Skip to main content
NCBI home page
Food Science & Nutrition logoLink to Food Science & Nutrition
. 2026 Sep 28;14(10):e72423. doi: 10.1002/fsn3.72423

Sunlight‐Induced Lipid Oxidation and Degradation of Edible Oil Mediated by Hemoglobin: Implications for Food System Sustainability

Mahdi Hajimohammadi 1,✉, Fadhel H Faraj 2, Mona Boghdachi 1, Asawer A Alwasiti 2, Zainab Y Shnain 2
PMCID: PMC13620216  PMID: 42812753

ABSTRACT

Lipid oxidation is one of the major factors affecting the quality, safety, and shelf life of edible oils. In this study, sunlight‐induced oxidation of edible oil was investigated in an aqueous system using erythrocyte‐derived hemoglobin (Hb) as a natural biocatalyst. Significant oxidation was observed only under visible‐light irradiation in the presence of Hb and oxygen. The peroxide value (PV) increased rapidly, reaching 9.7 meq O2/kg within 2.5 h, followed by the degradation of unsaturated fatty acids, indicating the occurrence of secondary oxidation reactions. Proton nuclear magnetic resonance (1H NMR) analysis showed a 31.2% decrease in vinylic protons and a 36.9% decrease in bis‐allylic protons, together with the appearance of an aldehydic signal at δ 9.5 ppm, confirming the formation of malondialdehyde (MDA). The generation of singlet oxygen (1O2) was supported by anthracene bleaching (33%) and its inhibition by sodium azide (NaN3), while the inhibitory effect of butylated hydroxytoluene (BHT) suggested the simultaneous involvement of radical‐mediated oxidation pathways. Compared with Rose Bengal and potassium permanganate (KMnO4), Hb showed higher photodegradation activity, likely due to the combined contribution of singlet oxygen generation and ferryl‐mediated (HbFe(IV)═O) hydrogen abstraction. Beyond advancing our understanding of the still underexplored mechanism of hemoglobin‐mediated photooxidation, these findings provide a scientific basis for exploring food‐derived hemoproteins as naturally abundant photosensitizers for the treatment of lipid‐rich wastewater.

Keywords: edible oil, food sustainability, heme‐mediated oxidation, hemoglobin, lipid oxidation, singlet oxygen

Sunlight‐activated hemoglobin (Hb) functions as a robust natural biocatalyst that promotes the degradation of edible oils through a dual, synergistic pathway. Photo‐excitation of Hb triggers simultaneous energy transfer, generating singlet oxygen (1O2) and electron transfer, forming highly reactive ferryl (HbFe(IV)═O) species. This mechanistic interplay accelerates lipid peroxidation and chain scission, leading to significant malondialdehyde (MDA) accumulation. These findings elucidate the underexplored hemoprotein mediated photooxidation mechanism, providing critical insights into food oil stability and potential strategies for the treatment of lipid‐rich wastewater.

graphic file with name FSN3-14-e72423-g004.webp

1. Introduction

Lipid oxidation and photooxidation are recognized as major factors influencing the quality, nutritional value, and shelf life of edible oils and other lipid‐containing foods (Looper and Vierck 2023; MacDougall 1982; Machado et al. 2023; Sebranek and Bacus 2007; Talbot 2016; Versino et al. 2023). Oxidative deterioration of lipids results in the formation of hydroperoxides, aldehydes, and other secondary oxidation products that adversely affect flavor, safety, and consumer acceptability (Agnez‐Lima et al. 2012; Domínguez et al. 2019; Droge 2002; Xi Huang and Ahn 2019; Kasaai 2025; Shetty et al. 2026). Among these oxidation markers, PV and MDA formation are widely recognized as key indicators of the primary and advanced stages of lipid peroxidation, respectively, in food systems (Abeyrathne et al. 2021). Since lipid photooxidation represents one of the major pathways contributing to oxidative deterioration, understanding its underlying mechanisms is essential for developing effective strategies to enhance food stability, optimize storage conditions, and improve processing practices (Afonso et al. 1999; Min and Boff 2002; Papuc et al. 2017).

In addition to storage‐related oxidation, edible oil processing and food manufacturing generate lipid‐rich effluents that pose significant environmental and technological challenges (Ahmad et al. 2020; Dordević et al. 2025; Jiang et al. 2015; Narra and Shahpasand 2025; Urrutia et al. 2024; C. Wang et al. 2025; Zhao et al. 2024). Conventional oxidation‐based treatments, such as ozonation and Fenton processes, can effectively degrade organic pollutants; however, they often require harsh chemical oxidants and energy‐intensive operating conditions (Alatabe et al. 2024; Ayoub 2022; Gogate and Pandit 2004; Malato et al. 2009). Consequently, there is growing interest in sustainable light‐driven strategies that harness naturally occurring biomolecules for environmentally friendly applications.

Hemoglobin, a heme‐containing protein abundant in animal tissues and slaughterhouse by‐products, is traditionally recognized for its role in oxygen transport in muscle foods (Nagababu and Rifkind 2004). However, heme proteins are also recognized as important mediators of lipid oxidation in meat and other food matrices (Meunier 2000). The heme prosthetic group, composed of an iron‐centered porphyrin structure, can participate in redox reactions that facilitate the generation and propagation of lipid radicals (Minning et al. 1999). Under light exposure, heme systems may further generate reactive oxygen species, including singlet oxygen, thereby accelerating oxidative processes (De Jager et al. 2017; Hajimohammadi, Verjani, et al. 2018). Previous studies have demonstrated that porphyrin and hemoglobin‐based systems can promote aerobic photooxidation of fatty acids and lipids under visible light (Hajimohammadi and Moradi 2025; Hajimohammadi and Nosrati 2018; Hajimohammadi et al. 2024; Hajimohammadi, Vaziri Sereshk, et al. 2018; Wu et al. 2024).

