Evaluation of pH‐treated fish sarcoplasmic proteins on rheological properties of fish myofibrillar protein mediated by microbial transglutaminase

Fish sarcoplasmic protein (SP) could be exploited in the water‐holding agent for fish protein gels, except that the gel strength is reduced. The adjustment of pH could modify protein interactions to overcome the inferior effect. Fish SP solutions were adjusted to pH 3 or 12, neutralised to pH 7 and lyophilised to be pH‐treated SPs. These SPs along with lyophilised untreated SP (Normal SP) were incorporated into fish myofibrillar protein (MP) with microbial transglutaminase (MTG). The denaturation temperature (T_d) of MP mixed with normal SP was 66 °C with the lowest shear stress value. The denaturation of MP mixed with pH‐treated SP reduced to be 57 °C, resulting in increased shear stress. The cooking loss of MP gel was reduced by adding pH‐treated SPs, while the breaking forces were similar to control. The result indicated that pH‐treated SPs could be used to reduce cooking loss of MTG‐mediated MP gels without affecting the gelling properties.     

Structural Changes and Functional Properties of Threadfin Bream Sarcoplasmic Proteins Subjected to pH-Shifting Treatments and Lyophilization

ABSTRACT: Structural changes and functional properties of threadfin bream (Nemipterus sp.) sarcoplasmic proteins (TB-SP) subjected to various pH conditions (pH 3, 5, 6.3, 9, and 12) after subsequent pH readjustment to pH 7 were investigated. Fourier transform infrared spectroscopy revealed the loss of α-helical and β-sheet structures of TB-SP after being subjected to pH 3 or pH 12 treatments. The extent of structural and conformational changes of TB-SP subjected to pH 3 was greater than alkaline pHs (pH 9, 12) and pH 5, respectively. The water holding capacity of lyophilized TB-SP treated at pH 3 and pH 12 increased about 6.5- and 5.4-fold, respectively, as compared to the crude counterpart. Both acid and alkaline pH treatments increased fat absorption capacity of lyophilized sample about 2-fold, but drastically decreased its solubility. The water soluble fraction of extremely acidic (pH 3→7) and alkaline (pH 12→7) samples exhibited higher oil binding capacity as measured by diphenylhexatriene fluorescence and emulsifying activity. A gel-like structure was formed when water-soluble fraction of crude TB-SP and those subjected to moderate pHs (pH 5, 9) at 2 mg/mL was prepared for the emulsion containing 50% oil (v/v). Functional properties of TB-SP varied, depending on the pH-adjustment process applied.     

pH-dependent characteristics of gel-like emulsion stabilized by threadfin bream sarcoplasmic proteins

Threadfin bream sarcoplasmic proteins (TBSP) were identified by 2-dimensional polyacrylamide gel electrophoresis and the major proteins were likely be pyruvate kinase, creatine kinase, aldolase, and carbonic anhydrase. Emulsifying and rheological properties of emulsion stabilized by TBSP at pH 3, 7.5, and 12 were investigated. Surface hydrophobicity of TBSP increased with pH. Emulsifying activity index was highest at pH 3 followed by pH 12 and pH 7.5. The mean droplet diameter (d_3,2) was in the order of pH 7.5 > pH 3 > pH 12 at oil volume fraction ranging from 0.1 to 0.5. A decrease of oil volume fraction resulted in a reduction of oil droplet diameter. At oil volume fraction of 0.5, TBSP-stabilized emulsion at pH 7.5 and 12 exhibited gel-like characteristics, while liquid emulsion was observed at pH 3, based on visual observation and dynamic rheological analysis. Storage modulus (G′) values of emulsion at all studied pH values increased with temperature, implying the development of protein gel network upon heating. The gel-like emulsion at pH 12 absorbed more protein and water than at pH 7.5. Protein retention and water holding capacity of emulsion increased with oil volume fraction. Hydrophobic interactions between proteins and oil droplets were likely responsible for the development of a gel-like emulsion.     

Identification of glutaminyl sites on β-lactoglobulin for threadfin bream liver and microbial transglutaminase activity by MALDI-TOF mass spectrometry

The cross-linking of β-lactoglobulin (BLG) was efficiently catalysed by microbial transglutaminase (MTG) but not by fish (threadfin bream) liver transglutaminase (FTG). BLG cross-linking was inhibited by 2 mM 5-(biotinamido) pentylamine (BPNH₂) and MTG incorporated BPNH₂ into BLG ∼5 times more than was FTG. The glutaminyl sites for the incorporation of BPNH₂ into BLG by FTG and MTG were identified using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS analyses showed that MTG and FTG incorporated 4 and 1 residues of BPNH₂ per molecule of BLG, respectively. The BPNH₂-tagged BLG was digested by trypsin and BPNH₂-tagged peptides were selectively purified by avidin-affinity chromatography. Amino acid sequences of BPNH₂-tagged peptides were identified by comparing their MALDI-TOF mass spectra with the theoretical mass profiles from the MASCOT database. The BPNH₂-modification sites catalysed by MTG were glutamine (Q)13, Q68, Q15 or Q20, Q155 or Q159, whilst FTG only incorporated BPNH₂ into BLG at Q68. The different reactivities between FTG and MTG might be due to the different accessibilities of these TGases to the Q residues as well as to differences in substrate specificities.     

Ca2+ Affects Physicochemical and Conformational Changes of Threadfin Bream Myosin and Actin in a Setting Model

The effect of Ca²⁺ on physicochemical and conformational changes of threadfin bream (TB) myosin and actin during setting at 25 and 40°C was investigated. Ca²⁺ ion at 10 to 100 mM induced the unfolding of myosin and actin as evident by an increase of surface hydrophobicity (S_o ANS) at 40 °C. Total SH groups also decreased with an increased Ca²⁺ concentration, suggesting that Ca²⁺ promoted the formation of disulfide bonds during setting at 40 °C. Both hydrophobic interactions and disulfide linkages were involved in formation of myosin aggregates at 40 °C and were enhanced by addition of 10 to 100 mM Ca²⁺. Myosin Ca‐ATPase activity decreased when Ca²⁺ was greater than 50 mM, indicating conformational changes of myosin head. Circular dichroism spectra demonstrated that Ca²⁺ reduced the α‐helical content of myosin and actin incubated at either 25 or 40 °C. Ca²⁺ induced conformational changes of TB myosin and actin incubated at 40 °C to a greater extent than at 25 °C.     

Thermal stability of fish natural actomyosin affects reactivity to cross-linking by microbial and fish transglutaminases

Natural actomyosin (NAM) from Pacific whiting (PW) showed thermal transition temperatures by circular dichroism at 31.8 and 43.1 °C, which were lower than those of threadfin bream (TB) NAM, 35.0 and 49.3 °C. Endothermic transitions of PW-NAM by differential scanning calorimetry were at 31.8, 42.1 and 75.3 °C, compared to 36.1, 50.9 and 78.4 °C for TB-NAM. Based on surface hydrophobicity, α-helical content, and solubility, PW-NAM unfolded to a greater extent than did TB-NAM when incubated at 25 °C for 4 h and 40 °C for 2 h, suggesting its lower thermal stability. Transglutaminase generally catalyzed more extensive cross-linking of PW-myosin heavy chain (MHC) than TB-MHC, and the MHC cross-linking mediated by microbial transglutaminase (MTG) was greater than by fish transglutaminase (FTG). Textural properties of PW-NAM gels increased approximately 3.6–6.1-fold and 1.3–1.5-fold in the presence of MTG and FTG, respectively.