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Diarrhetic shellfish poisoning (DSP) toxins pose a serious health risk for consumers of bivalves and other shellfish, as well as a huge economic burden for the bivalve-producing farmers. In this work, the aim was to utilize a solubilization-based protein-isolation method to produce a low-DSP toxin protein isolate from toxic blue mussels that are unsuitable for the whole shellfish market. A homogenate of whole mussel meat was solubilized at low pH (2.8) or high pH (11.1), followed by centrifugation and reprecipitation of the solubilized mussel proteins at the isoelectric pH. In a second centrifugation, precipitated proteins were collected. These processes resulted in 81 (acid solubilization) and 72% (alkaline solubilization) reduction in the initial DTX-1 toxin content of the mussel meat. No other DSP toxins were found in the protein isolates. Acid processing of mussel meat resulted in 50% reduction in the total lipid content, while alkaline treatment did not significantly affect the lipid content. The effect of citric acid and calcium chloride addition to the mussel meat-water homogenate on lipid and toxin content was also investigated. A poor correlation factor was surprisingly obtained between reductions in DTX-1 toxin and lipids in protein isolates from processed toxic mussels. Results from an analytical mass balance of the DTX-1 toxin during acid processing showed that 61% of this toxin ended up in the aqueous supernatant after the second centrifugation. The present study presents a promising alternative way of utilizing mussels for food production in periods when they are toxic.

The aqueous fraction (press juice, PJ) from herring muscle was recently shown to inhibit hemoglobin-mediated oxidation of washed fish mince lipids during ice storage. As a first step to evaluate potential in vivo antioxidative effects from herring PJ, the aim of this study was to investigate whether herring PJ retains its antioxidative capacity during a simulated gastrointestinal (GI) digestion. Press juice from whole muscle (WMPJ) and light muscle (LMPJ) was mixed with pepsin solution followed by stepwise pH adjustments and additions of pancreatin and bile solutions. Digestive enzymes were removed from samples by ultrafiltration (10 kDa). Before, during, and after digestion, samples were analyzed for their peptide content and for antioxidative properties with the oxygen radical absorbance capacity (ORAC) and the low-density lipoprotein (LDL) oxidation assays. From 0 to 165 min of digestion, the content of <10 kDa peptides in WMPJ and LMPJ samples increased 12- and 7-fold, respectively. Further, both samples got approximately 12.5 times higher ORAC values and gave rise to approximately 1.3-fold increased lag phase in Cu2+-induced LDL oxidation. The largest changes in peptide content, ORAC values, and LDL oxidation inhibition occurred between 30 and 75 min of digestion, indicating that these parameters might be interrelated. When comparing analytical data obtained after 165 min of digestion with data obtained from analyses of native nondigested PJs, it was found that the data on peptide content, ORAC, and LDL oxidation from digested PJs were 64-69%, 121-161%, and 112-115%, respectively, of those of nondigested PJs. The study thus showed that enzymatic breakdown of PJ proteins under GI-like conditions increases the peroxyl radical scavenging activity and the potential to inhibit LDL oxidation of herring PJs. These data provide a solid basis for further studies of uptake and in vivo activities of herring-derived aqueous antioxidants.

The antioxidative effect of herring (Clupea harengus) light muscle press juice (PJ) against hemoglobin-(Hb-) mediated oxidation of washed cod mince during ice storage was tested. The PJ was fractionated into low-molecular-weight (LMW; <1 [corrected] kDa) and high-molecular-weight (HMW; >1, >3.5, and > 50 kDa) fractions; it was preheated (10 min, 100 degrees C) and tested with or without removing heat coagulated proteins. Its antioxidative effect was compared with that given by endogenous levels of two tentative antioxidant candidates: ascorbic acid and uric acid. Oxidation was followed by determining rancid odor, peroxide value, and redness. Whole herring PJ and the LMW-PJ fraction significantly (p < 0.001) extended the oxidation lag phase of controls, from 2 up to 8 and 7 days, respectively. The HWM-PJ fractions were significantly (p < 0.05) less efficient than the whole and LMW-PJ samples, giving only 3.5-4.5 days of lag phase. Heat-treated PJ, with and without the heat-coagulated proteins, gave 7 and 5 days of oxidation lag phase, respectively. Heating different batches of the LMW-PJ fraction grouped the results into two categories: one where heating almost fully destroyed the antioxidative activity (fractions prepared from spring-caught herring) and another where heating had no or a minor effect (fractions prepared from fall-caught herring). The spring LMW-PJ had low ascorbic acid levels (18-42.6 microM), and 50-100% were destroyed by the heating. In fall LMW-PJ, the levels were 76.2-137.6 microM, and only 43-51% were destroyed. Ascorbic acid fortification of heated spring LMW-PJ to reach the levels found in the corresponding unheated spring LMW-PJ sample and the heated fall LMW-PJ gave back most of the antioxidative activity, which proved an important role of ascorbic acid for the antioxidative activity of LMW-herring PJ. This conclusion is drawn despite the fact that pure solutions with endogenous levels of ascorbic acid (giving 8.4-19.6 microM in final model) only very slightly delayed Hb-mediated oxidation of the washed cod mince.

