Nazlin K. Howell

Changes in the texture and structure of cod and haddock fillets during frozen storage

Abstract Changes in the texture of commercially important lean cod (which produces substantial formaldehyde), and haddock fillets (which produces negligible formaldehyde), stored at −10 and −30°C for up to 30 weeks were measured. For both species, hardness measured by large deformation rheology and elastic modulus ( G ′) by small deformation techniques, which reflect muscle toughening, increased with a higher storage temperature and prolonged time of storage, in a similar way. Differential scanning thermal analysis of both frozen cod and haddock muscle indicated alteration in the transition temperature and enthalpy due to a higher rate of protein denaturation at −10°C compared with muscle stored at −30°C. Protein denaturation and texture changes were accompanied by a decrease in protein solubility, an increase in hydrophobicity and the formation of non-covalently and covalently linked aggregates. The similarities in the texture and biochemical changes in both frozen cod and haddock indicates that formaldehyde is not a major factor in muscle toughening in frozen fish.

Studies on egg albumen and whey protein interactions by FT-Raman spectroscopy and rheology

Large deformation rheological studies of either egg albumen or whey protein isolate (15% protein w/w) gels made by heating at 90 °C for 30 min indicated a higher gel strength and Youngs modulus values for egg albumen compared to whey protein isolate. The proteins mixed in the ratio 10:5 whey/egg albumen indicated synergistic interactions producing greater than expected gel strength values. Small deformation rheological studies of egg albumen 15% (w/w) and whey protein isolate 15% (w/w) showed very low values of the storage modulus (G′) at 20 °C suggesting that both proteins do not easily aggregate at room temperature. Whey proteins gelled at 80 °C, whereas egg albumen proteins were already cross-linked at 20 °C (G′>G″). A mixture of 7.5% whey/7.5% egg albumen showed the highest G′ value at the end of both heating and cooling cycles but this value was lower than that of the individual proteins. Changes in the conformation, molecular structure and protein–protein interactions in whey and egg albumen (15% w/w in D2O pD7.2) and their mixtures (7.5:7.5 protein w/w) were investigated by FT-Raman spectroscopy. Upon heating to 90 °C for 30 min, individual whey and egg albumen proteins showed changes in the CH (1350 cm−1), CH2 (1450 cm−1) bending vibrations and Trp residues at 760 cm−1, reflecting involvement of hydrophobic interactions. Changes in the disulphide stretching vibrations were also observed. Peak intensity increased for β-sheet structures in the Amide 111′ region 980–990 cm−1 and decreased for helix structures (900–960 cm−1) for both egg albumen and whey proteins. Differences in the experimental and theoretical average spectra indicated changes in β-sheet structures, CH2 bending, carboxyl and hydrophobic groups in heated binary mixtures of egg albumen and whey proteins, providing evidence of protein–protein interactions, observed by large deformation rheology.

Electron spin resonance (ESR) study on free radical transfer in fish lipid–protein interaction

Oxidised lipids interact with proteins causing undersirable changes in the nutritional and functional properties, including a loss of amino acids, cross-linking and damage to proteins and DNA. Proteins including egg lysozyme, egg ovalbumin, fish myosin and amino acids arginine, lysine and histidine were exposed to oxidised lipids methyl linoleate and oil extracted from Atlantic mackerel (Scomber scombrus). A strong central singlet signal was induced in the proteins and amino acids which was detected by ESR spectroscopy and assigned to the carbon radical (g value range 2.0021–2.0049); with ovalbumin and fish myosin a downfield shoulder, attributed to the sulphydryl group (g value 2.014–2.017), was also observed. The above changes in the proteins were accompanied by an increase in fluorescence indicating the formation of cross-links. Synthetic antioxidants such as butyl hydroxytoluene (BHT) and butyl hydroxyanisole (BHA) as well as natural antioxidants ascorbic acid and α-tocopherol inhibited the development of both the free radical signal and fluorescence when added to the proteins prior to incubation with oxidised lipids; the central singlet signal attributed to the carbon radical was reduced by 10–50%. This paper clearly indicates direct free radical transfer from oxidised lipids to amino acids and proteins which results in protein denaturation and the formation of insoluble aggregates in fish flesh on storage. © 1999 Society of Chemical Industry

