Recovery of functional proteins from herring (Clupea harengus) light muscle by an acid or alkaline solubilization process

Proteins from herring (Clupea harengus) light muscle were extracted using acidic or alkaline solubilization; 92 and 89% of the initial muscle proteins were solubilized at pH 2.7 and 10.8, respectively, of which 96 and 94% were recovered during precipitation at pH 5.5. Consistency of the pH-adjusted muscle homogenates increased with increased raw material age and homogenization intensity; it declined following holding on ice. Some hydrolytic myofibrillar protein degradation occurred during cold storage of the acidified (pH 2.7) homogenates. With alkalized homogenates, hydrolysis was negligible. The total lipid content changed from 0.13 g/g of protein in the muscle to 0.04 g/g of protein in both the acid- and alkali-produced protein isolates. Corresponding values for the phospholipid content were from 0.037 to 0.02 g/g of proteins. Acid- and alkali-produced proteins made gels with equal strain and color. Stress values were equal or lower in acid- versus alkali-produced protein gels. When ice-stored raw material was used, strain and stress values of gels were reduced.     

Separation of membranes from acid-solubilized fish muscle proteins with the aid of calcium ions and organic acids

Calcium chloride, and to a lesser extent MgCl2, aided in the separation of membranes by centrifugation from cod (Gadus morhua) muscle homogenates solubilized at pH 3 in the presence of citric acid or malic acid but not lactic acid. Adding citric acid and Ca2+ before solubilizing the cod muscle homogenates was needed for the effect. At 1 mM citric acid, 70-80% of the phospholipid and 25-30% of the protein were removed at 10 mM Ca2+. At 8 mM Ca2+, citric acid showed an optimal effect on phospholipid removal at 5 mM with 90% of the phospholipid and 35% of the protein removed. The treatment with citric acid and Ca2+ was also effective in separating the membrane from solubilized herring (Clupea harengus) muscle homogenate. Ca2+ and citric acid might exert their influence by disconnecting linkages between membranes and cytoskeletal proteins.     

Changes in Salt Solubility and Microstructure of Proteins from Herring ( Clupea harengus ) after pH-Shift Processing

Salt solubility of pH-shift isolated herring ( Clupea harengus ) muscle proteins was studied in relation to pH exposure and microstructure using transmission electron microscopy (TEM). Using protein solubilization at pH 11.2 with subsequent precipitation at pH 5.5, salt solubility of the proteins decreased from 78 to 17%. By precipitating the alkali-solubilized proteins at the pH of native herring muscle, 6.5, salt solubility only decreased to 59%, proving that pH values between 6.5 and 5.5 affected protein salt solubility more than the pH cycle 6.5 → 11.2 → 6.5. Precipitation at pH 5.5 resulted in hydrogen bonds, hydrophobic interactions, and S-S bridges, whereas precipitation at pH 6.5 resulted only in the formation of hydrophobic interactions. The alkaline pH-shift isolation process severely rearranged the protein microstructure, with precipitation at pH 6.5 forming a finer, more homogeneous network than precipitation at pH 5.5. The former protein isolate also contained less lipid oxidation products and formed more deformable gels, without affecting protein yield.     

Separation of muscle membrane from alkali-solubilized fish muscle proteins

Treatment with Ca2+ and citric acid improved membrane removal from muscle homogenates solubilized at pH 10.5 by centrifugation at 4000 g for 15 min. The percentage of phospholipid removed from muscle homogenates increased with increasing Ca2+ concentrations at 1 mM citric acid. More than 85% phospholipid and 45% protein in the muscle homogenates were removed at Ca2+ concentrations of >20 mM in the presence of 1 mM citric acid. At 8 mM Ca2+, addition of citric acid at 5 mM improved phospholipid removal to approximately 78% from 58% in its absence. Because treatment with 8 mM Ca2+ alone can remove significant amounts of phospholipid, it is likely that Ca2+ played the major role in membrane removal in muscle homogenates solubilized at pH 10.5.     

