Ingrid Undeland

Methods to evaluate fish freshness in research and industry

Current work in a European concerted action project ‘Evaluation of Fish Freshness’ (AIR3 CT94-2283) focuses on harmonizing research activities in the area of fish freshness evaluation in leading fish laboratories in Europe (see Box 1). The overall aim of the concerted action project is to validate methods for the assessment of fish freshness and to discuss the freshness criteria for fish commercialized within the European Union. The projects participants are working in subgroups studying sensory analysis, microbiology, volatile compounds, proteins, lipids, adenosine triphosphate and physical measurements with respect to fish freshness evaluation. In this article, the different subgroups have summarized changes that occur in fish and methods to evaluate fish freshness as a first step towards the definition of criteria for fish freshness.

Comparison between methods using low-toxicity solvents for the extraction of lipids from herring (Clupea harengus)

Three alkane/alcohol/water-based lipid extraction systems were evaluated to determine which would be the best replacement for the frequently used chloroform/methanol/water system, nowadays known to be very toxic. All the methods were applied to samples of minced herring (Clupea harengus) differing in quality and composition. In addition to comparisons of total lipid yield, the extracted lipids were compared with respect to content of triglycerides, phospholipids, free fatty acids, α-tocopherol, lipid hydroperoxides and conjugated dienes. The content of phospholipids was found to differ most between the lipids extracted by the four methods. Here, the chloroform/methanol/water system was the most efficient, followed by heptane/ethanol/water/sodium dodecyl sulphate (SDS) and then iso-propanol/hexane. However, by decreasing the level of SDS, the efficiency of the heptane/ethanol/water/SDS system in extracting phospholipids was increased to the same level as that of the chloroform/methanol/water system. This decrease in SDS also resulted in a higher recovery of free fatty acids. The lack of correlation between yields of phospholipids and yields of lipid oxidation products throughout this study was surprising because of the often-described susceptibility of phospholipids to oxidation.

Influence of skinning on lipid oxidation in different horizontal layers of herring (Clupea harengus) during frozen storage

This study was conducted to evaluate the effect of skinning and of compositional differences on the oxidative stability of various horizontal layers from herring (Clupea harengus) during frozen storage. Herring fillets, with and without skin, were stored at -18°C for 0, 3, 9, 16 and 28 weeks. After each storage period, the fillets were divided horizontally into three layers: ‘under skin’, ‘middle part’ and ‘inner part’. Each layer was then extracted for total lipids, in which peroxide value (PV), absorbance at 234 nm (A234) and 268 nm (A268) as well as lipid-soluble fluorescent oxidation products (FP) were measured. Prior to storage, the fat content, fatty acid pattern and α-tocopherol were also analysed. During storage of skinless fillets, the under skin layer increased most in PV, A234, A268 and FP (P<0·05), followed by the inner and middle parts. In fillets stored with skin, the high oxidation rate of the under skin layer lipids was suppressed, but this layer still gave rise to the highest responses. Firstly, these results point to the protective properties of the skin and, secondly, to the unfavourable composition of the under skin layer: a lot of dark muscle; the silver surface; the highest fat content and the lowest level of α-tocopherol. Concerning the fatty acid pattern in the three layers, the amount of C20: 5, C18:1 and C20:1 in the fat gradually decreased from the under skin layer towards the inner part of the fillet, whereas the opposite was true for C22: 6.

Herring ( Clupea harengus) intake influences lipoproteins but not inflammatory and oxidation markers in overweight men.

Fish consumption is associated with a lower incidence of CVD and decreases in risk factors for atherosclerosis. Although fish contains other interesting components than fish oil, few studies focus on total fish composition and the influence food preparation might have on health-beneficial components. In the present cross-over intervention study the effect of a 6-week herring diet compared with a reference diet on CVD risk factors was investigated. Thirty-five healthy, but overweight, men (mean BMI 28.3 kg/m2) were randomised to a 6-week herring diet (150 g baked herring fillets/d, 5 d/week) or a reference diet (150 g baked lean pork and chicken fillets/d, 5 d/week). Diets were switched after a 12-week washout period. Plasma total cholesterol, TAG, HDL, HDL2, HDL3, LDL, C-reactive protein, IL-6, IL-18, intercellular adhesion molecule-1, oxidised LDL, oxygen radical absorbance capacity using perchloric acid (ORACPCA), whole-blood fatty acids, bleeding time and blood pressure were measured at the beginning and end of each dietary period. HDL was significantly higher after the herring diet period compared with after the reference diet period: 1.04 v. 0.99 mmol/l. TAG decreased after both diets, with no significant difference between the two diets. ORACPCA values did not indicate lower concentrations of non-protein plasma antioxidants, and oxidised LDL was not higher after the herring diet than after the reference diet. To conclude, a 6-week herring-rich diet significantly raised HDL compared with a diet of matched lean pork and chicken dishes. No adverse effects on in vivo oxidation or serum antioxidants were found after herring intake.

