Role of secondary structures in the gelation of porcine myosin at different pH values

Secondary structures, gelation properties and their relationships in porcine myosin were studied by circular dichroism, dynamic rheological measurement and scanning electron microscopy. Gelling of porcine myosin involved a change in myosin conformation with protein-protein and protein-water interactions. The gelation properties were strongly pH and temperature dependent. Near the pI (pH 5.5 and 6.0), porcine myosin could spontaneously coagulate at 15°C resulting partially from the presence of more β-sheets. Myosin at pH6.5-9.0 began to form a gel at temperatures greater than 38°C. Heating caused α-helices to partially turn into β-sheets and random coils. Subsequently, myosin aggregated and formed a gel network. The gel strength decreased and the water-holding capacity (WHC) increased with increasing pH. Correlation analysis indicated that both the unfolding of α-helices and the formation of β-sheets favored the gelation of porcine myosin. A high β-sheet fraction prior to heating resulted in a low WHC of resultant gel. A compact and uniform gel was also obtained at pH6.5.     

Study of the acid and thermal stability of β-lactoglobulin–ligand complexes using fluorescence quenching

The major protein in bovine milk whey, β-lactoglobulin (β-LG), has several binding sites for ligands. Its interactions with folic acid (a hydrophilic compound), resveratrol (amphiphilic) and α-tocopherol (hydrophobic) at neutral and acidic pH and after heating to 85 °C were studied using fluorescence quenching. Binding of folic acid occurs in a hydrophobic pocket in the groove between the α-helix and the β-barrel and is disturbed by decreasing the pH from 7.0 to 2.0. Resveratrol binds to the outer surface of β-LG near Trp19–Arg124 to form complexes that are stable at acidic pH. Acidification caused the release of α-tocopherol bound to the internal cavity but had no influence on that bound to a site at the surface of β-LG. The β-LG/folic acid complex was thermally stable. Thermal denaturing improved the affinity of the protein for resveratrol but decreased somewhat its affinity for α-tocopherol. These results should help guide the development of formulations based on β-LG as a carrier of a wide range of bioactive nutrients.     

Effects of high hydrostatic pressure on the structure of bovine α-lactalbumin

The effects of high hydrostatic pressure (HHP) processing (at 200 to 600 MPa, 25 to 55°C, and from 5 to 15 min) on some structural properties of α-lactalbumin was studied in a pH range of 3.0 to 9.0. The range of HHP processes produced a variety of molten globules with differences in their surface hydrophobicity and secondary and tertiary structures. At pH values of 3 and 5, there was a decrease in the α-helix content concomitant with an increase in β-strand content as the pressure increased. No changes in molecular size due to HHP-induced aggregation were detected by sodium dodecyl sulfate-PAGE. All samples showed higher thermostability as the severity of the treatment increased, indicating the formation of a less labile structure related to the HHP treatment.     

Direct nanoHPLC-ESI-QTOF MS/MS analysis of tryptic caseinophosphopeptides

Caseinophosphopeptides (CPPs) were generated following tryptic hydrolysis of sodium caseinate. Hydrolysate peptides were separated and identified using nano-HPLC ESI-QTOF MS/MS. Sequence coverage in the 3 h hydrolysate was 79.4%, 55.6%, 80.9% and 68.1% for α_s1-, α_s2-, β- and κ-casein (CN), respectively. Variable levels of serine phosphorylation in β-CN f1–25 were observed in the 3 h hydrolysate. Analysis of β-CN f1–25 4P demonstrated that this peptide was stable during the course of hydrolysis. The effect of heat treatment (75 °C, 45 min) at pH 6.0, 7.0 and 8.0 on the peptide profile of the 3 h hydrolysate was studied. Compared to pH 6.0 and 8.0, least modification in phosphopeptide profiles was observed for the hydrolysate sample heated at pH 7.0. Different dephosphorylation and oxidation patterns were also observed following heat treatment at the three pH values. These results demonstrate that heat treatment, in addition to pH, has a major effect on both the phosphorylated and non-phosphorylated peptide profiles of CN hydrolysates.     

Improvement of gelling properties of porcine blood plasma using microbial transglutaminase

The effect of microbial transglutaminase (MTGase) on the texture and water-holding capacity (WHC) of heat-induced gels prepared from porcine blood plasma at pH 5.5 was investigated. Different concentrations of commercial MTGase were added to plasma and incubated for several times under specific conditions of temperature and pH. From the results obtained, it can be postulated that enzymatic treatment enhances textural properties and WHC of plasma gels at pH 5.5, especially when incubated with 3% of the commercial product for 3 h at 30 °C and pH 7. This treatment increased by 0.4 N the hardness of gels and reduced by 3% the water released after gel centrifugation with respect to the control samples. SDS–PAGE confirmed that cross-linking took place when MTGase was added to plasma solutions. However, the results suggest that the sole addition of MTGase was not effective enough to improve the gelling properties of plasma proteins under acidic conditions.     