Preventing lipid oxidation during recovery of functional proteins from herring (Clupea harengus) fillets by an acid solubilization process

It has previously been found that a process based on solubilization at pH 2.7 gives high yields of herring muscle proteins with good functionality. In this study, the development of lipid oxidation during acid processing of herring mince was studied. It was tested how modifications of the process conditions and/or additions of antioxidants could prevent lipid oxidation during the actual process and then during ice storage of the protein isolates. Processing parameters evaluated were prewash of the mince, exposure time to pH 2.7, inclusion or exclusion of a high-speed centrifugation, and addition of antioxidants. Antioxidants tested were erythorbate (0.2%, 9.3 mM), sodium tripolyphosphate (STPP; 0.2%, 5.4 mM), ethylenediaminetetraacetic acid (EDTA; 0.044%, 1.5 mM), and milk proteins (4%). The first three antioxidants were added in the prewash or during the homogenization step, whereas milk proteins were added to the final precipitate. At time 0, all isolates were analyzed for pH, moisture content, and thiobarbituric reactive substances (TBARS). Selected isolates were also analyzed for lipid and protein content. Stability during ice storage was followed in terms of odor, TBARS, and color (a/b values). Extensive lipid oxidation took place using the "control" process without high-speed centrifugation. This was not significantly (p < or = 0.05) affected by a prewash or varied exposure time to pH 2.7. Including high-speed centrifugation (20 min, 10,000g) significantly (p < or = 0.05) reduced TBARS values, total lipids, a values and b values. Erythorbate alone, or in combination with STPP/EDTA, significantly (p < or = 0.05) reduced lipid oxidation during processing if added in the prewash or homogenization step. During ice storage, better stability was gained when antioxidants were added in both of these steps and when EDTA was used instead of STPP.     

Effect of chitosan and chitin on the separation of membranes from proteins solubilized by pH shifts using cod (Gadus morhua)

Studies with isolated membranes and isolated membranes suspended in muscle proteins solubilized at pH 3 showed that mixing chitosan and membranes at this low pH followed by a pH adjustment to 10.5 could sediment membranes effectively at 4000 g. In the solubilized muscle homogenate, the effectiveness of membrane removal by chitosan at 4000 g for 15 min was molecular weight dependent. About 80% of the phospholipids and 28% of proteins were sedimented from solubilized muscle homogenate by mixing muscle homogenate (10 g of muscle tissue homogenized with 90 mL of distilled water) with 10 mL of MW 310-375 k chitosan (10 mg/mL in 0.1 N HCl) before solubilizing it at pH 10.5, whereas 55% of the phospholipids and 12% of proteins were sedimented by mixing muscle homogenate with the MW 310-375 k chitosan before solubilizing the homogenate at pH 3. Low molecular weight chitosans (at MW 1k or 33k) showed little effect on membrane sedimentation under the same conditions. Chitin was not useful for removing membranes at either pH 3 or 10.5, whether added before or after pH adjustment.     

Properties and stability of oil-in-water emulsions stabilized by coconut skim milk proteins

Protein fractions were isolated from coconut: coconut skim milk protein isolate (CSPI) and coconut skim milk protein concentrate (CSPC). The ability of these proteins to form and stabilize oil-in-water emulsions was compared with that of whey protein isolate (WPI). The solubility of the proteins in CSPI, CSPC, and WPI was determined in aqueous solutions containing 0, 100, and 200 mM NaCl from pH 3 to 8. In the absence of salt, the minimum protein solubility occurred between pH 4 and 5 for CSPI and CSPC and around pH 5 for WPI. In the presence of salt (100 and 200 mM NaCl), all proteins had a higher solubility than in distilled water. Corn oil-in-water emulsions (10 wt %) with relatively small droplet diameters (d32 approximately 0.46, 1.0, and 0.5 mum for CSPI, CSPC, and WPI, respectively) could be produced using 0.2 wt % protein fraction. Emulsions were prepared with different pH values (3-8), salt concentrations (0-500 mM NaCl), and thermal treatments (30-90 degrees C for 30 min), and the mean particle diameter, particle size distribution, zeta-potential, and creaming stability were measured. Considerable droplet flocculation occurred in the emulsions near the isoelectric point of the proteins: CSPI, pH approximately 4.0; CSPC, pH approximately 4.5; WPI, pH approximately 4.8. Emulsions with monomodal particle size distributions, small mean droplet diameters, and good creaming stability could be produced at pH 7 for CSPI and WPI, whereas CSPC produced bimodal distributions. The CSPI and WPI emulsions remained relatively stable to droplet aggregation and creaming at NaCl concentrations of < or =50 and < or =100 mM, respectively. In the absence salt, the CSPI and WPI emulsions were also stable to thermal treatments at < or =80 and < or =90 degrees C for 30 min, respectively. These results suggest that CSPI may be suitable for use as an emulsifier in the food industry.     

