Cultivation of S. platensis

S. platensis powder was provided by DACO Agricultural Consulting Company, Cairo, Egypt. The strain was cultivated in an open raceway pond (60 m × 8 m × 0.35 m) equipped with a paddle wheel to ensure continuous mixing and nutrient distribution at a flow rate of 0.2 m s⁻¹. Cultures were maintained under outdoor sunlight and grown in modified Zarrouk medium (Zarrouk 1966), with the medium adjusted to pH 9.0 and temperature of 35 ± 2 °C. Biomass was harvested by filtration through a 54 μm nylon mesh and subsequently dried in a hot-air oven at 45 ± 2 °C.

Chemical composition of Spirulina powder

Dried Spirulina powder was analyzed by near-infrared (NIR) spectroscopy (DA1650, FOOS, Denmark) at the Central Laboratory, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt. The lipid, carbohydrate, and protein contents were measured (Table 1).

Table 1 Chemical composition of S. platensis powderγ-irradiation treatment

Before γ-irradiation, Spirulina powder samples (10 g each) were placed in sterile polyethylene bags and evenly distributed to form a layer with an approximate thickness of 2–3 cm, then sealed to prevent contamination. Irradiation was performed as a single exposure for each dose level (0, 2, 4, 6, 8, and 10 kGy) using cobalt-60 γ-radiation source (Gamma Chamber 4000, India) at the National Center for Radiation Research and Technology (NCRRT), Nasr City, Cairo, Egypt. Samples were positioned within a uniform irradiation zone of the chamber to minimize dose variation. The dose rate at the time of irradiation was 0.782 kGy h⁻¹, as determined using an alanine transfer dosimeter.

Microbial decontamination analysis

The pour-plate method was employed to assess the viability of aerobic bacteria, molds, and yeasts in Spirulina powder in accordance with ISO 4833-1 (ISO 2013). Briefly, 10 g of Spirulina powder were aseptically homogenized in a Stomacher (Laboratory Blender Stomacher 400, Seward Ltd., Worthing, UK) with 90 mL of sterile physiological saline solution (0.85% NaCl). Ten-fold serial dilutions were prepared using the same diluent, and aliquots (1.0 mL) were poured in triplicate onto 9 cm Petri dishes. Fifteen to twenty milliliters of Plate Count Agar (for total aerobic bacteria) and Potato Dextrose Agar (for molds and yeasts), tempered at 45–50 °C, were added and gently mixed. After solidification, plates were incubated at 30 °C for 72 h for total aerobic plate counts and at 25 °C for 5 days for molds and yeasts, as specified by the respective ISO standards. Microbial counts were expressed as log colony-forming units per gram (CFU g⁻¹) of sample. Enumeration of Enterobacteriaceae was performed according to ISO 21528-1 (ISO 2013) using red-bile-glucose agar, with plates incubated at 37 °C for 22–26 h.

Detection and enumeration of foodborne pathogenic bacteria

Foodborne bacterial pathogens evaluated included Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus cereus. Ten grams of each sample were homogenized in 90 mL of sterile buffered peptone water and then serially diluted. For P. aeruginosa, counts were performed using Pseudomonas selective agar medium according to ISO 13,720 (2010). A 0.1 mL sample of each dilution was spread on the surface of Pseudomonas agar medium, supplemented with the selective supplement SR0102, which contains cetrimide and nalidixic acid. Plates were incubated at 25 °C for 48 h. All colonies growing on the medium were counted. The Baird-Parker agar technique was used for S. aureus, according to ISO 6888-1 (ISO 2021), with samples incubated at 37 °C for 24–48 h; black colonies with clear halos were recognized as presumptive S. aureus. For B. cereus, diluted samples were plated onto Mannitol Egg Yolk Polymyxin agar in accordance with ISO 7932 (ISO 2004) and cultured at 30 °C for 24–48 h; pink colonies surrounded by a zone of precipitation were considered presumptive.

Radiation sensitivity pattern

The radiation sensitivity of bacterial pathogens isolated from Spirulina powder was calculated using the decimal reduction dose (D₁₀), defined as the absorbed γ-irradiation dose required to achieve a one-log (90%) reduction in the viable microbial population. The survival and resistance of the bacterial pathogens to γ-irradiation in liquid medium and in Spirulina powder were determined (Abd El-Al et al. 2022). For the liquid medium, a bacterial suspension of isolated pathogens was prepared by inoculating a single colony into sterile tryptic soy broth and incubating at 37 °C for 24 h. The resulting suspension, adjusted to an approximate concentration of 108 CFU/mL, was aliquoted into sterile tubes and exposed to γ-irradiation at doses ranging from 0 to 3 kGy. Following irradiation, the surviving bacterial populations were enumerated by plating appropriate dilutions onto selective media and incubating at optimal temperatures. For the substrate analysis, Spirulina powder samples were similarly inoculated with the same bacterial isolates, homogenized in sterile buffered peptone water, and subjected to the same irradiation doses. The surviving populations were also quantified through selective media plating. The D10-values for both liquid medium and substrate were calculated by plotting the log10 of the surviving bacterial counts against the irradiation dose and determining the slope of the resulting linear regression, allowing for the comparison of radiation sensitivity across different environments.

