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. 2025 Jul 2;15:23435. doi: 10.1038/s41598-025-08136-6

The impact of gamma and electron beam irradiation sterilization technology on the pigments and phenolic components of saffron

Mohammad Reshad 1, Seyed Pezhman Shirmardi 2, Hosein Afarideh 1, Samira Shahbazi 2, Hamed Askari 2,✉
PMCID: PMC12223047  PMID: 40604031

Abstract

Dried stigmas of saffron with the natural contaminant microflora were irradiated at five dose levels (1, 2, 3, 4 and 5 kGy) via a gamma and electron beam irradiator. The changes in the microbial population (total count, mold and yeast and coliform contamination), total phenolic content (TPC), antioxidant activity (AOA), bitterness, aromatic, and coloring strengths of saffron after radiation were investigated. Additionally, phenolic compounds and carotene glycosides were assayed via high–performance liquid chromatography (HPLC). Irradiation effectively eliminated microbial populations, with doses above 2 kGy resulting in no detectable fungi or bacteria. Additionally, radiation treatments had little effect on the crocin (color), picrocrocin (bitterness) and safranal (aroma) indices measured by the ISO 3632 procedure. On the other hand, the HPLC results revealed that major carotenoids (Picrocrocin, trans-4-GG-crocin, trans-3-Gg-crocin, cis-4-GG-crocin, trans-2-gg-crocin, trans-crocetin, and safranal) and phenolic compounds (catechol, salicylic acid, p-hydroxybenzoic acid, vanillin, gentisic acid, gallic acid, caffeic acid, syringic acid, ferulic acid, and p-coumaric acid) in saffron can experience undesirable changes in attributes due to high doses of radiation. Specifically, a gamma dose of 1 kGy and an electron beam dose of 2 kGy were found to be optimal for reducing the microbial load, with minimal damage to color, flavor, and phenolic and antioxidant compounds. Additionally, a high dose of radiation has a profound effect on the stability and concentration of phenolic compounds in saffron, potentially altering its antioxidant properties and overall quality. This study highlighted the effectiveness of low doses of gamma and electron beam irradiation in eliminating microbial loads and enhancing product quality.

Keywords: Saffron, Gamma radiation, Electron beam, Total phenolic content, Microbial quality, Apocarotenoids

Subject terms: Nuclear energy, Materials science

Introduction

Saffron (Crocus sativus L.) is a highly valued spice known for its unique flavor, color, and numerous health benefits1, primarily attributed to its rich phytochemical profile2. The spice contains bioactive compounds such as crocins, safranal, and carotenoids, which contribute to its therapeutic properties, including antioxidant, anti-inflammatory, and anticancer effects3–5. Ensuring the microbiological safety of saffron during processing and storage is crucial for maintaining its quality and health benefits1.

Saffron preservation through nonthermal methods has gained traction because of the need to maintain its quality while ensuring safety from microbial contamination. Various innovative techniques have been explored, including cold plasma treatment6, irradiation (gamma and electron beam), high-pressure processing, pulsed electric fields7, and supercritical carbon dioxide, which effectively reduce microbial loads while preserving the biochemical attributes of saffron. While nonthermal methods show promise, traditional thermal treatments, such as drying at 25–30 °C, remain widely used because of their simplicity and established effectiveness, despite their potential for quality degradation8,9. In contrast, all novel decontamination methods may not be universally applicable across all saffron varieties or production contexts, necessitating further research to optimize protocols for diverse conditions, and challenges remain in optimizing these techniques for large-scale applications and ensuring cost-effectiveness in commercial settings. Irradiation is a critical method for maintaining the quality of saffron, particularly its flavor, aroma, and color. Research indicates that irradiation can effectively decontaminate saffron while preserving its essential characteristics10, thereby extending the shelf-life of saffron without significantly altering its sensory and chemical characteristics during storage11. Gamma radiation is generally more effective at inactivating foodborne pathogens. For example, the D10 values for various bacteria exposed to gamma radiation were lower than those for e-beams, indicating a greater sensitivity of microbes to gamma rays. In studies, gamma radiation effectively reduced bacterial counts to undetectable levels at doses of approximately 4.0 kGy12. Both radiation types exhibit similar microbial lethality when the same dose is applied, regardless of the energy level or dose rate, suggesting that the total dose is the critical factor for microbial inactivation13. Studies indicate that while gamma radiation may cause more DNA damage and mutations in some strains, e-beam radiation tends to result in fewer genetic alterations14. In contrast, some researchers argue that the choice between gamma and e-beam radiation should consider factors such as cost, equipment availability, and specific microbial targets rather than solely focusing on efficacy. This perspective emphasizes the importance of context in selecting the appropriate sterilization method. However, certain doses may lead to changes in the chemical composition, particularly in the pigments responsible for saffron color and aroma15. The effects of radiation on the antioxidant properties of saffron have been explored in various studies, revealing nuanced interactions between radiation exposure and the bioactive compounds present in saffron, which include crocin, crocetin, picrocrocin, and safranal16,17. While some research indicates that irradiation does not significantly alter the chemical composition of saffron18, its impact on antioxidant properties remains a subject of interest. Research indicates that gamma irradiation does not significantly alter the sensory qualities or chemical characteristics of saffron at lower doses (up to 2.0 kGy)10,11,15. E-beam irradiation also preserves nutritional and antioxidant properties over extended storage periods19. While irradiation at lower doses (up to 2.0 kGy) does not significantly affect sensory quality, higher doses (5 kGy) may lead to slight deterioration in aroma and a decrease in glucosides, which are vital for saffron color15. The antioxidant properties of saffron remain largely intact, although some degradation of pigments may occur15.

While irradiation preserves key flavor components, excessive doses can diminish color strength, indicating a need for careful dose management8. Zareena et al.15 reported that a dose of 5 kGy may lead to slight quality deterioration in aroma and color but is effective for microbial decontamination. Additionally, Sales et al.18 reported that irradiation at 2 kGy enhances flavor and aroma without significantly degrading chemical properties. Irradiation at doses above 10 kGy can decrease the color strength (crocin) of saffron. Key components such as picrocrocin (bitterness) and safranal (aroma) remain largely unaffected at lower doses8. While irradiation is beneficial for microbial safety, excessive doses can compromise saffron quality. Thus, careful dose selection is essential for maintaining its prize attributes. Conversely, while irradiation offers numerous benefits, there are concerns regarding the potential degradation of certain sensitive compounds at higher doses, which necessitates ongoing research to optimize irradiation protocols for saffron preservation. Thus, the main objective of this research is to study the microbial and chemical changes caused by gamma- and electron beam irradiation of saffron, which are commercially used for packaging irradiated spices in Iran.