The use of washed cod light muscle minces in mechanistic studies of hemoglobin (Hb)-mediated fish lipid oxidation has largely increased in the past 5 years. Although cod light muscle has a low level of intrinsic lipid oxidation catalysts, a prerequisite for a good oxidation model system, we believe it cannot fully mimic the oxidation kinetics taking place in other fish species being more susceptible to lipid oxidation. The aim of this study was to systematically investigate whether washed mince model systems useful in Hb-mediated oxidation studies could be prepared also from herring (Clupea harengus) and salmon (Salmo salar) light muscles. The kinetics of oxidation in the washed models was measured during ice storage (+/-Hb), and the results were related to compositional differences. Minces from cod, herring, and salmon light muscles were washed 3 times with 3 volumes of water and buffer. A 20 microM portion of Hb and 200 ppm streptomycin was then added, followed by adjustment of pH and moisture to 6.3 and 86%, respectively. Samples with or without Hb were then stored on ice, and oxidation was followed as peroxide value (PV), rancid odor, redness (a*) loss and yellowness (b*). Prior to storage, all minces and models were also analyzed for total lipids, fatty acids, alpha-tocopherol, proteins, Hb, Fe, Cu, and Zn. Hb-mediated lipid oxidation appeared within 2 days on ice in all models. Small differences in the oxidation rates ranked the models as herring > cod > salmon. These differences were ascribed to more preformed peroxides and trace elements in the herring model, and more antioxidants in the salmon model. Controls, without Hb, stayed stable in all cases except herring, where a very slight oxidation appeared, especially if the herring raw material had been prefrozen. In conclusion, fattier fish like dark muscle species and salmonoids are useful for making washed mince model systems and would be a better choice than cod if there is an interest in the oxidation kinetics of such species.

The effectiveness of a white grape dietary fiber concentrate (WGDF) against hemoglobin-mediated oxidation of washed cod mince, with and without 10% added herring oil, was evaluated during ice storage. WGDF was added at two different levels: 2 and 4% based on final weight. An ethanol extract with the ethanol extractable polyphenols (EPP) and the ethanol-extracted grape dietary fiber residue were also tested as antioxidants in the washed cod mince. The addition of WGDF to the model system completely and significantly (p

An emerging model to test antioxidants for application in seafoods is washed cod mince fortified with hemoglobin (Hb) as a catalyst. This system has been used to test the antioxidative activity of certain muscle extracts and some pure compounds such as BHA, BHT, TBHQ, and propyl gallate during ice storage. However, the washed cod mince model has occasionally been resistant to Hb-mediated oxidation. This has been in cases when the moisture of the model has been minimized by washes at the protein isoelectric point (pH approximately 5.5) to allow for large additions of potentially antioxidative solutions. In this paper, noncontrollable and controllable factors for this intriguing occasional oxidation resistance were studied. Compositional analyses (lipid content, alpha-tocopherol, and lipid hydroperoxides) and structural analysis of a "normal" oxidizing model and a stable model were done to identify any differences among them. Some controllable factors related to the model preparation that were studied included different washing pH values (5.5-6.6), Hb concentrations (7.2 and 13.5 microM), final model moisture contents (75, 81, and 90%), and light exposure during ice storage (0 h, 3-4 h, or 24 h of light/day). Results revealed a 2-fold higher alpha-tocopherol content in the stable model than in the oxidizing model. Electron microscopy images showed a more and less disrupted myofibrillar structure in the stable and the oxidizing cod model, respectively. This indicated that "cold setting" (i.e., pre-gelation) of the stable model may have occurred and prevented Hb from diffusing freely in the model. Controllable factors that reduced lipid oxidation in the models were less Hb and lower moisture.