Spectroscopic analysesElectron spin resonance (ESR)

Electron spin resonance (ESR) signals corresponding to free radicals in dried Spirulina powder were recorded at ambient temperature using an X-band EMX spectrometer (Bruker, Germany) equipped with a standard rectangular cavity (ER 4102). The operating parameters applied during the measurements included microwave power (1.0 mW), modulation amplitude (8 G), modulation frequency (100 kHz), number of x-scans (1), x-axis resolution (1024 points), sweep width (4000 G), microwave frequency (9.71 GHz), time constant (81.92 ms), conversion time (19.7 ms), and sweep time (20.19 s). All parameters were optimized according to the sample characteristics to ensure reliable detection of paramagnetic species.

UV–Vis spectroscopy

UV–Vis spectra of Spirulina powder were recorded in phosphate buffer (pH 7) for phycobiliprotein and in methanol for chlorophylls and carotenoids, using a T60 spectrophotometer (PG Instruments, UK) over 200–800 nm.

Fourier transform infrared (FTIR) spectroscopy

Infrared spectra of the dried Spirulina powder were obtained using a BRUKER VERTEX 70 spectrophotometer (Germany) over the range of 400–4000 cm⁻¹ with a spectral resolution of 4 cm⁻¹. For analysis, samples were prepared as potassium bromide (KBr) pellets by mixing 1 mg of biomass with 100 mg of dry KBr.

Lipid peroxidation assay

Malondialdehyde (MDA) content was determined as an indicator of lipid peroxidation (Ohkawa et al. 1979). Control and irradiated biomass were homogenized in 50 mM potassium phosphate buffer (pH 7.5) and centrifuged at 4000 rpm for 15 min. About 0.2 mL supernatant was added to 1.0 mL chromogen solution (thiobarbituric acid, detergent, stabilizer), the mixture was heated in a boiling water bath for 30 min. Absorbance was measured at 354 nm, and MDA content was calculated as:

$$\:\text\text\text\:\left(\text\text\text\text\:}^\right)=\:\frac\ }}\:x\:100$$

Biomass pretreatment and extraction of high-value compoundsLipid and fatty acid methyl esters (FAME)

Lipids were extracted by the modified Bligh and Dyer method (1959). 0.5 g biomass was mixed with chloroform: methanol (1:1 v/v) and microwaved for 1 min. Water was added (final ratio 1:1:0.9 v/v/v). The lipid-containing chloroform layer was collected from the bottom of the separating funnel, rinsed with 5 mL of 5% NaCl, and dried to a constant weight in an oven at 60 °C. The lipid yield was expressed as mg/g dry biomass. Extracted lipids were transesterified following Christie’s method (1993). Fatty acid methyl esters (FAMEs) were analyzed on an HP 6890 gas chromatograph with a flame ionization detector (FID) at 300 °C and a BPX 70 capillary column (60.0 m × 0.32 mm × 0.25 μm). Air and hydrogen were supplied at flow rates of 400 and 35 mL/min, respectively. The oven temperature was adjusted to 120 °C for 1 min, then increased to 210 °C at 8 °C min⁻¹, 225 °C at 2 °C min⁻¹, and kept for 8 min. The injector was set to 250 °C with a split ratio of 20:1, and the carrier gas was nitrogen at 3.5 mL min⁻¹.

Protein

The protein extraction and determination were carried out according to Bradford (1976). Fifty milligrams of irradiated and non-irradiated Spirulina powder were resuspended in 5 mL of distilled water and sonicated at 10/10 s for 5 min on/off pulses at 4 °C with a frequency of 40 kHz. Then, 100 µL of each solution was pipetted into a test tube, and 5.0 mL of dye reagent was added, followed by a gentle vortexing. The samples were incubated for 10–30 min at room temperature, and the absorbance was measured at 595 nm using a V-200-RS spectrophotometer (LW Scientific, USA). Total protein (mg g⁻¹) = A₅₉₅/0.6021.