Building on these considerations, the present work aimed to elucidate the mechanism of sunlight‐induced lipid oxidation mediated by erythrocyte‐derived hemoglobin in an aqueous edible oil system. Its novelty lies in using structurally intact, erythrocyte‐derived hemoglobin as a native photosensitizer at the aqueous–oil interface under sunlight and bridging mechanistic photochemistry with biomimetic conditions. The work integrates singlet oxygen and radical/heme‐driven pathways within a single native system under visible light, enabling evaluation of their combined roles in lipid degradation. In addition to improving our understanding of the still underexplored mechanism of hemoglobin‐mediated photooxidation, this study provides a basis for exploring blood‐derived proteins as low‐cost, naturally abundant photosensitizers for the future treatment of lipid‐rich wastewater from edible oil processing.

Based on scavenger assays and spectroscopic analyses, we proposed that hemoglobin promotes lipid oxidation through a dual oxidative mechanism involving (i) singlet oxygen generation via energy transfer and (ii) a tentative heme iron‐mediated radical pathway. The combined contribution of these oxidative processes results in substantial degradation of unsaturated fatty acids under mild conditions. By connecting fundamental lipid oxidation chemistry with practical considerations in food stability and sustainable processing, this study advances the understanding of heme‐mediated oxidative reactions beyond their traditional biological functions.

2. Materials and Methods

2.1. Materials

Rose Bengal, anthracene, BHT, KMnO4, NaN3, acetonitrile (CH3CN), ethanol (EtOH), acetone ((CH3)2CO), methanol (MeOH), and dimethyl sulfoxide (DMSO) were purchased from Fluka and Merck and used as received without further purification. Commercially available edible oil (Oila, canola oil) was purchased from a local market and used without additional purification.

2.2. Preparation of Erythrocytes

Erythrocyte membranes were prepared according to the standard method described by Dodge et al. (1963). Defibrinated sheep blood was obtained from Kimiakavoshazma Company (Iran). Upon arrival, the samples were stored at 4°C and used within 48 h. Whole blood was centrifuged at 3000 rpm for 10 min at 4°C to separate the erythrocyte fraction from plasma. The collected erythrocytes were washed three times with phosphate‐buffered saline (PBS; 10 mM, pH 7.4) and subsequently resuspended in phosphate buffer (10 mM, pH 7.4). The erythrocyte suspension was adjusted to a protein concentration of approximately 1.0 mg mL−1 and freshly prepared suspensions were used for all photodegradation experiments. Hemoglobin concentration was normalized before each experiment by adjusting all samples to the same protein concentration using phosphate buffer (10 mM, pH 7.4). The hemoglobin concentration was further verified spectrophotometrically based on the Soret absorption band prior to analysis using a Shimadzu UV–visible spectrophotometer (UV‐2100) at 407 nm.

2.3. Photodegradation Procedure

A solution containing edible oil (2 × 10−2 v/v) and erythrocyte suspension (2 × 10−2 v/v) was irradiated using a solar simulator equipped with 276 LED lamps (1 W, 2.3 V; 380–780 nm; 59,660 lx; 8.74 mW cm−2) for 2.5 h at 25°C under atmospheric pressure in a water/acetonitrile (9:1, v/v) system. During irradiation, air was continuously bubbled into the reaction mixture through a glass diffuser to maintain aerobic conditions. Air was supplied at ambient pressure (1 atm) at an approximate flow rate of 50 mL min−1, while the reaction mixture was magnetically stirred throughout the experiment. Control experiments were conducted in the dark and in the absence of Hb. Aliquots were collected at defined time intervals for subsequent UV–Vis analysis. PVs were determined using the iodometric method described by Barthel and Grosch (1974) and expressed as meq O2 kg−1 oil. Instrument performance and analytical consistency were verified using standard peroxide solutions prior to analysis. Erythrocyte degradation was monitored using a Shimadzu UV–visible spectrophotometer (UV‐2100) at 407 nm. UV–visible absorption spectra were recorded with the same instrument equipped with 1 cm quartz cuvettes over the wavelength range of 200–800 nm at a spectral resolution of 1 nm. Baseline correction was performed using the corresponding solvent mixture as the reference.

2.4. Preparation of Samples for 1H NMR Analysis

A solution of edible oil (2 × 10−2 v/v) and erythrocyte suspension (2 × 10−2 v/v) was irradiated under the same conditions described in Section 2.3 for 6 h. 1H NMR spectra were recorded on a Bruker AMX 300 MHz spectrometer at 25°C using tetramethylsilane (TMS) as the internal standard. Spectra were acquired using a standard single‐pulse sequence with 32 scans, a relaxation delay of 1 s, and appropriate spectral width for proton detection. Chemical shifts are reported in ppm relative to TMS. The appearance of aldehydic resonances in the region of 9.4–9.6 ppm was used to confirm the formation of MDA.

2.5. Sample Preparation for Anthracene Bleaching Test

A solution containing anthracene (4 × 10−4 M), edible oil (2 × 10−2 v/v), and erythrocyte suspension (2 × 10−2 v/v) was irradiated under the same conditions described in Section 2.3, for 4 h. The resulting products were analyzed using a Shimadzu UV–visible spectrophotometer (UV‐2100) at 375 nm.

2.6. Sample Preparation for Investigation of Solvent Effects on Photooxidation Efficiency

A solution containing edible oil (2 × 10−2 v/v) and erythrocyte suspension (2 × 10−2 v/v) was irradiated under the same conditions described in Section 2.3, for 2.5 h in a water/solvent (9:1, v/v) system.

2.7. Effect of Standard Oxidants on Photooxidation Efficiency

Edible oil (2 × 10−2 v/v) was separately combined with erythrocyte suspension (2 × 10−2 v/v) or Rose Bengal (1 × 10−4 M) as a reference photosensitizer and then irradiated under the same conditions described in Section 2.3 for 2.5 h. In parallel, the reaction containing edible oil (2 × 10−2 v/v) and KMnO4 (1 × 10−4 M) was run under similar conditions but kept in the dark.

2.8. Statistical Analysis

All experiments were performed independently in triplicate (n = 3). Results were expressed as mean ± standard deviation (SD). Statistical analyses were conducted using SAS software (Version 9.4, SAS Institute Inc., Cary, NC, USA). Differences among experimental groups were assessed by one‐way analysis of variance (ANOVA), followed by Duncan's multiple range test for post hoc comparisons. Statistical significance was set at p < 0.05. In figures, different uppercase letters indicate statistically significant differences among groups based on Duncan's test.