Materials and methods

Saffron Preparation

Commercial samples of saffron (Crocus sativus L.) stigmata were obtained from Ghaen, Khorasan Province (Novin Zaferan ™). The samples were divided into equal parts and then packed in sterile petri dishes. (25 g per package). The samples were divided into 2 groups. One lot was kept as the nonirradiated control sample, and the other lot was subjected to γ-irradiation and electron beam radiation.

Irradiation

The irradiation process was carried out via two gamma irradiation (GMI) and electron beam irradiation (EBI) techniques. GMI treatment was performed with a cobalt-60 irradiator (Gammacell 220, MDS Nordion, Ottawa, Canada) at an exposure rate of 5.4 kGy/h at the Radiation Applications Research School, Nuclear Science and Technology Research Institute (NSTRI), AEOI, Tehran, Iran. EBI was performed via an electron accelerator machine with a beam energy of 10 MeV, a maximum beam current of 3 mA, and an effective beam width of 25 mm in Taft, Yazd Province, Iran, affiliated with the Atomic Energy Organization of Iran (AEOI). All the samples were exposed to GMI and EBI doses of 0, 1, 2, 3, 4, and 5 kGy in triplicate. Dosimetry was performed via a Red 4034 Perspex dosimeter (Harwell Technologies, UK)20.

Microbial analysis

After irradiation, serial dilutions of untreated and treated saffron samples were prepared in saline solution (tenfold serial dilutions), and the total count of microbial contamination was determined by culturing each sample on nutrient agar media. Additionally, the total count of coliform contamination was determined by culturing each sample on violet red bile (VRB) agar media. All media were incubated at 30 °C for 1–2 days. To count the total yeast and fungal populations, 100 µL of the prepared dilutions were inoculated onto the surface of potato dextrose agar (PDA) culture media supplemented with 50 ppm chloramphenicol and incubated at 25 °C for 3–5 days. After incubation, colonies were counted and reported as colony-forming units per gram (log CFU g−1).

Chemical characteristics of saffron-ISO/TS 3632-2:2003 analysis

First, the samples of saffron were powdered under aseptic conditions using a sterile porcelain mortar and then used for other experiments. The main characteristics of saffron were determined in terms of picrocrocin, safranal and crocin contents via a UV/VIS spectrophotometer (Genway 6715 UV/Vis, USA). By direct reading of the absorbance, the bitterness (Inline graphic257 nm, maximum absorbance of picrocrocin), aromatic strength (Inline graphic330 nm, maximum absorbance of safranal), and color strength (Inline graphic440 nm, maximum absorbance of crocin) were obtained according to ISO/TS 3632-2. Each value can be calculated as follows (Eq. 1):

graphic file with name 41598_2025_8136_Article_Equ1.gif 1

where D is the specific absorbance, m is the mass of the saffron sample in grams, and H is the moisture and volatile contents of the sample, expressed as a mass fraction. A 1 cm quartz cell was used as the sampling unit in the spectrophotometer. Each value was the average of three replicates21.

Saffron phenolic compound extraction

The extracts of untreated and treated saffron were obtained according to the methods reported by Budrat & Shotipruk22. Ten millilitres of 80% aqueous methanol were used to extract the phenolic compounds from 0.1 g of saffron. The mixture was sonicated at 45 °C for 30 min in an ultrasonic bath (Sonorex Digitech DT 1028 H, Bandelin, Germany). The methanolic extract was subsequently separated via centrifugation (at 7500 × g for 5 min) and used for total phenolic compound (TPC) and antioxidant activity evaluation.

Total phenolics content

The total phenolic content (TPC) was determined colorimetrically via the Folin‒Ciocalteu method, as described by Pinelo et al. 200423. Approximately 20 µL of the extract solution was diluted with 1160 µL of distilled water, and 100 µL of Folin-Ciocalteu reagent was added. After 10 min, 300 µL of aqueous sodium carbonate solution (20%, w/v) was added to the mixture, which was subsequently incubated at 40 °C for 60 min in the dark. The absorbance was measured at 765 nm via a UV/Vis spectrophotometer (Jenway, 6715 UV/Vis., Bibby Scientific Ltd., Dunmow, Essex, UK). Different concentrations of gallic acid solution were used to prepare a calibration curve, and the measured TPC was expressed as mg gallic acid equivalent (GAE) per g of saffron.

Determination of antioxidant activity via the DPPH free radical scavenging assay

DPPH was estimated via the Brand-Williams et al.24 method. Briefly, 1 mL of a fresh 1 mM 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution in methyl alcohol was mixed with 5 µL of the sample extracts and kept in the dark at room temperature for 30 min. The radical scavenging capacity was measured with a UV/Vis spectrophotometer (Genway 6715 UV/Vis, USA) at 515 nm. The percentage of DPPH radical scavenged was quantified via the following equation (Eq. 2):

graphic file with name 41598_2025_8136_Article_Equ2.gif 2

where A0 is the absorbance of the blank sample and A1 is the absorbance of the sample extracts 25.

Analysis of saffron phenolic components by HPLC

Extraction of phenolic compounds

The phenolic compounds were analyzed via HPLC extract preparation for high-performance liquid chromatography (HPLC) according to the procedure of Bourgou et al.26 with some modifications as follows: 0.1 g of fine powder from the leaves was extracted with 10 ml of absolute methanol for 24 h in the dark. The samples were subsequently centrifuged at 15,000 × g for 30 min, after which the resulting supernatant was filtered (0.45 μm), and the methanol was evaporated through a rotary evaporator. Finally, the extract was dissolved in 200 µl of methanol, and 20 µl was injected into the mixture for HPLC. A Knauer HPLC system equipped with a reversed-phase column (250 × 4.6 mm, C18-ODS3) was utilized. For phenolic acids, a linear gradient of acetonitrile (10–50%) in water (1% (v/v) acetic acid) was utilized as the mobile phase for a maximum elution time of 40 min at room temperature. The flow rate was 1.0 ml/min. Phenolic acids were detected at 280 nm. The quantitative determination of these compounds was performed utilizing external standards through the calibration curves of these compounds.

