Role of Gamma Irradiation in Enhancement of Nutrition and Flavor Quality of Soybean

Abstract

Soybean has the potential to be termed the “crop of the future” due to its significant capacity to address protein-energy malnutrition and hidden hunger, particularly in developing countries where diets are predominantly based on wheat and rice. Despite its substantial nutritional value, numerous health benefits, and its versatility in various food and industrial applications, soybean’s full potential remains underutilized due to inherent off-flavors and the presence of antinutritional factors (ANFs). Gamma irradiation is known to have a positive impact by inducing structural and chemical changes in biomolecules like carbohydrates, lipids, proteins, and other phytochemicals. This process leads to improved functionality and market demand by reducing ANFs and the off-flavor in soybeans. Scientifically, it has been demonstrated that low to moderate doses of gamma radiation, up to 10 kGy, can positively influence the antioxidant capacity of soybeans. This, in turn, helps control lipid and protein oxidation, reducing the generation of off-flavors and enhancing the quality and nutraceutical potential of soybeans.

1. Introduction

Soybeans hold the position of being the most widely cultivated oilseed crop, both in India and across the world. According to data from Our World in Data in 2021, soybeans were grown on around 129.52 million hectares of land globally. In 2021, global soybean production reached 371.69 million metric tons, and there are expectations that this figure will rise to 391.17 million metric tons by 2023. The major countries contributing to soybean production are the United States, Brazil, Argentina, China, and India.

Soybean seeds have a composition comprising roughly 27% complex carbohydrates, 40% protein, 20% oil, 8% moisture, and 5% minerals. This nutritional profile positions soybean as a valuable resource for addressing malnutrition and undernutrition, particularly in developing countries. Soybean’s protein quality, measured by the protein digestibility corrected amino acid score, is on par with protein sources derived from animals.

Irradiation involves the use of radiation on foods primarily for extended hygiene and safety purposes and others too. It is a low-cost, environment-friendly, non-thermal physical processing technology widely used in the food industry. It has been reported to reduce food allergenicity, effectively killing insects, molds, and bacteria [1, 2] and also to minimize the harmful substances such as biogenic amines [3] and anti-nutritional factors present in food [4]. Besides, when applied in the proper doses, it has no detrimental effect on the physicochemical, nutritional, and sensorial quality of food [5, 6, 7].

Irradiation is categorized into two types: ionizing radiation, which includes UV-C, γ-irradiation, electron beams, and X-rays, and non-ionizing radiation, which encompasses ultraviolet, visible light, and infrared. Ionizing radiation possesses higher energy levels compared to non-ionizing radiation. Among these irradiation methods, gamma irradiation is the most commonly used due to its exceptional penetration capabilities, as highlighted in a study by Liu et al. [8].

Generally, electron beam irradiation can only penetrate the surface of food to a depth of 50–150 mm, as noted by Fan et al. [9], while γ-irradiation can deeply and readily penetrate various materials, as mentioned by Ekezie et al. [10]. In industrial applications, the two primary sources of gamma radiation are cobalt-60 (⁶⁰Co) and iridium-192 (¹⁹²Ir). The energy of ionizing radiation is measured in electronvolts (eV), but due to the small size of an electronvolt, it’s common to express ionizing radiation energy in megaelectronvolts (MeV). Co-60 emits two γ-rays simultaneously with energies of approximately 1.17 and 1.33 MeV during its radioactive decay process. Additionally, ⁶⁰Co emits a low-energy electron (beta particle) with a maximum energy of around 0.3 MeV [9]. ¹⁹²Ir emits gamma rays at energies of 0.31, 0.47, and 0.60 MeV. Given that the radiation from ⁶⁰Co has roughly twice the energy of that from ¹⁹²Ir and possesses greater penetration power through materials, making it more hazardous and requiring more substantial shielding. The standard unit for measuring the absorbed dose of γ-radiation is the “rad” (Radiation Absorbed Dose). It is defined as a dose of 100 ergs of energy per gram of the given material. The International System of Units (SI) unit for measuring absorbed radiation is the gray (Gy), defined as joules per kilogram of mass. One gray is equivalent to 100 rads.

