Raman spectroscopic study of structural changes in Hake (Merluccius merluccius L.) muscle proteins during frozen storage

This paper examines changes in the structure and functionality of fish muscle proteins at frozen storage temperatures known to render very different practical storage lives (-10 and -30 degrees C). Apparent viscosity and dimethylamine (DMA) content showed drastic temperature-related differences during storage. Raman spectroscopy revealed the occurrence of some structural changes involving secondary and tertiary protein structures. The changes in secondary structure were quantified, showing an increase of beta-sheet at the expense of alpha-helix structure. The nuC-H stretching band near 2935 cm(-)(1) increased in intensity, indicating denaturation of the muscle proteins through the exposure of aliphatic hydrophobic groups to the solvent. These structural changes were more pronounced at -10 degrees C but occurred at both storage temperatures, whereas changes in apparent viscosity and DMA only occurred in storage at -10 degrees C. The possible utility of these structural changes for quality assessment is discussed.     

Ultrastructural changes and structure and mobility of myowater in frozen-stored hake (Merluccius merluccius L.) muscle: relationship with functionality and texture

The ultrastructural changes and the main Raman spectral features of water (3100-3500 and 50-600 cm(-)(1) ranges) in frozen-stored hake were studied with the aim of connecting these changes with loss of some functional properties such as water holding capacity, and with modifications of muscle texture. The following results were obtained: (a) The changes in the spaces between myofibrils can be related to modifications of shear resistance. (b) The behavior of the strong 160 cm(-)(1) band can be related to conformational transitions of muscle proteins, to changes in the structure of muscle water, and/or to alterations in protein-water interactions. (c) There were intensity changes in the nu(s)(OH) band that may be attributable to transfer of water to larger spatial domains during frozen storage.     

Characteristics of the salt-soluble fraction of hake (Merluccius merluccius) fillets stored at -20 and -30 degrees C

Natural actomyosin (NAM) extracted in 0.6 M NaCl from hake fillets stored at -20 and -30 degrees C for up to 49 weeks was studied. The extracted protein decreased as storage progressed and became poorer in myosin while the proportion of actin remained constant. Two major peaks composed of myosin plus actin and actin plus tropomyosin plus troponins were obtained by size exclusion chromatography. SDS-PAGE analysis of the protein retained in the precolumn filter showed that there was protein aggregated by covalent bonding. Surface hydrophobicity increased while Ca(2+)-ATPase activity, apparent viscosity, and SH groups decreased as storage progressed. The loss of Ca(2+)-ATPase activity was due mainly to denaturation of the extracted myosin, whereas the minimum viscosity values occurred earlier and were not directly due to the lower proportion of myosin in the extracts. Thus, the extracted NAM exhibited changes during frozen storage. The temperature-dependent difference was mainly quantitative due to a smaller amount of protein extracted at -20 degrees C.     

Ultrastructure of the myofibrillar component in cod (Gadus morhua L.) and hake (Merluccius merluccius L.) stored at -20 degrees C as a function of time.

Transmission electron microscopy and image analysis techniques were used to study the ultrastructure of the myofibrillar component in cod and hake muscle stored at -20 degrees C for varying periods of time. Cod muscle showed a deformation of the hexagonal array of thick filaments with the storage time, reflected in an increase in the eccentricity value, a parameter defined to measure changes in the ratio of maximum to minimum hexagon diameter, and an increase in the cross-linkings between the filaments. Degradation of cod thick filaments leading to detachment was also visible upon prolonged storage. In hake muscle significant changes were not found in the arrangement and morphology of thick filaments during frozen storage, suggesting a high incidence of intrafilament aggregation. The ultrastructural differences in the array of thick filaments between species were accompanied by a difference in the textural measurements.     

Chemical and sensory changes in Mediterranean hake (Merluccius merluccius) under refrigeration (6-8 degrees C) and stored in ice

Fresh Mediterranean hake (Merluccius merluccius var. mediterraneus), a species mainly caught off the shores of Spain, was stored at usual temperatures: in ice (commercial chain) and under refrigeration (home). Sensory and chemical analyses were performed throughout the storage time to determine the changes that took place and evaluate the effect of the storage temperature. Storage in ice resulted in a slight accumulation of volatile and biogenic amines in hake. When it was stored at 6-8 degrees C, a significant production of both trimethylamine and total volatile basic nitrogen (TVB-N) was observed, and biogenic amines were formed. Sensory analysis revealed that hake stored in ice was inedible after 29 days, the figure for refrigerated hake being 20 days. There was a nonsignificant correlation (p > 0.05) between TVB-N values and sensory score in hake stored at 0 degrees C. In all other cases, a significant correlation (p < 0.001) between volatile parameters and sensory analysis was found.     