3. Results

3.1. Photocatalytic Oxidation of Edible Oil Under Sunlight

Erythrocytes were employed in this study as a natural hemoglobin‐containing system that provides an intrinsic and biocompatible photosensitizing environment. The use of intact or lysed erythrocytes offers a physiologically relevant matrix for hemoglobin, allowing the preservation of its native structure and redox properties during light irradiation (Hajimohammadi et al. 2024; Wu et al. 2024). This strategy enables the direct investigation of hemoglobin‐mediated photodegradation of edible oils in aqueous systems under conditions that more closely resemble biological and environmental environments. Thus, erythrocytes serve not only as a natural source of hemoglobin but also as a functional model. The edible oil/Hb system was examined to elucidate the dual‐function photooxidative mechanisms driven by sunlight. Following 24 h of simulated sunlight irradiation, the edible oil/Hb mixture showed a gradual decrease in turbidity and yellow coloration. UV–Vis analysis revealed a reduction in the characteristic Soret absorption band of the heme‐containing system, corresponding to a 69.8% decrease in Soret‐band intensity and indicating substantial photochemical changes in the Hb chromophore during irradiation (Figure 1a). Control experiments performed (i) in the dark, (ii) in the absence of Hb, and (iii) under oxygen‐limited conditions showed negligible oxidation, demonstrating that the simultaneous presence of hemoglobin, oxygen, and light is required for efficient photooxidation (Table 1, Entries 1–4). These results confirm the essential contribution of Hb as a photosensitizer in the light‐driven degradation of edible oil. Furthermore, the similarity between the emission spectrum of the LED solar simulator and the solar radiation spectrum supports the relevance of this photocatalytic system under sunlight conditions (Table 1, Entry 5). Addition of BHT as a strong free radical quencher (Boulebd 2020; Durand et al. 2025) significantly reduced edible oil oxidation, supporting the involvement of carbon‐centered radicals generated through hydrogen abstraction (Table 1, Entry 6). Similarly, NaN3, a well‐established singlet oxygen scavenger (Li et al. 2001), markedly inhibited fatty acid oxidation, confirming the important contribution of 1O2 to the photooxidation process (Table 1, Entry 7).

FIGURE 1.

FIGURE 1

Open in a new tab

(a) UV–Vis spectral changes and erythrocyte remaining percentage of the edible oil–erythrocyte system during photooxidation under visible‐light irradiation. (b) Time‐dependent changes in (PV) during photooxidation. Different uppercase letters indicate statistically significant differences according to Duncan's multiple range test (p < 0.05).

TABLE 1.

Photooxidation of edible oils using erythrocytes as a hemoglobin‐based photocatalytic system under various reaction conditions a .

Entry Reaction condition PV (meq O2/kg)
1 Edible oil + erythrocyte + air + visible light 9.7
2 Edible oil + air + visible light Trace
3 Edible oil + erythrocyte + air Trace
4 Edible oil + erythrocyte + visible light Trace
5 b Edible oil + erythrocyte + air + sunlight 10.5
6 c Edible oil + erythrocyte + air + visible light + BHT 3.8
7 d Edible oil + erythrocyte + air + visible light + NaN3 3.1
Open in a new tab
a

Solution of edible oil (2 ⨯ 10−2 v/v) and erythrocyte (2 ⨯ 10−2 v/v) was illuminated by a solar simulator (276 power LED lamps [1 W, 2.3 V, 59,660 lux, 380–780 nm, 8.74 mW/cm2]) for 2.5 h at 25°C under 1 atm air bubble in a water/acetonitrile (9/1 v/v) solution.

b

The sunlight intensity was measured as 61,200 lux.

c

0.0016 mmol NaN3 was used.

d

0.0016 mmol BHT was used.

These scavenger experiments provide evidence for the coexistence of dual oxidative pathways involving both singlet oxygen and radical‐mediated reactions. Also, as shown in Figure 1b, the PVs increased progressively during the first 2.5 h of irradiation, reaching a maximum value of 9.7 meq kg−1. A subsequent slight decrease in PV was observed, which may be attributed to the decomposition of unstable primary hydroperoxides into secondary oxidation products. This trend indicates the continuous progression of lipid oxidative degradation within the oil matrix under the photocatalytic conditions.

3.2. Product‐Level NMR Evidence for Fatty Acid Oxidation Pathways

1H NMR analysis revealed light‐induced alterations in the fatty acid composition of edible oil in the presence of hemoglobin (Figure 2). To better understand the underlying oxidation mechanism, 1H NMR spectroscopy was employed to simultaneously detect multiple oxidation products and structural changes, including the formation of MDA (Guillén and Uriarte 2012; Hwang 2015, 2017; Liu et al. 2022; Ma et al. 2019). The vinylic proton signals (δ 5.2–5.4 ppm), which are susceptible to singlet oxygen‐mediated ene reactions (Bacellar and Baptista 2019), decreased by 31.2%, whereas the bis‐allylic proton signals (δ 2.7–2.8 ppm) decreased by 36.9%. The greater depletion of bis‐allylic protons is consistent with hydrogen‐abstraction reactions typically associated with radical‐mediated lipid oxidation pathways and may suggest the involvement of high‐valent heme intermediates (Davies and Guo 2014; Huang and Groves 2017). According to previous studies, these spectral changes indicate extensive oxidation at both double‐bond and bis‐allylic sites (Hwang 2017; Tynkkynen et al. 2012). Additionally, the appearance of a new proton resonance at δ 9.5 ppm (Figure 2b), confirms the formation of malondialdehyde (MDA), a well‐established marker of advanced lipid peroxidation and oxidative chain‐scission processes (Bacellar and Baptista 2019; Michel et al. 2008). Taken together, these spectral features demonstrated that 1O2‐mediated ene reactions (Huang and Groves 2017) and ferryl‐driven radical pathways (Huang and Groves 2017; Wilson and Reeder 2022) may operate concurrently in this photoreaction system.

FIGURE 2.