Reactive oxygen species (ROS) can cause oxidative stress, which has been linked to various diseases. It has been suggested that antioxidant-rich foods can reduce such oxidative stress. However, the lack of suitable model systems to screen for in vivo effects of food-derived antioxidants has prevented a clear consensus in this area. In this study, the aim was to use a single-cell model system (human monocyte) to evaluate whether certain pure antioxidants and complex muscle extracts (herring light muscle press juice, PJ) could prevent ROS formation under in vivo like conditions. ROS were excreted from the monocytes upon stimulation with phorbol myristate acetate and were then detected as isoluminol-enhanced chemiluminescence (CL). Adding 2000 units of catalase and 50 units of superoxide dismutase to the monocytes model lowered the CL response by 35 and 86%, respectively. Ascorbate (14.1 mM) lowered the response by 99%, alpha-tocoperhol (188 microM) by 37%, and Trolox (50 microM) by almost 100%. Crude herring PJ gave a dose-dependent reduction in the CL response. At 10, 100, and 1000 times dilution, the PJ reduced the CL signal by 93, 60.5, and 10.6%. PJ fractionated into low molecular weight (LMW) (<1000 Da) and high molecular weight (>3500 Da) fractions decreased the CL response by 52.9 and 71.4%, respectively, at a 100-fold dilution. Evaluation of the PJ samples in the oxygen radical absorbance capacity test indicated that proteins may be the primary radical scavenging compounds of PJ, whereas the ROS-preventing effect obtained from the LMW fraction may also be attributed to other mechanisms. Thus, this study proved that the monocyte assay can be a useful tool for studying whether food-derived antioxidants can limit ROS production under physiologically relevant conditions. It also showed that herring contains numerous aqueous compounds demonstrating antioxidative effects in the monocyte model system.

It has previously been found that a process based on solubilization at pH 2.7 gives high yields of herring muscle proteins with good functionality. In this study, the development of lipid oxidation during acid processing of herring mince was studied. It was tested how modifications of the process conditions and/or additions of antioxidants could prevent lipid oxidation during the actual process and then during ice storage of the protein isolates. Processing parameters evaluated were prewash of the mince, exposure time to pH 2.7, inclusion or exclusion of a high-speed centrifugation, and addition of antioxidants. Antioxidants tested were erythorbate (0.2%, 9.3 mM), sodium tripolyphosphate (STPP; 0.2%, 5.4 mM), ethylenediaminetetraacetic acid (EDTA; 0.044%, 1.5 mM), and milk proteins (4%). The first three antioxidants were added in the prewash or during the homogenization step, whereas milk proteins were added to the final precipitate. At time 0, all isolates were analyzed for pH, moisture content, and thiobarbituric reactive substances (TBARS). Selected isolates were also analyzed for lipid and protein content. Stability during ice storage was followed in terms of odor, TBARS, and color (a/b values). Extensive lipid oxidation took place using the "control" process without high-speed centrifugation. This was not significantly (p < or = 0.05) affected by a prewash or varied exposure time to pH 2.7. Including high-speed centrifugation (20 min, 10,000g) significantly (p < or = 0.05) reduced TBARS values, total lipids, a values and b values. Erythorbate alone, or in combination with STPP/EDTA, significantly (p < or = 0.05) reduced lipid oxidation during processing if added in the prewash or homogenization step. During ice storage, better stability was gained when antioxidants were added in both of these steps and when EDTA was used instead of STPP.

Unheated press juice (PJ) obtained from chicken breast muscle was a potent inhibitor of hemoglobin-mediated lipid oxidation in washed cod muscle. The <1 kDa fraction had a negligible effect on the rate of lipid oxidation. The high-molecular-weight (HMW) fraction was mildly inhibitory when added alone and highly inhibitory in the presence of <1 kDa components. Proteins of the HMW fraction were further fractionated by ammonium sulfate precipitation. Proteins in the 80% fraction were most inhibitory compared with other precipitated fractions on an equal protein basis. Inhibition by PJ was substantially decreased due to treatment with ascorbate oxidase. Adding ascorbate to the HMW fraction did not increase its inhibition, which suggested the presence of a complex ascorbate-reducing system in PJ consisting of HMW and low-molecular-weight (LMW) components. The ability of added ceruloplasmin to inhibit lipid oxidation was remarkably enhanced by addition of ascorbate or the <1 kDa fraction. Heated and centrifuged PJ had 8 times more LMW iron compared to unheated PJ. Adding heated PJ to washed cod containing hemoglobin slightly increased the rate and extent of lipid oxidation.

