Thermal and acidic denaturation of phycocyanin from Arthrospira platensis: Effects of complexation with λ-carrageenan on blue color stability.

The pH and temperature sensitivity of the natural blue pigment phycocyanin from Arthrospira platensis limits its application as food colorant. This study examines the effect of protein stabilization by the anionic polysaccharide λ-carrageenan on phycocyanins color appearance at pH 2.5-6.0, unheated and after heat treatments (70/90 °C). Electrostatic interactions, hydrophobic interactions, hydrogen bonds and disulfide-bridges were assessed by adding NaCl, urea and dithiothreitol (DTT) to the samples. Measurements of the zeta potential, transmittance and two-dimensional gel electrophoresis coupled to mass spectrometry confirmed electrostatic interactions around the zero surface charge of phycocyanin over a broad pH range (∼4.1-6.4). Despite a color shift towards turquoise, the color remained stable during heating, especially below of pH 3.5. Precipitation was inhibited over the entire pH range. Overall, electrostatic complexation of phycocyanin and λ-carrageenan is a promising technique to stabilize proteinaceous colorants, helping to reduce food waste and foster a shift to renewable materials.

Time-temperature-resolved functional and structural changes of phycocyanin extracted from Arthrospira platensis/Spirulina.

Arthrospira platensis, commonly known as Spirulina, gains increasing importance as alternative protein source for food production and biotechnological systems. A promising area is functional high-value algae extracts, rich in phycocyanin, a protein-pigment complex derived from A. platensis. This complex has proven functionality as the only natural blue colorant, fluorescent marker and therapeutic agent. The structure-function relationship is heat sensitive, making thermal processing in its production and its subsequent application a crucial aspect. In continuous high-temperature short-time treatments, it was shown how a purified phycocyanin (mixture of allophycocyanin and c-phycocyanin) disassembled and denatured between 50 and 70 °C. Three characteristic transition temperatures were allocated to specific quaternary aggregates. In contrast to sequential chemical denaturation, phycocyanin's chromophore and protein structure were simultaneously affected by thermal processing. Through a functionality assessment, the findings help optimize the efficiency of raw material usage by defining a processing window, enabling targeted process control resulting in desired product properties.

Influence of denaturation and aggregation of ß-lactoglobulin on its tryptic hydrolysis and the release of functional peptides.

Whereas previous studies showed that thermal pre-treatment of whey proteins promote their enzymatic hydrolysis, to date no correlation between the conformation of denatured protein and the release of individual peptides has been considered. Hence, in this study total denaturation of ß-lactoglobulin was performed at defined pH-values to enable the generation of different denatured particles. The denatured proteins were used as substrate for tryptic hydrolysis and the hydrolysis progress was characterised by the degree of hydrolysis (DH) and the release of functional peptides, detected using LC-ESI-TOF/MS. Denaturation and subsequent aggregation of ß-lactoglobulin, induced by thermal treatment at pH 5.1, altered the DH slightly, whereas the release of investigated peptides was significantly decreased. Contrary, denaturation at pH 6.8 and 8.0 led to formation of non-native monomers and reduced the DH to 75%, but showed promoting as well as reducing effects on the release of peptides, depending on their location within the protein.

Analysis of the effect of temperature changes combined with different alkaline pH on the ß-lactoglobulin trypsin hydrolysis pattern using MALDI-TOF-MS/MS.

Temperature and pH influence the conformation of the whey protein ß-lactoglobulin (ß-Lg) monomer, dimer, and octamer formation, its denaturation, and solubility. Most hydrolyses have been reported at trypsin (EC 3.4.21.4) optimum conditions (pH 7.8 and 37 °C), while the hydrolysate mass spectrometry was largely limited to peptides with <4 kDa. There are few reports on trypsin peptide release patterns away from optimum. This work investigated the influence of alkaline (8.65 and 9.5) and optimum (7.8) pH at different temperatures (25, 37.5, and 50 °C) on ß-Lg (7.5%, w/v) hydrolysis. Sample aliquots were drawn out before the addition of trypsin (blank sample) and at various time intervals (15 s to 10 min) thereafter. Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) was used to monitor peptide evolution over time with the use of two matrixes: α-cyano-4-hydroxycinnamic acid (HCCA) and 2.5-dihydroxyacetophenone (DHAP). Mass analysis showed that the N- and C-terminals (Lys(8)-Gly(9), Lys(100)-Lys(101), Arg(124)-Thr(125), Lys(141)-Ala(142), and Arg(148)-Leu(149)) of ß-Lg were cleaved early (15 s) implying the ease of trypsinolysis at the exposed terminals. Hydrolyses at 25 °C and pH 7.8 as well as at 50 °C and pH 9.5 were slowed down and ordered. Nonspecific chymotrypsin-like behavior occurred more at higher temperatures (50 °C) than at lower ones (25 and 37.5 °C). In addition to our earlier work in the acid pH region, it can be concluded that there is potential for controlled hydrolysis outside the trypsin optimum, where different target peptides with predictable biofunctionalities could be produced.