Trimethylamine and total volatile basic nitrogen determination by flow injection/gas diffusion in Mediterranean hake (Merluccius merluccius)

The reliability of flow injection/gas diffusion (FIGD) methods to determine trimethylamine (TMA-N) and total volatile basic nitrogen (TVB-N) in hake was studied in order to find an alternative and accurate, simple, cheap, and rapid method for non-protein nitrogen determination. FIGD methods involved extracting volatile amines with 7.5% trichloroacetic acid, followed by the injection of the extracts into the FIGD manifold, previously adjusted for TMA-N or TVB-N determinations. Each determination took approximately 2 min. Reliability was satisfactory in linearity, precision, recovery, and sensitivity. There was good correlation (p < 0.001) between FIGD and the classic official methods, for both TMA-N and TVB-N determinations, and also between FIGD and the gas chromatographic procedure described for TMA-N. These results proved that FIGD methods are simpler, cheaper, and faster than current official procedures. To check the suitability of FIGD procedures over a wide range of analyte concentrations, changes of both TMA-N and TVB-N and the P ratio values throughout the ice storage of hake were monitored. The usefulness of each of these potential freshness indicators for hake is discussed.     

Lipid oxidation in fillets of herring (Clupea harengus) during frozen storage. Influence of prefreezing storage.

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.     

High-pressure effects on the proteolytic enzymes of sea bass (Dicentrarchus labrax L.) fillets

High-pressure processing is a nonthermal technique ensuring food product safety and enabling a longer shelf life. The purpose of this experiment was to evaluate the effect of high pressure on the main proteolytic enzymes involved in fish muscle degradation during storage. Enzymes were extracted with sarcoplasmic proteins from Dicentrarchus labrax sea bass white muscle. Activity of cathepsins B, D, H, and L was quantified in protein extract, whereas calpain activity was evaluated after isolation from its endogenous inhibitor. High-pressure processing up to 500 MPa enhanced the activity of cathepsin B, H, and L, whereas the activity of cathepsin D increased up to 300 MPa and decreased above 300 MPa. With regard to calpain activity, high-pressure processing led to a decrease of activity, which was zero above 400 MPa. We suggest a leading explanation based on simultaneous deactivation of enzymes and an increase of liberation from lysosomes for cathepsins and on dissociation of subunits for calpains.     

Changes in volatile compounds of carrots (Daucus carota L.) during refrigerated and frozen storage

Carrots (Daucus carota L.) of cv. Bolero and cv. Carlo were processed into shreds and stored for up to 4 months at -24 degrees C (frozen storage), or the roots were stored for up to 4 months at 1 degrees C (refrigerated storage) followed by processing into shreds. Volatiles from the carrot shreds were collected by dynamic headspace technique and analyzed by GC-FID, GC-MS, GC-MS/MS, and GC-O to determine the volatile composition and aroma active components of carrots stored under different temperature conditions. A total of 52 compounds were quantified, of which mono- and sesquiterpenes accounted for approximately 99% of the total volatile mass. Major volatile compounds were (-)-alpha-pinene, beta-myrcene, (-)-limonene, (+)-limonene, (+)-sabinene, gamma-terpinene, p-cymene, terpinolene, beta-caryophyllene, alpha-humulene, and (E)- and (Z)-gamma-bisabolene. A considerable increase in the concentration of mono- and sesquiterpenes was observed during refrigerated storage, whereas the concentration of terpenoids was around the same level during frozen storage. GC-O revealed that the major volatiles together with (+)-alpha-pinene, (-)-beta-pinene, (+)-beta-pinene, 6-methyl-5-hepten-2-one, (-)-beta-bisabolene, beta-ionone, and myristicin had an odor sensation, which included notes of "carrot top", "terpene-like", "green", "earthy", "fruity", "citrus-like", "spicy", "woody", and "sweet".     

Flavonoids in baby spinach (Spinacia oleracea L.): changes during plant growth and storage

The variation in flavonoid concentration and composition was investigated in baby spinach (Spinacia oleracea L.) cv. Emilia sown on three occasions, each harvested at three growth stages at 6-day intervals. After harvest, leaves were stored in polypropylene bags at 2 or 10 degrees C. Flavonoids were analyzed by reversed phase HPLC. Twelve flavonoid peaks were detected. The main flavonoid, making up on average 43% of the total flavonoid concentration, was identified as 5,3',4'-trihydroxy-3-methoxy-6:7-methylenedioxyflavone-4'-glucuronide. Four other flavonoids each contributed 7-12% of the total flavonoid content. Total flavonoid content was relatively stable during normal retail storage conditions, although some of the individual flavonoid compounds showed considerable variation. The youngest plants had the highest flavonoid concentration, indicating that by harvesting the baby spinach a few days earlier than the current commercial stage of harvest, the flavonoid concentration in the product may be increased and the content of potentially health-promoting compounds enhanced.     