Chemical Interactions and Protein Conformation Changes During Silver Carp (Hypophthalmichthys Molitrix) Surimi Gel Formation

Chemical interactions and protein conformations changes during the formation of silver carp surimi gel were studied by textural analysis, chemical methods, laser Raman spectroscopy, and circular dichrosim. The optimum setting time at 40°C was 60 min. During surimi gel formation, ionic bonds and hydrogen bonds decreased significantly (P ≤ 0.05), while hydrophobic interactions, disulfide bonds, and non-disulfide covalent bonds increased significantly (P ≤ 0.05). Hydrophobic interactions, disulfide bonds, and non-disulfide covalent bonds were the main chemical interactions maintaining the stable structure of surimi gel. Secondary structural analysis of surimi protein showed that 94.11% α-helix existed in native myosin and it partly changed into β-turn and random coil during heating. Myosin gel was made up of 33.70% α-helix, 12.40% β-turn and 53.90% random coil. These three kinds of secondary structures were the main protein conformations in surimi gel.     

A method for the large-scale isolation of β-casein

A method for the large-scale isolation of β-casein from renneted skim milk was developed. The curd from renneted skim milk was dispersed in hot (?70 °C) water to inactivate residual chymosin. The heated curd was subsequently recovered by centrifugation, resuspended in water and incubated at 5 °C, during which β-casein dissociated from the curd; the suspension was centrifuged and the aqueous phase lyophilised. The isolated protein consisted mainly of β-casein, containing a minor amount of γ-caseins and traces of other caseins. Unless chymosin was fully inactivated by heating, some β-casein was hydrolysed at the Leu₁₉₂–Tyr₁₉₃ bond. The yield of β-casein increased with incubation time, up to ∼20% of the β-casein present in the milk after 24 h at 5 °C. Reducing milk pH to 5.5 or 6.0, prior to renneting, caused a high level of contamination with α_s-caseins. This isolation procedure can be easily scaled-up to an industrial process and the β-casein-depleted curd may be used for the manufacture of rennet casein or processed cheese.     

紫菜粉添加对鱿鱼鱼糜凝胶特性及其蛋白结构的影响

为改善秘鲁鱿鱼鱼糜凝胶品质,分析紫菜粉添加量对鱿鱼鱼糜凝胶感官评分、色泽、凝胶强度、持水性、微观结构及其肌原纤维蛋白组成和二级结构的影响,探究紫菜粉改善鱿鱼鱼糜凝胶品质的机理.结果表明:紫菜粉对鱿鱼鱼糜凝胶白度影响显著,添加后鱿鱼鱼糜凝胶色泽偏黄绿色;随着紫菜粉添加量的增加,鱿鱼鱼糜凝胶强度和持水性显著增加,并在添加量...     

Inactivation and structural change of horseradish peroxidase treated with supercritical carbon dioxide

The influence of supercritical carbon dioxide (SCCO₂) at 55 °C on inactivation of horseradish peroxidase (HRP) in buffer solution, pH 5.6, was studied while its structural change was analyzed by far UV-circular dichroism (CD) and tryptophan fluorescence spectroscopy. SCCO₂ treatment had significant effects on the residual activity of HRP, the least residual activity was only 12% at 30 MPa. HRP’s secondary and tertiary structures were changed. The α-helix relative content in the secondary structure decreased and the intrinsic relative fluorescence intensity (RFI) increased as the pressure of SCCO₂ treatment was elevated. The HRP’s inactivation closely corresponded to the loss of α-helix relative content and the increase of RFI. After a 7-day storage at 4 °C, the restoration of residual activity and the reversion of the α-helix relative content were observed while RFI resumed with exception of the 30 MPa treatment.     

