Microbial decontamination and enhanced extraction of value-added compounds from Spirulina platensis using gamma radiation
Hamdy Elsayed Ahmed Ali, Mohamed S. Abd El-AL, Marwa M. Moussa, Ola M. Gomaa, Kamal A. Hassan, Eman A. El-fayoumy
TL;DR
Gamma radiation effectively decontaminates Spirulina and boosts the extraction of valuable compounds like proteins and pigments.
Contribution
Gamma irradiation is shown to both decontaminate Spirulina and enhance the recovery of bioactive compounds.
Findings
Gamma irradiation at 6 kGy effectively decontaminates Spirulina biomass.
Irradiation at 6 kGy maximizes the extraction of lipids, proteins, and pigments.
Antioxidant activity peaks at 6 kGy, linked to increased phenolic content.
Abstract
Spirulina platensis has garnered increasing attention as a valuable source of proteins, lipids, and pigments, with applications in the food, feed, and nutraceutical industries. However, large-scale cultivation in open pond systems is liable to microbial contamination, posing risks to product quality and productivity. This study evaluated the effects of gamma (γ) irradiation up to 10 kilogray (kGy) as a dual strategy for microbial decontamination and enhanced extraction of bioactive compounds from dried Spirulina powder. A dose of 4 kGy resulted in a substantial reduction in microbial populations, while irradiation at 6 kGy reduced microbial counts to levels below the detection limit of the applied culture-based methods, indicating effective decontamination. The D₁₀-values obtained for Bacillus sp. (0.44 kGy), Staphylococcus aureus (0.41 kGy), and Pseudomonas aeruginosa (0.58 kGy)…
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Figure 9- —Egyptian Atomic Energy Authority
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Taxonomy
TopicsRadiation Effects and Dosimetry · Tannin, Tannase and Anticancer Activities · Chromatography in Natural Products
Introduction
The global population is expected to reach 9.8 billion by 2050, driving a projected increase of more than 50% in food demand (Searchinger et al. 2019). In response, plant-based diets have been promoted as sustainable alternatives to meat-based diets, offering benefits for public health, food security, and environmental sustainability. However, vital and sustainable supplements are required to bridge the gap between meat-based and plant-based diets. Microalgae are considered a perfect resource among plant-based resources. They have attracted considerable attention for their diverse applications in the food, feed, and nutraceutical industries (Sun et al. 2024). Spirulina platensis, in particular, is widely utilized as a functional ingredient and is considered a promising protein source for human consumption, livestock production, and aquaculture. Its biochemical composition—comprising 60–70% protein, 20–30% carbohydrates, 5–15% lipid, and essential vitamins—supports its use in high-value nutritional products (Withana et al. 2025). The presence of phycocyanin further enhances its commercial importance due to its antioxidant, antimicrobial, and anti-inflammatory properties (Bumandalai et al. 2024).
Large-scale production of Spirulina is predominantly conducted in open raceway ponds, where paddle wheels facilitate continuous mixing and aeration (Raeisossadati et al. 2019). Although they are considered cost-effective and energy-efficient compared with closed photobioreactors, open pond systems are highly vulnerable to contamination by bacteria, protozoa, and other predators. Such contamination compromises product safety, reduces biomass yield, and limits overall production efficiency (Wang et al. 2013; Molina et al. 2019). Ensuring microbial quality in Spirulina products is therefore essential to meet international food safety standards (Bumandalai et al. 2024).
Traditional decontamination methods—such as blanching, pasteurization, microwaving, and thermal processing— are commonly used to control microbial loads. However, these methods often degrade heat-sensitive biomolecules, including proteins, vitamins, and pigments such as phycocyanin, while also altering sensory properties such as flavor, texture, and color (Stramarkou et al. 2021). Furthermore, powdered products like Spirulina exhibit low water activity, which restricts heat transfer and reduces the effectiveness of conventional thermal treatments, allowing heat-resistant microorganisms to survive (Liu et al. 2022).
