Program

Chitosan-nisin (CS-nisin) microcapsules were prepared and characterized. Its preservation properties (chemical stability, microbiological stability, color and sensory properties) were examined during the storage of small yellow croaker (Pseudosciaena polyactis). The results showed that CS-nisin microcapsules were spherical in shape with particle size greater than 1μm. A significantly higher efficiency of CS-nisin microcapsules in inhibiting microorganism growth, lipid oxidation and protein degradation was observed, extending the shelf life of small yellow croaker during the storage by 6 to 9 days in comparision with CS, nisin treatment alone or the control according to the TVC assay, suggesting a better preservation effect of the microcapsules.

【Aim】Seaweeds are importantly economic species and contain varieties of active ingredients. In recent years, phlorotannins attract more attention for their functional activities. Undaria pinnatifida (U. pinnatifida) sporophyll is the by-product during the product processing. Therefore, it is meaningful to extract phlorotannins from U. pinnatifida sporophyll and determine its antioxidant and anti-inflammatory activities. And that will be helpful to develop functional food using phlorotannins.
【Methods】Phlorotannins were extracted by ethanol from U. pinnatifida sporophyll under the different condition. Single factor experiment was used to determine the total phenolic content in different processing conditions. The optimal conditions for extracting phlorotannins were obtained by response surface analysis. The antioxidant and anti-inflammatory activities of phlorotannins were determined using colorimetric methods and western-blotting analysis in RAW264.7 cells.
【Results】The optimum conditions for the phlorotannins extraction : heating temperature 170 ºC, heating time 5.2 h, ethanol concentration 52 %, and the yield of phlorotannins was 10.7±0.2 mg Gallic acid/g Dry weight. The phlorotannins showed good antioxidant and anti-inflammatory activities. The cell survival rate in the oxidative damage cell system involved in phlorotannins increased 1.29 times comparing with that of the H2O2 alone treated group. In the inflammatory cell model with phlorotannins, the concentration of NO decreased 88 % comparing with LPS treatment. It had no significant effect on the COX-2 expression between LPS and phlorotannins, but the iNOS expression was significantly inhibited by the phlorotannins comparing with LPS treatment. That means phlorotannins inhibited LPS-induced inflammatory through iNOS regulating pathway in RAW264.7 cells. This study has important theoretical significance for the development of functional health raw materials by using phlorotannins.

Real-time water dynamics in abalone during drying and rehydration processes was assessed using low field nuclear magnetic resonance (LF-NMR) relaxometry and magnetic resonance imaging (MRI) for the first time in this study. Four T2 components designated as strongly bound, lightly bound, immobilized, and free water were observed for abalone dried at 60 oC. Significant reduction of T2 relaxation time for all four components was found due to the heat-induced evaporation of water. MRI provided 2-Dimensional structure change of abalone dried at 60 oC for 16 h. A close correlation was observed between total area (ATotal) and the total moisture content (R2=0.982), and a prediction model with high correlation was established. Rehydration of dried abalone monitored by LF-NMR showed three T2 populations. The increased T2 relaxation time demonstrated an increased water mobility. MRI revealed the dynamics of water distribution during the rehydration process. High correlations between Texture profile analysis parameters (hardness, chewiness), rehydration ratio and NMR parameters of dried abalone were observed during the rehydration process.

Methodology of ice crystal analysis in hairtail samples was researched during three different freezing process, including conventional air freezing (CAF) at -20℃, refrigerator cryogenic freezing (RCF) at -80℃ and liquid nitrogen immersion freezing (LIF) at -196℃. An improved method based on cryo-section, a widely used histological tool, was applied to observe the formation of ice crystals in frozen hairtail meat, comparing with the traditional method of cryo-substitution. Results showed that cryosectioning without the need of paraffin-embedding is a highly effective tool for intuitionistic view of ice crystals, and greatly shortened the time from 1-2 weeks to 20-30 min. The equivalent diameter of the ice crystals determined by cryosectioning were 12.6±3.73, 3.62±1.91, and 1.22±0.39 µm for the samples subjected to CAF, RCF and LIF, respectively. Smaller and more regular intracellular ice crystals were observed in hairtail samples frozen by LIF compared with other treatments. In addition to these two sectioning methods, another two indirect methods of scanning electron microscopy (SEM) and X-ray micro computed tomography (X-ray CT) was analysed both based on the pores and spaces left by ice crystals through sublimation of ice. Two-dimensional representation of the microstructure of three freeze-dried samples was profiled by SEM. While X-ray CT technique was used to visualize the fiber-entangled networks in three dimensions (X, Y, and Z axes) of the three freeze-dried hairtails, and to determine the pore size distribution due to increase in ice crystal sizes. Significant differences were also observed in the water holding capacity in frozen samples of CAF (86.8%), RCF (87.4%), and LIF (89.2%). Methodology study of ice crystal characteristics will help our understanding of the crystallization phenomenon and its strong influence on the final quality of aquatic foods during freezing and storage.

