Structural, physicochemical and functional properties of cumin (Cuminum cyminum) proteins

Nowadays, there is an increasing number of plant candidates for protein extraction as an alternative for animal sources. The investigation of new plant protein sources and the study of the effects of processing methods on their structure, physicochemical, and functional properties obtain more attention and become necessary. Cumin (Cuminum cyminum) is a traditional and worldwide culinary spice. After the extraction of essential oils and oleoresin, a relatively important amount of defatted cumin residues is produced, which are rich in protein (28.33%) and constitute a good substrate for protein extraction. In this thesis, the composition of protein fractions from cumin seeds and their principal characteristics were studied, as well as the effects of different environmental conditions (pH and NaCl) and processing treatments (heat and high hydrostatic pressure treatment) on the structural, physicochemical and functional properties of cumin protein isolate (CPI) were assessed.
First of all, the protein fractions and CPI were extracted from defatted cumin residues, and their characteristics were investigated. The protein fractions of cumin seeds were albumin (62.29%), glutelin (25.16%) globulin (11.12%), and prolamin (1.43%). The CPI presented most bands of albumin, globulin and glutelin, and had more disulfide bonds than major protein fractions when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The glutamic acid and aspartic acid were the predominant amino acids in cumin proteins, furthermore, more hydrophobic and aromatic amino acids were found in CPI than in protein fractions, and the content of essential amino acids in CPI and globulin were higher than in albumin and glutelin. The glutelin displayed greater exposure of tyrosine and tryptophan residues and bigger particle size compared to albumin and CPI, while CPI presented more α-helix (14.4%) and less β-strand (30.7%) than albumin and glutelin. These differences in structure and amino acid composition for CPI, albumin, and glutelin significantly influenced their hydrophobicity (Ho) and zeta-potential (ζ) (p<0.05), and as a consequence their emulsifying and gelling property. Albumin exhibited the highest emulsifying activity (EAI) and stability (ESI) and the smallest emulsion particle size (4.29 μm), followed by CPI and glutelin. The CPI was efficient in forming a gel at 80.6 °C, and glutelin could form the hardest gel at 92.6 °C. CPI and albumin could thus be potential candidates as emulsifying agents, and CPI and glutelin could be used for gelation in food industry. Overall, CPI displayed better amino acid composition and characteristics than the major protein fractions of cumin seeds, and therefore a deep investigation of CPI would be carried out for the application in food industry.
Secondly, the effects of pH and NaCl on the structural, physicochemical, and functional properties of CPI were evaluated. The structure of CPI was greatly influenced by the pH adjustment and NaCl addition. The fluorescence intensity (FI) of CPI was reduced as pH reached a value close to 5.0 and the highest α-helix content (18.3%) in CPI was obtained at pH 5.0. The addition of NaCl led to a decrease in the α-helix content and an increase in the β-sheet for CPI. The ζ, Ho, and solubility of CPI were significantly reduced as pH reached 5.0 (p<0.05), whereas the addition of 0.5 and 1.0 M NaCl caused an increase in Ho and a decrease in solubility. CPI showed higher EAI and ESI at pH 7.0 and pH 9.0 with small emulsion droplet size, and the addition of 0.1 M NaCl produced an increase in ESI. At lower NaCl concentration (0.1 M), the EAI wasn’t affected, while the ESI was improved significantly (p<0.05). However, as pH changed to pH 5.0 or the addition of 0.5 M and 1.0 M NaCl, the emulsifying properties of CPI were reduced. All CPI emulsions showed pseudo-plastic behavior, and the apparent viscosity and viscosity coefficient (k) of the CPI emulsions at pH 5.0 were significantly higher than those for the other pH values, and increasing k values were found after the addition of NaCl. The desired emulsifying property of CPI might be obtained at neutral and alkali conditions with the addition of a lower concentration of NaCl.
Thirdly, effects of heat treatments at 65, 75, 85 and 95 °C for 30 min on the structure and the emulsifying properties of CPI were investigated. The structure of CPI was remarkably influenced as heating temperature increased while fluorescence intensity decreased. After heat treatments at different temperatures, more protein molecules were unfolded and more compact conformations were formed, the α-helix content of CPI was increased, while the β-sheet content was decreased. A significant increase in the Ho of CPI was found after heat treatments, and the Ho reached 1030.6 for heated CPI at 95 °C. Heat treatments induced the formation of high molecular mass aggregates of CPI, which led to a decrease in the EAI and ESI of heated CPI at different protein concentrations (0.1%, 0.5% and 1.0%). However, the protein absorption in emulsions stabilized by heated CPI gradually increased with heating temperature increasing. Both emulsions stabilized by native and heated CPI showed shear-thinning behavior and exhibited a decrease in their viscosities with protein concentration increasing. Thermal treatments produced different effects on the flow behavior of emulsions formed by various protein concentrations. Indeed, the flow index (n) for heated CPI emulsions decreased for a 0.1% concentration, but increased for protein concentrations of 0.5% and 1.0%. More heated CPI absorbed on the surface of the oil droplet will be helpful for the production of emulsion gel with higher protein concentrations.
Furthermore, the effects of high hydrostatic pressure (HHP) treatments (200, 400 and 600 MPa, 15 min) on the structural, physicochemical, and functional properties of CPI were studied. HHP treatments could result in the formation of more aggregates, more pores, irregular conformations, and bigger particle size from the results of SDS-PAGE, atomic force microscope (AFM), and scanning electron microscope (SEM). With pressure increasing, more protein aggregates with large protein molecular mass were formed which influenced the microenvironment of protein and led to a decrease in fluorescence intensity and an increase in α-helix content, a decrease in β-strand for CPI. Surface hydrophobicity of CPI significantly increased after HHP treatments, from 343.35 for native CPI to 906.22 at 600 MPa (p<0.05). HHP treatment at 200 MPa reduced the zeta-potential and solubility of CPI, while having a little effect at 400 and 600 MPa. After HHP treatments, the EAI and ESI of CPI were decreased with a significant increase in droplet size of emulsions (p<0.05). HHP-treated CPI at 400 and 600 MPa could form a heat-induced gel at lower Tgel (68.5 °C and 70.9 °C) with higher storage modulus (G') of 4100 Pa and 4170 Pa, respectively. HHP treatments might be a potential processing method in order to modify CPI making them potential candidates as gelation agent in food matrices.