Quantitative determination of saffron pigment components via HPLC

HPLC analysis was performed on a multisolvent delivery system (Waters Corporation, Milford, USA) equipped with a pump (Model 600E) and a multiple UV wavelength photodiode array detector (Model 996) linked to a computer system (Optiflex GX280, Dell Computer Corp.). Empower Software (Version 3.0; Waters Corporation, Milford, USA) was used for equipment control, data acquisition, and processing of the chromatographic information.

A Spherisorb RP C18 column (Waters, Milford, MA, USA; 25 cm length, 4.6 mm internal diameter, 10 lm particle size with a pore diameter of 80 A) was used for all analyses. A linear gradient of methanol (10–100%) in water (15% acetonitrile) was used as the mobile phase, with a flow rate of 1.0 ml/min and a maximum elution time of 60 min at room temperature. The analyses were performed in triplicate for each sample. Picrocrocin was detected at 250 nm, safranal at 310 nm and all crocins at 440 nm, whereas the internal standard was detected at the above three mentioned wavelengths27. For the external standards (catechol, salicylic acid, p-hydroxybenzoic acid, vanillin, gentisic acid, gallic acid, caffeic acid, syringic acid, ferulic acid, and p-coumaric acid), a calibration curve was prepared at final concentrations of 0.015, 0.031, 0.05, 0.062, 0.125, 0.25, and 1.0 mg/ml, respectively, in triplicate28. Quantitative determinations were made taking into account the molecular coefficient absorbance of each peak obtained at the wavelength of maximum absorbance of the respective ingredient as previously reported27, and the values are expressed in milligrams per gram of saffron stigma. The R2 range was from 0.9833 to 0.9990, and the slope R.S.D. values between the injected samples were lower than 5%.

Preparative analysis and spectrometric identification of pure compounds

A Spherisorb RP C18 column (Waters, 25 cm length, 20 mm internal diameter, 10 μm particle size with a pore diameter of 80 Å) was used for this analysis. The abovementioned linear gradient was used with a flow rate of 10 ml/min during an elution time of 60 min. The amount of sample injected was 50 µl of the test solution. Eluates across the peaks with tR values of 8.81 (picrocrocin), 9.44 (trans-4-GG-crocin), 10.81 (trans-3-Gg-crocin), 11.35 (cis-4-GG-crocin), 13.43 (trans-2-gg-crocin), 14.49 (trans-crocetin), and 16.24 (safranal) were collected via the technique of heart cutting29. Each collected peak was rapidly evaporated under vacuum using a centrifugal solvent evaporator system (CentriVap, Labconco, MO, USA) and further subjected to low-resolution fast atom bombardment mass spectrometry (LRFAB-MS) analysis. Positive-ion LRFAB-MS was registered via a glycerol matrix on an SX102A spectrometer (JEOL, Tokyo, Japan). Xenon was used as the collision gas. The accelerating voltage was 10 keV, and the resolving power was 3000. The mass spectra were determined in duplicate, and the pseudomolecular ion [M + Na+] was detected for each peak. The observed fragmentation pattern was used for crocin identification by comparison with previously reported spectra30.

Statistical analysis

All the experiments were independently repeated three times. Differences in all experiments were analyzed via a completely randomized design, and the results were subjected to analysis of variance (ANOVA), with means compared via the Duncan test and Duncan’s test (p < 0.05) via SPSS (version 16; IBM Corporation, Armonk, NY, USA) statistical software.

Results and discussion

Microbial analysis

The assessment of saffron hygienic status through total bacterial count revealed significant findings regarding the effectiveness of radiation treatments. The initial contamination level indicated a total count of 1.48 × 104 cfu/g, with notable yeast and coliform populations (5.15 × 102 and 9.09 × 101 cfu/g, respectively). All the gamma radiation treatments resulted in the absence of fungus and yeast in the saffron samples, and no microbial population was detected at doses greater than 2 kGy (Table 1). Additionally, the microbial population was significantly affected by electron radiation, and there was no fungal or total microbial population at doses above 1 kGy and 2 kGy, respectively. In all the gamma and electron radiation treatments, total coliform contamination was depleted in the saffron samples.

Table 1.

Disinfection of saffron samples with different doses of gamma and e-beam radiation (kGy). The data are presented as the means of triplicate samples. Different letters in each column indicate significant differences between different doses of radiation (p < 0.05, based on duncan’s test).

Microbial population Dose (kGy) Radiation treatment
Gamma E-beam
Total count (cfu/g) 0 1.48 a× 104 1.48 × 104
1 5.39 b× 102 1.06 × 103
2 N.D. *c 1.45 × 102
3 N.D. c N.D. c
4 N.D. c N.D. c
5 N.D. c N.D. c
Yeast & mold (cfu/g) 0 5.15a × 102 5.15a × 102
1 N.D. b 1.21b × 101
2 N.D. b N.D. c
3 N.D. b N.D. c
4 N.D. b N.D. c
5 N.D. b N.D. c
Total coliform (cfu/g) 0 9.09a × 101 9.09a × 101
1 N.D.b N.D. b
2 N.D. b N.D. b
3 N.D. b N.D. b
4 N.D. b N.D. b
5 N.D. b N.D. b
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*N.D.: not detected.

Additionally, other studies have shown that gamma radiation doses of 1–4 kGy significantly reduce microbial loads, with total coliform contamination deactivating11,18. Additionally, Belbe and Tofană31 demonstrated that gamma radiation was effective at reducing microbial populations at doses above 2 kGy. Ghoddusi & Glatz8 demonstrated that yeasts were more resistant to electron beam radiation and survived even at doses above 1 kGy, but overall microbial counts reached zero. While gamma irradiation effectively reduces microbial contamination in saffron, it may compromise pigment quality, raising concerns about the balance between the safety and preservation of saffron value.

