhibiting secretions of inflammation-related compounds in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Likewise, dMSA4 suppressed the LPS-induced gene and protein expression of inducible nitric oxide synthase, tumor necrosis factor-α, interleukin-6, and cyclooxygenase (COX)-2, while COX-1 expression was unaffected. Furthermore, oral administration of dMSA4 and the Mf–AO conjugate prior to proteolysis inhibited carrageenan-induced paw volume expansion in mice. These results indicate that AO conjugation using the controlled Maillard reaction in a dry state is a useful approach for enhancing the anti-inflammatory activity of salmon Mf as a nutraceutical food material.
KeywordsAlginate oligosaccharide Anti-inflammation Maillard reaction Muscle protein Salmon
Inflammation is an important host defense reaction by the immune system, which protects the body from harmful stimuli such as invasive pathogens. Immune cells including macrophages, natural killer cells, and dendritic cells eliminate foreign substances such as viruses and bacteria via inflammatory signaling cascades . However, recent reports have indicated that excess or chronic inflammation is associated with diseases such as inflammatory bowel disease, diabetes, cardiovascular disease, and neurodegenerative disease [2, 3, 4, 5], suggesting that the regulation of excess and chronic inflammation is important for health maintenance.
It has also recently been recognized that lifestyle, and diet in particular, is an important factor for mitigating excess inflammation , with increasing interest in the use of food components to regulate inflammation. A number of small organic molecules in food components have been shown to possess anti-inflammatory properties [7, 8, 9, 10, 11]. Likewise, some food proteins [12, 13] have been found to exhibit anti-inflammatory activity. These findings suggest that anti-inflammatory effects may be an additional health benefit of foods.
Global consumption of seafood has increased steadily over the past half-century , and fish as an important protein source is widely utilized as a raw material for processed seafood. Various processed foods are manufactured using fish myofibrillar proteins (Mf), given their excellent functional properties, including their emulsifying and gel-forming ability and water-holding capacity. There is also increasing interest in the health benefits of seafood, as many epidemiologic studies have provided evidence of the beneficial effects of fish consumption, and fish Mf and Mf-derived peptides have been shown to exert angiotensin-converting enzyme (ACE) inhibitory effects  and antioxidant activity , and to prevent skeletal muscle insulin resistance . Indeed, ACE-inhibitory peptides from fish Mf are marketed as functional materials defined as Food for Specified Health Uses (FOSHU) approved by the Japanese governmental Consumer Affairs Agency . However, few studies have been reported on the anti-inflammatory activity of Mf and Mf-derived peptides.
The functional properties of food proteins can be improved by conjugation with glycosyl units using the Maillard reaction , which commonly takes place between food components during food processing and preservation. This non-enzymatic reaction occurs between the reducing terminus of sugars and the amino groups of proteins, and is suitable for application in the modification of food proteins, as no chemical reagents are required. For example, the antioxidant activity of phosvitin , the radical-scavenging activity of soy protein , the antimicrobial activity of lysozyme , and the immunoreactivity of hazelnut allergen  were shown to be enhanced by conjugation with reducing glycosyl units including glucose, galactomannan, curdlan, and allose. Likewise, Isono et al. . reported that conjugation of chicken breast Mf with maltose increased its antioxidant activity.
The protein glycosylation described above proceeded via the Maillard reaction under conditions of elevated temperature and humidity (e.g. >50 °C and/or >65 % relative humidity). In our previous study , we demonstrated that peptides derived from fish Mf lyophilized with alginate oligosaccharide (AO) possessed anti-inflammatory activity, which was conferred by attaching a small amount of AO to the protein via the Maillard reaction during lyophilization. However, further study showed that the degree of AO modification in lyophilized Mf often varied even under identical lyophilization conditions, suggesting that the Maillard reaction under lyophilization was not well controlled. Thus it appeared that a controlled AO conjugation system should be adopted for the production of anti-inflammatory compounds from fish Mf.