FIGURE 2

Open in a new tab

(a) 1H NMR spectra of edible oil before and (b) after photooxidation in the presence of erythrocytes.

3.3. Anthracene Bleaching as a Probe for Singlet Oxygen

In the Hb–edible oil system, singlet oxygen generation was directly evaluated using anthracene as a well‐known 1O2 probe (Joseph and Kumar 2023). The anthracene absorption band at 375 nm underwent approximately 33% photobleaching after 4 h of irradiation under visible light, indicating the formation of the corresponding endoperoxide product (Figure 3). Importantly, the addition of NaN3 as a recognized 1O2 quencher significantly inhibited the bleaching process. This suppression confirms that anthracene degradation was primarily driven by reactions involving 1O2 rather than alternative radical‐mediated pathways. These findings demonstrate that hemoglobin can act as an effective natural photosensitizing mediator for singlet oxygen generation under mild sunlight conditions.

FIGURE 3.

FIGURE 3

Open in a new tab

(a) UV–Vis absorption spectra of anthracene before and after irradiation in the presence of erythrocytes. (b) Schematic representation of anthracene photooxygenation with 1O2. Different uppercase letters indicate statistically significant differences according to Duncan's multiple range test (p < 0.05).

3.4. Solvent Effects on the Efficiency of Photooxidation

The addition of polar organic solvents significantly influenced the oxidation efficiency of edible oil in the presence of hemoglobin. The observed oxidation activity followed the order: acetonitrile > ethanol > acetone > methanol > DMSO (Table 2). This trend is consistent with the reported solvent‐dependent lifetimes of singlet oxygen, with 1O2 exhibiting a longer lifetime in acetonitrile (~65 μs) compared with DMSO (~19 μs) (Chen et al. 2001). Furthermore, highly coordinating solvents such as DMSO may inhibit the formation of reactive ferryl heme intermediates (HbFe(IV)═O) (Hajimohammadi et al. 2011) and suppress associated radical/ferryl‐mediated oxidation pathways (Sasak et al. 2024). Therefore, the lower oxidation efficiency observed in DMSO may be attributed to its stronger quenching effects toward both singlet oxygen and ferryl intermediates.

TABLE 2.

Photooxidation of edible oil by hemoglobin in the presence of different solvents. a

Entry Solvent PV (meq O2/kg)
1 Acetonitrile 9.7
2 Ethanol 8.5
3 Acetone 7.6
4 Methanol 7.4
5 DMSO 2.6
Open in a new tab
a

Solution of edible oil (2⨯10−2 v/v) and erythrocyte (2 ⨯ 10−2 v/v) was illuminated by a solar simulator (276 power LED lamps [1 W, 2.3 V, 59,660 lux, 380–780 nm, 8.74 mW/cm2]) for 2.5 h at 25°C under 1 atm air bubble in a water/solvent (9/1 v/v) solution.

3.5. Comparison of Hemoglobin With Standard Oxidants

The photodegradation rate of edible oil based on PV (meq O2/kg) with different catalysts and oxidant followed the order Hb (9.7) > Rose Bengal (6.8) > KMnO4 (2.1). Rose Bengal acts as a selective generator of 1O2 (Fiegler‐Rudol et al. 2025), while KMnO4 mediates a classical electron‐transfer oxidation pathway (X. Wang et al. 2023). The superior performance of hemoglobin suggests that the synergistic contribution of 1O2 generation and ferryl‐mediated oxidation may results in enhanced oxidative activity compared with single‐pathway oxidants.

4. Discussion

4.1. Dual Oxidative Mechanism of Hemoglobin in Sunlight‐Induced Lipid Peroxidation

The present study was designed primarily to elucidate the mechanism of hemoglobin‐mediated sunlight‐induced lipid oxidation. The experimental results also provide preliminary evidence that this natural protein may be useful as a sustainable photosensitizer for lipid‐rich wastewater treatment. The findings of the present study suggest that hemoglobin promotes lipid oxidation through two possible pathways: 1O2‐mediated oxidation and radical‐mediated reactions. The contribution of singlet oxygen is supported by several observations, including anthracene bleaching, inhibition by NaN3 (a known 1O2 quencher), solvent‐dependent oxidation patterns consistent with 1O2 lifetime differences, and the formation of MDA accompanied by selective oxidation of vinylic sites. These results are reliable with established 1O2–driven lipid oxidation mechanisms in porphyrin‐based systems. In contrast, the formation of a ferryl (HbFe(IV)═O)‐type species was not directly confirmed in the present study. Its possible involvement is proposed based on indirect evidence, including the inhibitory effect of BHT, the decrease in bis‐allylic proton signals observed by 1H NMR, and the influence of DMSO on oxidation behavior. These observations are consistent with hydrogen‐abstraction reactions typically associated with high‐valent heme species. This interpretation is supported by previous studies on related heme systems, where ferryl intermediates have been detected, including UV–Vis observation of (TMPFe(IV)═O)2+ in metalloporphyrin models (Hajimohammadi et al. 2011) and mass spectrometric detection of Fe(IV)═O–protoporphyrin IX+ under oxidative conditions (Hajimohammadi, Verjani, et al. 2018). Also, hemoglobin exhibited higher oxidative efficiency than Rose Bengal and KMnO4, suggesting that its activity arises from the combined action of singlet oxygen chemistry and additional radical pathways rather than a single dominant mechanism.