Total phenolic content (TPC)

The total phenolic content (TPC) of saffron (mg equivalents of gallic acid/dry weight saffron) is significantly influenced by irradiation methods, as evidenced by our gamma and electron beam radiation results and shown in Fig. 1. The highest TPC was found in nonirradiated saffron, while both irradiation methods led to notable changes in phenolic levels, indicating a complex interaction between radiation and phenolic compounds. Gamma and e-beam radiation significantly affected the total phenolic content of saffron (p < 0.05).

Fig. 1.

Fig. 1

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Total phenolic content (TPC, mg/g) of saffron after gamma and e-beam irradiation (at 1–5 kGy). The data are presented as the means of triplicate samples. Different letters above the bars indicate significant differences between treatments (p < 0.05, based on Duncan’s test).

A significant decrease in phenolic content was observed in the methanolic extracts of saffron following 1, 2, 3, 4, and 5 kGy irradiation (p < 0.05) for both the gamma and e-beam irradiation treatments. The phenolic content decreased to 68. 81 (7.46% reduction) and 60.82 (14.92% reduction) mg/g for saffron irradiated with a dose of 5 kGy in the gamma and e-beam irradiation treatments, respectively, compared with the control. Similar studies indicate that doses of 2.5 to 5 kGy lead to a decrease in glycosides and an increase in aglycon forms of carotenoids, which may affect the overall phenolic profile of saffron38. In contrast, while irradiation can enhance certain beneficial properties, excessive doses may lead to the degradation of essential compounds, indicating the need for careful dose management in saffron processing. These findings indicate that e-beam radiation has a greater effect on phenolic compounds than does gamma radiation. Similar trends have been observed in other plants, where moderate doses of gamma irradiation (4–8 kGy) increased the phenolic content and antioxidant properties, whereas higher doses (10 kGy) resulted in a decrease33. This suggests a dose-dependent response in phenolic content across different species. An increase in the phenolic content of the extract was observed during research on the effect of gamma irradiation on almond skin with increasing doses of irradiation above 4 kGy. Compared with controls, Koseki et al.34 reported that irradiation doses between 10 and 30 kGy resulted in a reduction in the total phenolic compounds in dehydrated rosemary. Different plants have different phenolic compounds, which is attributed to differences in their effects. Radiation can cause the breakdown of larger phenolic compounds into smaller, less complex molecules, which may result in a net decrease in the total phenolic content32. Irradiation generates hydroxyl free radicals (°OH), which initiate the degradation of phenolic compounds by attacking the aromatic ring, leading to the formation of smaller byproducts35. The presence of oxygen during irradiation enhances the oxidative degradation of phenolic compounds, further contributing to their reduction36. While the degradation of phenolic compounds is a concern, certain doses of gamma irradiation can enhance the antioxidant activity of other saffron components, suggesting a complex relationship between irradiation and phenolic content.

Determination of antioxidant activity

The scavenging activity of saffron extracts, particularly in response to gamma and e-beam irradiation, significantly altered their antioxidant properties. The scavenging activity of the control and irradiated saffron extracts, expressed as the percentage of inhibition (% I) of the DPPH radical, was analyzed. The results showed that gamma and e-beam irradiation significantly changed the scavenging activity of the methanol extracts tested at all the radiation doses. The antioxidant activity of saffron samples that were irradiated with gamma and e-beam radiation is shown in Fig. 2 in comparison to that of nonirradiated samples. The statistical level of 0.05 was the level at which all the data were significantly different. The antioxidant activity significantly increased with increasing doses of gamma radiation. The irradiation of 1 kGy of saffron resulted in the highest amount of antioxidant activity being observed, and a slight decrease in antioxidant activity was observed after further increases in the radiation dose. Additionally, the electron beam had a significant effect on antioxidant activity, and the highest antioxidant activity was observed at doses of 1 and 3 kGy of electron beam radiation. There was no noticeable difference between the control sample and the e-beam radiation doses of 2 and 4 kGy.

Fig. 2.

Fig. 2

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Antioxidant activity (AOA, %) of saffron after gamma and e-beam irradiation (at 1–5 kGy). The data are presented as the means of triplicate samples. Different letters above the bars indicate significant differences between treatments (p < 0.05, based on Duncan’s test).

Saffron is rich in bioactive compounds such as crocins, picrocrocin, and safranal, which contribute to its strong antioxidant potential17,37. Gamma irradiation has been shown to increase the antioxidant activity of saffron, potentially through the release of bioactive compounds that can scavenge free radicals38. E-beam irradiation can preserve the nutritional and antioxidant properties of dried plants, including saffron, during extended storage periods. Studies have shown that irradiation helps maintain the integrity of bioactive compounds, reducing the degradation typically observed in nonirradiated samples19.

Chemical characteristics of saffron-ISO/TS 3632-2:2003 analysis

The ISO 3632 standard outlines the quality assessment of saffron through the measurement of specific absorbances at 257, 330, and 440 nm, which correspond to the compounds picrocrocin, safranal, and crocin, respectively. These compounds are critical for determining the bitterness, aromatic strength, and coloring strength of saffron, thus serving as indicators of its quality. The ISO 3632 standard provides a classification system based on the minimum required levels of these compounds, ensuring that saffron meets quality benchmarks39. Studies have shown that variations in these compounds can significantly affect the overall quality and market value of saffron40.

The results revealed that increasing the dose of gamma rays and electrons had a significant effect on the bitterness, aromatic strength and coloring strength of saffron (Figs. 3, 4 and 5). The bitterness index of saffron before irradiation was 118.61. According to the ISO 3236 standard, a bitterness strength greater than 80 is associated with excellent quality saffron (Fig. 3). The bitterness index changed after increasing the dose of gamma radiation to 2 kGy and was greater ​​at higher doses than that of unirradiated saffron. There was no significant difference between the samples irradiated with saffron at a dose of 1 kGy and the control samples. In general, samples irradiated with gamma rays presented higher values of the bitterness index than did the control. Additionally, irradiating saffron with an electron beam significantly affects the bitterness index, and increasing the radiation dose leads to a significant increase in the bitterness index. Doses of 3 to 5 kGy had no significant effect, and the highest level of the bitterness index was observed in this dose range.

Fig. 3.

