Sugar-mediated thermal stabilisation of C-phycocyanin from Arthrospira platensis

C-phycocyanin (C-PC), the major protein of cyanobacteria Arthrospira platensis, is a blue pigment that primarily transfers energy during photosynthesis.It has diverse biotechnological applications since it can be used in nutraceutical, cosmetics, pharmaceutical industries, and biomedical research.Its intensive blue colour and strong antioxidant activity give C-PC significant potential to replace synthetic colourants in the food industry. However, thermal treatment of food has detrimental effects on C-PC colour due to sensitivity to higher temperatures; therefore,the application of this natural colourant in food and other products is limited. Hence, improving C-PC stability is the major challenge for successful application in food and beverages colouring. In the light of this,we aim to investigate the thermal stability of C-PC in the presence of selected sugars (glucose, fructose and sucrose), commonly used in the food industry. Ex-situabsorption spectrophotometryshowed that 18% solution (w/v) of glucose, sucrose and fructose at pH 7,upon incubation at 65℃, exhibit 91.4, 52.9 and 52.5% of colour preservation, respectively.In situ fluorescence measurements revealed that free C-PC has a melting point of 55.4°C, while the presence of glucose and sucrose increases the melting point of C-PC to 64.4 and 61.4°C, respectively. On the other hand, fructose does not significantly influence the C-PC melting point. These results show that the thermal stability ofthe CPCsolution is substantially increased in the presence of sugars, while the type of sugar significantly determines the extent of the stabilisation effect. Overall, our study provides the strategy for enhancing the application potential of C-PC as natural food colourant, providing a food product with vivid blue colour and substantial antioxidant activities

Arthrospira platensis and Porphyra sp. – prospective serum- substitute in HEK293T cell culture

Both Arthrospira platensis (Spirulina) and Porphyra sp. (Nori) are algae known for their richness in vitamins, minerals, antioxidants, proteins, such as phycobiliproteins (PBPs). Тhanks to their exceptional nutritional properties they have potential to be considered as a high-quality substitute for fetal bovine serum (FBS) in cellular cultivation, which has numerous drawbacks since it can be involved in contamination development and its composition is still unclear. In this study we investigated the influence of Spirulina and Nori extracts on HEK293T cell line growth and viability in serum-reduced conditions. In DMEM/F12 medium supplemented with 0–10% FBS, the concentration-dependent effects of PBPs on cell proliferation were investigated. Cell viability and cytotoxicity were evaluated by MTT assay. During 3-day observation prior to MTT assay and MTT assay itself showed that HEK293T exhibited improvement in viability at lower PBP concentrations, while presence of higher concentrations resulted in inhibition of growth and change in morphology as a consequence of their cytotoxicity at higher concentrations. These findings suggest that PBPs have a positive outcome on cell growth at relevant doses. In general, in this study were obtained results proving the potential advantages of PBPs at lower doses on cell proliferation in serum-reduced conditions, but also HEK293T cells ability to adapt in non-standard cultivation set-up

Digestibilnost β-laktoglobulina nakon njegovog unakrsnog povezivanja dejstvom lakaze iz Trametes versicolor i polifenola iz jabuke