4.2. Proposed Pathway for Hemoglobin‐Mediated Photodegradation of Edible Oil

The proposed dual oxidative mechanism underlying hemoglobin‐mediated photodegradation of edible oil is illustrated in Figure 4. Upon light exposure, hemoglobin promotes lipid oxidation through the combined contribution of 1O2‐mediated and radical‐type pathways. The 1O2 pathway is proposed to initiate oxidation through ene‐type reactions at vinylic positions (δ 5.2–5.4 ppm, 1H NMR), leading to hydroperoxide formation and weakening of the lipid structure. In parallel, a ferryl heme–associated pathway may contribute to oxidation at bis‐allylic positions (δ 2.7–2.8 ppm, 1H NMR) through hydrogen abstraction reactions, promoting secondary radical reactions and fatty acid chain degradation. 1H NMR analysis also revealed the formation of MDA as an aldehydic product, demonstrating the cleavage of unsaturated fatty acids into smaller, more polar oxidation products. These compounds are products of lipid degradation and serve as molecular indicators of the progressive breakdown of the oil matrix during photooxidation. Overall, the synergistic contribution of 1O2‐mediated oxidation and possible ferryl‐associated radical processes may explain the high photodegradation efficiency of hemoglobin and supports its potential application in natural or biomimetic photo‐oxidative systems. In addition to the changes observed in the unsaturated regions, the glycerol proton signals at ~4.10 ppm exhibited noticeable perturbation, suggesting degradation of the –CH2–O–CO– ester linkages within the triglyceride backbone. Such alterations are characteristic of advanced lipid peroxidation, where radical‐driven β‐scission cleaves the ester bond connecting fatty acids to glycerol. This evidence is consistent with established mechanisms of lipid oxidation, in which high‐valent heme species such as ferryl intermediates can promote hydrogen abstraction and radical‐mediated oxidation processes that may contribute to triglyceride fragmentation (Kamal‐Eldin 2003; Porter et al. 1995). In contrast, singlet oxygen primarily reacts with C═C bonds through selective oxidation pathways and has limited reactivity toward ester moieties.

FIGURE 4.

FIGURE 4

Open in a new tab

Proposed mechanisms for hemoglobin‐mediated photooxidation of edible oil involving singlet oxygen generation and ferryl/radical oxidation pathway.

4.3. Environmental Implications

Hemoglobin is a biodegradable, food‐derived protein obtained as a by‐product of the meat processing industry. Its abundance, low cost, and renewable nature make it a promising material for valorizing animal‐processing waste within circular food systems. As a light‐responsive mediator, it enables sunlight‐driven oxidation of lipid‐rich effluents without the need for synthetic oxidants, offering a greener and more resource‐efficient approach for food industry wastewater treatment. These features highlight its potential for sustainable lipid degradation and waste management applications.

4.4. Study Limitations and Future Perspectives

Several limitations of the present study should be considered. First, this study used a simplified edible oil model rather than real industrial effluents; therefore, the influence of complex wastewater compositions on oxidation efficiency requires further investigation. Second, the long‐term stability of hemoglobin under continuous irradiation and the feasibility of scale‐up, including light penetration, oxygen transfer, reactor design, and protein recovery, remain to be evaluated. In addition, although the findings support a possible ferryl‐associated pathway, direct spectroscopic confirmation was not performed and should be addressed in future studies. Despite these limitations, this work highlights the potential of hemoglobin as a food‐derived photosensitizer for sustainable sunlight‐driven lipid oxidation and wastewater treatment applications.

5. Conclusions

This study primarily provides mechanistic insight into sunlight‐induced lipid oxidation mediated by erythrocyte‐derived hemoglobin. The collective experimental evidence supports the coexistence of singlet oxygen‐mediated oxidation and a likely radical‐associated pathway, although direct spectroscopic evidence for ferryl intermediates was not obtained. Beyond advancing understanding of hemoglobin‐mediated photooxidation, the results also suggest that blood‐derived proteins could serve as low‐cost, sustainable photosensitizers for future treatment of lipid‐rich wastewater. The synergistic interaction of these pathways promotes extensive oxidation of unsaturated fatty acids, resulting in hydroperoxide formation, MDA generation, and progressive lipid degradation. Beyond providing mechanistic insight into heme‐mediated lipid oxidation relevant to food storage and processing, these findings highlight the potential of food‐derived proteins as sustainable functional materials. Given its biodegradability, abundance as a meat industry by‐product, and low cost, hemoglobin represents a promising candidate for valorization within circular food systems. Future work should focus on protein stabilization, immobilization strategies, and bioinspired hybrid materials to improve photooxidative performance and enable scalable, solar‐driven applications for the treatment of lipid‐rich effluents in food processing industries.

Author Contributions

Mahdi Hajimohammadi: conceptualization, investigation, funding acquisition, writing – original draft, writing – review and editing, visualization, validation, methodology, software, formal analysis, project administration, data curation, supervision, resources. Fadhel H. Faraj: resources, writing – review and editing, funding acquisition, project administration. Mona Boghdachi: software, formal analysis, methodology, data curation. Asawer A. Alwasiti: conceptualization, resources, project administration, writing – review and editing. Zainab Y. Shnain: writing – review and editing, resources, project administration, funding acquisition.

Funding

This work was supported by Kharazmi University, 404703.

Ethics Statement

Defibrinated sheep blood used in this study was commercially obtained from Kimiakavoshazma Company (Iran). No animals were directly handled, housed, sampled, or subjected to experimental procedures by the authors. The study involved only commercially supplied biological material and did not include live animal experimentation. Therefore, institutional animal ethics approval was not required according to the applicable institutional guidelines.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