Instrumental measurement of redness loss (decrease in a* value) was evaluated as a tool to follow hemoglobin (Hb)-mediated lipid oxidation in fish muscle. Two washed cod mince model systems were used (prepared at pH 6.5 and 5.5), both fortified with 15 micromol/kg of trout Hb and adjusted to pH 6.5 and 81% moisture. The rate of oxidation was varied through pH alterations (pH 6.1 and 6.9) and addition of an antioxidative cod muscle press juice. During ice storage, TBARS, painty odor, and a* values were followed. In all "oxidizing" samples, a* values correlated well with TBARS and painty odor development; r = -0.95 and -0.77, respectively. In press juice containing samples, the correlation was lower (0.55 for a vs TBARS) because there was a slight a* value decrease even in the absence of measurable lipid oxidation. a* values distinguished between "oxidizing" and stable samples within 1 day, before any lipid oxidation products could be chemically detected. It was confirmed in an aqueous phosphate buffer model system that the redness loss corresponded to a buildup of brownish met-Hb at the expense of oxy- and deoxy-Hb. The a* value data were best used as a lipid oxidation index by calculating the rate of decrease (k value) in the "initial phase" of the redness loss (before accumulation of lipid oxidation products) or in the "differentiation phase" (during the exponential raise in TBARS/painty odor). Calibration to lipid oxidation products must, however, be made for each specific sample type. Washing method, pH, Hb-type, etc., all affected both k values and absolute a* readings. Small yellowness (b*) increases also occurred along with a value losses, possibly the result of polymerized Schiff bases.

Lipid pro-oxidative properties and deoxygenation/autoxidation patterns of hemoglobins from nonmigratory white-fleshed fish (winter flounder and Atlantic pollock) and migratory dark-fleshed fish (Atlantic mackerel and menhaden) were compared during ice storage at pH 7.2 and 6. A washed cod mince model system and a buffer model system were used for studying lipid changes and hemoglobin changes, respectively. TBARS and painty odor were followed as markers for lipid oxidation. At pH 6, all four hemoglobins were highly and equally active as pro-oxidants. At pH 7.2, pro-oxidation by all hemoglobins except that from pollock was slowed down, and activity ranked as pollock > mackerel > menhaden > flounder. The higher catalytic activities of the hemoglobins at pH 6 than at pH 7.2 corresponded with higher formation of deoxyhemoglobin and methemoglobin. Pollock had the most extensive formation of deoxy- and methemoglobin at both pH values, which could explain its high catalytic activity. The pro-oxidative differences among the other hemoglobins at pH 7.2 did not correlate with deoxygenation and autoxidation reactions. This indicates involvement of other structural differences between the hemoglobins such as differences in the heme-crevice volume. It is suggested that a biological reason for the species differences was their adaptations to different depths/water temperatures.

Fish muscle proteins can be isolated from a variety of low-value raw materials by solubilization in either acid or base. If the consistency of the resulting solution is sufficiently low, it is possible to recover most of the solubilized proteins and remove most of the lipids by centrifugation. Lipid removal should greatly stabilize the isolated proteins. In a previous investigation into the use of herring for production of these protein isolates, it was observed that this species had particularly high consistency values when the proteins were solubilized. This study was undertaken to determine the consistencies obtained with herring light muscle tissue over the pH range covered by the two processes, from about pH 2.7 to 10.8. Protein solubility was compared to consistency of the resultant solutions. Maximum consistencies of the homogenates, approximately 220 and approximately 175 mPa.s, were obtained at pH values of approximately 3.5 and 10.5, respectively. Consistency began to increase approximately when solubilization began. Storage of homogenates at pH 2.7 decreased the consistency over a 10 min time period. The magnitude of the consistency peaks at both acid and alkaline pH values increased when using ice-stored as well as frozen-stored herring, especially in the acid range. Protein solubility at pH <4 and pH >/=10.8 slightly decreased after post-mortem storage of the herring muscle. It is suggested that the observed changes in consistency result from the expansion and solvation of protein aggregates which eventually dissociate into smaller units, perhaps even monomers.

It was evaluated whether trout hemoglobin (Hb)-mediated oxidation of minced washed cod muscle lipids could be prevented by an aqueous isolate from cod and some other muscle sources. Lipid hydroperoxides and painty odor developed approximately 4 days faster in washed than unwashed cod mince. When adding back an aqueous fraction (press juice) isolated from unwashed mince to washed mince at 2-6-fold dilutions, development of hydroperoxides and painty odor was either delayed or completely prevented. The inhibitory substances were heat stable, and their effect was slightly reduced at reduced pH. The <1 kDa fractions of whole and heated press juices were as inhibitory as the unfractionated press juices. Inhibition by the unheated, heated, and ultrafiltered (30 kDa) press juices was lost after dialysis. These findings implied the presence of one or more highly effective aqueous low molecular weight antioxidants in cod muscle press juice. The same antioxidative properties were found in heated haddock, dab, and winter flounder muscle press juices but not in heated herring and chicken muscle press juices. Unheated chicken press juice was however highly inhibitory.