The formation of ice crystals and the change of moisture of Penaeus chinensis treated by ultrasonic assisted freezing (UF), immersion freezing (IF) or air refrigerator freezing (RF), and the effect of different exposure time were investigated. The result showed that the time to pass through the zone of maximum crystallization was 130 s for UF, showing an obviously higher rate in comparison with IF or RF. The tissue of cryostat section treated with cryofilms was clear with complete aperture structure. Wherein, the minimal tissue destruction was observed for samples treated by UF with the minimum diameter of crystal of 46.81um. Compared to the other groups, samples treated by UF had a higher water holding capacity and a lower dehydration. Furthermore, the result of Pearson analysis indicated that there was an extremely significant correlation between ice crystals area and moisture change for all the groups, and a significant correlation between the roundness and moisture change was also noticed for samples treated by UF, while no correlation was observed for those treated by IF or RF.

【Objective】In our research conducted to elucidate the factors that affect the quality deterioration of frozen surimi-based products, it has been shown that various factors affected the quality of heat-induced surimi gels upon freezing. On the other hand, Hiraoka et al. (2011) have reported that the quality of Jakotempura, which is a traditional fish paste product, prepared by Method B (setting → freezing → heating), was better than that of Jakotempura prepared by Method A (setting → heating → freezing), in terms of showing low thawing drip loss. However, the mechanism underlying the differences is still unclear. Therefore, this study aimed to elucidate this phenomenon by using heat-induced surimi gels as a model.
【Methods】Frozen surimi was thawed and ground with 30% water and 3% NaCl. After grinding with with/without 5% potato starch, the surimi paste was shaped and processed by Method A or Method B. For freezing, surimi gels were vacuum-packed, subjected to either quick freezing or slow freezing, and stored at −20 or −40 °C for 1 day or 4 weeks. Microscopic observations, the water-holding capacity and physical properties of thawed gels were evaluated.
【Results】For heat-induced surimi gels without starch, Method B was not more effective than Method A. On the other hand, in the case of heat-induced surimi gels with starch, Method B was significantly more effective than Method A, with lower thawing drip loss. Microscopic observations confirmed that the starch granules in the gel processed by Method A were destroyed after slow freezing. On the other hand, starch granules in samples processed by Method B maintained their shape. It suggested that the higher water-holding capacity obtained by Method B was due to the fact that starch granules were still intact after setting and freezing, which provided tolerance to freezing.

[Objectives] Frozen surimi are made from various fish species and several grades are on the market. However, their quality standards are not fully established. Suwari (sitting) treatment is important one in the manufacturing process of fish gel products. Furthermore, physical properties of sitting gel are quit different among various frozen surimi. Therefore, we focused the speed and the activation energy in the sitting reaction around 30 °C in this study. And it was examinated whether these values are suitable for quality evaluation or not.
[Methods] Frozen surimi of Alaska pollock (SA and RA grade), Blue grenadier (FA and KA grade) and Threadfin bream (SA and KA grade) were used in this study. According to general methods, meat pastes were prepared. Sitting reaction of meat paste was proceeded in three temperature ranges between 16 to 30 °C. The sitting speed (K s-1) was obtained from the breaking strength. And then, the activation energy (Ea) of the sitting reaction was calculated from the sitting speed of each temperatures.
[Results] In comparison with each Ea, Alaska pollock one was the highest value and Blue grenadier and Threadfin bream ones were almost same. Therefore, it is thought that the latter two surimi are easier to sit than that of Alaska pollock. On the other hand, in the case of different grades from the same fish, the sitting speed was clearly lower in the low grade surimi. From the these results, it was suggested that the Ea is good indicator for the quality among fish species. In addition, the sitting speed should also be good one, in particular among grades of the same fish.