Structural characterization of globulin from common buckwheat (Fagopyrum esculentum Moench) using circular dichroism and Raman spectroscopy

Raman and far-UV circular dichroism (CD) spectroscopy was used to study the conformation of globulin from common buckwheat (Fagopyrum esculentum Moench) (BWG) under the influence of various buffer environments and heat treatments. Secondary structural analysis of BWG by CD spectroscopy yielded 15.0% α-helical, 25.8% β-sheet, 28.9% β-turn and 30.3% random coil contents. Raman spectrum also showed β-sheets as the major secondary structure in native BWG. Chaotropic salts caused band shifts and intensity changes in Raman amide III vibration, indicating transitions from β-sheet to disordered structure following the lyotropic series of anions. Extreme pHs and several protein structure perturbants led to changes in CD and Raman spectral characteristics, demonstrating protein unfolding and denaturation. Increasing heating time at 100 °C induced the appearance of anti-parallel β-sheet (1235–1237 cm⁻¹) and caused a progressive increase in random coil content, suggesting protein denaturation and aggregation. Both non-covalent and covalent interactions play important roles in stabilizing the conformation of BWG.     

Pacific whiting (Merluccius productus) underutilization in the Gulf of California: Muscle autolytic activity characterization

In México’s Northwest coast, Pacific whiting (Merluccius productus) is considered an under-utilized species because of its high tendency to become parasitized, thus promoting a high proteolytic activity present in muscle tissue. Sample fish for study were separated in lots by degree of parasitism in “apparent” parasitized (APP) and advanced parasitized (ADP). Thus, pH and temperature conditions of mayor endogenous proteolytic activity in muscle were determined. Maximum activity was detected at 50 °C (pH 3.5–4.0) and at 60 °C (pH 6.75–7.0). Parasitism degree had a significant effect in enzyme activity at acidic pH (p < 0.05) being higher in APP at low temperature (30 °C). Higher temperatures (40–50 °C) favored (p < 0.05) activity in ADP muscle (same pH range) with the highest (p < 0.05) observed at 50 °C at pH 3.5. No much difference was observed at pH 7.0–8.0. Results suggest that pH around physiological conditions at 60 °C could be used as an advantage in fish protein hydrolysate production or as processing aid where a protein hydrolysis is required.     

Heat-induced protein structure and subfractions in relation to protein degradation kinetics and intestinal availability in dairy cattle

The objectives of this study were to reveal protein structures of feed tissues affected by heat processing at a cellular level, using the synchrotron-based Fourier transform infrared microspectroscopy as a novel approach, and quantify protein structure in relation to protein digestive kinetics and nutritive value in the rumen and intestine in dairy cattle. The parameters assessed included 1) protein structure α-helix to β-sheet ratio; 2) protein subfractions profiles; 3) protein degradation kinetics and effective degradability; 4) predicted nutrient supply using the intestinally absorbed protein supply (DVE)/degraded protein balance (OEB) system for dairy cattle. In this study, Vimy flaxseed protein was used as a model feed protein and was autoclave-heated at 120°C for 20, 40, and 60 min in treatments T1, T2, and T3, respectively. The results showed that using the synchrotron-based Fourier transform infrared microspectroscopy revealed and identified the heat-induced protein structure changes. Heating at 120°C for 40 and 60 min increased the protein structure α-helix to β-sheet ratio. There were linear effects of heating time on the ratio. The heating also changed chemical profiles, which showed soluble CP decreased upon heating with concomitant increases in nonprotein nitrogen, neutral, and acid detergent insoluble nitrogen. The protein subfractions with the greatest changes were PB1, which showed a dramatic reduction, and PB2, which showed a dramatic increase, demonstrating a decrease in overall protein degradability. In situ results showed a reduction in rumen-degradable protein and in rumen-degradable dry matter without differences between the treatments. Intestinal digestibility, determined using a 3-step in vitro procedure, showed no changes to rumen undegradable protein. Modeling results showed that heating increased total intestinally absorbable protein (feed DVE value) and decreased degraded protein balance (feed OEB value), but there were no differences between the treatments. There was a linear effect of heating time on the DVE and a cubic effect on the OEB value. Our results showed that heating changed chemical profiles, protein structure α-helix to β-sheet ratio, and protein subfractions; decreased rumen-degradable protein and rumen-degradable dry matter; and increased potential nutrient supply to dairy cattle. The protein structure α-helix to β-sheet ratio had a significant positive correlation with total intestinally absorbed protein supply and negative correlation with degraded protein balance.     