Changes in the antioxidative property of herring (Clupea harengus) press juice during a simulated gastrointestinal digestion

The aqueous fraction (press juice, PJ) from herring muscle was recently shown to inhibit hemoglobin-mediated oxidation of washed fish mince lipids during ice storage. As a first step to evaluate potential in vivo antioxidative effects from herring PJ, the aim of this study was to investigate whether herring PJ retains its antioxidative capacity during a simulated gastrointestinal (GI) digestion. Press juice from whole muscle (WMPJ) and light muscle (LMPJ) was mixed with pepsin solution followed by stepwise pH adjustments and additions of pancreatin and bile solutions. Digestive enzymes were removed from samples by ultrafiltration (10 kDa). Before, during, and after digestion, samples were analyzed for their peptide content and for antioxidative properties with the oxygen radical absorbance capacity (ORAC) and the low-density lipoprotein (LDL) oxidation assays. From 0 to 165 min of digestion, the content of <10 kDa peptides in WMPJ and LMPJ samples increased 12- and 7-fold, respectively. Further, both samples got approximately 12.5 times higher ORAC values and gave rise to approximately 1.3-fold increased lag phase in Cu2+-induced LDL oxidation. The largest changes in peptide content, ORAC values, and LDL oxidation inhibition occurred between 30 and 75 min of digestion, indicating that these parameters might be interrelated. When comparing analytical data obtained after 165 min of digestion with data obtained from analyses of native nondigested PJs, it was found that the data on peptide content, ORAC, and LDL oxidation from digested PJs were 64-69%, 121-161%, and 112-115%, respectively, of those of nondigested PJs. The study thus showed that enzymatic breakdown of PJ proteins under GI-like conditions increases the peroxyl radical scavenging activity and the potential to inhibit LDL oxidation of herring PJs. These data provide a solid basis for further studies of uptake and in vivo activities of herring-derived aqueous antioxidants.     

Solubility improvement of shellfish muscle proteins by reaction with glucose and its soluble state in low-ionic-strength medium

When myofibrillar proteins of scallop striated adductor muscle were reacted with glucose through the Maillard reaction, the change in the solubility of myofibrillar proteins in 0.05-0.5 M NaCl solutions during glycosylation and their soluble states were investigated. The solubility in low-ionic-strength media increased greatly with the progress of the Maillard reaction. The solubility in 0.1 M NaCl reached 83% when more than 60% of lysine residues in myofibrillar proteins were modified by glucose. However, the excess progress of the Maillard reaction impaired the improved solubility of myofibrillar proteins in a low-ionic-strength medium. Myosin, actin, and paramyosin in glycosylated myofibrillar proteins were solubilized independently regardless of NaCl concentration. In addition, the glycosylated myosin lost its filament-forming ability and existed as a monomer in 0.1 M NaCl.     