This research was supported by a grant from Kharazmi University, Tehran, Iran (No. 404703).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Abeyrathne, E. D. N. S. , Nam K., and Ahn D. U.. 2021. “Analytical Methods for Lipid Oxidation and Antioxidant Capacity in Food Systems.” Antioxidants 10, no. 10: 1587. 10.3390/antiox10101587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Afonso, S. , Enriquez de Salamanca R., and Batlle A. d. C.. 1999. “The Photodynamic and Non‐Photodynamic Actions of Porphyrins.” Brazilian Journal of Medical and Biological Research 32: 255–266. 10.1590/S0100-879X1999000300002. [DOI] [PubMed] [Google Scholar]
  3. Agnez‐Lima, L. F. , Melo J. T., Silva A. E., et al. 2012. “DNA Damage by Singlet Oxygen and Cellular Protective Mechanisms.” Mutation Research/Reviews in Mutation Research 751, no. 1: 15–28. 10.1016/j.mrrev.2011.12.005. [DOI] [PubMed] [Google Scholar]
  4. Ahmad, T. , Belwal T., Li L., et al. 2020. “Utilization of Wastewater From Edible Oil Industry, Turning Waste Into Valuable Products: A Review.” Trends in Food Science & Technology 99: 21–33. 10.1016/j.tifs.2020.02.017. [DOI] [Google Scholar]
  5. Alatabe, M. J. A. , Ghorbanpour M., and Farzi A.. 2024. “Adsorption and Advanced Oxidation Processes for Sustainable Treatment of Oil Produced Water and Organic Wastewater. A Review.” Russian Journal of Applied Chemistry 97, no. 10: 751–771. 10.1134/S1070427224100045. [DOI] [Google Scholar]
  6. Ayoub, M. 2022. “Fenton Process for the Treatment of Wastewater Effluent From the Edible Oil Industry.” Water Science & Technology 86, no. 6: 1388–1401. 10.2166/wst.2022.283. [DOI] [PubMed] [Google Scholar]
  7. Bacellar, I. O. , and Baptista M. S.. 2019. “Mechanisms of Photosensitized Lipid Oxidation and Membrane Permeabilization.” ACS Omega 4, no. 26: 21636–21646. 10.1021/acsomega.9b03244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Barthel, G. , and Grosch W.. 1974. “Peroxide Value Determination—Comparison of Some Methods.” Journal of the American Oil Chemists' Society 51, no. 12: 540–544. 10.1007/BF02636025. [DOI] [Google Scholar]
  9. Boulebd, H. 2020. “Comparative Study of the Radical Scavenging Behavior of Ascorbic Acid, BHT, BHA and Trolox: Experimental and Theoretical Study.” Journal of Molecular Structure 1201: 127210. 10.1016/j.molstruc.2019.127210. [DOI] [Google Scholar]
  10. Chen, Y. , Xu S., Li L., Zhang M., Shen J., and Shen T.. 2001. “Active Oxygen Generation and Photo‐Oxygenation Involving Temporfin (m‐THPC).” Dyes and Pigments 51, no. 2–3: 63–69. 10.1016/S0143-7208(01)00071-7. [DOI] [Google Scholar]
  11. Davies, S. S. , and Guo L.. 2014. “Lipid Peroxidation Generates Biologically Active Phospholipids Including Oxidatively N‐Modified Phospholipids.” Chemistry and Physics of Lipids 181: 1–33. 10.1016/j.chemphyslip.2014.03.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. De Jager, T. , Cockrell A., and Du Plessis S.. 2017. “Ultraviolet Light Induced Generation of Reactive Oxygen Species.” In Ultraviolet Light in Human Health, Diseases and Environment, vol. 993, 15–23. Advances in Experimental Medicine and Biology. 10.1007/978-3-319-56017-5_2. [DOI] [PubMed] [Google Scholar]
  13. Dodge, J. T. , Mitchell C., and Hanahan D. J.. 1963. “The Preparation and Chemical Characteristics of Hemoglobin‐Free Ghosts of Human Erythrocytes.” Archives of Biochemistry and Biophysics 100, no. 1: 119–130. 10.1016/0003-9861(63)90042-0. [DOI] [PubMed] [Google Scholar]
  14. Domínguez, R. , Pateiro M., Gagaoua M., Barba F. J., Zhang W., and Lorenzo J. M.. 2019. “A Comprehensive Review on Lipid Oxidation in Meat and Meat Products.” Antioxidants 8, no. 10: 429. 10.3390/antiox8100429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dordević, D. , Vítězová M., Vítěz T., Dordevic S., Hamšíková M., and Kushkevych I.. 2025. “Enhancing Wastewater Treatment Efficiency: Utilising Saponification Products for Sustainable Cleaning Processes.” Environmental Microbiology Reports 17, no. 4: e70124. 10.1111/1758-2229.70124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Droge, W. 2002. “Free Radicals in the Physiological Control of Cell Function.” Physiological Reviews 82, no. 1: 47–95. 10.1152/physrev.00018.2001. [DOI] [PubMed] [Google Scholar]
  17. Durand, E. , Laguerre M., Bourlieu‐Lacanal C., Lecomte J., and Villeneuve P.. 2025. “Navigating the Complexity of Lipid Oxidation and Antioxidation: A Review of Evaluation Methods and Emerging Approaches.” Progress in Lipid Research 97: 101317. 10.1016/j.plipres.2024.101317. [DOI] [PubMed] [Google Scholar]
  18. Fiegler‐Rudol, J. , Lipka B., Kapłon K., et al. 2025. “Evaluating the Efficacy of Rose Bengal as a Photosensitizer in Antimicrobial Photodynamic Therapy Against Candida albicans : A Systematic Review.” International Journal of Molecular Sciences 26, no. 11: 5034. 10.3390/ijms26115034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gogate, P. R. , and Pandit A. B.. 2004. “A Review of Imperative Technologies for Wastewater Treatment I: Oxidation Technologies at Ambient Conditions.” Advances in Environmental Research 8, no. 3–4: 501–551. 10.1016/S1093-0191(03)00032-7. [DOI] [Google Scholar]
  20. Guillén, M. D. , and Uriarte P. S.. 2012. “Monitoring by 1H Nuclear Magnetic Resonance of the Changes in the Composition of Virgin Linseed Oil Heated at Frying Temperature: Comparison With the Evolution of Other Edible Oils.” Food Control 28, no. 1: 59–68. 10.1016/j.foodcont.2012.04.024. [DOI] [Google Scholar]