The objective of this study was to elucidate the efficacy of the controlled Maillard reaction with alginate oligosaccharide (AO) in a dry state, with controlled temperature and humidity, in developing fish protein material with excellent anti-inflammatory properties. Here, Mf was prepared from spawned-out chum salmon Oncorhynchus keta, which is an underutilized protein resource because of its unacceptable soft texture and unpleasant taste and smell. AO was selected as the glycosyl unit to attach to the target protein as it has been found to be superior in enhancing the food functionalities of fish Mf [26, 27]. The chum salmon Mf was mixed with AO and sorbitol (protein denaturant), and the mixture was incubated under controlled temperature and humidity conditions to prepare the Mf–AO conjugates containing different quantities of AO. The effect of the AO conjugation on enhancing the anti-inflammatory activity of Mf was evaluated using lipopolysaccharide (LPS)-stimulated macrophages and carrageenan-induced paw edema in mice as a model of acute inflammation. The effect on gene and protein expression related to secretion of inflammation-related compounds in macrophages was also examined.
Materials and methods
Spawned-out chum salmon (average weight 3 kg) was obtained from the Hekirichi River hatchery of Hokkaido Prefecture (Hokuto, Hokkaido, Japan). They were immediately chilled, gutted, dressed, and frozen at −25 °C until use. AO (mean degree of polymerization = 6) prepared by alginate-lyase degradation was supplied from Hokkaido Mitsui Chemical Industry Co., Ltd. (Sunagawa, Hokkaido, Japan). The RAW 264.7 murine macrophage cell line was purchased from American Type Culture Collection via Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan). Male ICR mice (age 7 weeks) were obtained from Charles River Japan, Inc. (Yokohama, Japan). Pepsin from porcine mucosa, trypsin from bovine pancreas, and lipopolysaccharide (LPS) from Salmonella typhimurium were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and non-essential amino acids were purchased from Life Technologies (now Thermo Fisher Scientific, Carlsbad, CA, USA). Anti-murine tumor necrosis factor-α (TNF-α), anti-murine interleukin-6 (IL-6), biotinylated anti-murine TNF-α, biotinylated anti-murine IL-6, horseradish peroxidase (HRP)-conjugated streptavidin, and 3,3',5,5'-tetramethylbenzidine (TMB) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The recombinant TNF-α and IL-6 were purchased from BioVision, Inc. (Milpitas, CA, USA). Anti-murine β-actin rabbit IgG, anti-murine iNOS rabbit IgG, and peroxidase-conjugated goat anti-rabbit IgG antibody were purchased from MBL International Corporation (Nagoya, Japan), Cell Signaling Technology Japan, K.K. (Tokyo, Japan), and Bio-Rad Laboratories (Hercules, CA, USA), respectively. Anti-murine COX-1 rabbit IgG and anti-murine COX-2 rabbit IgG were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Immobilon Western Chemiluminescent HRP Substrate was purchased from EMD Millipore (Billerica, MA, USA). Indomethacin and carrageenan were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan), with the exception of the following: bovine serum albumin fraction V (BSA; Merck KGaA, Darmstadt, Germany), Bio-Safe Coomassie G-250 stain (Bio-Rad Laboratories, Hercules, A, USA), interferon-γ (IFN-γ; PeproTech, Inc., Rocky Hill, NJ, USA).
Preparation of salmon Mf
The salmon Mf was prepared as previously described . Briefly, the thawed salmon muscle was minced and washed, and the protein suspension was homogenized. The homogenate was centrifuged to collect the Mf pellet, which was mixed with a cryoprotectant (sorbitol) or AO as follows: sorbitol and AO were added at amounts equal to or half of the total protein weight, respectively, and the protein–sugar mixtures were lyophilized by freeze-drying. In this study, two types of lyophilized samples were prepared, Mf mixed with sorbitol (MS) and Mf mixed with sorbitol and AO (MSA), and samples were stored at −30 °C until use.