Fish oil is an excellent source of long-chain polyunsaturated fatty acids‎, such as ‎icosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3). To increase the value of food with high functionality, new products containing added fish oil have been developed in recent years. ‎Previously, we investigated the effects of emulsifying fish oil into heat-induced surimi gels and found that positive effects on quality characteristics, such as color, water-holding capacity, and gel-forming ability and on the prevention of quality deterioration by freezing. To apply this technology in the surimi-based product industry, this study was aimed to clarify the effect of ‎emulsifying fish oil on the quality of ‎the commercial product sasa-kamaboko.
Frozen surimi was ground with water, salt, and other seasonings to obtain surimi paste. Fish oil (0 (control), 2.5, and 5.0%) was emulsified by vigorous mixing under a vacuum. Emulsified surimi paste was shaped and heated by far infrared rays and baked to obtain sasa-kamaboko. The products were cooled, individually vacuum-packed, and frozen both rapidly and slowly. Measurement of water-holding ‎capacity, oil particle sizes, physical properties, and whiteness before and after frozen storage was conducted to evaluate the products. Microscopic observation and sensory evaluation were also performed.
The emulsified product displayed higher water-holding capacity and whiteness compared to the control. The texture of the emulsified product showed high scores in sensory evaluation. Microscopic observation revealed that the sizes of ice crystals in the frozen emulsified products were smaller and frozen damage of thawed products was lower compared to in control products. These results showed that the emulsification of fish oil positively affects not only the quality of commercial surimi-based products, but also on the quality changes during frozen storage.

The fish proteins, recovered from the skeletal muscle of blue scad (Decapterus maruadsi) by acidic- and alkaline- aided isoelectric solubilization/precipitation (ISP), were compared based on their recovery yields, amino acid compositions and gel-forming abilities. As results, the recovery yields of ISP protein were 65.0% and 84.6 % by acidic and alkaline treatment, respectively. No significant differences were found between acidic and alkaline treatment in their protein and amino acid compositions. However, the pronounced proteinase activity was detected in alkaline ISP protein, which also occurred modori phenomenons during heat-induced gelling processing. To further clarify the mechanism, a water soluble serine proteinase was purified from blue scad skeletal muscle to homogeneity by ammoniun sulfate fractionation and a series of chromatographies including DEAE-Sepharose, SP-Sepharose, Q-HP. It was purified to 796- fold with and a yield of 1.4 %. The molecular weight of purified proteinase was estimated to be 60 kDa by SDS-PAGE. Based on myofibrillar protein zymography, the optimum temperature and pH of purified proteinase were 40 ^oC and 9.0, respectively. The fluorogenic substrate analysis showed that obtained proteinase can specifically hydrolyze Boc-Gln-Arg-Arg-MCA, which belongs to the serine proteinase family. Purified proteinase was strongly inhibited by serine proteinase inhibitors and partially inhibited by pepstatin inhibitors and cathepsin proteinase inhibitors. These results suggest that purified proteinase was serine proteinase, which is probably involved in modori phenomenons of alkaline-aided ISP protein from blue scad.