Gamma (γ) irradiation has been used for food decontamination since the 1960 s and is now regarded as one of the safest, most efficient, and environmentally friendly technologies for improving food microbiological quality (Ognyanov et al. 2022). International guidelines recommend that food irradiation doses not exceed 10 kGy (CODEX STAN). Despite its proven safety, the use of γ-irradiation for microbial decontamination of Spirulina powder remains insufficiently explored. This is largely due to concerns regarding possible irradiation-induced degradation of sensitive biomolecules, uncertainties related to oxidative and structural changes in the dry biomass matrix, and the lack of integrated studies simultaneously assessing microbial safety alongside the preservation of functional and nutritional quality. Although γ-irradiation has demonstrated effectiveness in sterilizing low-density herbal materials such as powders and decoction syrups, its impact on bioactive components is strongly dose- and species-dependent. Several studies report that optimized γ-irradiation doses can preserve or even enhance herbal quality; for example, recommended irradiation levels preserved active compound content and antioxidant activity in Cnidii Rhizoma and Alismatis Rhizoma (Baek et al. 2019), while turmeric powder also showed no significant degradation following irradiation (Aghamohseni et al. 2025). In contrast, other reports indicate that irradiation at 7 kGy, although effective in controlling microbial contamination in Shengmai Yin preparations, significantly reduced schisandrin content and altered chromatic properties (Wang et al. 2022). Therefore, establishing species-specific irradiation tolerance thresholds and understanding component-specific response patterns are essential prerequisites for optimizing γ-irradiation treatments, particularly for nutritionally and biochemically complex matrices such as S. platensis.
The rigid peptidoglycan-based cell wall of S. platensis provides substantial resistance to environmental stress but also makes the extraction of intracellular bioactive compounds challenging (Vladimirescu 2010). Effective cell disruption is therefore essential for improving extraction efficiency by breaking down cell walls and membranes to release valuable biomolecules (Rahman et al. 2022). γ-irradiation is a highly penetrative form of electromagnetic radiation capable of inducing polysaccharide degradation, depolymerization, and partial disruption of algal cell walls, making it a promising pretreatment strategy (Correa et al. 2021). Compared with mechanical or chemical disruption methods, γ-irradiation offers advantages including low temperature, short reaction time, minimal chemical use, and reduced environmental impact (Foteinis et al. 2018). Previous studies have shown that moderate γ doses can enhance the extraction of valuable compounds without compromising product quality. For example, γ-irradiation has been found to improve lipid extraction from Haematococcus pluvialis while preserving astaxanthin content (de Moraes et al. 2023).
To the best of our knowledge, this is the first study to evaluate γ-irradiation as a dual approach for microbial decontamination and cell disruption of S. platensis powder cultivated in raceway ponds, intending to enhance the extraction of high-value biomolecules. This study assessed the impact of γ-irradiation on microbial reduction, physicochemical properties, and the recovery of protein, carbohydrates, lipid, pigments, and bioactive compounds for nutraceutical and biotechnological applications.
Materials and methods
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 powderBiomass compositionConcentrationUnitProtein49.01%Carbohydrates27.98%Lipid4.71%Fiber3.87%Ash5.46%Moisture8.97%
γ-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 10^8^ 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 D_10_-values for both liquid medium and substrate were calculated by plotting the log_10_ 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 analyses
Electron 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:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:\mathrm{M}\mathrm{D}\mathrm{A}\:\left(\mathrm{n}\mathrm{m}\mathrm{o}\mathrm{l}\:{\mathrm{g}}^{-1}\right)=\:\frac{A\text{}\ {sample}}{A\:\mathrm{standard}}\:x\:100$$\end{document}Biomass pretreatment and extraction of high-value compounds
Lipid 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:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Chl\;a\;content\;(mg\;g^{-1})=15.65\;\times\;A_{666}-7.34\;\times\;A_{653}$$\end{document} \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Chl\;b\;content\;(mg\;g^{-1})=27.05\times A_{653}-11.2\times A_{666}$$\end{document} \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{c}Total\;carotenoid\;content\;(mg\;g^{-1})\\=\frac{1000\times A_{470}-(2.86\times A_{666})-(1.29\times A_{653})}{245}\end{array}$$\end{document}Where A_666_, A_653_, and A_470_ 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:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$APC\;content\;(mg\;g^{-1})=\frac{A_{650}-(0.208\times A_{620})}{5.09}$$\end{document} \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$PC\;content\;(mg\;g^{-1})=\frac{A_{620}-(0.474\times A_{650})}{5.34}$$\end{document} \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$PE\;content\;(mg\;g^{-1})=\frac{A_{650}-(2.41\times PC))-(0.849\times APC)}{9.62}$$\end{document}Where A_650_, A_620_, and A_560_ 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 assays
DPPH 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.
