Effect of freezing on theβ-hydroxyacyl-CoA-dehydrogenase (HADH) activity of fish meat

Changes in the β-hydroxyacyl-CoA-dehydrogenase (HADH) activity of squid (Loligo vulgaris), mackerel (Scomber scombrus), tuna (Thunnus alalunga), sea bream (Pagellus centrodontus), sole (Solea solea), hake and small hake (Merluccius merluccius) meat due to freezing treatment at ?10° C, ?18° C, ?35° C, ?80° C or ?196° C were investigated. With the exception of the small hake, the HADH activity of aqueous extracts from meat was significantly higher (p<0.0001) in all frozen/thawed fish species studied than in unfrozen animals because during freezing there was a release of HADH. HADH activity values of frozen/thawed squid, unfrozen mackerel, frozen/thawed and unfrozen sea bream and unfrozen hake were affected by the storage time in crushed ice.     

Use of β-hydroxyacyl-CoA-dehydrogenase (HADH) activity to differentiate frozen from unfrozen fish and shellfish

 Further work on an enzymic method to differentiate frozen from unfrozen fish and shellfish is reported. The method is based on the release of the β-hydroxyacyl-CoA-dehydrogenase (HADH) from mitochondria during freezing. Enzymic activity was evaluated in fresh and frozen thawed samples from sole (Solea solea), sea bream (Pagellus centrodontus), hake (Merluccius merluccius), gilt headed bream (Sparus aurata), sea bass (Dicentrarchus labrax), salmon (Salmo salar), prawn (Penaeus japonicus) and Norwegian lobster (Nephrops norvegicus). Changes in the HADH activity of fresh and frozen thawed samples were compared after freezing at –196  °C for 15 min. Two values were obtained: U (by dividing: HADH activity of samples frozen at –196  °C, then thawed/HADH activity of unfrozen samples) and F (by dividing: HADH activity of samples frozen at –18  °C, thawed, then frozen at –196  °C /HADH activity of samples frozen at –18  °C, then thawed). Statistical analysis showed significant differences (P≤0.05) between both quotients for gilt headed bream, salmon, sea bream, sole and prawn, and an arbitrary limit was set at 2 to differentiate frozen thawed from unfrozen samples. The application of this limit made it possible to discriminate the unfrozen from the frozen thawed state of around 90% of the total samples analysed. Best results were obtained for prawn (100% of samples differentiated). In the present paper, a laboratory routine is proposed based on the comparison of the HADH activity of a sample analysed straight away and that of a sample frozen at –196  °C and then thawed. The reported method is simple and fast. The entire laboratory procedure can be performed in 45 min. Received: 20 July 1998 / Revised version: 2 November 1998     

β-hydroxyacyl-CoA-dehydrogenase (HADH) differentiates unfrozen from frozen-thawed crawfish (Procambarus clarkii) and trout (Salmo gairdneri) meat

An enzymatic method for differentiation of unfrozen from thawed crawfish ( Procambarus clarkii ) and trout ( Salmo gairdneri ) has been developed. The method is based on the release of the β-hydroxyacyl-CoA-dehydrogenase (HADH) during freezing. The HADH activity mean values were 7.9 and 78.9 for unfrozen and thawed trout, respectively. In the case of crawfish, these values were 12 and 70 respectively. Statistical analyses showed significant differences ( P 0.001) in both species. Trout could be distinguished with a high confidence level, but for crawfish this level was slightly lower.     