beta-Lactoglobulin (BLG) is an important nutrient of dairy products and an important allergen in cow's milk allergy. The aim of this study was to investigate the potential of laccase to cross-link BLG in the presence of an apple phenolic extract (APE) and to characterize the obtained products for their digestibility by pepsin and pancreatin. The composition of the apple phenolics used for cross-linking was determined by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The apple phenolic extract contained significant amounts of quercetin glycosides, catechins and chlorogenic acid. The laccase cross-linked BLG in the presence of apple phenolics. The polymerization rendered the protein insoluble in the reaction mixture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the cross-linking reaction mixture revealed a heterogeneous mixture of high molecular masses (cross-linked BLG), with a fraction of the BLG remaining monomeric. Enzymatic processing of BLG by laccase and apple polyphenols as mediators can decrease the biphasal pepsin pancreatin digestibility of the monomeric and cross-linked protein, thus decreasing its nutritional value. In addition, reduced BLG digestibility can decrease its allergenic potential. Apple polyphenols can find usage in the creation of new, more functional food products, designed to prevent obesity and hypersensitivity-related disorders.β-Laktoglobulin (BLG) je važan nutrijent mlečnih proizvoda i važan alergen kod alergija na kravlje mleko. Cilj ove studije je bilo ispitivanje potencijala lakaze da unakrsno poveže BLG u prisustvu fenolnog ekstrakta jabuke (APE), kao i karakterizacija dobijenih proizvoda sa aspekta njihove digestibilnosti pepsinom i pankreatinom. Kompozicija fenola jabuke korišćenih za unakrsno povezivanje određena je pomoću LC-ESI-MS. Fenolni ekstrakt jabuke sadrži znatne količine glikozida kvercetina, katehine i hlorogensku kiselinu. BLG je unakrsno povezan lakazom u prisustvu fenola jabuke, pri čemu je polimerizacija učinila BLG nerastvornim u reakcionoj smeši. SDS-PAGE analiza pokazala je da reakciona smeša sadrži heterogenu smešu velikih molekulskih masa (unakrsno povezan BLG), kao i deo zaostalog monomernog BLG. Enzimsko procesovanje BLG lakazom, u prisustvu polifenola jabuke kao medijatora, može smanjiti bifaznu pepsin-pankreatinsku digestibilnost kako monomernog, tako i unakrsno povezanog BLG, i na taj način smanjiti njegovu nutritivnu vrednost. Takođe, smanjena digestibilnost BLG može smanjiti njegov alergeni potencijal. Polifenoli jabuke mogu se koristiti za kreiranje novih, funkcionalnijih prehrambenih proizvoda, dizajniranih za prevenciju kako gojaznosti, tako i bolesti vezanih za preosetljivost

Stabilization of C-phycocyanin by immobilization in alginate beads

C-Phycocyanin (C-PC), the major protein of cyanobacteria Arthrospira platensis, is a phycobiliprotein with potent biological activity. It has several beneficial effects, including anti-oxidant, anti-inflammatory, immunomodulatory, and anti-cancer. A significant challenge for the broader application of C-PC in the food industry is its stability in food processing conditions, such as increased light exposure, temperature, and high pressure and drying. This work aimed to investigate if the immobilization of C-PC onto alginate beads could improve its stability. C-PC was immobilized by dropping the solution of C-PC and 1% alginate (final concentration) in the solution of 2% CaCl2. Both protein/alginate mixture and CaCl2 were kept at pH 4. Immobilized CPC was treated for 30 min at 65°C, by high pressure up to 4500 bar, and incubated under light exposure for a month. Alginate beads with immobilized C-PC were also left to dry in the fridge and kept for a month. C-PC was extracted from alginate beads by immersing them in 20 mM phosphate buffer, pH 7. The stability of C-PC was assessed by a color change and UV-VIS spectroscopy. Immobilized C-PC was stable under all tested conditions, with only small aggregation and color change appearing after high-pressure treatment. Immobilization of C-PC by alginate thus shows promise for its efficient stabilization under food processing conditions

Examination of C-phycocyanin interactions with selected vitamins

C-phycocyanin (C-PC) is a photosynthetic protein from Arthrospira platensis (cyanobacteria). Due to its intense blue colour, which is very rare in nature, C-PC has industrial applications as a food colourant as a substitute for synthetic food colourants. Disadvantages of C-PC as a food colourant are its poor stability at high temperatures (during thermal treatment of the food) and its sensibility to change pH value. The binding of food-derived small molecules, such as vitamins, could stabilize the structure of C-PC at high temperatures and wide pH ranges. In this study, we characterized the binding of selected vitamins to C-PC, purified from the commercial powder of Arthrospira platensis. We used hydrophilic vitamins (B1, B2, B7, B9, B12), lipophilic vitamins (A, D3) and provitamin (β-carotene). Fluorescent spectroscopy showed a decrease in fluorescence of CPC i n t he p resence o f v itamin A, v itamin D3 a nd β -carotene (lipophilic molecules) compared to the control. In contrast, the fluorescence of C-PC in the presence of hydrophilic vitamins showed minimal change. The protein fluorescence quenching approach demonstrated hydrophobic (pro)vitamins binding affinities ranging from 0.02 to 5.9 x 105 M-1, with the ability of hydrophobic (pro)vitamins to bind at the different sites on C-PC. UV-VIS spectrophotometry showed that the binding of hydrophobic (pro)vitamins does not affect the protein colour, while CD spectroscopy revealed that the binding of chosen molecules does not significantly influence the secondary structure of C-PC. Overall, this study demonstrated C-PC's significant potential in binding hydrophobic (pro)vitamins, while further research is required to test if these ligands could improve CPC stability