Results
Microbial decontamination
Although microscopic examination is commonly used for preliminary contamination screening during Spirulina cultivation, this study focused on culture-based microbiological analyses of the final dried product, as post-harvest steps such as drying, handling, and packaging may introduce cross-contamination and require quantitative assessment of viable microorganisms. Therefore, the impact of γ-irradiation on the microbial quality of Spirulina powder was evaluated by measuring total bacterial, mold, yeast, and Enterobacteriaceae counts (Table 2). Non-irradiated samples (0 kGy) exhibited high microbial levels: total bacteria (4.18 ± 0.55 log CFU g⁻¹), molds and yeasts (4.85 ± 0.45 log CFU g⁻¹), and Enterobacteriaceae (3.30 ± 0.58 log CFU g⁻¹). Irradiation at 2 kGy resulted in slight decreases in bacterial and fungal counts (4.11 ± 0.44 and 4.71 ± 0.36 log CFU g⁻¹, respectively), while Enterobacteriaceae were reduced to 2.00 ± 0.67 log CFU g⁻¹. At 4 kGy, total bacteria decreased by approximately 2 logs (2.30 log CFU g⁻¹), and Enterobacteriaceae approached < 1 log CFU g⁻¹. Complete inactivation (< 1 log CFU g⁻¹) of all microbial groups was achieved at 6 kGy and maintained at higher doses.
Table 2. Effect of γ-irradiation on microbial load (log CFU/g) of S. platensis powderIrradiation Dose (kGy)Microbiological analysesTotal bacterial count (CFU/g)Total molds and yeasts count (CFU/g)Enterobacteriaceae (CFU/g)04.18 ± 0.554.85 ± 0.453.30 ± 0.5824.11 ± 0.444.71 ± 0.362.0 ± 0.6742.30 ± 0.653.17 ± 0.84< 16< 1< 1< 18< 1< 1< 110< 1< 1< 1
The impact of γ-irradiation on foodborne pathogenic bacteria in Spirulina powder is presented in Fig. 1. In non-irradiated samples, S. aureus recorded the highest count (2.70 log CFU/g), followed by P. aeruginosa (2.30 log CFU/g), while B. cereus was absent. Irradiation at 4 kGy reduced pathogen counts markedly, although S. aureus remained detectable. Significant reduction of all tested pathogens was observed at 6 kGy.Fig. 1. Effect of γ-irradiation on pathogenic bacterial levels in S. platensis powder (heatmap analysis)
Three bacterial groups were isolated from Spirulina powder, namely P. aeruginosa, S. aureus, and members of the genus Bacillus. Colonies presumptive for the Bacillus cereus group were not detected on Mannitol Egg Yolk Polymyxin (MYP) agar; therefore, Bacillus sp. were reported in the present study. Radiation sensitivity (D_10_-values) of pathogenic bacteria was further assessed in both tryptone soya broth (TSB) and Spirulina powder (Fig. 2). Initial bacterial count ranged between 8.48 log CFU/mL in TSB and 8.70 log CFU/g in Spirulina powder. Bacillus sp. exhibited D_10_ values of 0.50 kGy in Spirulina powder and 0.44 kGy in TSB. P. aeruginosa revealed D_10_-values of 0.39 kGy in Spirulina powder and 0.36 kGy in TSB. S. aureus showed higher sensitivity in broth (0.41 kGy) compared to Spirulina powder (0.46 kGy).Fig. 2. Radiation sensitivity patterns of pathogenic bacteria in tryptone soya broth and S. platensis powder: (A) Bacillus sp., (B) Pseudomonas aeruginosa, (C) Staphylococcus aureus
Spectroscopic analyses and lipid peroxidation
ESR analysis revealed a dose-dependent increase in radical signal intensity in Spirulina powder following γ-irradiation (0–10 kGy), indicating the accumulation of stable radiation-induced radicals within the dry biomass matrix (Fig. 3). The control (0 kGy) exhibited only a weak baseline signal, whereas irradiation induced a progressively stronger central resonance, detectable from 2 kGy and further increasing with doses up to 10 kGy. UV–Vis spectra of carotenoids, chlorophylls, and phycobiliproteins revealed no significant alterations after γ-irradiation, confirming that pigment structural stability was maintained (Fig. 4).Fig. 3ESR spectra of S. platensis powder exposed to different γ-irradiation dosesFig. 4UV–Vis spectra of S. platensis powder exposed to different γ-irradiation doses: (A) chlorophyll a and carotenoids, (B) phycobiliprotein