Effect of freezing and frozen storage of doughs on bread quality

The effects of freezing and storage in frozen conditions on bread quality, crumb properties, and aggregative behavior of glutenins were analyzed. The effect of different additives on bread quality was also studied. The results obtained showed that freezing and storage at -18 degrees C decreased the bread quality. Samples stored in frozen conditions supplemented with diacetyl-tartaric acid ester of monoglycerides, gluten, and guar gum produced breads of greater volume and more open crumb structure than those prepared with the base formulation (without additives). All additives analyzed increased the proof time. Crumb firmness increased with dough frozen storage and bread aging time at 4 degrees C. A decrease in the amount of glutenin subunits of high molecular mass was observed by electrophoresis analysis of the SDS-soluble proteins aggregates extracted from the frozen dough. This result suggested that the protein matrix of bread underwent depolymerization during storage in frozen conditions.     

常规冷冻冻藏对猪肉保水性和组织结构的影响

调查了在生产中常规冷冻工艺条件下冻结的肉块,经-18℃冷库冻藏一定时间之后,在固定解冻条件下解冻后,肉的保水性、蛋白溶解度和组织结构的变化规律。结果表明:随着冻藏时间的延长,冷冻猪肉的保水性逐渐降低,主要表现为:解冻汁液流失率、蒸煮损失率和加压失水率等逐渐升高。肉样的蛋白溶解度逐渐降低,主要是全蛋白和肌原纤维蛋白的溶解度降低。随着冻藏时间的延长,冰晶在冻结肉样中逐渐长大,导致肌束受压聚集。这可能是肉的保水性降低的一个重要原因。冻藏1个月对于肉样的各种品质特性影响不大,2个月后指标的变化比较明显,5个月后指标的变化非常明显。     

Structural changes in natural actomyosin and surimi from ling cod (Ophiodon elongatus) during frozen storage in the absence or presence of cryoprotectants

Surimi and natural actomyosin (NAM) from ling cod (Ophiodon elongatus) were subjected to frozen storage in the absence or presence of cryoprotectants (sorbitol, sucrose, lactitol, and Litesse, either individually or in combination). Effects of frozen storage were studied for NAM frozen at -10 degrees C for 10 days and for surimi after eight freeze-thaw cycles. A commercial blend cryoprotectant (4% sucrose and 4% sorbitol), individual cryoprotectants at 8%, and optimal blends at 4, 5.5, 6, and 8%, were effective in maintaining the gel strength of surimi and NAM gels. Surimi or NAM frozen in the absence of cryoprotectants or with only 4% individual cryoprotectants, showed increased percent alpha-helical content by Raman analysis. Increased disulfide content was also observed in the treatment without cryoprotectants by the Raman SS stretching band and by chemical determination. Tyrosine residues were in a buried environment before and after freezing for all treatments, and surface hydrophobicity measured by 1-anilinonaphthalene-8-sulfonate decreased after frozen storage in the absence of cryoprotectants.     

Validation of a tRNA-Glu-cytochrome b key for the molecular identification of 12 hake species (Merluccius spp.) and Atlantic Cod (Gadus morhua) using PCR-RFLPs, FINS, and BLAST

The goal of this study was to develop a diagnostic key for hake meat to solve the limitations of previous identification methodologies, mainly related to the high degradation of the DNA recovered from processed foods. We describe the development of two molecular tools based on polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphisms of the cytochrome b gene, respectively, to identify DNA from 12 hake species in commercial products. The first assay is an exclusion test consisting of the PCR amplification of a 122 bp fragment using nested primers interspecifically conserved in Merluccius spp. and in Gadus morhua. This 122 bp amplicon, being the shortest one so far designed for hake DNA, is a useful traceability tool for highly degraded samples because its sequence contains enough interspecific diagnostic variation to identify 10 hake species and cod and has been successfully amplified from most commercial products so far tested. The second identification key follows a positive outcome of the exclusion test and consists of the PCR amplification of a 464-465 bp fragment and its digestion with three restriction enzymes whose targets map at interspecifically nonconserved sites of the cytochrome b. The key presented here has passed through a rigorous methodological calibration including its testing for genus specificity, its validation on a large number of authenticated sample types from each species range, and its implementation with a maximum likelihood method for the assignment of unknown samples. Together, these two procedures constitute the most complete molecular key so far developed for Merluccius spp., which is optimal for routine identification of hakes in large commercial samples at a reasonable cost-time ratio.     

Evaluation of the quality of frozen minced red hake: use of fourier transform near-infrared spectroscopy.