Conjugation of Mf with AO
Since denaturation of salmon Mf and the loss of its salt solubility occurs in the dry state under conditions of high humidity (>65 % relative humidity) , a low-humidity condition was selected for this study. Thus AO conjugation of MSA was performed in a controlled dry state at 60 °C and 35 % relative humidity in a humidity cabinet (model PR-1G; Tabai ESPEC Corp., Tokyo, Japan) for 0, 2, 4, and 6 h (referred as to MSA0, MSA2, MSA4, and MSA6, respectively).
Enzymatic digestion of MS and MSA
Salmon Mf samples (MS and MSAs) were suspended in 50 mM NaCl and then precipitated in 60 % saturated ammonium sulfate at pH 7.5 to remove sorbitol and unbound AO. After dialysis against 50 mM NaCl, the protein suspensions were digested by pepsin (pH2.0) and trypsin (pH8.0) at 37 °C for 3 h each (enzyme Mf = 1:100 [w/w]). The digested MS and MSA samples were boiled for 15 min, lyophilized, and stored at −30 °C until use. The corresponding enzymatically digested samples as described above are denoted as dMS and dMSA0, dMSA2, dMSA4, and dMSA6, respectively. Before use in cell culture experiments, the digested Mf samples were dissolved in phosphate buffered saline (pH 6.8) and filtered through a 0.22-µm membrane.
Electrophoretic analysis of AO-conjugated protein
The conjugation of MSA with AO was visualized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), according to the method described by Laemmli , with 4.0 and 7.5 % acrylamide slab gels used as the stacking and resolving gels, respectively. Samples were prepared as follows: 0.1 ml of the protein solution was added to 0.2 ml of 20 mM Tris–HCl (pH 8.0) containing 2 % SDS, 8 M urea, and 2 % β-mercaptoethanol and was heated for 2 min, after which 10 μl was loaded onto the gel. Coomassie Brilliant Blue R-250 and Lillie's Cold Schiff’s Reagent were used for protein and carbohydrate staining , respectively.
Measurement of protein concentration and amount of AO bound to Mf
The protein concentration of each sample was determined by the Biuret method  using bovine serum albumin as a standard, and the AO concentration was determined by the phenol–sulfuric acid method . The amount of AO bound to the protein was expressed as µg/mg of the protein. The assays were carried out after removal of sorbitol and unreacted AO as described above.
Measurement of remaining lysine content
Following reaction with AO, the protein was precipitated with 15 % trichloroacetic acid (TCA) by incubation at room temperature for 30 min to remove the unbound AO. After removal of TCA and AO by centrifugation at 2,100×g for 30 min, the precipitated protein was resuspended in 50 mM phosphate buffer (pH 9.5) containing 2 % SDS. The available lysine content was determined by spectrophotometric analysis using o-phthalaldehyde and N-acetyl-l-cysteine . The available lysine content was calculated using the following equation: available lysine content (g/g protein) = 146.19 × absorbance at 335 nm/(6830 × protein concentration [mg/ml]), where 146.19 is the molecular weight of lysine and 6830 is the molar absorptivity of the OPA-lysine derivative.
Analysis of molecular weight distribution of digested Mf
The digested and lyophilized Mf samples were dissolved in 50 mM NaCl at a concentration of 10 mg/ml, filtered through a 0.45-µm membrane, and analyzed by gel permeation chromatography (Superdex Peptide 10/300 GL; GE Healthcare Life Sciences, Little Chalfont, UK). Samples were eluted using a flow rate of 0.5 ml/min, and eluents were monitored at 220 nm.
RAW264.7 cells were cultured in DMEM containing 10 % heat-inactivated FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.1 mM non-essential amino acids at 37 °C in 5 % CO2.
Analysis of NO production
RAW264.7 cells (200 µl, 2 × 105 cells/well) were seeded in 96-well plates, allowed to adhere for 2 h, and washed twice with phosphate-buffered saline prior to treatment. Cells were cultured in 200 µl of fresh 10 % FBS-DMEM (without phenol red) containing dMSAs (0–500 μg/ml), IFN-γ (0.5 ng/ml), and LPS (2.5 ng/ml) for 24 h. The secretion of nitric oxide (NO) was directly measured in the culture supernatants using the Griess method  with sodium nitrite as a standard.