Volatile compounds released from herring fillets (Clupea harengus) during 15 days of storage on ice have been measured with a commercial hybrid gas-sensor array system. Using partial least-squares regression modeling, the sensor responses were correlated with data from chemical analyses (lipid oxidation products and antioxidants) and sensory analyses (odor). Eight of the 16 sensors proved significant in the correlation studies: 6 metal oxide semiconductor field effect transistor (MOSFET) sensors and 2 Taguchi type sensors. Correlation coefficients for chemical and sensory data ranged from 0.9 to 0.98 and from 0.49 to 0.92, respectively, with 0.92 referring to both "sharp/acrid" and "rancid" odors. Prediction errors ranged from 8 to 14% and from 11 to 25% for the chemical and sensory measures, respectively. That the prediction errors for oxidation product formation (5-9%) were close to the analytical errors of the chemical reference methods indicated close to "optimum" performance of the gas-sensor system. The sensor system predicted the storage time of the herring with a 1-day error. Results illustrate high potential of the gas-sensor technology in rapid nondestructive quality determination of ice-stored herring.

Proteins from herring (Clupea harengus) light muscle were extracted using acidic or alkaline solubilization; 92 and 89% of the initial muscle proteins were solubilized at pH 2.7 and 10.8, respectively, of which 96 and 94% were recovered during precipitation at pH 5.5. Consistency of the pH-adjusted muscle homogenates increased with increased raw material age and homogenization intensity; it declined following holding on ice. Some hydrolytic myofibrillar protein degradation occurred during cold storage of the acidified (pH 2.7) homogenates. With alkalized homogenates, hydrolysis was negligible. The total lipid content changed from 0.13 g/g of protein in the muscle to 0.04 g/g of protein in both the acid- and alkali-produced protein isolates. Corresponding values for the phospholipid content were from 0.037 to 0.02 g/g of proteins. Acid- and alkali-produced proteins made gels with equal strain and color. Stress values were equal or lower in acid- versus alkali-produced protein gels. When ice-stored raw material was used, strain and stress values of gels were reduced.

Hemoglobin-mediated lipid oxidation in washed, minced cod muscle was related to the triacylglycerol to membrane lipid ratio. The same rapid development of thiobarbituric acid reactive substances (TBARS) and painty odor occurred with and without the presence of up to 15% menhaden oil. Without hemoglobin, development of TBARS and painty odor was slow, despite a high amount of hydroperoxides in samples with oil added (1135 micromol/kg muscle). This suggested that hemoglobin reacted by cleaving preformed hydroperoxides into secondary oxidation products. Nearly doubling the hemoglobin concentration approximately doubled the extent of lipid oxidation with and without added oil. This indicated that hemoglobin was limiting for the oxidation reaction. The noneffect of added oil suggests that membrane lipids and/or preformed membrane lipid hydroperoxides provided sufficient substrate in hemoglobin-catalyzed oxidation of washed minced cod muscle. Fe(2+-)ADP did not induce any oxidation of washed minced cod with/without added oil. Results suggest that lipid oxidation in fatty fish may be more related to the quantity and type of the aqueous pro-oxidant and the membrane lipids than to variations in total fat contents.

Fillets of herring (Clupea harengus) were kept on ice for 0, 3, 6, and 9 days prior to storage at -18 degrees C for 0, 21, 42, 63, and 84 days. At each storage point, peroxide value (PV), absorbance at 268 nm (A(268)), fluorescent products (FP), alpha-tocopherol, glutathione peroxidase (GSH-px) activity, and ascorbic acid were measured. As shown by regression analyses, samples held for 6 days on ice formed oxidation products at the highest rate during frozen storage, followed by, for PV and FP, the 9-day samples. These data indicate that severe changes that negatively affect the oxidation process took place in the herring muscle between 3 and 6 days after catch. Both the initial antioxidant levels and the rate of antioxidant loss at -18 degrees C decreased with increased prefreezing holding time, the latter being most obvious for GSH-px activity and ascorbic acid. alpha-Tocopherol showed the largest losses and had disappeared entirely from the 6- and 9-day samples at the end of the frozen storage. Partial least-squares regression analysis of the data showed that ice storage had a greater effect than frozen storage on changes in PV, A(268), FP, alpha-tocopherol, and ascorbic acid. For GSH-px activity, frozen storage had the greatest effect.