Partial purification and characterization of cysteine proteinase inhibitor from chicken plasma.

A high-molecular-weight cysteine proteinase inhibitor (CPI) was purified from chicken (Gallus gallus) plasma using polyethylene glycol (PEG) fractionation and affinity chromatography on carboxymethyl-papain-Sepharose-4B. The CPI was purified 96.8-fold with a yield of 28.9%. Based on inhibitory activity staining for papain, CPI was shown to have an apparent molecular mass of 122 kDa. No inhibitory activity was obtained under reducing condition, indicating that CPI from chicken plasma was stabilized by disulfide bonds. CPI was stable in temperature ranges from 40 to 70 degrees C for 10 min; however, more than 50% of the inhibitory activity towards papain was lost within 30 min of heating at 90 degrees C. CPI was stable in the presence of salt up to 3%. The purified CPI exhibited the inhibitory activity toward autolysis of arrowtooth flounder (Atheresthes stomias) and Pacific whiting (Merluccius productus) natural actomyosin (NAM) in a concentration-dependent manner.

Capillarity proposed as the predominant mechanism of water and fat stabilization in cooked comminuted meat batters.

Fat- and nonfat-containing meat gels structurally became coarser and porous by partial substitution of whey protein isolate for myofibrillar protein, creating a weaker texture plus greater cook loss (CL: fat+water) and expressible water (EW). Microstructure examinations revealed a tendency for fat to coalesce during cooking of the more coarse-structured gels. This tendency was unaffected by fat pre-emulsification prior to addition, arguing against a strong role of an interfacial protein film in stabilizing fat. Instead, a gel structure with evenly distributed small pores leads to lower CL and EW, thus controlling both water- and fat- holding since fat cannot readily permeate small water-filled hydrophilic pores. Only when large pores or continuous fissures are structurally present can water be released, allowing liquid fat to also migrate and coalesce. This changes the current paradigm of understanding regarding the mechanism of fat/water-holding in comminuted meat products: gel capillarity (gel structure), not fat emulsifying ability of protein, is the likely determining factor.

Stability of omega-3 fatty acids in fortified surimi seafoods during chilled storage.

Physical, chemical, and sensory properties of cooked surimi seafood gels (crab analogue) fortified with omega-3 fatty acids (FA) were monitored during chilled storage. Three sources of stabilized omega-3 FA (fish oil concentrate, menhaden oil, and a purified marine oil) were each incorporated into gels to an omega-3 FA content of 1.5 or 2.5%, w/w. Omega-3 FA stability, development of off-flavors, and changes in color and texture were monitored during chilled storage for 2 months. Gels with fish oil concentrate developed fishy flavor and aroma within 30 days and were eliminated from the study. Gels containing menhaden oil and purified marine oil exhibited little change in sensory properties or oxidation products throughout 2 months of storage. Relative polyene index values (ratio of polyunsaturated to saturated fatty acids) indicated that the omega-3 FA were stable at both levels of addition. Omega-3 fortified gels were whiter than control gels, and gel texture was modified when menhaden and purified oils were added but not significantly affected by the level of omega-3 addition.

Extracellular production of a functional soy cystatin by Bacillus subtilis.

A recombinant Bacillus subtilis producing soy cystatin was developed by subcloning with a soy cystatin gene cloned in Escherichia coli. An active form of cystatin against the cysteine protease from Pacific whiting fillets contaminated with Myxosporidia parasite was constitutively expressed and secreted extracelluarly into the medium. Two gene fragments of signal peptides from kerA and sacB were introduced and compared for secretion efficiency of cystatin. The secretion level of active cystatin improved with the signal peptide of kerA when compared to that of sacB. Inhibitor activity was reduced rapidly after peak expression of the target protein at 36 h of fermentation. The addition of 1% glucose, a suppressor of protease, into the medium sustained the increase of the cystatin activity during fermentation. This study introduced a potential new method for fermentation production of cystatin.

Capillary pressure as related to water holding in polyacrylamide and chicken protein gels.