Effect of freezing/thawing conditions and long‐term frozen storage on the quality of mashed potatoes

The effects of freezing temperature (−80, −40 or −24 °C) and thawing mode (microwave or overnight at 4 °C) on quality parameters of mashed potatoes made from tubers (cv Kennebec) and from potato flakes were examined, as was the effect of long‐term frozen storage on the quality of mashed potatoes. Mashed potatoes were tested for texture profile analysis (TPA) and cone penetration, oscillatory and steady rheometry, colour, dry matter, Brix and sensory analyses. In natural mashed potatoes, TPA hardness and oscillatory parameters showed that processing resulted in a softer product than the fresh control. The parameters were lower in the samples thawed at 4 °C than in those thawed by microwave at all the freezing temperatures used, which may be ascribed to gelatinisation of the starch released from damaged cells. Differences from the freshly prepared product decreased when the samples were frozen at −80 °C and thawed by microwave. No difference was found in sensory acceptability between samples frozen at −80 and −40 °C, which probably reflects the panellists' mixed preferences for air‐thawed versus microwave‐thawed samples. Increasing the time in frozen storage led to a natural mash with a firmer texture, higher L*/b* value and Brix; nonetheless, panellists found the samples at 0, 3 and 12 months of frozen storage equally acceptable. In commercial mash, penetration and oscillatory parameters showed that processing made for a firmer product than the fresh control, probably owing to retrogradation of gelatinised starch. Thawing mode had a significant effect on parameters, which were lower in the samples thawed at 4 °C. The structure and quality of commercial mash was more detrimentally affected by freezing and, therefore, we would not recommend either freezing or frozen storage of this mashed potato in the used conditions. Natural mash made from Kennebec potatoes should be frozen quickly and thawed by microwave in the conditions described to obtain a product more similar to that freshly made. If the samples are frozen by air blasting at −40 °C, the product can withstand frozen storage for one year. Copyright © 2005 Society of Chemical Industry     

Differentiation between fresh beef and thawed frozen beef

In order to prevent meat retailers offering thawed, imported frozen beef as fresh domestic beef, the method of Gottesmann and Hamm for differentiating between fresh and frozen/thawed meat is recommended. The principle of this method is based on the fact that freezing and thawing of meat results in a release of the enzyme β-hydroxyacyl CoA-dehydrogenase (HADH) from the mitochondrion into the sarcoplasm; an elevated HADH activity in the muscle press-juice indicates freezing and thawing of tissue. The HADH colour test of Gottesmann and Hamm was modified by replacing the electron-transmitter meldolablue by resazurin which results in a much higher colour stability after reaction with fresh meat extracts.     

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.     

Viability of Lactobacillus plantarum in Orange Juice under Low pH and Temperature Conditions

A strain of Lactobacillus plantarum, isolated from fermenting orange juice (OJ), was inoculated into unpasteurized or reconstituted OJ and held under low pH/low temperature conditions. Viability decreased more rapidly at subfreezing temperatures near – 5°C than at lower or higher temperatures. Viability decreased 0.37 log CFU/mL/hr at - 6.6°C and 0.05 log CFU/mL/hr at - 18°C. Rates of population decrease in frozen samples at - 5°C were about three times greater than in unfrozen samples at the same temperature. An inverse linear relationship existed between rate of population decrease (log CFU/mL/day) at - 7°C and OJ pH, with the rate of decrease at pH 4.1, about 1 log cycle lower than at pH 3.5 (0.024 and 0.60, respectively).     

Frozen herring as raw material for spice‐salting

One batch of herring (Clupea harengus) was spice‐salted fresh and as thawed after 32 days of frozen storage at −24 °C. After salting, samples of both groups were sent to participating laboratories in Iceland, Denmark, Norway, Germany and England. The herring was kept at 5 ± 1 °C and sampled three times during a 26 week storage period. The development of taste and texture characteristics (determined by sensory evaluation and instrumental texture measurements), formation of low‐molecular‐weight nitrogen compounds and changes in proteolytic activity were followed in both groups. The sensory evaluation results showed that thawed salted herring ripened in a similar manner to herring salted fresh, but at a faster rate. Instrumental texture analysis showed a faster rate of tenderising in thawed salted herring. Proteolytic activity measured as general activity and with specific synthetic substrates was higher in the thawed salted herring. The formation of trichloroacetic acid‐soluble nitrogen and free amino acids was faster in the previously frozen herring. The results show an accelerated but similar rate of ripening in thawed spice‐salted herring in comparison with fresh salted herring. © 2000 Society of Chemical Industry     