Gelation of protein recovered from whole Antarctic krill (Euphausia superba) by isoelectric solubilization/precipitation as affected by functional additives

This study demonstrated that the novel isoelectric solubilization/precipitation can be applied to recover functional muscle protein in a continuous mode from whole Antarctic krill. Protein recovered from whole krill had a much lower ash content than whole krill, suggesting good removal of inedible impurities (shell, appendages, etc.). Lipids were retained to a higher degree with krill protein solubilized at acidic rather than basic pH. The viscoelastic modulus (G') showed that recovered krill protein failed to form heat-induced gel unless beef plasma protein (BPP) was added. Therefore, protease inhibitors are suggested for development of krill-derived products. Even with BPP, the G' decreased between 45 and 55 degrees C. However, krill protein solubilized at acidic pH had a higher decrease of the G' than the protein solubilized at basic pH, likely due to krill endogenous cathepsin L. Krill protein-based gels developed from protein solubilized at basic pH, especially pH 12.0, had better texture (torsion and Kramer tests and texture profile analysis) than acidic counterparts, possibly due to higher proteolysis and denaturation at acidic pH. Gels made from protein solubilized at acidic pH were brighter and whiter likely due to a higher lipid content.     

Solubilization of Fe (III) from humic-Fe complexes,humic/Fe-oxide mixtures and from poorly ordered Fe-oxide by organic acids - consequences for P adsorption

The effect of organic acids on the solubility of Fe from humic Fe complexes, poorly ordered Fe-oxides and humic/Fe-oxide mixtures was investigated at pH 4 and 7. After reaction, the suspensions were filtered through a 20 000 D membrane and Fe and humic concentrations were determined in the filtrate. Among four organic acids, citric acid had the strongest effect on Fe-solubility and solubilized up to 25% of total Fe from the humic complexes at pH 4. Humic substances were solubilized by citrate too, the proportions being higher at pH 7 than at pH 4. Malic and tartaric acid solubilized substantial but lower amounts of Fe from humic complexes than citric acid, whereas phthalic acid did not solubilize Fe and humics. Citrate added to poorly ordered Fe-oxide and its mixtures with humics, aged for 65 days, increased 20 000 D filtrable Fe, the effect being higher for humic/Fe-oxide complexes. Adding citrate to humic/Fe-oxide mixtures and to Fe-oxide strongly decreased the P sorption, the extent, being higher for the former adsorbent.     

Preparation and characterization of papain-modified sesame (Sesamum indicum L.) protein isolates

Defatted sesame meal ( approximately 40-50% protein content) is very important as a protein source for human consumption due to the presence of sulfur-containing amino acids, mainly methionine. Sesame protein isolate (SPI) is produced from dehulled, defatted sesame meal and used as a starting material to produce protein hydrolysate by papain. Protein solubility at different pH values, emulsifying properties in terms of emulsion activity index (EAI) and emulsion stability index (ESI), foaming properties in terms of foam capacity (FC) and foam stability (FS), and molecular weight distribution of the SPI hydrolysates were investigated. Within 10 min of hydrolysis, the maximum cleavage of peptide bonds occurred as observed from the degree of hydrolysis. Protein hydrolysates have better functional properties than the original SPI. Significant increase in protein solubility, EAI, and ESI were observed. The greatest increase in solubility was observed between pH 5.0 and 7.0. The molecular weight of the hydrolysates was also reduced significantly during hydrolysis. These improved functional properties of different protein hydrolysates would make them useful products, especially in the food, pharmaceutical, and related industries.     

Salts and pH Induced Changes in Functional Properties of Amaranth (Amaranthus tricolor L.) Seed Meal

Amaranth meal is a rich source of proteins, carbohydrates, and minerals with a low amount of anti‐nutritional factors. It exhibits good functional properties. The effect of NaCl and NaHCO₃ salts and pH level on the functional properties of amaranth meal was studied. The water absorption capacity and protein solubility were improved in the presence of the salts. Protein solubility was high at extreme pH values and minimum at pH 4. Foaming capacity was poor in the presence of the two salts, while foam stability was better at lower concentrations of NaCl (0.2–0.6M). Changes in pH had a pronounced effect on the foaming properties of amaranth meal. Salts did not change the emulsification properties but significantly increased the relative viscosity of amaranth seed meal at higher concentrations of NaCl and NaHCO₃ (0.6–1.0M). Relative viscosity was highest at pH 10 and lowest at pH 4.     