FTIR spectra of irradiated and control Spirulina biomass showed dose-dependent changes in several characteristic absorption bands (Fig. 5). The broad band at 3300–3400 cm⁻¹, assigned to O–H and N–H stretching vibrations, gradually decreased in intensity and was no longer detected at irradiation doses ≥ 4 kGy. Bands at 2925–2850 cm⁻¹, corresponding to aliphatic C–H stretching vibrations, showed only minor intensity changes across the irradiation doses. The amide I band (~ 1650 cm⁻¹) and amide II band (~ 1540 cm⁻¹) exhibited slight peak shifts and intensity variations after irradiation. Additionally, changes in band intensity were observed in the polysaccharide-associated region between 1150 and 1000 cm⁻¹ with increasing irradiation dose. Detailed FTIR band assignments and peak shifts are provided in Table S1. Exposure of Spirulina powder to γ-irradiation significantly affected lipid peroxidation. A notable decrease (p < 0.05) in MDA content was observed at 6 kGy (57.15 ± 1.08 nmol g⁻¹ dried biomass) compared with the control (0 kGy; 125.61 ± 2.23 nmol g⁻¹) (Fig. 6).Fig. 5FTIR spectra of *S. platensis *powder exposed to different γ-irradiation doses, showing major functional groups associated with proteins, lipids, and polysaccharidesFig. 6Effect of γ-irradiation on malondialdehyde (MDA) content in S. platensis powder. Results represent mean ± standard deviation of three replicates. Different small letters indicate a significant difference (*p < *0.05)
FTIR revealed minor shifts in the C–H and carbohydrate absorption bands, as well as the disappearance of the O–H stretching band (3300–4000 cm⁻¹) at irradiation doses ≥ 4 kGy. These changes indicate structural rearrangements within polysaccharides and hydroxyl-containing biomolecules (Fig. 5).
High-value compounds recovery
As illustrated in Fig. 7, exposure of Spirulina powder to γ-irradiation significantly influenced the yield of major macromolecules. The maximum levels of lipid (60.83 ± 1.38 mg g⁻¹), protein (547.43 ± 2.97 mg g⁻¹), and carbohydrates (275.6 ± 2.5 mg g⁻¹) were obtained at 6 kGy. γ-Irradiation had no significant effect on the ratios of saturated fatty acids (SFAs) and unsaturated fatty acids (UFAs) (Table 3). The fatty acid profiles showed only minor variations compared with the control. Pretreatment with 10 kGy resulted in higher concentrations of palmitic acid (C16:0), linoleic acid (C18:2), and linolenic acid (C18:3), while oleic acid (C18:1) remained unchanged. Stearic acid (C18:0) was absent in all irradiation treatments. In addition, lauric acid (C10:0) decreased by about 50% at all doses except 4 kGy relative to the control.Fig. 7. Effect of γ-irradiation on extraction yields of (A) lipid, (B) protein, and (C) carbohydrates from S. platensis powder. Results represent mean ± standard deviation of three replicates. Different small letters indicate a significant difference (*p < *0.05)
Table 3. Fatty acid profiles of lipid extracted from S. platensis powder exposed to different γ-irradiation dosesFatty acidArea (%)γ–irradiation dose (kGy)0246810Lauric acid (C10:0)4.402.894.083.522.272.32Myristic acid (C14:0)2.562.812.722.632.84ndPalmitic acid (C16:0)35.5436.0235.8035.8734.8137.19Palmitoleic acid (C16:1)6.246.356.276.326.216.53Stearic acid (C18:0)1.15ndndndndndOleic acid (C18:1)8.038.828.378.688.528.81Linoleic acid (C18:2)17.6818.9918.6518.8718.3519.08Linolenic acid (C18:3)16.2916.7116.6316.7116.8817.52SFAs43.6541.7242.642.0239.9239.51USFAs48.2450.8749.9250.5849.9651.94PUFAs33.9735.735.2835.5835.2336.6*nd: *not detected