Examining fatty acid interactions with Arthrospira platensis-derived C-phycocyanin

Cultured meat requires less land and water and is less polluting, but still costly. The critical challenge in cultivated meat science is identifying and developing bovine serum albumin alternatives as the key component in cell media. Phycobiliproteins (PBPs) from micro- and macroalgae are promising candidates for albumin replacement due to their high abundance and well-known excellent antioxidative and metal-binding activities of covalently attached tetrapyrrole chromophores. Considering the importance of fatty acids (FA) binding by albumin for cell cultivation, the additional prerequisites for developing PBPs as albumin replacement components is their validation for the ability to bind FA. This study aims to examine the ability of C-phycocyanin (C-PC), the major PBP of microalgae Arthrospira platensis, to bind seven fatty acids (stearic, palmitic, oleic, elaidic, linoleic, linolenic and docosahexaenoic acid). For this purpose, we employed various optical spectroscopy techniques (fluorescence, CD, and VIS absorption spectroscopy). The protein fluorescence quenching approach demonstrated FA binding affinities ranging from 0.42 to 2.4 x 105 M−1, with the ability of FA to bind at different sites on C-PC. Fatty acid binding induces substantial changes in the VIS absorption spectra of C-PC, indicating the FA are attached in the vicinity of C-PC chromophores. On the other hand, CD spectroscopy did not show significant effects of FA binding on C-PC secondary structure content. Overall, this study revealed C-PC's significant potential in binding FA, the critical prerequisite to replacing albumin for developing animal-free cell media for meat cultivation

Exploring if Porphyra sp. extract functions as serum substitute in HT29 cell culture

This study investigates the impact of Porphyra sp. extracts on HT29 cell line growth and viability at reduced serum conditions. The concentration-dependent effects of phycobiliproteins (PBPs) on cell proliferation were examined over various time intervals. Lower concentrations of PBPs (20 μg/mL) demonstrated an increase in HT29 cell viability after 48 hours and 5 days of cultivation at reduced serum concentration (final serum concentration was in the range from 5 to 8%). This suggests a potential positive influence on cell proliferation, likely due to their antioxidant properties. Conversely, higher concentrations of PBPs exhibited inhibitory effects on cell growth, possibly due to cytotoxicity at elevated levels. Remarkably, when HT29 cells were cultured solely in algal extract without fetal calf serum (FCS), complete growth inhibition was observed after 72 hours. This finding underscores the insufficient nutrient and growth factor provision of PBPs alone for sustaining cell viability. Morphological differences observed in cells cultured with 70 μg/mL of PBPs indicated potential alterations in cellular morphology. Notably, 70 μg/mL of PBPs in RPMI medium with 5% FCS displayed growth inhibition compared to the control (5% FCS). Furthermore, we assessed HT29 cell adaptability to changes in FCS concentration and PBP supplementation. Cells incubated under varying FCS and PBP conditions were subcultured into RPMI medium with lower FCS concentration and PBPs from Porphyra. The viability of cells following subculturing indicated sustained adaptability to reduced FCS levels. Overall, this study provides valuable insights into the concentration-dependent effects of PBPs from Porphyra extracts on HT29 cell growth and viability. The findings underscore the potential benefits of PBPs at lower concentrations for cell proliferation at reduced serum conditions and reveal the adaptability of HT29 cells to changing culture conditions

Examining fatty acid interactions with Arthrospira platensis-derived C-phycocyanin