Carbohydrates

Total carbohydrates were determined by the phenol–sulfuric acid method (Pak and Simon 2004). In 20 mL screw tubes, 50 mg of each irradiated and non-irradiated Spirulina powder was resuspended in 10 mL of distilled water, 100 µL of 10% phenol solution (w/v) was added to the mixture, and it was thoroughly mixed. Subsequently, 50 µL of sulfuric acid (98%) was slowly poured into the side of the tube, followed by gentle blending, and the mixture was left standing for 10 min at room temperature in the dark. The mixture was then cooled to 25 °C in an ice bath and centrifuged at 4000 rpm for 10 min. The absorbance was measured at 485 nm using a V-200-RS spectrophotometer (LW Scientific, USA) against a reagent blank. A calibration curve was prepared under a similar set of conditions using standard solutions of D-glucose. Carbohydrates (mg g⁻¹) = (A₄₈₅ − 0.1499)/0.0024.

Chlorophylls and carotenoids

Chlorophyll a & b and total carotenoid contents were estimated via Lichtenthaler and Wellburn’s method (1983). Ten mL of 96% methanol was added to 1 g of irradiated and non-irradiated Spirulina powder, the mixture was well-homogenized at 1000 rpm for 1 min, and the residue was frequently re-extracted until the homogenate became colourless. The mixture was centrifuged at 4000 rpm for 10 min, and the supernatant was collected. The absorbance was recorded at 653, 666, and 470 nm using a V-200-RS spectrophotometer (LW Scientific, USA). The concentration of pigments was calculated using the equations below:

$$Chl\;a\;content\;(mg\;g^)=15.65\;\times\;A_-7.34\;\times\;A_$$

$$Chl\;b\;content\;(mg\;g^)=27.05\times A_-11.2\times A_$$

$$\beginTotal\;carotenoid\;content\;(mg\;g^)\\=\frac-(2.86\times A_)-(1.29\times A_)}\end$$

Where A666, A653, and A470 are the absorbances of Chl a, Chl b, and carotenoids at the mentioned wavelengths, respectively.

Phycobiliprotein

The phycobiliprotein were extracted according to Patel et al. (2005). 1 g of irradiated and non-irradiated Spirulina powder was suspended in 100 ml of 0.1 M sodium phosphate buffer (pH 7), and the mixture was centrifuged at 6000 rpm for 15 min at 4 °C. A clear supernatant layer containing phycobiliprotein was collected. The absorbance of allophycocyanin (APC), phycocyanin (PC), and phycoerythrin (PE) was determined spectrophotometrically (V-200-RS spectrophotometer, LW Scientific, USA) at wavelengths of 650, 620, and 560 nm, respectively. Contents of APC, PC, and PE were calculated via the following equations:

$$APC\;content\;(mg\;g^)=\frac-(0.208\times A_)}$$

$$PC\;content\;(mg\;g^)=\frac-(0.474\times A_)}$$

$$PE\;content\;(mg\;g^)=\frac-(2.41\times PC))-(0.849\times APC)}$$

Where A650, A620, and A560 are absorbances of APC, PC, and PE at the stated wavelengths, respectively.

Bioactive compounds

One gram of γ-irradiated Spirulina powder was extracted with 100 mL absolute ethanol. The extraction was repeated three times, and the combined supernatants were collected, filtered, and concentrated using a rotary vacuum evaporator at 40–45 °C. The yield of extractable compounds (crude extracts) was quantified and expressed as mg g⁻¹ dry weight (DW).

Antioxidant and phenolic assaysDPPH radical scavenging activity

The free radical scavenging activity of the ethanolic extracts was assessed using DPPH, as described by Yen and Chen (1995). Two mL of 0.16 mM DPPH in methanol was mixed with 2 mL of extract (200 µg mL⁻¹) and incubated in the dark at room temperature for 30 min. The absorbance was measured at 517 nm, and antioxidant activity was estimated as:

Antioxidant activity (%) = (Ac − At/Ac) * 100.

where At and Ac represent the absorbance of the samples and the DPPH control, respectively.

Total phenolic content

Using the technique outlined by Taga et al. (1984), the total phenolic content of the ethanolic extracts was calculated and expressed as gallic acid equivalents per gram of dry weight (mg GAE/g extract). Briefly, 500 µL of each sample was combined with 100 µL of 50% Folin–Ciocalteu reagent, followed by 2 mL of 2% Na₂CO₃, vortexed for 1 min, and then incubated for 30 min at room temperature. At 720 nm, the absorbance was then measured.

Statistical analysis

All experiments were performed as triplicates. One-way ANOVA was used to evaluate significant differences between variables at a 95% confidence level (p < 0.05). Tukey’s test was then used to identify pairwise differences between treatment levels. Minitab software (version 18, Minitab Inc., USA) was used to perform statistical analyses.