UNLABELLED: The ability of food gels to hold water affects product yield and organoleptic quality. Most researchers believe that water is held by capillarity such that gels having smaller mean pore diameter and a more hydrophilic surface hold water more tightly. To date, however, only qualitative evidence relating pore size to water holding (WH) properties has been provided. The present study sought to provide quantitative confirmation of this hypothesis. Scanning electron microscopy coupled with image analysis was used to measure pore size, and water contact angle with the gel surface was measured by the captive bubble method, in both model polyacrylamide gels and heat-induced protein (minced chicken breast) gels. These were related to water lost during cooking of meat pastes to form gels (cooking loss (CL)), as well as water lost upon centrifugation (expressible water (EW)) or by capillary suction (CSL) of all prepared gels, as inverse measures of WH. As predicted by the Young-Laplace equation for calculating capillary pressure, the presumed mechanism of WH, gels with lower water losses exhibited a more hydrophilic surface (smaller contact angle). Yet, both lower CL and CSL correlated with larger mean pore diameter of gels, not smaller as had been expected. Polyacrylamide gels varied more in WH than did prepared meat gels, yet only the capillary suction method was sensitive enough to detect these differences. PRACTICAL APPLICATION: The ability of gels to hold water is important for economics of processing, food quality, and food safety. This study investigated the prevailing theory for how gels hold water, capillarity. Both the pore sizes of gel microstructures and the degree of hydrophilicity of the polymers comprising each gel were quantitatively assessed and related to water holding (WH) properties, and this was the first report using such methodologies. It appeared that the degree of hydrophilicity was much more important explaining WH properties than pore size, and that future research of this kind should be carried out.

Rapid heating of Alaska pollock and chicken breast myofibrillar proteins as affecting gel rheological properties.

Surimi seafoods (fish/poikilotherm protein) in the U.S.A. are typically cooked rapidly to 90+°C, while comminuted products made from land animals (meat/homeotherm protein) are purposely cooked much more slowly, and to lower endpoint temperatures (near 70 °C). We studied heating rate (0.5, 25, or 90 °C/min) and endpoint temperature (45 to 90 °C) effects on rheological properties (fracture, small strain) of washed myofibril gels derived from fish (Alaska pollock) compared with chicken breast at a common pH (6.75). This was contrasted with published data on gelation kinetics of chicken myosin over the same temperature range. Heating rate had no effect on fracture properties of fish gels but slow heating did yield somewhat stronger, but not more deformable, chicken gels. Maximum gel strength by rapid heating could be achieved within 5 min holding after less than 1 min heating time. Dynamic testing by small strain revealed poor correspondence of the present data to that published for gelling response of chicken breast myosin in the same temperature range. The common practice of reporting small-strain rheological parameters measured at the endpoint temperature was also shown to be misleading, since upon cooling, there was much less difference in rigidity between rapidly and slowly heated gels for either species.

Effects of heating rate and pH on fracture and water-holding properties of globular protein gels as explained by micro-phase separation.

UNLABELLED: The effect of heating rate and pH on fracture properties and held water (HW) of globular protein gels was investigated. The study was divided into 2 experiments. In the 1st experiment, whey protein isolate (WPI) and egg white protein (EWP) gels were formed at pH 4.5 and 7.0 using heating rates ranging from 0.1 to 35 °C/min and holding times at 80 °C up to 240 min. The 2nd experiment used one heating condition (80 °C for 60 min) and probed in detail the pH range of 4.5 to 7.0 for EWP gels. Fracture properties of gels were measured by torsional deformation and HW was measured as the amount of fluid retained after a mild centrifugation. Single or micro-phase separated conditions were determined by confocal laser scanning microscopy. The effect of heating rate on fracture properties and HW of globular protein gels can be explained by phase stability of the protein dispersion and total thermal input. Minimal difference in fracture properties and HW of EWP gels at pH 4.5 compared with pH 7.0 were observed while WPI gels were stronger and had higher HW at pH 7.0 as compared to 4.5. This was due to a mild degree of micro-phase separation of EWP gels across the pH range whereas WPI gels only showed an extreme micro-phase separation in a narrow pH range. In summary, gel formation and physical properties of globular protein gels can be explained by micro-phase separation. PRACTICAL APPLICATION: The effect of heating conditions on hardness and water-holding properties of protein gels is explained by the relative percentage of micro-phase separated proteins. Heating rates that are too rapid require additional holding time at the end-point temperature to allow for full network development. Increase in degree of micro-phase separation decreases the ability for protein gels to hold water.