  21. Hajimohammadi, M. , and Moradi S.. 2025. “High Inhibitory Effect of 2‐Nitrobenzaldehyde on the Photodegradation of Erythrocytes, the Production of Singlet Oxygen, and Meat Color Change.” Journal of Food Processing and Preservation 2025, no. 1: 8039409. 10.1155/jfpp/8039409. [DOI] [Google Scholar]
  22. Hajimohammadi, M. , and Nosrati P.. 2018. “Scavenging Effect of Pasipay (Passiflora Incarnate L.) on Singlet Oxygen Generation and Fatty Acid Photooxygenation.” Food Science & Nutrition 6, no. 6: 1670–1675. 10.1002/fsn3.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hajimohammadi, M. , Safari N., Mofakham H., and Deyhimi F.. 2011. “Highly Selective, Economical and Efficient Oxidation of Alcohols to Aldehydes and Ketones by Air and Sunlight or Visible Light in the Presence of Porphyrins Sensitizers.” Green Chemistry 13, no. 4: 991–997. 10.1039/C0GC00910E. [DOI] [Google Scholar]
  24. Hajimohammadi, M. , Sheikh Mahboobi F., and Wu H.. 2024. “Suppressing Effect of Flavonoid Compounds on Lipids Photooxidation of Sheep Red Blood Cells and Oleic Acid Photooxidation.” Food Science & Nutrition 12, no. 12: 10405–10411. 10.1002/fsn3.4539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hajimohammadi, M. , Vaziri Sereshk A., Schwarzinger C., and Knör G.. 2018. “Suppressing Effect of 2‐Nitrobenzaldehyde on Singlet Oxygen Generation, Fatty Acid Photooxidation, and Dye‐Sensitizer Degradation.” Antioxidants 7, no. 12: 194. 10.3390/antiox7120194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hajimohammadi, M. , Verjani M. K., Ghasemi H., Safari N., and Knoer G.. 2018. “Selective Conversion of Aldehydes to Carboxylic Acids by Hemoglobin and Air.” Journal of Porphyrins and Phthalocyanines 22, no. 8: 679–685. 10.1142/S1088424618500840. [DOI] [Google Scholar]
  27. Huang, X. , and Ahn D. U.. 2019. “Lipid Oxidation and Its Implications to Meat Quality and Human Health.” Food Science and Biotechnology 28: 1275–1285. 10.1007/s10068-019-00631-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Huang, X. , and Groves J. T.. 2017. “Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins.” Chemical Reviews 118, no. 5: 2491–2553. 10.1021/acs.chemrev.7b00373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hwang, H.‐S. 2015. “NMR Spectroscopy for Assessing Lipid Oxidation.” Lipid Technology 27, no. 8: 187–189. 10.1002/lite.201500037. [DOI] [Google Scholar]
  30. Hwang, H.‐S. 2017. Advances in NMR Spectroscopy for Lipid Oxidation Assessment. Springer. [Google Scholar]
  31. Jiang, D. , Xin C., Li W., et al. 2015. “Quantitative Analysis and Health Risk Assessment of Polycyclic Aromatic Hydrocarbons in Edible Vegetable Oils Marketed in Shandong of China.” Food and Chemical Toxicology 83: 61–67. 10.1016/j.fct.2015.06.001. [DOI] [PubMed] [Google Scholar]
  32. Joseph, S. , and Kumar S. A.. 2023. “Recent Advances in the Photophysical Detection and Delivery of Singlet Oxygen.” Coordination Chemistry Reviews 496: 215408. 10.1016/j.ccr.2023.215408. [DOI] [Google Scholar]
  33. Kamal‐Eldin, A. 2003. Lipid Oxidation Pathways. AOCS Publishing. 10.1201/9781003040316. [DOI] [Google Scholar]
  34. Kasaai, M. 2025. “Oxidative and Hydrolytic Deteriorations of Lipids and Several Alternative Pathways for Their Protections: An Overview.” Food Nutrition Chemistry 3, no. 1: 238. 10.18686/fnc238. [DOI] [Google Scholar]
  35. Li, M. , Cline C., Koker E., Carmichael H., Chignell C., and Bilski P.. 2001. “Quenching of Singlet Molecular Oxygen (1O2) by Azide Anion in Solvent Mixtures.” Photochemistry and Photobiology 74, no. 6: 760–764. 10.1562/0031-8655(2001)074<0760:qosmoo>2.0.co;2. [DOI] [PubMed] [Google Scholar]
  36. Liu, J. , Wang Y. J., Huang X. Y., Li X. Z., Ma C. G., and McClements D. J.. 2022. “NMR Analysis of Lipid Oxidation in Flaxseed Oil‐in‐Water Emulsions.” Journal of Agricultural and Food Chemistry 70, no. 27: 8417–8429. 10.1021/acs.jafc.2c00951. [DOI] [PubMed] [Google Scholar]
  37. Looper, J. , and Vierck K. R.. 2023. “Determining the Effectiveness of Rosemary Essential Oil on the Shelf Life of Ground Beef Under Different Lighting Conditions.” Discovery, the Student Journal of Dale Bumpers College of Agricultural, Food and Life Sciences 24, no. 1: 1–20. [Google Scholar]
  38. Ma, L. , Liu G., and Liu X.. 2019. “Amounts of Malondialdehyde Do Not Accurately Represent the Real Oxidative Level of All Vegetable Oils: A Kinetic Study of Malondialdehyde Formation.” International Journal of Food Science & Technology 54, no. 2: 412–423. 10.1111/ijfs.13952. [DOI] [Google Scholar]
  39. MacDougall, D. B. 1982. “Changes in the Colour and Opacity of Meat.” Food Chemistry 9, no. 1: 75–88. 10.1016/0308-8146(82)90070-X. [DOI] [Google Scholar]
  40. Machado, M. , Rodriguez‐Alcalá L. M., Gomes A. M., and Pintado M.. 2023. “Vegetable Oils Oxidation: Mechanisms, Consequences and Protective Strategies.” Food Reviews International 39, no. 7: 4180–4197. 10.1080/87559129.2022.2026378. [DOI] [Google Scholar]
  41. Malato, S. , Fernández‐Ibáñez P., Maldonado M. I., Blanco J., and Gernjak W.. 2009. “Decontamination and Disinfection of Water by Solar Photocatalysis: Recent Overview and Trends.” Catalysis Today 147, no. 1: 1–59. 10.1016/j.cattod.2009.06.018. [DOI] [Google Scholar]