The impact of γ-irradiation on food depends on whether it is in a dry or aqueous state. In solid foods, the food molecules directly absorb radiation energy, leading to structural changes. In the case of aqueous foods, when exposed to γ-radiation, water generates hydroxyl radicals and hydrated electrons, which subsequently react with biomolecules to form covalent bonds [11]. Proteins in soybeans are influenced by γ-irradiation through mechanisms such as conformational changes [12] and the promotion of amino acid oxidation, peptide bond cleavage, and the formation of disulfide bonds [13]. γ-irradiation has a long history as a food preservation method. Currently, it is widely acknowledged that foods irradiated at doses below 10 kGy are safe and pose no toxicity risks to humans [7, 14]. The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) have also stated that the maximum absorbed dose delivered to foods should not exceed 10 kGy, except when it is necessary to achieve a legitimate technological purpose [15]. However, it’s important to note that the specific irradiation dose can vary based on the type of food, its intended use, and regulations in different countries. For instance, in the United States, the irradiation dose for dry spices/seasonings is 30 kGy, and for frozen packaged meats, it is 44 kGy.

Hence, the impact of γ-irradiation on the nutritional aspects and flavor of soybean seeds and their processed products has been extensively explored in the literature. This study could potentially serve as a valuable resource for preserving the nutritional value of soybeans and enhancing their bioavailability and scientific acceptance to its worldwide consumers.

2. Impact of γ-irradiation processing on inherent properties of soybean

Ionizing radiation is a highly efficient method for controlling pathogens, preventing spoilage, and enhancing the storage life of various food products, including spices, herbs, fresh fruits, vegetables, leguminous seeds, and cereals. Research conducted by the World Health Organization has confirmed that food irradiation does not raise any significant concerns regarding toxicity, microbiology, or nutritional content [16]. However, it does induce chemical changes that may impact the overall quality of the products. It is generally considered safe and nutritionally sound to consume food irradiated at doses up to 10 kGy, with some products even approved for higher doses [17].

The effects of γ-irradiation on various inherent properties of soybeans are summarized in Figure 1 and extensively discussed in subsequent sections.

Figure 1.

Effects of γ-irradiation on soybean. FAs: Fatty acids, FFAs: Free fatty acids, ROS: Reactive oxygen species, TAB: Thiobarbituric acid, Co- cobalt, Cs- Cesium. Recommended dose of γ-irradiation is 0.5–10 kGy. Down and up arrow represent decrease and increase respectively.

2.1 Physicochemical profile

Exposing soybean to γ-irradiation levels of up to 10 kGy has been observed to effectively preserve essential nutritional aspects. Soybean oil extracted from soybeans subjected to gamma radiation up to 10 kGy exhibited no significant changes in various physicochemical characteristics, including lipid content, fatty acid composition, acid value, peroxide value, and trans fatty acid content [18]. However, there was a clear trend of prolonged induction periods with higher radiation doses. Beyond the 10 kGy thresholds, a notable increase in n-hexanal content was detected, indicating its potential as an indicator of excessive radiation exposure in soybeans. Additionally, as the irradiation dose increases, there is a tendency towards an extended period of resistance to oxidation, which is known as the “induction period.” However, it’s worth noting that when irradiation doses exceed 10 kGy, there is a noticeable increase in the levels of n-hexanal, which can potentially serve as an indicator of excessive irradiation in soybeans. In the case of peanut seeds, an irradiation dose of 1 kGy is considered suitable. Nevertheless, a higher dose of 10 kGy has been found to significantly reduce the fat and protein content of peanuts while increasing their water activity. On the other hand, moisture, ash, and total sugar contents are unaffected by γ-irradiation [8]. γ-irradiation accelerates lipid oxidation, leading to increased values for fatty acid, peroxide, carbonyl, and malondialdehyde, while reducing lipase activity. Additionally, γ-irradiation induces changes in fatty acid and amino acid composition. Hazelnut kernels irradiated with a 0.5 kGy dose maintain high quality in terms of free fatty acids, peroxide content, and vitamin E [19]. The impact of γ-irradiation on protein properties and structures was examined, with doses up to 10 kGy tested. [20], studied the impact of mildew and gamma irradiation (using ⁶⁰Co-γ rays) on the digestibility, composition, and protein structure of soybeans. Mildew reduced pepsin digestibility and amino acid content while altering the protein’s secondary structure. In fresh soybeans, ⁶⁰Co-γ irradiation in the range of 10 to 30 kGy had no significant effect on amino acids and pepsin digestibility, except for changes in protein secondary structure. However, in mildew soybeans, γ-irradiation reduced amino acid content and caused structural changes in the protein. As the radiation dose increased, there was a partial reduction in protein concentration, resulting in decreased solubility in water. This could be attributed to alterations in protein conformation, potentially causing hydrophobic amino acid residues originally hidden within the protein to become exposed on its surface, thus increasing the protein’s hydrophobic nature. The irradiated samples displayed diminished emulsifying abilities and heightened surface hydrophobicity [21, 22, 23]. Furthermore, this dose does not adversely affect the sensory characteristics of hazelnut kernels. However, vitamin E levels decrease with increasing irradiation doses.