Fig. 3

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The effects of gamma and e-beam irradiation (1–5 kGy) on the bitterness index of saffron. The data are presented as the means of triplicate samples. Different letters above the bars indicate significant differences between treatments (p < 0.05, based on Duncan’s test).

Fig. 4.

Fig. 4

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The effects of gamma and e-beam irradiation (1–5 kGy) on the aromatic strength of saffron. The data are presented as the means of triplicate samples. Different letters above the bars indicate significant differences between treatments (p < 0.05, based on Duncan’s test).

Fig. 5.

Fig. 5

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Effect of gamma and e-beam irradiation (at 1–5 kGy) on the coloring strength of saffron. The data are presented as the means of triplicate samples. Different letters above the bars indicate significant differences between treatments (p < 0.05, based on Duncan’s test).

The effects of gamma and electron radiation on the aromatic strength of saffron are shown in Fig. 4. All the data were significantly different at the 0.05 level, and the increase in radiation dose led to an increase in this index compared with that of the control sample. Compared with the control, a gamma radiation dose of 1 kGy did not have a significant effect. Gamma rays at doses ranging from 2 to 5 kGy resulted in the greatest aromatic strength. These changes included an 8.70–15.90% increase in the aromatic strength of saffron. Additionally, increasing the electron beam dose increased the aromatic strength of the irradiated saffron samples. The highest aromatic strength was observed at doses of 4 (14.66% increment) and 5 kGy (17.59% increment) of e-beam radiation. There was no significant difference between e-beam doses of 1 to 3 kGy.

Figure 5 shows the effects of gamma and electron radiation doses on saffron coloring strength as a function of radiation dose. All the obtained results were significantly different at the 0.05 level. Increasing the dose of gamma radiation led to an increase in the coloring strength of the irradiated saffron samples. The greatest amount of color strength was observed at doses of 3 (17.73% increase), 2 and 4 kGy. Saffron irradiated with a dose of 1 kGy of gamma radiation was not significantly different from the control sample. Additionally, increasing the electron beam radiation dose led to an increase in the coloring strength at doses of 1 (10.84% increase), 5 and 4 kGy. The saffron samples irradiated with doses of 2 and 3 kGy e-beam radiation were not significantly different.

The irradiation of saffron with gamma and electron beams has been shown to enhance its quality attributes, such as bitterness, aromatic strength, and coloring strength, without negatively impacting these characteristics according to ISO 3632 standards. However, concerns have been raised regarding the accuracy of the ISO method in measuring safranal levels compared with high-performance liquid chromatography (HPLC), suggesting a need for more precise analytical techniques to ensure saffron quality as global demand increases.

Analysis of saffron pigment components by HPLC

The chemical compositions of the different radiated saffron samples (gamma & electron beam radiation) were determined via reversed-phase C18 HPLC. The chromatographic conditions employed allowed the identification of 7 major components (picrocrocin, trans-4-GG-crocin, trans-3-Gg-crocin, cis-4-GG-crocin, trans-2-gg-crocin, trans-crocetin, and safranal) in each sample, and well-resolved baseline separation was obtained. Each component was identified by comparison of its retention time as previously described in the literature27,41 as well as by LRFAB-MS analysis through the detection (m/z) of its corresponding pseudomolecular ion (M + H)[+ 30,41.

Figure 6a-l shows twelve chromatograms of gamma- and electron beam-irradiated saffron samples, one with the standard concentration of analyzed glycosidic carotenoids (Fig. 7a), the second as nonirradiated (Fig. 7b, c) and the other with gamma- and electron beam irradiation (Fig. 7e-l). The peak identification was as follows: peak number 1 detected at 250 nm was picrocrocin; peak number 7 (310 nm) was safranal; and peak numbers 2 to 6 (440 nm) were trans-4-GG-crocin, trans-crocin 3, trans-3-Gg-crocin, cis-4-GG-crocin, trans-2-gg-crocin, and trans-crocetin, respectively. The impact of gamma and electron beam radiation on saffron pigments, particularly through high-performance liquid chromatography (HPLC), revealed significant alterations in the chemical composition of saffron.

Fig. 6.

Fig. 6

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HPLC chromatograms of pigments extracted from saffron samples: (a) standards, (b) nonirradiated sample and (c, d) 1 kGy, (e,f) 2 kGy, (g, h) 3 kGy, (i, j) 4 kGy and (k, l) 5 kGy gamma-irradiated and electron beam-irradiated saffrons. Picrocrocin (1), trans-4-GG-crocin (2), trans-3-Gg-crocin (3), cis-4-GG-crocin (4), trans-2-gg-crocin (5), trans-crocetin (6), safranal (7).

Fig. 7.

Fig. 7

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HPLC chromatograms of the phenolic components of the saffron samples: (a) standard sample, (b) nonirradiated sample and (c, d) 1, (e, f) 3, and (g, h) 5 kGy gamma-irradiated and electron beam-irradiated saffron samples. Catechol (1), salicylic acid (2), p-hydroxybenzoic acid (3), vanillin (4), gentisic acid (5), gallic acid (6), caffeic acid (7), syringic acid (8), ferulic acid (9), and p-coumaric acid (10) were used.

The nonradiated saffron had the highest total concentration of apocarotenoids (32.825 mg/g saffron). In nonradiated saffron, the highest concentrations of trans-4-GG-crocin, safranal, trans-3-Gg-crocin and picrocrocin were present in the analyzed apocarotenoids. According to our analysis, different radiated saffron samples (Tables 2 and 3) did differ in the concentration of each component.

Table 2.

HPLC-based quantitative analyses of 7 apocarotenoids (mg/g) from different gamma-irradiated (1–5 kGy) saffrons.