Oxidation of Cod Liver Oil during Gastrointestinal in Vitro Digestion

Oxidation of cod liver oil rich in long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) was investigated during a gastrointestinal (GI) in vitro digestion. The digestion stimulated TBA-reactive substances (TBARS) formation in both the gastric and intestinal steps, whereas levels of lipid hydroperoxides remained nearly constant. The presence of digestive compounds was decisive for the TBARS development because TBARS did not change when the cod liver oil was subjected only to the temperature and pH gradient of the GI model. Preformed oxidation products in the cod liver oil resulted in further elevated TBARS levels during the digestion. Addition of hemoglobin (11.5 μM) to emulsified cod liver oil dramatically increased TBARS and lipid hydroperoxide levels during GI digestion, whereas 1 mg α-tocopherol/g oil did not show any protection against oxidation. Specific concern thus needs to be taken in the design of foods containing LC n-3 PUFA to preserve these lipids and avoid harmful oxidation, both before and after consumption.

Quantification of total fatty acids in microalgae: comparison of extraction and transesterification methods.

Determination of microalgaes’ fatty acid content is often done with chloroform and methanol according to the Bligh and Dyer extraction, though faster methods exist. A number of comparisons between the Bligh and Dyer and faster methods have resulted in contradicting data, possibly due to differences in algae used and the different versions of the Bligh and Dyer method applied. Here, various forms of direct-transesterification (D-TE) and two-step transesterification (2-TE), including three versions developed in our lab, are compared with the original Bligh and Dyer (Can J Biochem Physiol 37: 911–917, 1959) extraction and two modifications thereof (Lee et al. J AOAC Int 79:487–492, 1996, and our own acidified version) on microalgae with different cell walls: Isochrysis galbana, Nannochloropsis oculata, and Phaeodactylum tricornutum. In total, fatty acid extracts from 11 methods were separated and quantified by gas chromatography with mass spectrometry. Results show that, for N. oculata and P. tricornutum, methods based on chloroform–methanol underestimated the fatty acid content compared with the 2-TE and D-TE methods, which gave similar results. Moreover, D-TE methods are faster than chloroform–methanol methods and use chemicals that are less toxic. Of the D-TE methods, the ones using hydrochloric acid and sulfuric acid recovered the most fatty acids, while boron trifluoride recovered slightly less. The main qualitative difference between the fatty acids recovered was that the chloroform–methanol methods recovered less saturated fatty acids in P. tricornutum.

Protein Isolation from Gutted Herring (Clupea harengus) Using pH-Shift Processes

Herring ( Clupea harengus ) and other pelagic fish species are mainly used for fish meal and oil production and not for human consumption. In this study, acid pH-shift processing and alkaline pH-shift processing were used to isolate proteins from whole gutted herring with the aim to investigate the potential use of herring proteins as a food ingredient. The acid and alkaline processes gave rise to similar protein yields, 59.3 and 57.3%. The protein isolates from both processes had a significantly (p < 0.05) whiter color and higher protein and lower lipid contents than the starting material. The removal of ash was >80% for both processes, with a trend (p = 0.07) toward higher removal during the alkaline process. Also, Ca and Mg removal was significantly (p < 0.05) higher during the alkaline process. The isolated proteins from the acid process contained myosin degradation products and had a lower salt solubility than proteins from the alkaline process. Both protein isolates had an amino acid profile meeting the recommendations for adults according to FAO/WHO/UNU and could produce a surimi gel of medium strength. The results show that pH-shift processing could be a valuable method for the production of functional food proteins from gutted herring.