In oil seeds like soybean, peanut, and sesame, the proportions of induced unsaturated fatty acids are dependent on the irradiation dose, generally increasing with higher doses for different seed types [24]. The natural compounds found in soybeans, including isoflavones, Bowman-Birk factor, tocopherols, lecithin, lunasin, saponins, and others, have shown significant health benefits in combating conditions like cancer, atherosclerosis, diabetes, and osteoporosis, as evidenced by Kumar et al. [25] and Messina [26]. This has led to soybeans being recognized as a “functional food.” However, the global consumption of soybeans as a dietary source remains low, with only around 10% of soybean production being used as food, primarily in East and South East Asia. One of the reasons for this limited consumption is the presence of anti-nutritional factors like phytic acid, Kunitz trypsin inhibitor, and allergens, including Gly m Bd 68 K, a subunit of β-conglycinin, which can deter people from incorporating soybeans into their diets. Meinlschmidt et al. [27, 28] conducted research on the impact of γ-irradiation (at doses of 3–100 kGy) on the immunogenecity of soybean protein isolate and observed a remarkable reduction of 91–100% in the soluble protein fraction’s allergenicity. γ-irradiation achieves this by introducing structural changes, promoting protein aggregation or degradation, and destroying exposed epitopes [29]. The aversion to including soybeans in one’s diet is often linked not only to allergens but also to the characteristic beany flavor associated with soybean products. This off-flavor is primarily generated through three processes: automatic, photosensitized, and enzymatic oxidations. Among these, enzymatic degradation, specifically the breakdown of unsaturated fatty acids catalyzed by lipoxygenases (LOX) in soybean seeds, is the primary pathway for producing compounds responsible for the beany flavor [30]. This process involves the catalytic oxidation of polyunsaturated fatty acids containing 1,4-Z, Z-pentadiene structures, such as linoleic and α-linolenic acid, by soybean seed lipoxygenases. The resulting hydroperoxides are then further metabolized by hydroperoxide lyases, leading to the production of volatile aldehydes and ketones that give soy products their distinctive off-beany flavor [21, 22]. The beany flavor of soybean is associated with more than 20 volatile compounds, including fatty aldehydes, fatty alcohols, fatty ketones, furans, furan derivatives, and aromatic compounds [30]. Of these, hexanal and (Z)-3-hexenal are the most significant contributors [31]. Hexanal has a scent reminiscent of cut grass and green notes, with an incredibly low odor threshold of 0.0045 ppb, and it results from the enzymatic oxidation of linoleic acid. (Z)-3-Hexenal, with a green beany flavor, also strongly influences the soybean’s overall flavor profile and has an extraordinarily low odor threshold of 0.00012 ppb. It is formed through the oxidation of α-linolenic acid [32]. Apart from these volatile compounds, certain non-volatile compounds like flavonoids, saponins, phenolic acids, and specific amino acids can also contribute to the formation of the beany odorants [30]. The content of soluble dietary fiber in soybean increases when exposed to higher irradiation doses, ranging from 400 kGy to 1200 kGy. The combination of γ-irradiation and micronization (grinding to produce soft mass/powder) demonstrates the most effective means of enhancing dietary fiber degradation while also positively influencing its physical and chemical characteristics [33, 34]. As a result, this method is considered an optimal approach for enhancing the quality of soybean overall nutrition. These investigations collectively underscore the promise and potential utility of γ-irradiation as a technology for maintaining and enhancing the physicochemical properties and nutritional value of soybean.