Analysis of TNF-α and IL-6 production by ELISA
The dMSAs (0–500 μg/ml) and LPS (2.5 ng/ml) were added to RAW264.7 cells seeded in 96-well plates and cultured in fresh 10 % FBS-DMEM as described above. After cultivation for 24 h, the culture supernatants were analyzed by sandwich enzyme-linked immunosorbent assay (ELISA) to measure TNF-α and IL-6 concentrations, as previously described . Briefly, the culture supernatants (50 µl) were added to 96-well ELISA plates (AGC Techno Glass Co., Ltd., Tokyo, Japan) coated with anti-murine TNF-α or anti-murine IL-6 as primary antibodies, and biotinylated anti-murine TNF-α or biotinylated anti-murine IL-6 as secondary antibodies. The concentrations of TNF-α and IL-6 were calculated by standard curves using recombinant cytokines.
Analysis of iNOS and COX protein expression by immunoblotting
RAW264.7 cells (3.0 ml, 3 × 106 cells/well) were seeded in 6-well plates for 2 h, and then cultured in 2.7 ml of fresh 10 % FBS-DMEM containing dMSAs (0–500 μg/ml), IFN-γ (0.1 ng/ml), and LPS (5.0 ng/ml) for 24 h. Total protein was extracted from the cells using lysis buffer (62.5 mM Tris–HCl, 2 % SDS, 10 % glycerol, 50 mM DTT, 0.01 % bromophenol blue), and the samples were heated at 95 °C for 5 min. Samples were subjected to Laemmli SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; EMD Millipore, Billerica, MA, USA) using a semi-dry blotting system (ATTO Corporation, Tokyo, Japan). The membrane was incubated for 1 h at room temperature in blocking buffer (5 % nonfat dry milk dissolved in 20 mM Tris–HCl, pH 7.6, containing 0.1 % Tween and 140 mM NaCl [TTBS]) and then incubated with the primary antibody at 4 °C overnight. The four primary antibodies were diluted with blocking buffer as follows: anti-murine β-actin rabbit IgG (1:10,000), anti-murine iNOS rabbit IgG (1:1000), anti-murine COX-1 rabbit IgG (1:500), and anti-murine COX-2 rabbit IgG (1:10,000). After washing three times with TTBS, the membrane was incubated with peroxidase-conjugated goat anti-rabbit IgG antibody (1:20,000) at 37 °C for 3 h. After three washes with TTBS, bound antibodies were detected with Immobilon Western Chemiluminescent HRP Substrate and visualized using a CCD camera (ATTO Corp., Japan). The intensity of each band was measured using an image analyzer (CS Analyzer; ATTO Corp., Japan) and was normalized with respect to β-actin (internal control). The resulting iNOS and COX-1 and -2 protein expression is presented as a percentage relative to control (untreated) cells.
Analysis of gene expression of inflammatory mediators
RAW264.7 cells (12.0 ml, 1.0 × 106 cells/ml) were seeded into plastic dishes (diameter: 100 mm) and allowed to adhere for 2 h. Cells were then cultured in fresh 10 % FBS-DMEM containing dMSAs (0–500 µg/ml), IFN-γ (0.1 ng/ml), and LPS (5.0 ng/ml) for 24 h. Extraction of total RNA from the culture cells, reverse transcriptase polymerase chain reaction (RT-PCR), and the primers used in the experiment were described in the previous work . The PCR products were electrophoresed on 2.0 % agarose gel. After staining with ethidium bromide, the DNA bands were detected at 312 nm, and the reverse images are presented. The intensity of each DNA band was measured using the image analyzer and was normalized with respect to β-actin (internal control). The resulting mRNA expression is presented as a percentage relative to control (untreated) cells.