Fish and cardiovascular health

The antiatherogenic and antithrombotic effects of fish-oil-derived n-3 (omega-3) polyunsaturated fatty acids have given these compounds a dominating role in explaining the tentatively beneficial effects of fish, e.g. in the prevention of cardiovascular disease (CVD). As a result, the words ‘‘omega-3’’ and ‘‘fish’’ are often lumped together in both experimental studies and reviews. There are, however, strong reasons for separating fish lipids from whole fish muscle. The fish muscle matrix is highly complex, and many other compounds therein have been suggested as bioactive. This review summarizes data from epidemiological and intervention studies addressing effects of fish consumption per se on CVD. Potential roles of fish in CVD protection, and some risks connected to excessive fish eating, i.e. biocides and oxidation, are also discussed. Twelve out of 18 prospective cohort studies, two out of two case_/control studies and two out of three ecological studies indicated reduced coronary mortality among subpopulations eating more fish, often at low fish intakes. Two cohort studies indicated increased risk for cardiovascular mortality. Out of 16 epidemiological studies in total, six prospective cohort studies, two case_/control studies and one ecological study indicated a reduced risk of coronary morbidity with higher fish consumption, and one cohort study indicated an elevated risk of coronary morbidity. Two intervention studies on men with coronary heart disease produced different results, one indicating decreased total mortality, with lower coronary morbidity, but the other indicating an elevated risk for coronary mortality without affecting total morbidity. Thus, ample epidemiological data favour fish intake for reducing mortality and morbidity in cardiovascular disease, although evidence from intervention studies is inconsistent. Among risk factors for CVD that have been proven to be affected by a fish-containing diet are high-density lipoprotein cholesterol, serum triglycerides and blood clotting, the former increasing and the latter two decreasing. Keywords: cardiovascular; coronary health; diet; fish; n-3 PUFA

Protein isolation from blue mussels (Mytilus edulis) using an acid and alkaline solubilisation technique -process characteristics and functionality of the isolates

BACKGROUND The pH shift method was developed to isolate proteins from low-value raw materials by solubilisation at high or low pH followed by precipitation. In this paper the application of the pH shift method on isolated mussel (Mytilus edulis) meat and whole mussels is reported. RESULTS Highest protein solubilisation was achieved at pH values of 2.6 and 12. The optimum precipitation pH values were established as around 5.8 following acid solubilisation and 5.2 following alkaline solubilisation. Protein recoveries were 430 and 580 g kg(-1) with the acid and alkaline processes respectively. Using whole crushed mussels, the corresponding recoveries were 310 and 480 g kg(-1). Process modifications to further improve protein recovery resulted in only a marginal increase. Lipid oxidation was not induced during pH shift processing, but heavy proteolysis occurred during the acid process version. Proteolysis could not be prevented by porcine plasma protein. Alkali-produced proteins performed better in all functionality tests compared with acid-produced proteins. The acid process removed slightly more lipids, recovered relatively more cysteine, methionine and lysine and resulted in whiter isolates. CONCLUSION The pH shift method can be successfully used to extract functional proteins from mussels and add value to blue mussels unsuitable for human consumption (with or without shells).

Herring (Clupea harengus) supplemented diet influences risk factors for CVD in overweight subjects

Objective:To assess the effect of a 4-week herring diet compared to a reference diet on biomarkers for cardiovascular disease in obese subjects.Design:Randomized crossover trial.Setting:Department of Internal Medicine, Sahlgrenska University Hospital.Subjects:Fifteen healthy obese men and women (age 24–70 years) included, 13 completed.Intervention:Subjects were randomly assigned to four weeks of herring diet (150 g baked herring fillets/day 5, days/week) or reference diet (pork and chicken fillets) and switched diets after 2 weeks washout. P-total cholesterol, p-TAG, p-HDL, p-HDL2, p-HDL3, p-LDL, p-apolipoprotein A, p-apolipoprotein B, p-Lipoprotein (a), p-fibrinogen, p-C- reactive protein and p-antioxidative capacity were analysed at 0,2,4,6,8 and 10 weeks.Results:P-HDL was significantly higher after the herring diet period compared to after the reference diet period; 1.22 vs 1.13 mmol/l (P=0.036). There was a small, but not statistically significant, decrease in TAG but no effect on other biomarkers. TEAC and FRAP, but not ORAC-values, indicated that plasma antioxidants may have been reduced. CRP tended to be lower after the herring diet compared to after the reference diet.Conclusions:Consumption of oven-baked herring (150g/day, 5 days/week) for 4 weeks, compared to consumption of pork and chicken fillets, significantly increased p-HDL. Patients with insulin resistance and obesity, who commonly have low HDL, may therefore benefit from addition of herring to the diet.Sponsorship:Region Västra Götaland, National board of fisheries (Dr 223-2451-01), Sweden (EU structural funds), The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) (Grant No 2001-1246).