2.2 Antioxidants

The antioxidant properties of soybeans can be significantly enhanced through γ-irradiation. The seed coat color has a prominent effect on the antioxidant response to γ-radiation. In a study conducted by Tewari and colleagues [35], soybeans were exposed to low doses of gamma rays (0.25 kGy, 0.5 kGy, 0.75 kGy, and 1.0 kGy). The findings indicated that soybean varieties with darker seed coat colors displayed greater susceptibility to γ-irradiation. A significant observation was the marked rise in the levels of tocopherols and anthocyanins in dark-colored soybean varieties. Further, at a radiation dose of 0.25 kGy, there was a notable increase in the concentration of isoflavones. Additionally, γ-radiation had a significant reducing effect on parameters associated with undesirable flavors, including seed lipoxygenase activity, thiobarbituric acid number, and carbonyl value in the dark-colored soybean varieties.

Radiation dose appears to play a crucial role in the extractable phenolics found in legume and cereal seeds, which may also influence the antioxidant properties of soybeans. However, it’s essential to note that γ-irradiation is not currently employed to intentionally boost the antioxidant properties of soybeans. Nevertheless, under certain favorable radiation conditions, an increase in the content of extractable phenolics has been detected. Kumari and co-workers [36], conducted a study in which γ-irradiation resulted in a decrease in non-extractable phenolics in both yellow and black-coated soybean cultivars. Remarkably, a substantial increase in the total extractable flavonoids was noted in irradiated samples, which included soybeans [37], peanut skins [38], and quinoa subjected to electron beam irradiation [39]. On the contrary, some researchers have reported a significant enhancement in anthocyanin content in pigmented rice and soybeans as a result of γ-irradiation Taken together, these findings imply that γ-irradiation can impact the antioxidant properties of soybeans and other food products. However, the precise effects may vary depending on factors such as the type of seed, radiation dose, and other environmental conditions. Soybeans are rich in isoflavones, and their levels are influenced by genetic factors. Isoflavones, a type of polyphenol, serve as antioxidants that can help prevent osteoporosis, reduce the risk of atherosclerosis, and protect against cardiovascular disease. The impact of γ-irradiation on isoflavones has been predominantly studied in soybeans. The specific isoflavones, like genistein, daidzein, and glycetein, showed a significant increase in concentration during irradiation processing up to 10 kGy. Additionally, measures of antioxidant potential, such as DPPH (1,1-diphenyl-2-picrylhydrazyl) scavenging activity and enhanced hydroxyl radical scavenging, exhibited a positive correlation with the radiation dose, suggesting that using radiation as a food preservation method can have beneficial nutritional implications [37, 40, 41]. However, at 50 kGy, isoflavone levels in the samples showed a lower, though not statistically significant change [42]. Interestingly, aglycons and isoflavone glucosides have displayed diverse responses to γ-irradiation. Variyar and group [41] noted that γ-irradiation (0.5–5 kGy) remarkably reduced the overall content of isoflavones, particularly the glycosides, while enhancing the content of isoflavone aglycons in 80% methanol extracts of soybeans. However, there have been reports where γ-irradiation treatments had no significant impact on the isoflavone content of soybeans when the radiation dose was below 10 kGy [40]. This variation could potentially be linked to the choice of solvent utilized for extraction. Furthermore, reduction in the overall isoflavone content was solely noted in yellow soybeans subjected to γ-irradiation at a dose of 1 kGy. This decline in non-extractable phenolics can be linked to the lower presence of protective anthocyanins, which serve as guards against radiolytic degradation, particularly when compared to black soybeans [43]. Among black soybeans, the metal chelating (Fe²⁺) activity and reducing power exhibited a decreased sensitivity to γ-irradiation. Conversely, yellow and green-coated soybeans demonstrated a notable enhancement in these attributes at radiation doses of 0.5 and 2 kGy. Moreover, Krishnan et al. [44] and Bansal et al. [45] reported that an effective improvement in extractable antioxidant activities and the preservation of nutraceutical properties was achieved with the application of a lower dose, specifically 0.5 kGy.