Long-term preservation of egg and tissue homogenates for determination of organochlorine compounds: freezing versus freeze-drying

Storage of wet egg homogenates at temperatures from -18 degrees to -28 degrees C was more suitable for long-term preservation than freeze-drying. Changes in residue levels of heptachlor epoxide, oxychlordane, dieldrin, hexachlorobenzene,p,p'-DDE, mirex, and PCBs were not significant over a 3-year period in fresh herring gull egg homogenates stored at -18 degrees to -28 degrees C. Compounds with gas chromatographic retention times shorter than hexachlorobenzene vaporized during freeze-drying at a rate proportional to their volatility. Evaporative losses of components with vapor pressures less than hexachlorobenzene did not occur in naturally contaminated herring gull eggs after storage at room temperature for up to 1 year. Higher losses of all compounds, up to 25% for p,p'-DDE, occurred in freeze-dried whole-body herring gull homogenates. Easily dehydrochlorinated compounds were rapidly degraded in freeze-dried chicken egg homogenate at room temperature: The half-life of p,p'-DDT and p,p'-DDD was about 20 days, and that of alpha- and gamma-hexachlorocyclohexane was much less than 16 days. About one-third of oxychlordane in herring gull eggs was lost in 1 year under these conditions, but none was lost after freeze-drying when the homogenate was stored at -18 degrees to -28 degrees C.     

Effect of acetic acid added to cooking water on the dissolution of proteins and activation of protease in rice

The effect of acetic acid on the dissolution of proteins in rice was studied to elucidate the mechanism for the textural change induced by the acid by chemical and SDS-PAGE analyses of the rice proteins in the soaking solution. More proteins were extracted with 0.2 M acetic acid (pH 2.7) than with water (pH 6.8). The effect of acetic acid on the protein dissolution increased with increasing temperature. Immunoblotting confirmed that, when rice was soaked in acetic acid, glutelin was dissolved into the soaking solution and degraded by aspartic proteinase. Aspartic proteinase degraded glutelin much more than it did albumin and globulin. It was found that the combined amount of albumin and globulin dissolved into the acetic acid solution was much larger than that of glutelin, despite the smaller amounts present of albumin and globulin than of glutelin. Metal ions were extracted more with acetic acid than with water. In addition, carboxypeptidase was activated under the acidic condition and resulted in an increase in the amount of free amino acids. The main effect of acetic acid on the dissolution of rice proteins was enhancement of the solubility of albumin, globulin, and glutelin, the effect of proteases being minor.     

Soybean glycinin subunits: Characterization of physicochemical and adhesion properties

Soybean proteins have shown great potential for applications as renewable and environmentally friendly adhesives. The objective of this work was to study physicochemical and adhesion properties of soy glycinin subunits. Soybean glycinin was extracted from soybean flour and then fractionated into acidic and basic subunits with an estimated purity of 90 and 85%, respectively. Amino acid composition of glycinin subunits was determined. The high hydrophobic amino acid content is a major contributor to the solubility behavior and water resistance of the basic subunits. Acidic subunits and glycinin had similar solubility profiles, showing more than 80% solubility at pH 2.0-4.0 or 6.5-12.0, whereas basic subunits had considerably lower solubility with the minimum at pH 4.5-8.0. Thermal analysis using a differential scanning calorimeter suggested that basic subunits form new oligomeric structures with higher thermal stability than glycinin but no highly ordered structures present in isolated acidic subunits. The wet strength of basic subunits was 160% more than that of acidic subunits prepared at their respective isoelectric points (pI) and cured at 130 degrees C. Both pH and the curing temperature significantly affected adhesive performance. High-adhesion water resistance was usually observed for adhesives from protein prepared at their pI values and cured at elevated temperatures. Basic subunits are responsible for the water resistance of glycinin and are a good starting material for the development of water-resistant adhesives.     