Carrageenan-induced paw edema in mice
In vivo anti-inflammatory activity was measured using a carrageenan-induced paw edema model in mice. Male ICR mice (7 weeks, 8 mice/group) were housed in a temperature- and humidity-controlled environment, and provided ad libitum access to food and water. Mice were then subjected to the experiment following 16 h of fasting. All chemicals and samples used in the animal experiment were dissolved or suspended in sterile saline. Mice in the test group received oral administration of MSA4 or dMSA4 (300 mg/kg of body weight). Negative-control mice received sterile saline only and positive-control mice received indomethacin (10 mg/kg of body weight), both via an oral route. After a period of resting for 1 h, edema was induced in the right hind paw by an intraplantar injection of 40 µl of 1 % (w/v) carrageenan solution, and the paw volume was measured and the increasing rate of paw edema (%) calculated by the method described in the previous work . The experimental protocol was approved by the Committee for Animal Care and Use of Hokkaido University, and the animal experiment was performed in accordance with the Hokkaido University guidelines for the care and use of laboratory animals.
Data are presented as mean ± SEM. Statistically significant differences were assessed by the Tukey–Kramer method, at p < 0.05.
Characterization of Mf after conjugation with AO and molecular mass distribution of the digested peptides
Available lysine content and AO bound to MS and MSA
Available lysine content (%)
AO bound to protein (µg/mg)
Suppressive effect of digested Mf–AO conjugates on NO and pro-inflammatory cytokine secretion in macrophages
Effect of dMSA on the protein and gene expression of inflammation-related compounds and COXs
Anti-inflammatory effects of dMSA4 in a model of acute inflammation in mice
As presented in Table 1, the amount of AO attached to Mf increased with the incubation of the lyophilized Mf–AO mixture under controlled dry conditions at 60 °C and 35 % relative humidity, and the enhanced anti-inflammatory activity of Mf was observed with the progress of the AO conjugation. The results of this work clearly indicate that the Maillard reaction under a controlled dry state is useful for the production of anti-inflammatory fish Mf.
AO conjugation during lyophilization was observed in MSA0, which contained 8.9 µg/mg of AO, whereas 19 µg/mg of AO was conjugated to Mf during lyophilization in the previous work . The variance of the degree of AO conjugation suggests that the Maillard reaction under low-temperature conditions was not precisely controlled. In addition, the anti-inflammatory effect of dMSA0 (Fig. 3a) was reduced in comparison with that observed in the previous work , indicating that the level of AO conjugation in the lyophilization process affected the functional improvement of Mf. Therefore, utilization of AO conjugation with lyophilization is unsuitable for developing an anti-inflammatory compound from fish Mf.
To investigate the enhanced anti-inflammatory activity with the progress of AO conjugation, digested Mf conjugated with different amounts of AO (Table 1) was subjected to in vitro macrophage assay. When the digested Mf samples were subjected to anti-inflammatory assays in LPS-stimulated RAW264.7 cells (Figs. 3 and 4), results clearly showed that conjugation of Mf with AO enhanced the suppressive effects on inflammation-related compounds (NO, TNF-α, and IL-6) of digested salmon Mf peptides. Some studies have reported cytotoxicity of the Maillard reaction products, such as glyceraldehyde-related products and methylglyoxal 3-deoxyglucosone [18, 34], but 1,000 µg/ml of the digested Mf samples in this work had no negative effect on the viability of the RAW 264.7 cells after cultivation for 48 h (data not shown). Additionally, AO that is not attached to Mf showed no inhibition of inflammation-related compound production in LPS-stimulated RAW 264.7 cells (Fig. 4). Therefore, the result of Fig. 3 would indicate that conjugation of Mf with AO enhanced the anti-inflammatory activity of digested salmon Mf peptides. The results described above raise the possibility that the anti-inflammatory compounds contained in the digested Mf samples are digested and/or AO-conjugated peptides. As shown in Fig. 3a, dMS, which was not conjugated with AO, showed anti-inflammatory activity, suggesting that the pepsin-trypsin digestion produced anti-inflammatory peptides from salmon Mf. Additionally, a marked decrease in the production of inflammation-related compounds upon treatment with dMSA2, dMSA4, or dMSA6 suggests the involvement of AO-conjugated peptides in enhancing anti-inflammatory activity. Indeed, the existence of multiple anti-inflammatory peptides in dMSA0 has been suggested using isoelectric focusing chromatography . Structural changes in AO-conjugated protein occurring with the progression of the Maillard reaction may also be related to the anti-inflammatory effect, as intermediate products of the Maillard reaction, such as 5-hydroxymethyl-2-furfural and 5-hydroxymethyl-2-furoic acid, have anti-inflammatory properties . The identification of the compounds responsible for the anti-inflammatory activity observed here is the next step in revealing the relationship between AO conjugation and increased anti-inflammatory activity.