[Objectives] The gel product made from squid muscle shows unique texture, which is different of kamaboko made from fish muscle. It is suggested that the difference between these two products is caused of paramyosin specifically existing in invertebrate muscle. Nevertheless, it has not been reported the primary structure of squid paramyosin. Therefore, in this study, the primary structures of paramyosin from several kinds of squids were determined by cDNA cloning and the structural characteristics were investigated.
[Materials and Methods] Freeze mantle muscle from several kinds of squid Ommastrephes bartramii, Dosidicus gigas, Sepia esculenta and Gonatus onyx were used in this study. Firstly, total RNA was prepared from each mantle muscles, and cDNA was synthesized by using a reverse transcriptase. Primers were designed from the conserved sequences of several kinds of mollusks previously reported. PCR, 3’ RACE and 5’ RACE method was performed to obtain a DNA fragment, and the fragment was sequenced. The deduced amino acid sequences were aligned using ClustalW. And then, the phylogenetic tree was constructed using the neighbor-joining method.
[Results] Three cDNAs encoding paramyosin from the O. bartramii, D. gigas and G. onyx were cloned. The O. bartramii paramyosin cDNA was cloned up to 2,605 bp and contains an ORF of 2,574 bp encoding 858 amino acids. The D. gigas one was cloned up to 2,691 bp and contains an ORF of 2,640 bp encoding 880 amino acids. The G. onyx one was cloned up to 2,609 bp and contains an ORF of 2,574 bp encoding 858 amino acids. The phylogenetic analysis including three paramyosins obtained in this study, showed that three paramyosins formed a group, which was near to that of octopus.

Mackerels are commonly consumed both fresh and as processed fish products in Japan. Two mackerel species are used in processed fish products in Chiba Prefecture: spotted mackerel Scomber australasicus and Pacific mackerel S. japonicus. Muscle softening is often observed in spotted mackerel, especially in frozen-thawed fish, which reduces its commercial value. Recently, we have developed frozen sashimi products made from spotted mackerel. Fish muscle quality is influenced by seasonal variation of chemical composition. To improve fish quality and commercial value, the deterioration in fish meat quality must be characterized so that post-mortem muscle softening can be prevented. In this study, seasonal variation of breaking strength and chemical composition was compared in these two mackerel species. Chemical composition and biochemical properties such as muscle toughness, gonadosomatic index, water content, crude fat, pH, and cathepsin B and L activity were measured. In spotted mackerel, muscles were softer in May and September. Furthermore, in both species, gonadosomatic indexes were high in April and May, muscle water content was high and crude fat content low in February, and Cathepsin B and L activity was high from April to June. Significant positive correlation between breaking strength and pH (r=0.34) and significant negative correlation between breaking strength and gonadosomatic index (r=-0.27) were detected in spotted mackerel; positive correlation between breaking strength and pH (r=0.57) and negative correlation between breaking strength and cathepsin L and B activity (r=-0.23) were detected in Pacific mackerel. These findings suggest that the toughness of spotted mackerel and Pacific mackerel meat is strongly influenced by pH value of muscle.

【Aim】 The Ruditapes philippinarum is nutrient-rich and widely-distributed, but little attentions had been paid on the identification and characterization of bioactive peptides in the bivalve. In the food industry, although the preparations of protein hydrolysates are common and usually use food-grade materials, proteins and peptides may produce allergic reactions after ingest in the human body due to their special amino acid sequence.
【Methods】 In this study, the Ruditapes philippinarum enzymolysised by trypsin under conditions as following, E:S 3:100 (w/w), pH 9.0, 45 ℃ for 4 h. Using a combination of UPLC separation and a ESI-Q-TOF-MS/MS spectrometer, after separation and detection, followed by data processing, sequence-similarity database searching, a Swiss-Prot database and a Ruditapes philippinarum sequence database were used. The potential allergenicity of the peptides was assessed in silico by two methods.
【Results】 Totally 966 unique peptides were identified by non-error tolerant database searching, 173 peptides matched 55 precursor proteins were highly conserved cytoskeleton protein. The rest 793 peptides were identified from the Ruditapes philippinarum sequence database. Results showed that 510 peptides were labeled as allergens and 31 peptides were potential allergens, 425 peptides were predicted to be non-allergens. The abundant peptide information would contribute to further studying the structure and potential function of the Ruditapes philippinarum. And further vitro studies would be performed in order to demonstrate and ensure correct production of the hydrolysates if used in food industry of the Ruditapes philippinarum.