Peak no. Retention time
(min)		 Compound name
(mg/g)		 Dose of irradiation (kGy)
0 1 2 3 4 5
1 8.81 Picrocrocin 4.540*a ±0.24 3.511b ± 0.15 2.520c ± 0.18 1.970d ± 0.11 1.499e ± 0.10 1.202e ± 0.11
2 8.44 trans-4-GG-crocin 10.502a ± 0.68 9.128b ± 0.32 7.054c ± 0.28 6.357d ± 0.40 4.841e ± 0.26 4.027f ± 0.15
3 10.81 trans-3-Gg-crocin 6.594a ± 0.13 5.373b ± 0.16 4.652c ± 0.12 3.323d ± 0.21 2.382e ± 0.16 2.366e ± 0.08
4 11.35 cis-4-GG-crocin 0.494a ± 0.05 0.322bc ± 0.08 0.317bc ± 0.06 0.339b ± 0.03 0.231cd ± 0.05 0.172d ± 0.03
5 13.43 trans-2-gg-crocin 1.555a ± 0.012 0.931b ± 0.05 0.894b ± 0.11 0.726c ± 0.07 0.679c ± 0.04 0.370d ± 0.04
6 14.49 trans-crocetin 0.451a ± 0.05 0.384ab ± 0.07 0.317b ± 0.08 0.162c ± 0.03 0.200c ± 0.02 0.119c ± 0.03
7 16.24 Safranal 8.689a ± 0.51 8.229a ± 0.44 6.667b ± 0.23 4.403c ± 0.16 3.375d ± 0.18 2.514e ± 0.07
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* Different letters in each row indicate significant differences between different doses of radiation (p < 0.05, based on Duncan’s test).

Table 3.

HPLC-based quantitative analyses of 7 apocarotenoids (mg/g) from different e-beam-irradiated (1–5 kGy) saffrons.

Peak no. Retention time
(min)		 Compound name
(mg/g)		 Dose of irradiation (kGy)
0 1 2 3 4 5
1 8.81 Picrocrocin 4.540*a ±0.24 2.797b ± 0.12 1.767c ± 0.10 1.323d ± 0.08 1.019e ± 0.09 0.617f ± 0.06
2 8.44 trans-4-GG-crocin 10.502a ± 0.68 7.536b ± 0.38 5.670c ± 0.19 3.524d ± 0.20 3.124de ± 0.16 2.529e ± 0.11
3 10.81 trans-3-Gg-crocin 6.594a ± 0.13 3.580b ± 0.10 3.166c ± 0.09 2.156d ± 0.10 1.630e ± 0.06 1.064f ± 0.05
4 11.35 cis-4-GG-crocin 0.494a ± 0.05 0.263c ± 0.05 0.322b ± 0.03 0.168d ± 0.02 0.106e ± 0.02 0.078e ± 0.02
5 13.43 trans-2-gg-crocin 1.555a ± 0.012 0.867b ± 0.08 0.592c ± 0.04 0.429d ± 0.06 0.263e ± 0.02 0.207e ± 0.05
6 14.49 trans-crocetin 0.451a ± 0.05 0.259b ± 0.05 0.199c ± 0.02 0.150c ± 0.02 0.08d ± 0.01 0.076d ± 0.02
7 16.24 Safranal 8.689a ± 0.51 7.538b ± 0.30 5.192c ± 0.23 3.540d ± 0.17 2.734e ± 0.12 2.219f ± 0.09
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* Different letters in each row indicate significant differences between different doses of radiation (p < 0.05, based on Duncan’s test).

Table 2 shows the concentration of each detected compound in the different gamma-irradiated saffron samples. Although the irradiation of food products up to 10 kGy induces no nutritional problems31, saffron is abundant in carotenoid and phenolic compounds that are targets of hydroxyl and superoxide free radicals generated during irradiation, leading to oxidation and degradation32.

These results indicate that high doses of radiation can also induce undesirable changes in the apocarotenoid compound attributes of saffron, making them unacceptable for consumer acceptance. This variation could be the result of the radiation processes used, which could affect the concentration of glycosidic carotenoids, as they are sensitive components to oxidation41. Gamma irradiation, particularly at doses of 2.5 and 5 kGy, leads to a notable decrease in glycoside content and an increase in aglycon forms of carotenoids, primarily crocins, which are responsible for saffron color32. Increasing the dose of gamma radiation caused a significant (p < 0.05) decrease in the concentration of apocarotenoid compounds. Table 2 also shows that picrocrocin, a precursor of saffron aroma components (safranal and 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde), was present in large amounts in nonradiated saffron (4.54 mg/g), and increasing the dose of gamma radiation caused a significant decrease in the picrocrocin concentration. The results revealed that gamma radiation of saffron at a dose of 1 kGy led to a 22.6% decrease in the picrocrocin concentration. Compared with nonradiated saffron, gamma-irradiated saffron had no significant difference in safranal concentration at a dose of 1 kGy. Since picrocrocin is the precursor of safranal, a decrease in the concentration of picrocrocin at a dose of 1 kGy has led to its conversion to safranal. As a result of these conversions, the effect of a radiation dose of 1 kGy on the safety of the control and irradiated samples was not significant.

The irradiation process generates free radicals and other reactive species, which can accelerate chemical reactions, leading to the degradation of picrocrocin and favoring the formation of safranal. According to some studies, these radicals can promote the hydrolysis of the glycosidic bond in picrocrocin, releasing glucose and converting the aglycone part into safranal42. While lower to moderate doses of irradiation facilitate the conversion of picrocrocin to safranal, higher doses (> 2 kGy) can adversely affect saffron quality (Table 2). At very high doses, not only does it lead to additional degradation of picrocrocin, but it may also convert safe compounds into other undesirable compounds due to excessive bond cleavage and radical reactions, diminishing overall quality and flavor43. The natural form of crocins is unstable and can be greatly degraded when isolated41 or irradiated with different doses of gamma radiation. The maximum degradation of the apocarotenoid compounds was observed with high doses of gamma radiation (4 and 5 kGy), which resulted in a 50% reduction in all the apocarotenoid compounds. The stability of crocins is compromised under high radiation, leading to further degradation44. Excessive irradiation can lead to the formation of undesirable compounds from safranal, negatively impacting saffron’s flavor profile43. Overall, the results showed that irradiation at 1 kGy can significantly enhance the conversion of picrocrocin to safranal in saffron, with moderate doses offering the best balance in improving sensory qualities while preserving essential attributes. However, caution must be exercised regarding the dosage; higher doses may lead to detrimental effects on safranal and overall saffron quality. Understanding the mechanisms at play and adhering to optimal irradiation conditions can help harness the benefits of this technology for saffron processing.