Effects of temperature,solid content and pH on the stability of black carrot anthocyanins

Anthocyanin stability of black carrots was studied at various solid contents (11, 30, 45 and 64° Brix) and pHs (4.3 and 6.0) during both heating, at 70–90 °C, and storage at 4–37 °C. Monomeric anthocyanin degradation fitted a first-order reaction model. Degradation of monomeric anthocyanins increased with increasing solid content during heating, while it decreased during storage. For example, at pH 4.3, half-life periods for anthocyanins at 30, 45 and 64° Brix were, respectively, 8.4, 6.9 and 5.2 h during heating at 80 °C and 18.7, 30.8 and 35.9 weeks during storage at 20 °C. At 30–64° Brix, increasing pH from 4.3 to 6.0 enhanced the degradation of anthocyanins during heating. The effect of pH on thermal stability of anthocyanins was also studied at six different pHs (2.5–7.0) in citrate-phosphate buffer solutions and significant decrease in anthocyanin stability was observed at pHs above 5.0. Higher activation energies (E_a) were obtained during heating than during storage with increasing solid contents. At 30–64° Brix, E_a values ranged from 68.8 to 95.1 kJ mol⁻¹ during heating and from 62.1 to 86.2 kJ mol⁻¹ during storage. Q₁₀ values at 20–37 °C were as high as 3.1 at 45° Brix and 3.6 at 64° Brix.     

Functional properties of protein fractions obtained from commercial yellow field pea (Pisum sativum L.) seed protein isolate

Commercial pea protein isolate was separated into water-soluble (WS), salt-soluble (SS), alkaline-soluble (AS) and ethanol-soluble (ES) fractions. AS fraction was the most abundant, constituting about 87% of the proteins in PPI followed by WS, SS and ES fractions in decreasing order. ES fraction consistently formed emulsions with a narrow range of smaller oil droplet sizes (0.6–19 μm) at pH 4.0, 7.0 or 9.0 compared to a wider range of sizes for emulsions stabilised by WS, SS and AS fractions. Emulsions formed with ES fraction were also the most stable (p < 0.05) over the 3 h test period at all the pH values used in this work. The WS fraction had significantly highest (p < 0.05) protein solubility and foaming capacity at all the pH values when compared to solubility of PPI, SS, and ES. Except for AS and ES fractions, foaming capacities of the protein fractions were higher at pH 9.0 than at pH 4.0 or 7.0.     

In situ thermal denaturation of myofibre sub-type proteins studied by immunohistofluorescence and synchrotron radiation FT-IR microspectroscopy

The thermal denaturation of proteins in skeletal muscle was studied and characterised for the first time taking into account the in situ metabolic and contractile fibre types. From serial histological sections, collagen, elastin, various type I, IIa and IIx fibres and type I–IIa and IIa–IIx hybrids were identified by immunohistofluorescence. Histological sections were incubated in buffer solutions at increasing temperatures (40, 50, 60, 70 and 80 °C). Protein secondary structure was investigated by synchrotron radiation FT-IR microspectroscopy on connective tissue and in muscle fibres rigorously identified for sub-type. Whatever the target protein components, increasing temperature resulted in a decrease in α-helix secondary structure and an increase in β-sheet structure. This phenomenon was more pronounced for intracellular proteins than for connective tissue. Although hybrid fibres were generally somewhat less sensitive to unfolding than the pure types, the amplitude of the thermal denaturation of intracellular proteins was practically independent of fibre type.     

Biocontrol of Fusarium species by a novel lectin with low ecotoxicity isolated from Sebastiania jacobinensis

A lectin from Sebastiania jacobinensis bark was isolated using a combination of acetone precipitation, ammonium sulphate fractionation, ion exchange and gel filtration chromatographies. The lectin purified, with a molecular mass of 52.0 kDa and composed of two subunits of 24 kDa, is a glycoprotein with a neutral carbohydrate content of 6.94%. The lectin shows maximum activity over the pH range 4.0–7.5 and heat stability up to 70 °C. Our results show that the lectin is an incompetitive inhibitor for trypsin, with a Ki of 0.39 ± 0.02 μM. Fluorescence spectroscopy indicated the existence of a hydrophobic surface. The percentages of secondary structure are 75% α-helix, 10% β-sheet, 5% β-turn and 10% unordered. Lectin inhibits the mycelial growth of Fusarium moniliforme and Fusarium oxysporum with an IC₅₀ value of 123 ± 0.5 and 303 ± 0.9 μg, respectively. Artemia salina Leach and embryos of Biomphalaria glabrata are not affected by the lectin, indicating low environmental toxicity. Alternative viewpoints are presented that might hopefully help in future efforts to develop safer and more effective microbial control agents.     