We have examined the molecular mechanism of anti-inflammatory activity of dMSA, and found that dMSA4 exerted its anti-inflammatory activity by suppressing LPS-induced gene and protein expression. Interestingly, dMSA4 had no effect on COX-1 expression as opposed to suppression of COX-2 (Fig. 5). COX enzymes (COXs) are responsible for converting arachidonic acid to prostaglandins . COX-1 is constitutively expressed in most tissues, whereas COX-2 is induced by stimulation with mitogens such as LPS or pro-inflammatory cytokines, resulting in the production of inflammation-enhancing prostaglandins . Concurrent inhibition of COX gene expression is often caused by non-steroidal anti-inflammatory drugs, causing adverse effects such as gastric ulceration and renal toxicity . Hence, selective inhibition of COX-2 is required in anti-inflammatory compounds expected to avoid gastrointestinal and renal damage. dMSA4 may be a potential COX-2 selective inhibitor.
The expression of inflammation-related genes is regulated by signaling cascades in cells. For example, NF-κB, one of the transcriptional factors controlling the expression of inflammatory mediators, is activated by stimuli such as LPS, pro-inflammatory mediators, and oxidative stress . Most anti-inflammatory compounds exert their anti-inflammatory effects by inhibiting NF-κB signaling cascades [8, 40, 41, 42]. dMSA may also inhibit the activation of NF-κB signaling such as phosphorylation of NF-κB p65 or IκBα, as dMSA simultaneously reduced the expression of several NF-κB activated genes including TNF-α, IL-6, iNOS, and COX-2.
Finally, we carried out a test of carrageenan-induced edema in mice to examine the possible use of dMSA as an anti-inflammatory food material. Administration of dMSA4 showed an anti-inflammatory effect, regardless of digestion (Fig. 6), suggesting that anti-inflammatory peptides included in MSA4 could be released by enzymatic digestion (pepsin and trypsin) in vivo in the gastrointestinal tract. The fact that the oral administration of AO-conjugated salmon Mf reduced localized acute inflammation indicates its potential as a nutraceutical. The result shown in Fig. 6 indicates that the digestion process of Mf–AO conjugates is not essential for the exertion of anti-inflammatory effects. However, ingestion of the Mf–AO conjugate after enzymatic digestion is desirable for attaining effective anti-inflammatory activity, as gastrointestinal digestion tends to be affected by the health condition of organisms.
The anti-inflammatory mechanisms of dMSA4 in mice may be related to the inhibition of inflammation-related compounds, as the excess expression of pro-inflammatory cytokines iNOS and COX-2 are involved in the development of carrageenan-induced edema [43, 44, 45]. The pepsin-trypsin digestion may be important for the stable production of anti-inflammatory compounds, as protein digestion is often altered by protein denaturation during the storage process.
In conclusion, conjugation with AO using the Maillard reaction under controlled temperature and humidity conditions is a useful method for producing salmon Mf with excellent anti-inflammatory properties. Since conjugation with AO also improves the solubility, thermal stability, and emulsion-forming ability of fish Mf [27, 46, 47, 48], this work could contribute to the development of an edible anti-inflammatory fish product.
We deeply appreciate the technical assistance of Mr. Yutaka Shimizu, Ph.D., Hokkaido University, for instruction on immunoblotting strategy. We deeply appreciate Hideki Kishimura, Associate Professor, Hokkaido University, for instruction on PCR strategy. We thank Hokkaido Mitsui Chemical Industry Co., Ltd., for supplying alginate oligosaccharide. Part of this work was supported by the Japan Science and Technology Agency, A-STEP Program (FS stage), 2010.
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