The concentrations of apocarotenoid metabolites, specifically trans-crocetin and cis-4-GG-crocin, are significantly influenced by saffron treatment. The reduction in the cis-4-GG-crocin concentration was not significantly affected by increasing the radiation dose from 1 to 3 kGy. However, 4 kGy was the radiation dose that caused a greater than 50% decrease in the cis-4-GG-crocin concentration.

Trans-3-Gg-crocin is the carotenoid metabolite that is the most sensitive to high doses of gamma radiation, degrading more than 77% of the carotenoid metabolite at doses higher than 3 kGy. Among all the apocarotenoid compounds, trans-2-gg-crocin and cis-4-GG-crocin were the most sensitive to gamma radiation at a dose of 1 kGy, with decreases of 40% and 35%, respectively.

The levels of apocarotenoid metabolites in nonradiated saffron were significantly reduced due to the increase in the dose of electron beam radiation, and the results are shown in Table 3. Compared with samples subjected to gamma irradiation, electron beam-irradiated samples presented greater reductions in the levels of apocarotenoid compounds at different doses of radiation. Saffron’s electron beam radiation at a higher dose of 3 kGy resulted in a more than 65% decrease in the concentration of apocarotenoid metabolites. The maximum degradation of apocarotenoid compounds in electron beam-irradiated samples was observed at a high dose of electron beam radiation (5 kGy), which resulted in a 75–89% reduction in different apocarotenoid compounds.

A study by Rastkari et al.32 highlighted the significant impact of irradiation on saffron pigments, particularly crocetin digentiobiosyl ester, which decreased from 40.033 g/kg to 4.396 g/kg at a dose of 5 kGy. This reduction indicates the degradation of key colorants essential for the commercial value of saffron. These findings suggest that while irradiation effectively reduces microbial contamination, it also adversely affects the pigment profile of saffron. Other studies corroborate that irradiation doses above 5 kGy can lead to substantial losses in crocin content, affecting saffron’s overall quality45. HPLC profiles indicated a significant decrease in the absorbance of both the n-butanol and ethyl acetate fractions, suggesting radiation-induced breakdown of carotene glycosides. Other studies have utilized HPLC to separate and quantify saffron components, including picrocrocin and various crocin isomers, enhancing the understanding of saffron’s chemical properties46,47.

Analysis of saffron phenolic components by HPLC

Some phenolic acids in the nonradiated and radiated (gamma and electron beam radiation) saffron were determined via HPLC. The chromatographic conditions employed allowed the identification of 10 major phenolic components (catechol, salicylic acid, p-hydroxybenzoic acid, vanillin, genic acid, gallic acid, caffeic acid, syringic acid, ferulic acid, and p-coumaric acid) in each sample, and a well-resolved baseline separation was obtained.

The results demonstrated that gamma and electron radiation changed the content of phenolic compounds in the saffron as a function of radiation dose. In this work, the lowest and highest concentrations of phenolic compounds in nonradiated saffron were observed for gallic acid and salicylic acid, respectively. The results revealed that the contents of all the phenolic compounds decreased with increasing doses of radiation (Fig. 7a-h; Tables 4 and 5).

Table 5.

Relative distribution of phenolic compounds (µg/g) in electron beam-irradiated saffron identified via HPLC.

Peak No. Retention time
(RT) min		 Compound name
(µg/g)		 Dose of irradiation (kGy)
0 1 3 5
1 7.21 Catechol 0.465*a ±0.05 0.309b ± 0.05 0.157c ± 0.02 0.110c ± 0.02
2 8.01 Salicylic acid 1.612a ± 0.10 0.941b ± 0.13 0.679c ± 0.05 0.368d ± 0.09
3 8.12 p-Hydroxy benzoic acid 0.265a ± 0.06 0.160b ± 0.02 0.152b ± 0.01 0.085c ± 0.02
4 9.04 Vanillin 0.452a ± 0.08 0.206b ± 0.02 0.190b ± 0.02 0.076c ± 0.02
5 9.32 Gentisic acid 0.392a ± 0.02 0.253b ± 0.04 0.095c ± 0.01 0.062c ± 0.01
6 10.05 Gallic acid 0.181a ± 0.02 0.088b ± 0.01 0.035c ± 0.01 0.031c ± 0.01
7 10.25 Caffeic acid 0.852a ± 0.07 0.609b ± 0.03 0.407c ± 0.06 0.214d ± 0.03
8 12.04 Syringic acid 0.557a ± 0.11 0.322b ± 0.03 0.241bc ± 0.03 0.133c ± 0.01
9 12.53 Ferulic acid 1.271a ± 0.13 0.725b ± 0.09 0.455c ± 0.04 0.302c ± 0.06
10 21.95 p-Coumaric acid 0.541a ± 0.05 0.225b ± 0.04 0.130c ± 0.02 0.095c ± 0.02
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* Different letters in each row indicate significant differences between different doses of radiation (p < 0.05, based on Duncan’s test).

Catechol was the first metabolite produced by the chromatography column and was detected at 7.21 min. This phenolic metabolite significantly decreased with increasing gamma radiation dose. The concentrations of catechol at doses of 1 and 3 kGy gamma rays were not significantly different. The SA concentration in the irradiated saffron samples decreased significantly with increasing gamma radiation dose. The greatest reduction was observed at a dose of 5 kGy of gamma radiation compared with that of the control. Additionally, other phenolic compounds, such as vanillin, gentisic acid, gallic acid, caffeic acid, syringic acid, ferulic acid, and p-coumaric acid, showed similar changes at different doses of gamma radiation.

Table 4.