Aqueous extracts from some muscles inhibit hemoglobin-mediated oxidation of cod muscle membrane lipids

It was evaluated whether trout hemoglobin (Hb)-mediated oxidation of minced washed cod muscle lipids could be prevented by an aqueous isolate from cod and some other muscle sources. Lipid hydroperoxides and painty odor developed approximately 4 days faster in washed than unwashed cod mince. When adding back an aqueous fraction (press juice) isolated from unwashed mince to washed mince at 2-6-fold dilutions, development of hydroperoxides and painty odor was either delayed or completely prevented. The inhibitory substances were heat stable, and their effect was slightly reduced at reduced pH. The <1 kDa fractions of whole and heated press juices were as inhibitory as the unfractionated press juices. Inhibition by the unheated, heated, and ultrafiltered (30 kDa) press juices was lost after dialysis. These findings implied the presence of one or more highly effective aqueous low molecular weight antioxidants in cod muscle press juice. The same antioxidative properties were found in heated haddock, dab, and winter flounder muscle press juices but not in heated herring and chicken muscle press juices. Unheated chicken press juice was however highly inhibitory.     

Isolation and quantitation of zeins in waxy and amylose-extender and wild flint and dent maize endosperm using a new solvent sequence for protein extraction

Protein distribution in endosperm of maize grains differing by their texture, flint or dent, and by their genotype, wild or waxy or amylose-extender, was examined by the successive use of 0.5 M NaCl, 0.5 M NaCl plus 0.6% 2-mercaptoethanol (2ME) at neutral and then alkaline pH, and 55% 2-propanol plus 0.6% 2ME as extractants. Proteins extracted in the presence of 2ME were characterized by their size polymorphism and amino acid composition. Proteins isolated with NaCl plus 2ME at neutral pH corresponded with a mixture of gamma-zein (27 kDa) and glutelin-like proteins. Proteins isolated with NaCl plus 2ME at pH 10 were a mixture of gamma-zeins (27 and 16 kDa) and beta-zeins (14 kDa). Alcohol-soluble proteins consisted of alpha-, beta-, and delta-zeins, alpha subunits being predominant. Zein quantitation was improved by weighing the nitrogen percentage of extracts by their zein content, as estimated from the data on amino acid composition. The data reported by Wolf et al. (Cereal Chem. 1975, 52, 765) were integrated to the results of this work to suggest the occurrence of an inverse correlation between amylose in starch and zeins in proteins.     

Effect of low pH on the susceptibility of isolated cod (Gadus morhua) microsomes to lipid oxidation

During the extraction of muscle to produce protein isolates by acid or alkali solubilization, membranes are exposed to abnormally low or high pH. Low but not high pH treatment induces rapid oxidation of membrane phospholipids in the presence of hemoglobin. The goal of this research work was to study the oxidative stability of microsomes under the conditions met during acid solubilization. Isolated microsomes from cod muscle were used as a model system. At pH 5.3 or lower, 99% of isolated cod membranes sedimented at low centrifugation speeds. Isolated membranes that were exposed to pH 3.0 were less susceptible to hemoglobin-mediated lipid oxidation. Cod hemoglobin exposed to pH 3 was rendered less pro-oxidative than the untreated cod hemoglobin. However, when microsomes and hemoglobin were together exposed to low pH, oxidation was promoted. Citric acid and calcium chloride, as well as press juice isolated from cod muscle, were able to inhibit lipid oxidation of microsomal suspensions.     

Development of yellow pigmentation in squid (Loligo peali) as a result of lipid oxidation

The impact of lipid oxidation on yellow pigment formation in squid lipids and proteins was studied. When the squid microsomes were oxidized with iron and ascorbate, thiobarbituric acid reactive substance were observed to increase simultaneously with b values (yellowness) and pyrrole compounds concomitantly with a decrease in free amines. Oxidized microsomes were not able to change the solubility, sulfhydryl content, or color of salt-soluble squid myofibrillar proteins. Aldehydic lipid oxidation products were able to decrease solubility and sulfhydryl content of salt-soluble squid myofibrillar proteins but had no impact on color. Aldehydic lipid oxidation products increased b values (yellowness) and pyrrole compounds and decreased free amines in both squid phospholipid and egg yolk lecithin liposomes. The ability of aldehydic lipid oxidation products to change the physical and chemical properties of egg yolk lecithin liposomes increased with increasing level of unsaturation and when the carbon number was increased from 6 to 7. These data suggest that off-color formation in squid muscle could be due to nonenzymatic browning reactions occurring between aldehydic lipid oxidation products and the amines on phospholipids headgroups.