Pigment extraction efficiency was also enhanced by γ-irradiation. The highest concentrations of chlorophylls (11.40 ± 0.1 mg g⁻¹), carotenoids (3.48 ± 0.3 mg g⁻¹), and phycobiliprotein (125.20 ± 0.7 mg g⁻¹) were observed at 6 kGy (Fig. 8). Moreover, γ-irradiation significantly (p < 0.05) enhanced the antioxidant activity and phenolic content of Spirulina extracts, with peak values observed at 6 kGy (69.61 ± 1.47% and 29.88 ± 0.49 mg GAE/g extract, respectively; Fig. 9).Fig. 8. Effect of γ-irradiation on extraction yields of (A) chlorophyll a, (B) carotenoids, and (C) phycobiliprotein from S. platensis powder. Results represent mean ± standard deviation of three replicates. Different small letters indicate a significant difference (*p < *0.05)Fig. 9. Antioxidant activity (%) and phenolic content (mg GAE/g extract) of ethanolic extracts of S. platensis powder treated with different γ-irradiation doses. Results represent mean ± standard deviation of three replicates. Different small letters indicate a significant difference (*p < *0.05)
Discussion
Role of γ-irradiation in Spirulina decontamination and quality preservation
The high microbial loads observed in untreated Spirulina powder indicate potential safety concerns, consistent with reports that microalgal products are prone to contamination due to cultivation and handling practices (Wang et al. 2013). The significant reductions in total aerobic bacterial counts, molds, yeasts, and Enterobacteriaceae demonstrate its effectiveness as a microbial decontamination strategy. At irradiation doses of 6 kGy and above, counts of bacteria, molds, yeasts, and Enterobacteriaceae were reduced to levels below the detection limit of the applied culture-based methods (< 1 log CFU g⁻¹), indicating effective microbial control and microbiological safety of Spirulina powder. Comparable dose-dependent decreases in microbial populations following γ-irradiation of Spirulina-based products were reported, with higher irradiation doses significantly suppressing bacterial populations and delaying regrowth during storage (Tang et al. 2015). Similarly, effective microbial decontamination at 6 kGy has been documented in algal and plant powders (Armah et al. 2023).
Pathogen-specific responses further highlight the effectiveness of γ-irradiation. The predominant contaminants, S. aureus and P. aeruginosa, showed progressive dose-dependent reductions, with counts falling below the detection limit at irradiation doses of 6 kGy. The absence of B. cereus aligns with its variable prevalence in food products. Importantly, the presence of S. aureus at initial levels is of concern due to its toxin-producing capability (Maia et al. 2020), but irradiation effectively controlled this risk. These findings are consistent with earlier work showing the ability of γ-irradiation to suppress both spoilage and pathogenic microorganisms in food systems (Robichaud et al. 2021).
The D_10_-values obtained reveal species-specific radiation resistance patterns. Bacillus sp. showed moderate resistance, reflecting the protective nature of the nutrient-rich Spirulina matrix. By contrast, S. aureus and P. aeruginosa were more sensitive, particularly in broth, which may reflect the absence of protective compounds. These variations are consistent with previous findings that microbial resistance to irradiation depends on intrinsic species characteristics, food composition, and irradiation conditions (Abd El-Al et al. 2022).
