The crude extract of phycobiliproteins with the purities of 1.24 for C-PC and 0.445 for APC was subjected to two further steps of downstream processing. As previously demonstrated (Antecka et al. 2022), the foam fractionation process can be successfully used to concentrate C-PC from the crude solution. In addition, it does not require the addition of surfactants or pH adjustment, but only air, which greatly reduces the cost of operation. In this step, the recovery yielded over 47% and the purity of C-PC was increased by 1.36 times. The obtained C-PC had a purity of 1.69 and can be classified as a food or cosmetic colorant (Wu et al. 2016; da Silva Figueira et al. 2018), whereas, the purity of APC after the FF process was equal to 0.516 with the recovery of 17.4% and the purification factor of 1.16. The resulting condensate was then subjected to preparative liquid chromatography (FPLC), which is considered the most efficient method for separating C-PC from solution (Ledakowicz et al. 2024). Of the four columns tested, the highest purity of C-PC and its complete separation from allophycocyanin was obtained on a Q Resource column in a system with 10 mM acetate buffer, pH 5.0, and elution with 1 M NaCl in a 0–18% gradient. Total protein recovery was approximately 23.9% for C-PC and 8.60% for APC, while the purification factors were approximately 3.60 and 7.26, respectively. After a single pass through the Resource Q column and a relatively slow elution with a linear gradient, two separate fractions containing C-phycocyanin and one containing allophycocyanin were obtained, as shown by the two main peaks labelled C-PC I and C-PC II, and an allophycocyanin peak labelled APC (Fig. 1).

Chromatogram of phycobiliproteins from Synechococcus PCC 6715 obtained by anion exchange chromatography on a Resource Q column. Absorbance at 280, 616 and 652 nm (solid lines), NaCl gradient (dashed lines)

The two fractions of deep blue-colour C-PC and greenish-blue-colour APC were collected separately and subjected to stability testing. The results of the entire purification procedure are presented in tables (Table 1) for C-PCs and (Table 2) for APC. The protein recovery yields were 17.4% for C-PC I and 6.52% for C-PC II at a purity of 4.66 and 4.25, respectively and 8.60% for APC at a purity of 3.23.

Extensive work has been carried out to investigate the effect of temperature, pH, time and light on the stability of purified C-PCs and APC. The individual, detailed results are presented in the following subsections below.

Temperature is one of the most important factors affecting the stability of pigment proteins present in cyanobacteria. Chentir et al. (2018) reported a dramatic decrease in the concentration of C-PC from Spirulina at temperatures above 50 °C. However, for C-PC from Synechococcus this critical factor does not seem to be as destructive as for C-PC from mesophilic species. In this experiment the purified PBPs were subjected to heat stress from 30 to 64 °C for 5 h. The results obtained are shown in Fig. 2.

Stability and purity of C-PCs and APC during 5 h incubation at different temperatures; a stability of C-PC I, b purity of C-PC I, c stability of C-PC II, d purity of C-PC II, e stability of APC, f purity of APC. Measurements were performed in triplicate (standard deviations < 5%)

Both fractions of C-PC (Fig. 2a, c) were found to be stable at temperatures of up to 50 °C, with no significant changes in the concentration values observed. However, when the temperature was increased from 40 to 64 °C, the residual concentrations of C-PC I after 5 h of incubation decreased from 99% at 40 °C to 96% at 50 °C, 88% at 60 °C and 79% at 64 °C. In addition, fraction C-PC I appears to be slightly more stable than fraction C-PC II, as the residual activity after 5 h at 64 °C was 79% instead of 72% for C-PC II. The degree of purity of the isolated C-PCs (Fig. 2b, d) varied with time, as did their concentration. During short-term exposure at temperatures as high as 60 °C, the thermal decomposition of C-PC is not as apparent, probably influenced by the helical secondary structure of the phycocyanin. Interestingly, the second pigment APC was much less stable than C-PC (Fig. 2e, f). Its concentration decreased slightly already at 40 °C, and after 5 h of incubation the residual activity was 89%, with rapid denaturation at temperatures above 60 °C.

The pH of the solvent is also a critical factor in the application of bioproducts, as proteins are sensitive to changes in pH. Therefore, in order to determine the optimal pH range for purified PBPs, samples were placed in acetic buffers with different pH values ranging from 3 to 12. The results (Fig. 3) show that the C-PC remains unchanged over a wide range of pH from 3 to 10, only a strongly alkaline pH causes immediate discoloration of the pigment. In contrast, the second product, APC, is much more sensitive to pH changes, remaining unchanged only at pH 5 and 9. At pH values of 4, 7 and 10 the pigment concentration retained about 70% of its initial value, while pH 3 and 12 caused immediate protein degradation.