  42. Meunier, B. 2000. “Models of Heme Peroxidases and Catalases.” In Biomimetic Oxidations Catalyzed by Transition Metal Complexes, edited by Meunier B., 171–214. World Scientific. 10.1142/9781848160699_0004. [DOI] [Google Scholar]
  43. Michel, F. , Bonnefont‐Rousselot D., Mas E., Drai J., and Thérond P.. 2008. “Biomarkers of Lipid Peroxidation: Analytical Aspects.” Annales de Biologie Clinique 66, no. 6: 605–620. 10.1684/abc.2008.0283. [DOI] [PubMed] [Google Scholar]
  44. Min, D. , and Boff J.. 2002. “Chemistry and Reaction of Singlet Oxygen in Foods.” Comprehensive Reviews in Food Science and Food Safety 1, no. 2: 58–72. 10.1111/j.1541-4337.2002.tb00007.x. [DOI] [PubMed] [Google Scholar]
  45. Minning, D. M. , Gow A. J., Bonaventura J., et al. 1999. “Ascaris Haemoglobin Is a Nitric Oxide‐Activated ‘Deoxygenase’.” Nature 401, no. 6752: 497–502. 10.1038/46822. [DOI] [PubMed] [Google Scholar]
  46. Nagababu, E. , and Rifkind J. M.. 2004. “Heme Degradation by Reactive Oxygen Species.” Antioxidants & Redox Signaling 6, no. 6: 967–978. 10.1089/ars.2004.6.967. [DOI] [PubMed] [Google Scholar]
  47. Narra, S. , and Shahpasand M.. 2025. “Developing a Chemical Process for Optimizing Oil Extraction From Cooking Oil Secondary Waste.” Recycling 10, no. 2: 35. 10.3390/recycling10020035. [DOI] [Google Scholar]
  48. Papuc, C. , Goran G. V., Predescu C. N., and Nicorescu V.. 2017. “Mechanisms of Oxidative Processes in Meat and Toxicity Induced by Postprandial Degradation Products: A Review.” Comprehensive Reviews in Food Science and Food Safety 16, no. 1: 96–123. 10.1111/1541-4337.12241. [DOI] [PubMed] [Google Scholar]
  49. Porter, N. A. , Caldwell S. E., and Mills K. A.. 1995. “Mechanisms of Free Radical Oxidation of Unsaturated Lipids.” Lipids 30, no. 4: 277–290. 10.1007/bf02536034. [DOI] [PubMed] [Google Scholar]
  50. Sasak, K. , Nowak M., Wlodarczyk A., Sarniak A., and Nowak D.. 2024. “Effect of Selected Organic Solvents on Hydroxyl Radical‐Dependent Light Emission in the Fe2+−EGTA‐H2O2 System.” Molecules 29, no. 23: 5635. 10.3390/molecules29235635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sebranek, J. , and Bacus J.. 2007. “Natural and Organic Cured Meat Products: Regulatory, Manufacturing, Marketing, Quality and Safety Issues.” American Meat Science Association White Paper Series 1: 1–15. [Google Scholar]
  52. Shetty, S. , Singh D. K., Chudasama M. H., Pradhan R. C., and Perumal T.. 2026. “Advances in Active/Intelligent Packaging Systems for the Detection and Prevention of Lipid Oxidation in Foods.” Food Reviews International 42, no. 6: 4543–4586. 10.1080/87559129.2026.2631782. [DOI] [Google Scholar]
  53. Talbot, G. 2016. “The Stability and Shelf Life of Fats and Oils.” In The Stability and Shelf Life of Food, edited by Subramaniam P., 461–503. Elsevier. 10.1016/B978-0-08-100435-7.00016-2. [DOI] [Google Scholar]
  54. Tynkkynen, T. , Mursu J., Nurmi T., Tuppurainen K., Laatikainen R., and Soininen P.. 2012. “NMR Protocol for Determination of Oxidation Susceptibility of Serum Lipids and Application of the Protocol to a Chocolate Study.” Metabolomics 8, no. 3: 386–398. 10.1007/s11306-011-0323-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Urrutia, R. I. , Aagaard T. F., Gutierrez V. S., González J. O. W., Frechero M. A., and Volpe M. A.. 2024. “Co‐Production of Bioinsecticide and Biochar From Sunflower Edible Oil Waste: A Preliminary Feasibility Study.” Bioresource Technology Reports 26: 101836. 10.1016/j.biteb.2024.101836. [DOI] [Google Scholar]
  56. Versino, F. , Ortega F., Monroy Y., Rivero S., López O. V., and García M. A.. 2023. “Sustainable and Bio‐Based Food Packaging: A Review on Past and Current Design Innovations.” Food 12, no. 5: 1057. 10.3390/foods12051057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wang, C. , Xiao W., Li Q., Tian X., Xu J., and Ding K.. 2025. “Oil Removal From Wastewater With Biomass‐Derived Hydrochars Laboratory Insights.” Scientific Reports 15, no. 1: 22176. 10.3390/foods1205105710.1038/s41598-025-07240-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wang, X. , Guo D., Zhang J., Yao Y., and Liu Y.. 2023. “Insights Into the Electron Transfer Mechanisms of Permanganate Activation by Carbon Nanotube Membrane for Enhanced Micropollutants Degradation.” Frontiers of Environmental Science & Engineering 17, no. 9: 106. 10.1007/s11783-023-1706-0. [DOI] [Google Scholar]
  59. Wilson, M. T. , and Reeder B. J.. 2022. “The Peroxidatic Activities of Myoglobin and Hemoglobin, Their Pathological Consequences and Possible Medical Interventions.” Molecular Aspects of Medicine 84: 101045. 10.1016/j.mam.2021.101045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wu, H. , Tatiyaborworntham N., Hajimohammadi M., Decker E. A., Richards M. P., and Undeland I.. 2024. “Model Systems for Studying Lipid Oxidation Associated With Muscle Foods: Methods, Challenges, and Prospects.” Critical Reviews in Food Science and Nutrition 64, no. 1: 153–171. 10.1080/10408398.2022.2105302. [DOI] [PubMed] [Google Scholar]
  61. Zhao, Z.‐Q. , Zhang T., Shi T.‐Q., Barcelo D., and Zheng H.‐B.. 2024. “Biodegradation and High‐Value Utilization of Waste Cooking Oil: Strategies and Mechanisms for Producing Lipopeptide Biosurfactants Using Bacillus subtilis YZQ‐2.” Journal of Environmental Chemical Engineering 12, no. 6: 114549. 10.1016/j.jece.2024.114549. [DOI] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Articles from Food Science & Nutrition are provided here courtesy of Wiley

RESOURCES