Spectroscopic findings and oxidative stability
The ESR findings demonstrate that γ-irradiation induces a clear accumulation of stable radicals in Spirulina powder, with the highest signal intensity observed at 10 kGy. However, this increase in radical signals did not correspond to enhanced oxidative damage, as lipid peroxidation decreased significantly at irradiation doses ranging from 4 to 10 kGy (Fig. 6). This indicates that radical formation within the dry biomass matrix does not necessarily lead to oxidative stress, possibly due to the protective effects of endogenous antioxidant compounds.
Consistent with this observation, UV–Vis spectra confirmed pigment stability, with carotenoids, chlorophylls, and phycobiliproteins remaining intact after irradiation. These findings agree with earlier reports showing only minor spectral shifts in carotenoids and chlorophyll (420–440 nm) and phycocyanin (671 nm) following irradiation (Brasoveanu et al. 2005). The low water content in dried Spirulina biomass may further limit indirect radiation-induced damage by reducing free radical formation (Osaili et al. 2008).
FTIR analysis revealed subtle structural changes, particularly the disappearance of O–H stretching bands at irradiation doses ≥ 4 kGy, indicating modifications in hydroxyl-containing biomolecules such as polysaccharides. These molecular rearrangements may contribute to enhanced biomolecule accessibility and are associated with the observed improvements in antioxidant activity. Hamed et al. (2025) reported that gamma irradiation induces chain scission and cross-linking in polysaccharides, resulting in structural modifications detectable by FTIR, including changes in hydroxyl and glycosidic functional groups. Such molecular alterations are closely associated with modifications in physicochemical properties and enhanced biological activities, particularly antioxidant capacity.
Together, the spectroscopic evidence indicates that γ-irradiation of Spirulina powder leads to stable radical accumulation and minor structural modifications without compromising pigment integrity. Instead, irradiation appears to promote structural adaptations that preserve bioactive compounds and limit oxidative damage.
Despite the increase in free radical formation detected by ESR, the reduction in MDA levels at 6 kGy indicates that γ-irradiation does not result in elevated oxidative stress in dried Spirulina biomass. Instead, moderate irradiation appears to activate protective mechanisms that stabilize lipids. This effect may be attributed to the antioxidant activity of carotenoids, phenolic compounds, and other bioactive molecules, which can scavenge free radicals and interrupt lipid peroxidation pathways (Dobosz et al. 2022).
Mechanistically, γ-irradiation can depolymerize bound phenolics and flavonoids, increasing their extractable forms and enhancing radical-scavenging capacity, for example, through hydrogen donation to peroxyl radicals (Amiri et al. 2023). The observed increases in phenolic content and antioxidant activity are consistent with earlier reports on irradiated plant materials such as fennel seeds and soybean, where bound phenolics were released through radiation-induced cleavage of hydrogen bonds and glycosidic linkages (Jameel and Mohammed 2021; Ahmed and Hassan 2023). These newly released phenolics expand the antioxidant pool, thereby reducing oxidative damage.
FTIR data further support this interpretation by demonstrating structural modifications in polysaccharides and hydroxyl-rich compounds following irradiation, potentially increasing antioxidant extractability and facilitating radical immobilization (Helal et al. 2023). Collectively, ESR, MDA, FTIR, phenolic, and carotenoid data suggest that γ-irradiation at 6 kGy induces biochemical and structural adaptations that enhance antioxidant defenses and preserve both pigment and lipid stability.
Impact of γ-irradiation on high-value compound extraction
Enhanced extraction of lipid, protein, carbohydrates, and pigments at moderate irradiation doses suggests that γ-irradiation improves accessibility of cellular components. This stimulatory effect aligns with previous reports in cyanobacteria; for example, Moisescu et al. (2019) observed up to a 1.16-fold increase in chlorophyll a and carotenoid content in Synechocystis PCC 6803 at 0.1 kGy. The underlying mechanism likely involves the physicochemical effects of γ-rays, which possess high energy and strong penetration. Interaction with residual water generates reactive free radicals that can attack and modify macromolecules, including polysaccharides and other cell wall constituents (Blanco et al. 2018). These radicals can cleave covalent bonds, weakening the rigid cell wall and creating cracks and pores, thereby loosening the structure (Sofi et al. 2024; Jeong et al. 2024). Such modifications enhance solvent permeability, facilitating the release and extraction of intracellular biomolecules, including pigments, proteins, and lipids, and contributing to increased extraction yields. Although γ-irradiation enhanced lipid and protein yields, the fatty acid profile remained largely stable, with only minor variations observed in the relative ratio of individual fatty acids, including palmitic, linoleic, and linolenic acids, indicating limited radiation-induced modification of lipid composition. Oleic acid content remained unchanged, while stearic acid was not detected, and lauric acid showed a reduction at most irradiation doses. The absence of stearic acid and the decrease in lauric acid suggest that certain fatty acids may exhibit higher sensitivity to radiation-induced alterations.