Relative distribution of phenolic compounds (μg/g) in gamma-irradiated saffron identified via HPLC

Peak No. Retention time
(min)		 Compound name
(µg/g)		 Dose of irradiation (kGy)
0 1 3 5
1 7.21 Catechol 0.465*a ±0.05 0.313b ± 0.02 0.257b ± 0.02 0.158c ± 0.02
2 8.01 Salicylic acid 1.612a ± 0.10 1.018b ± 0.12 0.767c ± 0.07 0.550d ± 0.02
3 8.12 p-Hydroxy benzoic acid 0.265a ± 0.06 0.245a ± 0.03 0.160b ± 0.01 0.083c ± 0.01
4 9.04 Vanillin 0.452a ± 0.08 0.307b ± 0.01 0.142c ± 0.01 0.127c ± 0.01
5 9.32 Gentisic acid 0.392a ± 0.02 0.246b ± 0.01 0.177c ± 0.02 0.137d ± 0.01
6 10.05 Gallic acid 0.181a ± 0.02 0.130b ± 0.01 0.069c ± 0.01 0.067c ± 0.01
7 10.25 Caffeic acid 0.852a ± 0.07 0.677b ± 0.06 0.490c ± 0.03 0.369d ± 0.03
8 12.04 Syringic acid 0.557a ± 0.11 0.393b ± 0.02 0.264c ± 0.02 0.200c ± 0.02
9 12.53 Ferulic acid 1.271a ± 0.13 0.882b ± 0.05 0.543c ± 0.03 0.458c ± 0.06
10 21.95 p-Coumaric acid 0.541a ± 0.05 0.298b ± 0.02 0.249b ± 0.03 0.134c ± 0.01
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* Different letters in each row indicate significant differences between different doses of radiation (p < 0.05, based on Duncan’s test).

In general, the results revealed that the lowest decrease in the concentration of phenolic compounds among the different doses of gamma rays or electron beams occurred at a dose of 1 kGy. Among the phenolic compounds present in saffron irradiated with gamma and e-beam radiation at a dose of 1 kGy, p-coumaric acid was the most sensitive to radiation and presented the greatest percentage reduction at the lowest dose of radiation.

Conclusions

This study highlighted the effectiveness of low doses of gamma and electron beam irradiation in eliminating microbial loads and enhancing product quality. Specifically, a gamma dose of 1 kGy and an electron beam dose of 2 kGy were found to be optimal for reducing the microbial load. This suggested that lower doses can achieve significant results, which is a notable finding compared with the higher doses typically reported in other studies. While gamma irradiation remains the superior method, electron beam irradiation can be considered a highly efficient method compared with other methods.

The relationship between radiation dose and antioxidant activity has been explored in various studies, particularly those focused on saffron and other plant extracts. Research indicates that both gamma irradiation and electron beam irradiation can enhance antioxidant properties, with specific optimal doses yielding the best results. In this study, gamma radiation of 1 kGy resulted in the highest amount of antioxidant activity being observed, and a slight decrease in antioxidant activity was observed after further increases in the radiation dose. Additionally, the electron beam had a significant effect on antioxidant activity, and the highest antioxidant activity was observed at doses of 1 and 3 kGy of electron beam radiation. While these studies highlight the benefits of irradiation in enhancing antioxidant properties, it is essential to consider potential adverse effects at higher doses, which may counteract the benefits observed at lower doses.

The results showed that irradiation of saffron with gamma and e-beams does not have a negative effect on the bitterness, aromatic strength or coloring strength of saffron corresponding to the standard of ISO 3632, and in general, irradiation leads to increases in these indicators. While the ISO method is widely used, some research suggests that it may not accurately measure safranal levels compared with high-performance liquid chromatography (HPLC) methods, indicating a need for further evaluation of the ISO standards48. The ISO method for assessing safranal and crocin levels has been criticized for its inaccuracy, particularly in measuring safranal. HPLC provides a more reliable measurement of saffron’s chemical constituents, highlighting discrepancies in ISO results48. While irradiation appears beneficial for saffron quality, the reliance on ISO standards may hinder accurate assessments, necessitating a shift toward more robust analytical methods such as high-performance liquid chromatography (HPLC) to maintain product integrity on a growing market.

This discrepancy highlights the importance of adopting more precise analytical techniques to ensure the quality and authenticity of saffron, particularly as its demand continues to rise globally. As the saffron market expands, establishing rigorous quality control measures that can reliably differentiate between irradiated products and nonirradiated products becomes increasingly crucial.

The HPLC results revealed that the attributes of the apocarotenoid compounds in saffron can undergo undesirable changes due to high doses of radiation, making them unacceptable for consumers. Additionally, the HPLC results demonstrated that gamma and electron radiation changed the content of phenolic compounds in the saffron as a function of radiation dose. These findings suggest that gamma radiation has a profound effect on the stability and concentration of phenolic compounds in saffron, potentially altering its antioxidant properties and overall quality. This alteration in phenolic content may influence the therapeutic efficacy of saffron, as these compounds are known for their health benefits and role in combating oxidative stress. Further research is needed to explore the mechanisms behind these changes and to assess how varying radiation doses might alter the bioactive profile of saffron, ultimately affecting its applications in food and medicine. Understanding the implications of these alterations is crucial for optimizing the use of saffron in various industries, particularly as a natural antioxidant and therapeutic agent. Conversely, some studies suggest that alternative methods, such as hurdle technology combining irradiation with other preservation techniques, may offer enhanced stability and sensory quality without the risks associated with higher irradiation doses18. While gamma irradiation is often viewed as superior, electron beam treatment presents a viable alternative, particularly in specific applications such as pest control and food preservation, where it can be equally effective at slightly higher doses.

Understanding the optimal balance of these doses is essential for maximizing the benefits of irradiation while minimizing any potential negative impacts on food quality. Achieving this balance requires extensive research and experimentation to identify the precise irradiation parameters that can effectively reduce microbial contamination without compromising the desirable attributes of food products. This ongoing investigation into irradiation techniques not only enhances food safety but also paves the way for innovative preservation methods that can cater to consumer preferences for fresh and nutritious options.

Acknowledgements

This study was supported by the Nuclear Science and Technology Research Institute (NSTRI), Atomic Energy Organization of Iran (AEOI).

Author contributions

M.R.: Conceptualization, Writing - review & editing; SP. Sh.: Conceptualization, Methodology, Investigation, Writing- review & editing; H.A.: Conceptualization, Writing - review & editing, Supervision, Project administration. Compliance with Ethical Standards; S. Sh.: Conceptualization, Methodology, Investigation, Formal analysis, Writing- original draft, Writing - review & editing, Visualization, Funding acquisition, Supervision, Project administration.; H.A.: Conceptualization, Methodology, Investigation, Formal analysis, Writing- original draft, Writing - review & editing, Visualization, Supervision. All authors reviewed the manuscript.

Funding

This study was funded by the Nuclear Science and Technology Research Institute (NSTRI), Atomic Energy Organization of Iran (AEOI).

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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