Antioxidative Properties of Herring (Clupea harengus) Press Juice in Food, In Vitro and In Vivo Model Systems

Doktorsavhandling, 2007

Some species like herring (Clupea harengus) are currently under-utilized due to their small size, dark colour and susceptibility to lipid oxidation. Our hypothesis has been that the high susceptibility of herring to oxidation makes it well equipped with natural antioxidants, both fat and water soluble ones. The aim of this thesis has been to isolate the water soluble antioxidants in terms of a herring light muscle extract (press juice, PJ) and to test the capacity of this PJ to inhibit oxidation in different model systems ranging from food to in vivo conditions. Attempts were also made to identify the tentative compounds responsible for the antioxidative properties. Herring PJ was fractionated into low molecular weight (LMW; <1 kDa) and high molecular weight (HMW; >1, 3.5, 50 kDa) fractions. Some samples were also heat treated (100 degree C, 10 min). The antioxidaitve properties of the PJ samples were tested and compared with pure antioxidants using a hemoglobin (Hb) containing washed cod mince during ice storage, in the oxygen radical absorbance capacity (ORAC) assay, in a Cu induced low density lipoprotein (LDL) assay and in a human monocyte model. The herring PJs were also exposed to simulated in vitro gastrointestinal (GI) digestion to evaluate possible changes in the ORAC and LDL oxidation assay responses. During optimization of the washed cod mince model, it was found that the starting cod mince should not contain > 20 mg alpha-tocopherol/kg and that cold setting (i.e pre-gelation) as well as too low moisture (<75%) of the washed cod models should be avoided. All these factors delayed oxidation. PJ and LMW-PJ strongly inhibited Hb mediated development of rancid odour, peroxide value (PV), and redness loss of the washed cod mince. All the HMW-PJ tested were significantly (p < 0.05) less effective than native and LMW-PJ. Heating the PJ only slightly reduced its activity, while heating the LMW-PJ revealed a heat labile nature of the LMW-antioxidants. Several ascorbic acid analyses of seasonally different batches of heated and non-heated LMW-PJ used in the washed cod mince revealed that the initial level of ascorbic acid played a role for the antioxidative capacity of LMW-PJ. Higher starting levels gave higher antioxidant capacity after heating. Thus we expect that ascorbic acid in synergy with other LMW compounds contribute to the antioxidative properties of herring PJ. The ORAC assay run at 37 degree C and pH 7.4 ranked the PJs as follows, whole herring PJ (WMPJ) > PJ > HMW-PJ > LMW-PJ. These results indicate a major contribution of the PJ protein fraction to peroxyl radical scavenging. All the PJs except the LMW-PJ also extended the Cu induced LDL oxidation lag phase and decreased the rate of LDL oxidation. Herring PJs also showed a capacity to prevent phorbol myristate acetate (PMA) induced reactive oxygen species (ROS) formation in a human monocyte model. The results here ranked the PJs as PJ > HMW-PJ >LMW-PJ. In vitro GI digestion of herring PJs for 165 min, increased the ORAC values of the protein containing PJs up to approx 12.5 fold and extended the LDL oxidation lag phase approx 1.3 fold. Along with this there was a significant increase in formation of peptides that were < 10 kDa. When comparing all analytical data of non-digested PJs with those of PJ’s digested for 165 min, the peptide content, ORAC values and LDL oxidation had increased by 64-69%, 121-161%, and 112-115%, respectively. This work revealed a strong antioxidative activity of herring PJ, however antioxidative properties of the PJ fractions are very different in different model systems. Non-protein compounds like ascorbic acid efficiently protected the washed cod mince against Hb-mediated oxidation during cold storage. However, such compounds had little effect in in vitro and in vivo models where instead the PJ proteins had activity. Using the ORAC and LDL-models, it was also seen that the antioxidative properties of the PJ-proteins increased after a simulated GI digestion.