Aldehyde formation in frozen mackerel (Scomber scombrus) in the presence and absence of instant green tea

The effect of frozen storage on lipid peroxidation in Atlantic mackerel (Scomber scombrus) stored for up to 26 weeks at -10 or -80°C (control), with and without green tea antioxidants, was investigated. Hydroperoxides (PV) and aldehydes (TBARS) were measured by HPLC and LC-MS and hexanal by GC. There was an increase in peroxide value which was associated with an increase in aldehydes, followed by hexanal increase with storage time and at a higher temperature of -10°C compared with samples stored at -80°C. Although TBARS is a common assay used to follow malondialdehyde formation, other aldehyde products can also react with thiobarbituric acid to give the red chromogen. Analysis of aldehyde-TBA adducts by LC-MS confirmed the presence of malondialdehyde and, in particular, we report the production of gluteraldehyde for the first time in stored frozen fish. Green tea (at 250ppm) substantially slowed down the oxidation process, whereas at 500ppm it was less effective.

Gelation properties of soya and whey protein isolate mixtures

Abstract Gelation properties of commercially available soya and whey protein isolates (15–18% w/w) were tested in isolation and when mixed in ratios ranging from 17:1 to 1:17 soya/whey to give a final protein concentration of 18% protein (w/w) in distilled water. The isolates and mixtures were investigated by both small and large deformation rheological analysis, which indicated phase separation of the two proteins systems. Phase inversion occurred at 5:1 ratio (soya/whey) where the lowest shear modulus value, by large deformation testing, was 3.5 compared to 5.5 for 18% (w/w) soya and 10.7 for 18% (w/w) whey proteins in distilled water. The G ′ value obtained by small deformation testing, also showed the lowest value at the 5:1 ratio (soya/whey proteins). In addition, gels exhibited maximum syneresis at this ratio indicating instability at the point of inversion. Electron microscopy in combination with immunocytochemistry using anti-soya or anti-whey antibodies also indicated phase separation of the two types of proteins. Phase separation was considered to occur due to differences in the molecular sizes of the proteins and physical–chemical differences including hydrophobicity as verified by the cis -parinaric fluorescent probe technique.

High-resolution NMR and magnetic resonance imaging (MRI) studies on fresh and frozen cod (Gadus morhua) and haddock (Melanogrammus aeglefinus)

Changes in the fish muscle from cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) were investigated by high-resolution NMR and magnetic resonance imaging (MRI). Water- and salt-soluble extracts from fish stored at −20°C and −30°C were analysed by high-resolution proton NMR and enabled the identification of metabolites including trimethylamine oxide, trimethylamine (TMA) and dimethylamine. It was not possible to detect formaldehyde by NMR either in the stored fish samples or in spiked water or salt extracts even at high levels of formaldehyde addition, probably due to polymerisation. Systematic and controlled storage trials indicated the presence of dimethylamine at around 9 months for samples stored at −20°C, whereas no changes were detected at the control storage temperature of −30°C. A comparison of cod and haddock fillets stored for 1 year at −20 and −30°C confirmed the production of dimethylamine only in cod stored at −20°C. It was interesting to note that ‘fresh’ cod and haddock samples purchased from a local supermarket showed high levels of TMA indicating a breakdown of trimethylamine oxide to TMA by bacteria. TMA was not detected in the fish fillets especially obtained for the storage trials. MRI of fresh cod and fish stored at −8 and −30°C indicated that the fish half stored at −8°C exhibited dense lines or arches which are indicative of gaps in the tissue due to possible breakdown of the connective tissue. The images of fish stored at −30°C did not indicate any differences compared with the fresh fish. MRI also showed the presence of frozen and unfrozen areas in the fish non-destructively.