Seafood allergy has been a world public food safety issue with the increasing number of allergic patients. Tropomyosin (Tm), a myofibrillar protein of 35-38 kDa, has been demonstrated to be a major allergen in various crustaceas and mollusc. In order to investigate the effect of sterilization treatments on the change of Tm, 4 sterilization treatments including boiling, autoclaved sterilization, microwave radiation and high hydrostatic pressure (HHP) were performed to shrimp muscle and balls of Pacific white shrimp (Litopenaeus Vannamei). According to the SDS-PAGE, Western blotting and inhibition ELISA results, low reduction was observed in the Tm of shrimp ball samples (<36.5%), however, Tm in shrimp muscle is almost not reduced after boiling. Whereas, slight reduction was observed in the Tm of shrimp muscle after microwave radiation, but little effect was found in the Tm of shrimp ball samples after microwave radiation. On the other hand, Tm reduction was most significant in autoclaved shrimp muscle and balls, over 90% after treating for 20 min. Second was the HHP ball samples (>77.10%), however, the content and allergenicity of Tm in the shrimp muscle was stable after autoclaved sterilization. In conclusion, compared with the boiling and microwave radiation treatments, autoclaved sterilization and high hydrostatic pressure are capable of the development of lower allergenic shrimp products.

Ingestion of marine invertebrate species, including shrimp, squid and abalone, causes food allergy. The major allergen in invertebrate foods is tropomyosin (TM), which consists of the α-helical coiled coil structure throughout almost the whole molecule. TM, like other ordinary allergens, shows higher resistance against digestion in gastric than intestine. The reason of such difference in digestibility is unknown. We therefore performed constant-pH molecular dynamics simulation at pH 1 and pH 7 (10 ns × 3) for TM of kuruma shrimp Marsupenaeus japonicus. The trajectories were then analyzed by TWISTER to obtain the coiled-coil radius and phase per residue. As a result, the average values of the coiled-coil radius at pH 1 and pH 7 were 4.83 Å and 4.80 Å, respectively. The differences in the radius were observed around the acidic cores: the radius around Asp137 was larger at pH 7 than at pH 1, while the radius around Glu218 was lower at pH 7 than at pH 1. The average values of the coiled-coil phase per residue at pH 1 and 7 were -3.38° and -3.18°, respectively. The coiled-coil structure around Asp137 at pH 7 was less twisted than that at pH 1. In addition, the phases around Glu218 at pH 7 were more fluctuated than those in other region. These differences between pH 1 and pH 7 were mainly found at the two acidic cores, even though longer simulations are needed to confirm the observed difference. The pH-dependence of the overall coiled-coil structure or its rigidity was little, except those found at the two acidic cores, and thus it seems to be difficult to explain the difference in the digestibility between the stomach and small intestine by the pH-dependence of overall structural properties of the coiled-coil.

Thermal disintegration of surimi gel structure is called modori. Many previous studies have already found that modori results from degradation of myofibril proteins, especially myosin heavy chain (MHC) by heat-activated proteases. Most previous studies on modori have used SDS-PAGE analysis, rheological analysis and sensory evaluation to confirm the relation between MHC degradation and disintegration of surimi gel. On the other hand, several studies have examined the effects of protease inhibition on surimi gel properties. All these methods however have limitations on analysis of MHC degradation in surimi. Here we show a novel approach using proteomics to exploring dynamics of MHC degradation in surimi during heating.
In-gel digestion coupled with mass spectrometric analysis (GeLC-MS/MS) is one of general methods for proteomics. Combination of densitometry on PAGE and GeLC-MS/MS allows quantitative proteomics. We then used GeLC-MS/MS and densitometry on PAGE to investigate dynamics of MHC degradation in deepsea bonefish (Pterothrissus gissu) surimi during heating at 35°C. 30, 50 and 75 kDa MHC fragments were found in proteinaceous materials extracted from the heated surimi by treatment with a low ionic strength buffer. In addition, an apparent time-dependent increase of MHC fragments was observed. Analysis of changes in urea-extracted proteinaceous materials showed that a time-dependent increase of 150 and 170 kDa MHC fragments was observed; both of 100 kDa> and 100 kDa< MHC fragments quickly increased in the surimi during heating. Slow increase in 300, 340 and 440 kDa MHC dimers were also found. The 300 and 340 kDa MHC dimers seem to be polymerized MHC fragments. Polymerization of MHC fragments contributes to structural inhomogeneity and consequently causes fragile gel structure. These results indicate that MHC degradation prior to polymerization is critical for disintegration of deepsea bonefish surimi gel.