The effect of thermal treatment on β‐glucosidase inactivation in vanilla bean (Vanilla planifolia Andrews)

The conditions for enzyme activity (pH and temperature) and kinetic parameters for the thermal inactivation of β‐glucosidase enzyme in vanilla beans have been investigated. The maximum enzyme activity was detected at pH 6.5 and 38 °C. The values obtained for V_max and K_m were 62.05 units and 2.07 mm, respectively. When hot water treatment (the most practical method of vanilla bean killing) was applied, β‐glucosidase treated at pH 6.0 and 60 °C for 3 min lost 51% of activity, while at 70 °C for 90 s the enzyme lost 60% of activity and at 80 °C for 30 s the enzyme lost 48% of its activity. When vanilla beans were cured in an oven at 60 °C for 36 to 48 h all β‐glucosidase activity was lost.     

Synthesis and degradation of adenosine triphosphate in cod (Gadus morhua) at subzero temperatures

This study has demonstrated that the extraction step is very important when analysing ATP and its degradation products. An important factor is whether the sample is fresh, frozen or thawed when homogenised since thawing of the sample will lead to rapid loss of ATP. During frozen storage it was found that ATP in cod (Gadus morhua) was stable at −40 °C in small samples for at least 12 weeks. At −20 °C it was found that ATP content increases initially and thereafter falls. It was demonstrated that degradation of ATP in small samples occurs faster at 0 °C than at −2 and −5 °C. Furthermore, it was found that in whole cod ATP could be synthesised at a significant rate at −7 °C. © 1999 Society of Chemical Industry     

Extraction of Milk Clotting Enzyme from Sodom Apple (Calotropis procera)

A milk clotting enzyme with low proteolytic activity was extracted with ammonium sulfate, at 0.40-0.65 saturation, from sodom apple leaves. The enzyme with apparently a basic isoelectric point was activated by cysteine and was more active at 65°C than at 35°C. Milk clotting activity increased with pH at 65°C, with the enzyme being almost twice as active at pH 6.4 as at pH 5.4-5.7. Storage at 4°C for 15 days resulted in a 30-50% loss in enzyme activity.     

Freezing, thawing and cooking effects on quality profile assessment of green beans (cv. Win)

Results are presented of the effect of freezing followed by thawing (air and water immersion, both at environmental temperature) and cooking (traditional boiling in a covered pot) on quality profile (in terms of objective texture, colour, chlorophylls and pheophytins and sensory attributes) and structure of green beans (cv. Win). Freezing was carried out at three different rates by forced convection with liquid nitrogen vapour. Kramer shear cell (KSC) and Warner–Bratzler (WB) tests were used for objective assessment of the texture. The highest parameter values occurred in beans frozen at the highest rate and air-thawed at the slowest rate. Also, minimum alteration of the rheological behaviour of cooked beans was achieved by freezing at the highest rate. The best parameter for assessing the texture of frozen green beans after thawing and cooking was the Warner–Bratzler slope (S _WB). Coefficients of softening estimated for S _WB in the thawed beans showed that the texture of the beans frozen at −24 °C was almost four and almost five times softer than that of the beans frozen at −70 °C, for air and water thawing respectively. Frozen and thawed green beans were darker than fresh control, whereas freezing prior to cooking produced lighter-coloured beans than direct cooking. The freezing rate affected colour parameters differently depending on the process that followed. When beans were thawed, increasing the freezing rate produced lighter-coloured beans, whereas when beans were cooked, increasing the rate produced darker-coloured beans. No difference was found in sensory assessments between cooked samples frozen at −24 °C, −35 °C and −70 °C, which probably reflects the panellists' mixed preferences for quickly and slowly frozen samples. Scanning electron microscopy (SEM) revealed different degrees of mechanical damage to tissue structure, which accounted for the rheological behaviour of the beans.     