Purification and characterisation of two pectinmethylesterase from persimmon (Diospyros kaki)

Two pectinmethylesterase isoforms, PME I and PME II, have been separated and purified from persimmon using chromatography techniques. Both isoforms presented differences in molecular weight (PME I: 51 kDa, PME II: 30 kDa), isoelectric point (PME I: 8.4, PME II: 6.9) and K m values (PME I: 54 μg ml -1 , PME II: 31 μg ml -1 ). They differed in their optimum pH and thermal stability, PME being the more thermostable isoform. Both isoforms exhibited similar behaviour with respect to Ca 2+ and Na + concentrations and were strongly inhibited by CaCl 2 concentrations of over 80 mM and by NaCI concentrations of over 500 mM. Both isoforms were activated by low concentrations of polygalacturonic acid and were competitively inhibited by D-galacturonic acid and high concentrations of polygalacturonic acid.

Protein-Protein Interactions

Protein—protein interactions occur in many chemical and physical processes which are involved in the complex molecular organisation of plant and animal cells. Most of these interactions affect the processing, storage and behaviour of proteins in food. There are various reasons for studying protein interactions, including acquiring knowledge of structure—function relationships of proteins; optimising the use of product constituents; improving quality, cost reduction and new protein applications.

Angiotensin I-converting enzyme (ACE) inhibitory activity and structural properties of oven- and freeze-dried protein hydrolysate from fresh water fish (Cirrhinus mrigala).

The angiotensin I-converting enzyme (ACE) inhibitory activity and structural properties of oven-dried (OD-FPH) and freeze-dried (FD-FPH) protein hydrolysates derived from fresh water fish (Cirrhinus mrigala) muscle, using papain, were investigated. Amino acid profiles indicated a higher proportion of hydrophobic residues in OD-FPH and hydrophilic residues in FD-FPH samples. Fourier transform infrared (FT-IR) spectra revealed random coil structure in OD-FPH and β-sheet in FD-FPH samples. The approximate molecular weight of peptides in OD-FPH and FD-FPH was in the range of 7030-339Da. The IC50 values for ACE inhibition by OD-FPH and FD-FPH samples were found to be 1.15 and 1.53mg of proteinml(-1), respectively. The ACE-inhibitory activity of OD-FPH was more stable (during sequential digestion, using pepsin and pancreatin) than that of FD-FPH sample. The study suggested that the ACE inhibitory activity of protein hydrolysate was not affected by oven-drying.

Antioxidant and ACE Inhibitory Bioactive Peptides Purified from Egg Yolk Proteins

Protein by-products from the extraction of lecithin from egg yolk can be converted into value-added products, such as bioactive hydrolysates and peptides that have potential health enhancing antioxidant, and antihypertensive properties. In this study, the antioxidant and angiotensin converting enzyme (ACE) inhibitory activities of peptides isolated and purified from egg yolk protein were investigated. Defatted egg yolk was hydrolyzed using pepsin and pancreatin and sequentially fractionated by ultrafiltration, followed by gel filtration to produce egg yolk gel filtration fractions (EYGF). Of these, two fractions, EYGF-23 and EYGF-33, effectively inhibited the peroxides and thiobarbituric acid reactive substance (TBARS) in an oxidizing linoleic acid model system. The antioxidant mechanism involved superoxide anion and hydroxyl radicals scavenging and ferrous chelation. The presence of hydrophobic amino acids such as tyrosine (Y) and tryptophan (W), in sequences identified by LC-MS as WYGPD (EYGF-23) and KLSDW (EYGF-33), contributed to the antioxidant activity and were not significantly different from the synthetic BHA antioxidant. A third fraction (EYGF-56) was also purified from egg yolk protein by gel filtration and exhibited high ACE inhibitory activity (69%) and IC50 value (3.35 mg/mL). The SDNRNQGY peptide (10 mg/mL) had ACE inhibitory activity, which was not significantly different from that of the positive control captopril (0.5 mg/mL). In addition, YPSPV in (EYGF-33) (10 mg/mL) had higher ACE inhibitory activity compared with captopril. These findings indicated a substantial potential for producing valuable peptides with antioxidant and ACE inhibitory activity from egg yolk.