The quality of frozen minced red hake was assessed by FTNIR spectroscopy in transmission mode. The region 1530-1866 nm was best correlated to dimethylamine (DMA) concentration, which is an accepted quality index for frozen gadoid fish. Partial least-squares (PLS) analysis showed that this technique predicts the DMA content sufficiently well for quality assurance (SD/SEP > 3). The PLS calibration was equally successful when five narrow spectral regions were chosen instead of the continuous spectrum, indicating that a cheap filter instrument may be developed for industrial use.     

Optimizing angiotensin I-converting enzyme inhibitory activity of Pacific hake (Merluccius productus) fillet hydrolysate using response surface methodology and ultrafiltration

The in vitro angiotensin I-converting enyzme (ACE) inhibitory activity of Pacific hake hydrolysates was investigated as a function of hydrolysis conditions, starting material variability, and ultrafiltration. Hake fillets were hydrolyzed using Protamex protease under various conditions of pH, hydrolysis time, and enzyme-to-substrate ratio (% E/S) according to a response surface methodology (RSM) central composite design. The hydrolysate produced at pH 6.5, 125 min, and 3.0% E/S had an IC 50 of 165 +/- 9 microg of total solids/mL. ACE-inhibitory activity was not significantly different (P < 0.05) for hydrolysates produced using higher time-enzyme combinations within the model or from fish of different catches. Ultrafiltration (10 kDa molecular mass cutoff) resulted in an IC50 value of 44 +/- 7 microg of peptides/mL, 2.5 times more potent than the commercial product PeptACE Peptides (IC50 = 114 +/- 8 microg of peptides/mL). These results suggest that hydrolysates prepared with minimal fractionation from Pacific hake, an undervalued fish, may be a commercially competitive source of ACE-inhibitory peptides.     

Controlled atmosphere storage of wild strawberry fruit (Fragaria vesca L.)

Controlled atmosphere storage technology to extend the shelf life of "Reina de los Valles" wild strawberry fruit (Fragaria vesca L.) was studied. Fruits were stored at 3 degrees C for three weeks in different atmosphere compositions: 0.05% CO2/21% O2 (air), 3% CO2/18% O2, 6% CO2/15% O2, 10% CO2/11% O2, and 15% CO2/6% O2. The effect of gas composition on soluble solids content, titrable acidity, pH, off-flavor, aroma volatiles, and consumer preference was monitored. The result showed that the 10% CO2/11% O2 combination can efficiently prolong the shelf life of wild strawberries by maintaining the quality parameters within acceptable values, through inhibiting the development of Botrytis cinerea, without significantly modifying consumer acceptance.     

Effect of storage on fructan and fructooligosaccharide of onion (Allium cepa L.)

The purpose of this study was a comparative examination of the fructan and fructooligosaccharide (FOS) content of different varieties of onions (Allium cepa L. cv. Sturon, Hysam, Durco, Grano de Oro, and Caribo) and the changes produced during their commercial storage. In fresh onions, the Grano de Oro variety presented a remarkably different behavior, showing low contents of total fructans and FOS and high levels of reducing sugars. In the other varieties, Sturon, Hysam, Durco, and Caribo, fructans were the main carbohydrates, the lowest polymerized FOS being the major oligomer. Storage period caused in these varieties important increased levels of free fructose attributed to fructan hydrolysis. Maleic hydrazide treatment had no significant effect in avoiding the hydrolysis of fructans during storage conditions for the Sturon variety. Varieties with >16% dry matter or 15% soluble solids contents could be stored for 6 months at 0 degrees C and 60-65% relative humidity.     

Production and characterization of rabbit polyclonal antibodies to almond (Prunus dulcis L.) major storage protein.

Rabbits were immunized with purified almond major protein (AMP), the primary storage protein in almonds. Rabbit anti-AMP polyclonal antibodies (PA) could detect AMP when as little as 1-10 ng/mL were used to coat microtiter plates in a noncompetitive enzyme linked-immunosorbent assay (ELISA). Competitive inhibition ELISA assays detected the AMP down to 300 ng/mL. PA recognized the AMP in protein extracts from all U.S. major marketing cultivars of almonds (Mission, Neplus, Peerless, Carmel, and Nonpareil) with specific reactivity of 52.6-75% as compared to that of the AMP alone. Immunoreactivity of protein extracts prepared from commercial samples of blanched almonds, roasted almonds, and almond paste was respectively reduced by 50.0%, 56.6%, and 68.4% (noncompetitive ELISA) when compared to the immunoreactivity of the AMP. Moist heat (121 degrees C, 15 min) pretreatment of the AMP reduced the PA reactivity by 87% (noncompetitive ELISA). Exposing AMP to pH extremes (12.5 and 1.5-2.5) caused a 53% and 57% reduction in PA reactivity, respectively (noncompetitive ELISA). PA showed some cross-reactivity with the cashew major globulin, and to a lesser extent, the Tepary and Great Northern bean major storage protein (7S or phaseolin). The presence of almonds in a commercial food was detected using PA in a competitive ELISA.