Study On Relationship Between The Pork Myofibrillar Protein Structure And It's Functionality Of Heat

The gel-forming properties of myofibrillar proteins are essential to the development of muscle-based products in that it contributes to textural properties, shaping the product, retaining water, and holding other food components in the product. During gelation, the molecular transition of the protein from its native state to the denatured state involves conformational changes in the quaternary, tertiary and secondary structures, which are influenced by pH, protein concentration, ionic conditions, vary muscle sources and additives. These changes determine the final structure and textural properties of the protein gels. The effect of temperature, ionic strength, microbial transglutaminase (MTG) and polyphosphate on the functional properties and biochemical characteristics of pork myofibrillar protein (PMP) gel were studied by combined low field nuclear magnetic resonance (NMR), Raman spectroscopy and circular dichroism spectra (CD) technique. The aim was to provide more insight into the functional properties of myofibrillar proteins, allowing the manipulation of processing conditions in order to obtain products with the desired structural and textural attributes. The detailed contents and results are shown as follows.1 Study on WHC and gel strength of PMP heat-induced gelationThe effect of temperature, NaCl, MTG and polyphosphate on WHC and hardness of PMP gel was investigated. The results showed the WHC of PMP gel decreased significantly with increasing of the temperature, however the hardness improved significantly and reached maximum at 60℃. The addition of NaCl produced a significant increase in the WHC and hardness of PMP gel. The enzymatic protein preparations had significantly higher WHC and hardness in comparison with the control system. The WHC and hardness increased from 81.4% and 36.70 g of control system to 95.6% and 63.24 g of the PMP gel containing 2 U·g-1 protein, respectively. However no further changes were observed for WHC when the level of MTG increased, and the hardness reached the maximum at the 4 U·g-1 MTG. The addition produced a significant increase in WHC for all polyphosphates and in hardness for HMP whereas decrease in hardness for PP and TPP.2 Study on functional properties of PMP gel by low field NMR methodThe effect of temperature, NaCl, MTG and polyphosphate on T2 relaxation times and microstructure of PMP gel was estimated by low field NMR and scanning electron micrograph (SEM). The results showed the NMR decay curve was fitted to three component which are attributed to bound (T21), immobile (T22) and free water state (T23) and the T2 relaxation times (peak time) of these three components were T21,1.15-7.05 ms; T22,231.01-403.70 ms and T23,1629-2477.08 ms, respectively. The position of the major component T22 clearly shifted lower relaxation times with increasing temperature, indicating limited water proton mobility, a fine and porous microstructure was observed due to the increasing temperature. The distributed water proton NMR T2 relaxation of different levels of NaCl after heat treatment characterized by two minor populations with relaxation times centered around 0.76-6.14 ms (T21) and 16.30-43.29 ms (T22a), a major population with a relaxation time of 76.65-174.75 ms (T22b) which was very broad. In addition, a broad, less well-defined population also appeared in the region between 1332.85 and 1629.75 ms (T23). The position of the major component clearly shifted higher relaxation times with increasing NaCl concentration, and integrated peak area proportion increased, which could explain the increasing WHC. Homogenous microstructure with decreasing pore size were introduced after the addition of NaCl and heat treatment. The distributed water proton NMR T2 relaxation of different levels of MTG characterized by two minor populations with relaxation times centered around 0.7-1.1 ms (T21) and 26-35 ms (T22a), a major population with a relaxation time of 170-230 ms (T22b) that was very broad. In addition, a broad, less well-defined population also appeared in the region between 1400 and 1800 ms (T23). The enzymatic preparation had significantly lower values of spin-spin time (T2). The major population T2 relaxation time was reduced from 226 ms (peak value) of the PMP gel containing no MTG to 188 ms of the PMP gel containing 2 U·g-1 protein. However no further decrease was shown when the dosage of MTG increased. The principal component analysis (PCA) also revealed the sample with MTG has shorter relaxation time. The position of the major relaxation component T22b clearly shifted towards longer relaxation times with increasing concentration of polyphosphate indicating increase of water proton mobility. And the integrated peak area proportion of the component increased with the adding of polyphosphate, which revealed that the increased WHC.3 Study on changes of the protein structure by Raman spectroscopy and CD The effect of different temperature, NaCl, MTG and polyphosphates on Raman spectroscopy and CD of PMP gel was examined. The normalized intensity of the band located near 545 cm-1 which is assigned to disulfide bonds in the trans-gauche-trans conformations decreased, indicating changes in disulfide bond stretching. The ratio of 1850/1830 ranged from 1.13 to 2.56 during heat-induced gelation, suggesting that the tyrosine residues of the samples were mainly exposed and able to participate in moderate or weak hydrogen bonding. Modifications in the amideⅠ(1650-1680 cm-1) and amideⅢ(1200-1300 cm-1) regions indicated a significant decrease in a-helix content, accompanied by a significant increase inβ-sheet,β-turn and random coil contents. The normalized intensity of the band located near 545 cm-1 which is assigned to disulfide bonds in the trans-gauche-trans conformations first decreased, and then increased, indicating changes in disulfide bond stretching. The ratio of 1850/1830 ranged from 1.24 to 2.19 due to different NaCl levels, suggesting that the tyrosine residues of the samples were mainly exposed and able to participate in moderate or weak hydrogen bonding. No significant differences of the protein secondary structure estimated were found as a function of NaCl concentration, maybe effect of NaCl on secondary structure before heat treatment was balanced by the heat treatment. The normalized intensity of the band assigned to C-H stretching vibration increased significantly, and there was a trend towards an increase in the integrated area of this band, although no significant changes were observed. Raman spectroscopy analysis indicated the occurrence of secondary structure and microenvironment changes due to MTG. Modifications in the amideⅠand amideⅢregions indicated a significant decrease in a-helix content, accompanied by a significant increase in other structures and a fine and porous microstructure was observed which limited the mobility of water proton and resulted in shorter T2 relaxation time due to the addition of the enzyme to PMP gel. The content of a-helix increased at the expense ofβ-sheet after the level of HMP reached 0.25%. No significant difference of secondary structure was demonstrated with adding of PP. However, a significant increase of content of a-helix andβ-sheet was observed after the TPP reached 0.15% and 0.2%, respectively.4 Study on the relationship of the protein structure and functional properties of PMP gel by PCA and correlation matrixThe relationship of the protein structure and functional properties of PMP gel was determined by PCA and correlation matrix. The PCA results of original parameters showed the first three principal components could explain 99.59% of the total variance, significant correlations were found between the WHC, hardness, T2 relaxation times and protein secondary structural changes of myofibrillar protein, the low and high temperature treatment located in the opposite position of the PCA score plots. The PCA results suggested the samples with low levels NaCl located in the right side of the PCA score plots, characterized by low WHC, hardness, T23, T24, peak3area proportion and polarity, whereas, the high levels NaCl located in the left side and characterized by the opposite properties. The correlation of the parameters was displayed in the PCA loading score plot. A strong correlation was observed between WHC, NMR T2 relaxation characteristics and secondary structure, and hardness strong related to NMR T2 relaxation characteristics, tryptophan buried or exposed, which indicated strong correlation between structure and functionality. No strong correlation between WHC, hardness and protein secondary structure was observed after phosphates were added.To sum up, a decreasing in a-helix content at the expense ofβ-sheet and shorter T22b relaxation time with the increasing of temperature and MTG resulted in higher hardness. The WHC improved with NaCl and phosphate added, which has no significant correlation with the content of protein secondary structures. The decrease of gel pore size leads to the decline of T2 relaxation times and mobility of immobile water for the samples of different temperature and MTG dosage.