Rapid heating of Alaska pollock and chicken breast myofibrillar protein gels as affecting water-holding properties.

The gelation response of salted muscle minces to rapid versus slow heating rates is thought to differ between homeotherm and poikilotherm species. This study investigated water-holding (WH) properties of pastes prepared from refined myofibrils, at equal pH, of chicken breast versus Alaska pollock both during [cook loss (CL)] and following [expressible water (EW)] their cooking by rapid [microwave (MW)] versus slow [water bath (WB)] heating and whether such properties were related to gel matrix structure parameters and water mobility. Results did not confirm the industrial experience that pastes of meat from homeotherms benefit from slower cooking. Gels of equally high WH ability (low CL or EW) were made by rapid heating when the holding time did not exceed 5 min prior to cooling, which was sufficient for completion of gelation. Reduced CL and EW correlated with larger and smaller amplitudes of T21 and T22 water pools, respectively, measured by time-domain nuclear magnetic resonance (TD-NMR).

Autolytic degradation of skipjack tuna during heating as affected by initial quality and processing conditions.

UNLABELLED: Several factors were studied as affecting protein degradation and texture of skipjack tuna muscle following ambient pressure thermal processing (precooking). These included degree of mushy tuna syndrome (MTS) evidenced in the raw meat, raw meat pH, abusive thawing/holding, and precooking temperature/time. Slurries and intact pieces from frozen skipjack tuna, either tempered for 2 h or thawed and held at 25 °C for 22 h (abusive treatment) were heated at temperatures ranging from 40 to 80 °C for up to 2 h, and also at 90 °C for 1 h, with or without prior adjustment of pH to 5 or 7 to favor cathepsin or calpain activity, respectively. Proteolysis of precooked samples was monitored by Lowry assay and SDS-PAGE; cooked texture of intact meat was measured using a Kramer shear press and by sensory profile analysis. Proteolysis maximally occurred in slurries of skipjack tuna muscle that had been abusively stored (22 h at 25 °C) and adjusted to pH 5 prior to heating at 55 °C. Intact pieces of tuna abusively thawed/held for 22 h with subsequent heating at 55 °C also evidenced the most proteolysis and were the least firm in texture. Raw fish that evidenced higher severity of MTS when raw displayed higher levels of proteolysis prior to cooking, which were further increased after cooking at 55 °C. PRACTICAL APPLICATION: The kinetic data presented here can be used to optimize processing conditions for skipjack tuna canning to minimize textural degradation and optimize quality.

Diffusion of active proteins into fish meat to minimize proteolytic degradation.

Proteases in fish muscle often cause undesired softening of intact meat pieces during refrigerated storage or slow cooking. Several food-grade proteinaceous inhibitors can overcome this softening if properly delivered to the intracellular sites where proteases are located. Fluorescence recovery after photobleaching (FRAP) and laser scanning confocal microscopy (LSCM) were used to measure the translational diffusion of fluorescein isothiocyanate (FITC)-labeled protease inhibitors into intact muscle fibers of halibut. Diffusion coefficients (D) of alpha-2-macroglobulin (720 kDa), soybean trypsin inhibitor (21 kDa), and cystatin (12 kDa) were measured in both muscle fibers and dilute aqueous solutions. On the time scale of the observation (35 min), cystatin and soybean trypsin inhibitor diffused through the cell membrane (sarcolemma) and sarcoplasm, but at a considerably slower rate (>10-fold difference) than in dilute aqueous solution. alpha-2-Macroglobulin did not diffuse into muscle cells within the time frame of the experiment, but did completely penetrate the cell during overnight exposure. The present study thus shows a clear dependence of D on protein inhibitor size when moving within intact skeletal muscle fibers. Low molecular weight protease inhibitors such as cystatin can be effectively diffused into intact fish muscle cells to minimize proteolytic activity and meat softening.