The significant enhancement of antioxidant activity and phenolic content observed at 6 kGy suggests that moderate γ-irradiation can stimulate the release of bioactive compounds in Spirulina. Although studies directly addressing γ-irradiated Spirulina are limited, comparable responses have been reported in closely related cyanobacteria and microalgal systems. For instance, moderate γ-irradiation significantly enhanced total antioxidant activity and polyphenolic content in several microalgae, with Scenedesmus obliquus showing the highest response at 300 Gy, followed by Arthrospira platensis at 700 Gy and Chlorella vulgaris at 200 Gy (Helal et al. 2023). Similar dose-dependent increases in antioxidant activity and phenolic compounds have also been reported in γ-irradiated fennel seeds and soybean, indicating a conserved stress-induced response across photosynthetic organisms (Ahmed and Hassan 2023; Štajner et al. 2007). The positive correlation between phenolic content and antioxidant activity observed in the present study is consistent with earlier findings in Spirulina-based systems (Madkour et al. 2024).
The present study focuses on two main goals: (1) to address the significant gap in the literature on gamma irradiation and *Spirulina *powder, and (2) to simultaneously decontaminate *Spirulina *powder and enhance the extraction of high-value bioactive compounds. Most previous studies focused on living or freshly harvested biomass, examining physiological responses, growth, pigment composition, or antioxidant activity, but did not assess post-harvest microbial safety or sterilization of dry Spirulina powder, which is essential for food, nutraceutical, and biotechnological applications. To date, only one study has applied gamma irradiation for the sterilization of Spirulina-derived powder (Tang et al. 2015). That work was limited to total bacterial count, did not evaluate pathogenic microorganisms, and focused mainly on protein integrity, neglecting lipids, fatty acids, and other bioactive compounds. No spectroscopic analyses (FTIR, UV–Vis) or radical-based techniques were used to assess structural or functional changes. In contrast, this study is the first comprehensive investigation demonstrating the dual functionality of gamma irradiation on dry Spirulina powder by providing effective sterilization, supported by extended microbiological evaluation, and enhancing the extraction of high-value bioactive compounds. The use of FTIR, ESR, UV–Vis spectroscopy, and lipid peroxidation analysis delivers molecular- and functional-level insight into irradiation-induced changes across multiple biochemical constituents. This integrated approach distinguishes the present work and establishes gamma irradiation as a multifunctional technology for improving both microbial safety and bioactive compound recovery from *Spirulina *powder.
Conclusion
The present study uniquely demonstrates the application of γ-irradiation as a dual-purpose strategy for ensuring microbial safety and enhancing the extraction of high-value biomolecules from S. platensis powder. Moderate doses of γ-irradiation (6 kGy) reduced microbial loads to levels below the detection limit of the applied culture-based methods, with species-specific radiosensitivity observed among the tested bacteria. Spectroscopic analyses confirmed the preservation of biomolecular structures at doses up to 10 kGy, while irradiation at 6 kGy enhanced the extraction of high-value compounds and increased antioxidant activity. The fatty acid profiles of extracted lipids exhibited only minor variations across the applied γ-irradiation doses compared to the control. Overall, γ-irradiation provides a safe, efficient, and biotechnologically viable approach for producing microbiologically stable Spirulina biomass with enhanced functional properties, supporting its application in food, nutraceutical, and pharmaceutical industries.
Supplementary Information
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The reference list from the paper itself. Each links out to its DOI / PubMed record.
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