Transaminase (AST/GOT and ALT/GPT) Activity in Ground Beef as a Means of Determining End-point Temperature

Thermal processing conditions [rate of temperature increase, 0.35°C or 3.5°C/min; end-point temperature (EPT), 57.2, 71.1, and 79.4°C; dwell time, 0 and 30 min; and enzyme extraction medium (deionized water, 0.9% saline, and pH 7 buffer)] affected glutamic oxalacetic transaminase (GOT; aspartate aminotransferase, E.C. 2.6.1.1) and glutamic pyruvic transaminase (GPT; alanine aminotransferase; E.C. 2.6.1.2) activity in thermal treated ground beef samples. There was greater loss of GOT activity in samples heat processed at 0.35°C/min than samples heat processed at 3.5°C/min with depletion nearly to zero between 71.1 and 79.4°C. More GOT activity was noted when samples were extracted with 0.9% saline solution. GPT activity was low in all samples and was present after heating to 79.4°C.     

Differentiation of Frozen and Unfrozen Beef Using Near-Infrared Spectroscopy

Freezing and thawing affects the quality of meat. The present paper focuses on using near-infrared (NIR) diffuse reflectance spectroscopy to detect whether beef has been frozen and thawed. Intact beef and drip or centrifuged meat juice of M longissimus dorsi slices from 40 cattle were used as samples. The meat juices were analysed using dry extract spectroscopy by infrared reflection (DESIR). From centrifuged juice 80 samples were classified 100% correctly, using crossvalidation, into frozen or unfrozen beef by the K nearest neighbours method. This was obtained by high-order principal components from 400–2500 nm spectra. Other multivariate techniques, smaller wavelength ranges and detecting refrozen, thawed beef also gave results between 90 and 100%. Analyses of drip loss, exudative properties, water-holding capacity and dry matter of meat juice supported the interpretation of the NIR measurements. The results showed that NIR might be used as a screening method to differentiate unfrozen and frozen beef. © 1997 SCI.     

Effects of dehydration temperatures on colour and polyphenoloxidase activity of Amasya and Golden Delicious apple cultivars

During dehydration at different temperatures (60, 70 and 80 °C) of Amasya and Golden Delicious apple cultivars, changes in colour, polyphenoloxidase activity (PPO), browning index and hydroxymethylfurfural (HMF) content were studied. Effects of dehydration time on the L* values of both cultivars were not significant. Only Amasya samples dehydrated at 80 °C were found to have significantly higher L* values than the remaining samples. In all cases, Golden Delicious samples had higher L* values than those of the Amasya cultivar. a* values of samples increased within the first hour of dehydration and then remained almost unchanged. The enzyme activity showed a rapid decrease in the first hour of dehydration and residual enzyme activities (%) reached 9.8%, 5.3% and 4.5% for Amasya, and 17.4%, 10.3% and 4.6% for the Golden Delicious cultivar at 60, 70 and 80 °C, respectively. In all final samples the residual enzyme activities were around 1%. The highest browning values were observed at the second hour of dehydration at 60 and 70 °C and the first hour of dehydration at 80 °C. In all conditions Amasya apples had a higher browning tendency. The presence of HMF was detected only at the 4th hour of dehydration at 80 °C for both cultivars. Effects of dehydration temperature on colour and PPO of the final product were insignificant. However, an important effect of temperature was determined on the browning index of Golden Delicious samples. The lowest browning tendency was measured on samples dehydrated at 80 °C. The results showed that cultivar and dehydration temperature had considerable effect on the browning of apple slices. Copyright © 2006 Society of Chemical Industry     

Isolation of Casein by Ultrafiltration and Cryodestabilization

Casein was isolated from skim milk by ultrafiltration, storage of the retentate at -8°C for l-4 wk, and centrifuging of thawed samples at 5,000 ×g for 10 min. The extent of cryodestabilization of casein was dependent on the extent of ultrafiltration and storage time at -8°C. Ultrafiltration to a 4× or 6× volume concentration (VCR) ratio resulted in greater than 95% recovery of casein. Casein thus precipitated could be washed with water at 0°C without any significant loss of casein. Removal of lactose caused accelerated casein cryodestabilization in ultrafiltered samples as compared to control samples. Casein precipitated by this method is dispersible in water, whereas acid precipitated casein is not.     