Effects of microwave heating,steaming, boiling and baking on the structure and functional properties of quinoa (Chenopodium quinoa Willd.) protein isolates

The effects of microwave heating (MW), steaming (SM), boiling (BL) and baking (BK) on the structure and functional properties of quinoa protein isolates (QPI) were investigated. SDS-PAGE showed the 20–30 kDa band strength of QPI-BL was enhanced, which indicated the disulphide bond was broken and protein molecules dissociated. Due to the recombination of subunits, some large molecular weight or insoluble polymer components of QPI-SM and QPI-BK did not pass through the gel. Microwave heating and boiling showed negative effects for α-helix and positive effects for β-sheet, which implied the molecular structure was transformed from ordered to disordered, the secondary structure became loose. The reduction of free SH (sulfhydryl) and surface hydrophobicity implied that aggregation and cross-linking of protein molecules for QPI-SM and QPI-BK. QPI, QPI-MW and QPI-BL exhibited better solubility, which was related to the water holding capacity (WHC) and emulsification. For QPI-SM and QPI-BK, functional properties (including solubility, WHC and gel-forming ability) decreased due to molecular aggregation. Heat treatments significantly affect the structure and functional properties of QPI, the current research showed that microwave heating and boiling might be better heat treatment methods for QPI and would help the development of quinoa protein products.     

High‐pressure processing‐induced conformational changes during heating affect water holding capacity of myosin gel

This study aimed to investigate the effects of high‐pressure processing (HPP) (0.1‐400 MPa for 9 min) on the water holding capacity (WHC) of heat‐induced rabbit myosin gel and structural changes during thermal treatment (25–75 °C). HPP at 100 MPa significantly increased the WHC (P < 0.05) and formed more regular and homogeneous three‐dimensional network. Myosin tails at 100 MPa unfolded completely during the thermal treatment, which was beneficial to form a high WHC gel network. However, myosin pressurised at 200 MPa and above formed a weak gel. Their heads were already aggregated before heating, preventing from subsequent thermal denaturation and aggregation. With the temperature increasing, unfolding of myosin tails was not sufficient for a filamentous network formation. These results suggested that HPP could modify the myosin structure and affect the gel formation during heating. The 100 MPa was the optimum pressure level for the WHC of rabbit myosin gel.     

Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus)

Trypsin from the viscera of Sardina pilchardus was purified by fractionation with ammonium sulphate, heat treatment and Sephadex G-100 gel filtration with a ninefold increase in specific activity and 9% recovery. The molecular weight of the enzyme was estimated to be 25,000 Da on SDS–PAGE. This enzyme showed esterase-specific activity on Nα-benzoyl-l-arginine ethyl ester (BAEE). The purified enzyme was inhibited by benzamidine, a synthetic trypsin inhibitor, and phenylmethylsulphonyl fluoride (PMSF) a serine-protease inhibitor, but was not inhibited by the β-mercaptoethanol. The optimum pH and temperature for the enzyme activity were pH 8.0 and 60 °C, respectively. The relative activity at pH 9.0 was 95.5% and the enzyme showed pH stability between 6.0 and 9.0. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECQKYSQ. S. pilchardus trypsin, which showed high homology to other fish trypsins, had a charged Lys residue at position 9, where Pro or Ala are common in fish trypsins. The enzyme was strongly inhibited by Zn²⁺ and Cu²⁺.     

Heat-induced denaturation/aggregation of porcine plasma and its fractions studied by FTIR spectroscopy

The aim of the present work is the in depth study of the protein aggregation mechanisms of whole porcine plasma and its fractions (serum, albumin and globulins) during heating using FTIR spectroscopy. Also, 2D correlation spectroscopy (2D COS) was used to establish the sequence of events during heat-induced gelation for all fractions. The results indicate that serum albumin quickly aggregates from 70 °C through non-native intramolecular β-sheets while globulins show lower susceptibility to protein aggregation. When found together, the aggregation pattern strongly depends on the composition of the protein mixture. That makes the great difference between plasma (serum albumin + globulins + fibrinogen) and serum (serum albumin + globulins) behavior, with the aggregation degree at the end of the thermal process being enhanced in the presence of fibrinogen - and achieving a similar level to that of serum albumin - while minimized in its absence. Attending on the low content of fibrinogen in plasma, our results suggest a great fibrinogen ability to alter the thermal serum albumin and globulins behavior by modifying the negative interactions established between them when no more proteins are found in the media. Moreover, it is noteworthy the slow plasma aggregation pattern at the beginning of the thermal process relative to serum albumin, this way allowing a higher protein unfolding. This could be related to the high heat-induced gel properties of plasma. Also, 2D COS indicates that the sequence of events is very similar for the all analyzed samples, with α-helix being more thermo-labile than native β-sheet structure.