Cultured meat requires less land and water and is less polluting, but still costly. The critical challenge in cultivated meat science is identifying and developing bovine serum albumin alternatives as the key component in cell media. Phycobiliproteins (PBPs) from micro- and macroalgae are promising candidates for albumin replacement due to their high abundance and well-known excellent antioxidative and metal-binding activities of covalently attached tetrapyrrole chromophores. Considering the importance of fatty acids (FA) binding by albumin for cell cultivation, the additional prerequisites for developing PBPs as albumin replacement components is their validation for the ability to bind FA. This study aims to examine the ability of C-phycocyanin (C-PC), the major PBP of microalgae Arthrospira platensis, to bind seven fatty acids (stearic, palmitic, oleic, elaidic, linoleic, linolenic and docosahexaenoic acid). For this purpose, we employed various optical spectroscopy techniques (fluorescence, CD, and VIS absorption spectroscopy). The protein fluorescence quenching approach demonstrated FA binding affinities ranging from 0.42 to 2.4 x 105 M−1, with the ability of FA to bind at different sites on C-PC. Fatty acid binding induces substantial changes in the VIS absorption spectra of C-PC, indicating the FA are attached in the vicinity of C-PC chromophores. On the other hand, CD spectroscopy did not show significant effects of FA binding on C-PC secondary structure content. Overall, this study revealed C-PC's significant potential in binding FA, the critical prerequisite to replacing albumin for developing animal-free cell media for meat cultivation

Exploring if Porphyra sp. extract functions as serum substitute in HT29 cell culture

This study investigates the impact of Porphyra sp. extracts on HT29 cell line growth and viability at reduced serum conditions. The concentration-dependent effects of phycobiliproteins (PBPs) on cell proliferation were examined over various time intervals. Lower concentrations of PBPs (20 μg/mL) demonstrated an increase in HT29 cell viability after 48 hours and 5 days of cultivation at reduced serum concentration (final serum concentration was in the range from 5 to 8%). This suggests a potential positive influence on cell proliferation, likely due to their antioxidant properties. Conversely, higher concentrations of PBPs exhibited inhibitory effects on cell growth, possibly due to cytotoxicity at elevated levels. Remarkably, when HT29 cells were cultured solely in algal extract without fetal calf serum (FCS), complete growth inhibition was observed after 72 hours. This finding underscores the insufficient nutrient and growth factor provision of PBPs alone for sustaining cell viability. Morphological differences observed in cells cultured with 70 μg/mL of PBPs indicated potential alterations in cellular morphology. Notably, 70 μg/mL of PBPs in RPMI medium with 5% FCS displayed growth inhibition compared to the control (5% FCS). Furthermore, we assessed HT29 cell adaptability to changes in FCS concentration and PBP supplementation. Cells incubated under varying FCS and PBP conditions were subcultured into RPMI medium with lower FCS concentration and PBPs from Porphyra. The viability of cells following subculturing indicated sustained adaptability to reduced FCS levels. Overall, this study provides valuable insights into the concentration-dependent effects of PBPs from Porphyra extracts on HT29 cell growth and viability. The findings underscore the potential benefits of PBPs at lower concentrations for cell proliferation at reduced serum conditions and reveal the adaptability of HT29 cells to changing culture conditions

Dietary fatty acids as a new binding partner of C - phycocyanin: a fluorimetric study

C-Phycocyanin (C-PC) is a phycobiliprotein from cyanobacteria, where it harvests light energy that is then transferred to chlorophylls during photosynthesis. It has an intense blue color due to a covalently bonded tetrapyrrole chromophore, and owing to this property is used in the food industry as a good natural alternative for food coloring. In addition to its coloring properties, C-PC has anti-inflammatory, antioxidant, anti-cancer, and immune-enhancing effects that qualify it as a dietary supplement already included in various formulations, mainly Spirulina extract powders. Since it is used as a food colorant and as a dietary supplement, it may interact with food ingredients, affecting its stability, digestibility, or antioxidant properties. Palmitic acid and linoleic acid (which can be metabolized to linolenic acid) are abundant in meat, milk, and edible oils, so that they could interact with C-PC. C-Phycocyanin isolated from the cyanobacterium Arthrospira platensis (Spirulina) was incubated with increasing concentrations of these three fatty acids, and its fluorescence intensity was monitored. Incubation resulted in a fluorescence quenching effect, indicating that binding had occurred. The binding equations indicated that the association constants were of the same order of magnitude and that the number of approximate binding sites was more than one (Ka = 4.64 x 10⁴ M-¹, n = 1.5 for linoleic acid; Ka = 2.88 x 10⁴ M–¹, n = 1.9 for linolenic acid; Ka = 0.44 x 10⁴ M–¹, n = 0.8 for palmitic acid). This moderate interaction between C-PC and fatty acids could influence its behavior as a nutraceutical and food colorant