Effect of pH on the concentration of purified PBPs: C-PC I, C-PC II and APC. Measurements were performed in triplicate (standard deviations < 5%)

Although knowing the optimum pH for a protein is very useful, from a practical point of view, its stability under given conditions is even more important. Therefore, to determine the stability of the PBPs, samples were incubated for 5 h at given pH values ranging from 3 to 12. The results for C-PCs (Fig. 4a, c) show that both its fractions remain stable at pH 3 to 10. In alkaline solutions (pH 11), the concentration of C-PC immediately decreased by about 50% and then it remained stable for 5 h, while pH 12 caused an immediate decolorization of the pigment. As with the temperature stability, fraction C-PC II was more sensitive to pH changes than fraction C-PC I.

Stability and purity of isolated C-PCs and APC during 5 h incubation at different pH values; a stability of C-PC I, b purity of C-PC I, c stability of C-PC II, d purity of C-PC II, e stability of APC, f purity of APC. Measurements were performed in triplicate

The purity of both fractions of C-PC (Fig. 4b, d) varied similarly to their concentration during incubations at different pH values, confirming the absence of other proteins in the samples. APC appeared to be much more pH-dependent, as its concentration remained unchanged only at pH 5–9 (Fig. 4e, f), whereas strongly acidic (pH 3) or alkaline (pH 11–12) pH induced rapid pigment degradation. However, all the results obtained suggest that PBPs from Synechococcus are more pH stable than C-PC from Spirulina, which is only stable at pH 5–7.5 when the hexameric form predominates (Pez Jaeschke et al. 2021).

Appropriate storage conditions are another parameter that guarantees the preservation of the valuable properties of the proteins. Therefore, the long-term stability of purified PBPs and the effect of three different temperature storage conditions were investigated and the results are shown in the figures (Fig. 5).

Long-term stability and purity of isolated C-PCs and APC during one month of storage at three different temperatures; a time stability of C-PC I, b purity of C-PC I, c time stability of C-PC II, d purity of C-PC II, e time stability of APC, f purity of APC. Measurements were performed in triplicate (standard deviations < 5%)

The results (Fig. 5a, c) show little difference between the C-PC fractions, C-PC I was stable after 1 month of storage at 4 and 25 °C and the C-PC II only at 4 °C. The concentration of both fractions decreased significantly when stored at 45 °C. Interestingly, the purity of isolated C-PCs (Fig. 5b, d) remains unchanged only at 4 °C. This proves that the lower temperature helps maintain the stability of the phycocyanin for a longer period of time. For comparison, in the case of C-PC from Spirulina even ambient temperature caused a 20% loss in concentration after 10 days of incubation (Pez Jaeschke et al. 2021).

In the case of APC (Fig. 5e, f) a decrease in both concentration and purity was observed regardless of temperature, but at 45 °C APC was completely inactivated after 6 days of incubation. At temperatures 4 and 25 °C, a systematic decrease in APC concentration was observed, so that after 30 days of incubation the residual concentrations were approximately 50%. Interestingly, the purity was almost unchanged at 4 °C, indicating a similar decrease in both absorbance values, at 652 and 280 nm.

With respect to light exposure (Fig. 6), a large difference was also observed between the C-PC fractions and the APC. APC was much more sensitive to photochemical degradation than C-PC. After 10 h of exposure C-PCs still had about 86% of their initial concentration, while APC had only about 10%.

Photostability of purified PBPs during 10 h of light exposure in front of the UV–VIS lamp (600 W); a residual concentrations of PBPs, b purity of PBPs. Measurements were performed in triplicate (standard deviations < 5%)

SDS-PAGE analysis of purified PBPs (Fig. 7) shows that both isolated phycocyanin fractions contain α-subunits of 15.0 kDa and β-subunits of 16.4 kDa, confirming their identity by native-PAGE. The molecular weight of allophycocyanin is 15.5 kDa. These experimental results indicated that the purification procedure used produced reliable, highly pure PBPs.

SDS PAGE of Synechococcus PCC 6715 phycobiliproteins after two-step of purification. Line 1. Ladder: PageRuler 10–180 kDa (ThermoFisher Scientific); line 2. Crude extract; line 3. C-PC I; line 4. C-PC II; line 5. APC