Assessment of Previous Heat Treatment of Laboratory Heat-Processed Meat and Poultry using a Commercial Enzyme System

The APIZYM enzyme system was assessed with respect to: enzyme activity in aqueous and saline extract filtrates from raw and heat processed (60° and 71.1°C) beef; effect of rate of heating (0.2–0.3°C, 0.4–0.5°C, and 0.9–1.0°C/min) to internal temperatures of 66.7°, 67.8°, 68.9°, 70.0°, and 71.1°C and holding times of 0, 15, 30, 45, and 60 min at these temperatures on residual enzyme activity; reduction of enzyme assay incubation time; and reproducibility of the system. Results showed that slightly higher enzyme activity in heated samples was obtained with 0.9% saline extraction compared to water. Greater enzyme activity was observed in filtrates from pork samples heat-processed at a fast rate than at a slow rate. Enzyme activity could be detected after 2 hr incubation, but 4 hr were needed for the enzymes to react with their respective substrates. The system was found to be reproducibile. The results of this study also indicated the potential of using leucine aminopeptidase as an indicator enzyme for determining the adequacy of heat treatment of meat and poultry products.     

Lipid Oxidation in Ground Beef Patties as Affected by Time-Temperature and Product Packaging Parameters

Oxidative rancidity in fresh and stored ground beef samples was measured using a thiobarbituric acid (TBA) assay with antioxidant protection. The independent variables were fat concentration (15 or 30%), package type (polyethylene or vacuum-packaged), freezer storage temperature (-12.2°, -23.3° or -34.4°C) and storage time (4, 8, 12, 16, or 20 weeks). At the end of each storage time samples were thawed and TBA values were determined on the samples before and after cooking. TBA values increased during the first 12 to 16 weeks after which time it decreased for both the cooked and uncooked samples. The higher fat samples, packaged in polyethylene, had higher TBA values for both cooked and uncooked patties. Uncooked patties stored at - 12.2°C had higher TBA values than those stored at -23.3°C or -34.4°C but cooked sample TBA values showed no dependence on storage temperature.     

Isolation, Purification and Physicochemical Characterization of Polyphenoloxidases (PPO) from a Dwarf Variety of Banana (Musa cavendishii, L)

A procedure for isolating and purifying polyphenoloxidase from banana pulp is described. Maximum activity was obtained from the inner portion of the pulp by extraction with 0.2M sodium phosphate buffer, pH 7.0, containing 1% insoluble PVP and 0.25% Triton X-100. The enzyme was purified 38.8-fold after acetone precipitation, re-extraction of the precipitate with phosphate buffer containing Triton X-100, storage in the freezer for 7 days (-40°C), chromatography on Sephadex G-100 and electrophoresis on polyacrylamide gel. The purified enzyme (four isozymes) gave a poly-peptide molecular weight (SDS-gel) of 31,000 ± 1,000 and molecular weight on sucrose gradient ultracentrifugation of 62,000 ± 2,000. The isoelectric point, determined by isoelectric focusing on polyacrylamide gel, was 5.2. The enzyme was stable at 55°C for 30 min; it lost mole than 90% of the activity after 5 min at 85°C and was completely inactivated at 95°C for the same time. The absorption spectrum of the purified enzyme showed a maximum at 278 nm and a shoulder at 340-350 nm. This preparation, when submitted to paper chromatography revealed the presence of a fluorescent compound with R_f= 0.57, which was identical with chlorogenic acid.