Radiation-induced transformations within the same class of phenolic compounds, such as the conversion of glycosides to aglycons, are largely attributed to the breaking of glycosidic bonds—a process known as deglycosylation. For example, γ-irradiation of soybeans has been shown to increase isoflavone aglycons and decrease their glucosides due to the deglycosylation of isoflavone glucosides [41]. They also showed that the increased DPPH scavenging activity of isoflavones in γ-irradiated soybeans was attributed to higher levels of the free form, such as isoflavone aglycon, despite a reduction in both the total isoflavone content and its glucosides. The hydroxyl radical scavenging activity, as evidenced by its ability to inhibit deoxyribose oxidation, stayed at a high level up to a radiation dose of 2.0 kGy in soybeans. This suggests that γ-irradiation contributes to an increase of antioxidant activity. Medium doses of gamma irradiation (1–10 kGy) were reported to enhance total phenolic and tannin contents, as well as DPPH scavenger activity, in soybean seeds, while reducing protein oxidation. Although gamma irradiation had minimal impact on lipid peroxidation and soluble protein content, a significant decrease in protein oxidation was observed at a 10 kGy dose, suggesting improved antioxidant activity depending of radiation dose [46]. In another study, Mata-Ramírez and group [47] conducted an assessment of the quantification and bioactivity of isoflavones in soybean callus subjected to UV-light-induced stress. Their results suggested that exposure to UV-C light stress resulted in elevated levels of genistein-O-glucosyl-malonate and genistein-O-glucoside in soybean callus, leading to improvements in both antioxidant and anti-inflammatory activities [48]. This suggests that the application of both tissue culture techniques and UV-B radiation treatment can be an effective means of obtaining bioactive compounds. The increased antioxidant activity observed in soybean genotypes following exposure to low doses of gamma irradiation may be associated with the production of free flavonoid. Research has shown that these liberated flavonoids display stronger antioxidant characteristics in comparison to glycosides. Variyar et al. [41] proposed that the radiation-induced degradation of glycosides leads to the liberation of free isoflavones. Given that isoflavones are a type of phenolic compound, the rise in their concentration at lower doses of gamma irradiation corresponds with the concurrent increase in total phenolic content. This, in turn, contributes to the improvement in antioxidant activity.

The improvement in soybean antioxidant activity was linked to an increase in the overall phenolic content. It was proposed that γ-irradiation could boost phenolic content by stimulating the activity of phenylpropanoid pathway enzymes. The increase in isoflavones was theorized to result from either the conversion of malonyl derivatives into free glycosides or an augmented biosynthesis. Using the epithelial cell line BEAS-2B, [43] explored the effect of three different doses of γ-radiation, for instance 0.25, 0.5 and 1.0 kGy, on the anti-proliferative and cytoprotective effect of black and yellow soybean extracts that were characterized in terms of total phenolics, and flavonoids and the anthocyanin cyanidin 3- glucoside. It was reported that γ-irradiation at 1 kGy dose enhanced the cyanidin 3- glucoside content by 33%, which correlated well with a 78% decrease in reactive oxygen species (ROS). Also, irradiation up to 0.5 kGy enhanced the total phenolic content in black and yellow soybeans. The flavonoids, particularly daidzein, showed an increase at radiation doses of 0.25, 0.5, and 1.0 kGy in black soybeans, and at 0.25 and 0.5 kGy in yellow soybeans. The rise in total phenolic and anthocyanin content in irradiated soybeans was attributed to an enhancement in the activity of enzymes like flavonoid glucosyltransferase and phenylalanine ammonia-lyase or to the cleavage of glycosidic linkages in bound procyanidins, leading to their free monomeric form.

Medium doses of γ-radiation have been shown to have a positive effect on the antioxidant potential of seeds that, in turn, combats the free radical-induced lipid and protein oxidation, thus minimizing off-flavor production [35, 44, 49]. Kumari and co-workers [36] revealed that γ-irradiation at 2.0 kGy dose improved the protein quality of soybeans by reducing protein oxidation and increasing protein solubility. The report indicated a 37% reduction in protein oxidation, as measured by the carbonyl number, in black soybeans and a 28% reduction in yellow soybeans. They noted that increasing the radiation dose to 5 kGy resulted in elevated protein oxidation levels, similar to those of untreated soybeans. Further, was noted that γ-irradiation up to a 5 kGy dose reduced the activity of LOX isozymes, which improved protein quality by limiting interactions between lipid hydroperoxides and proteins. Additionally, γ-irradiation up to a 5 kGy dose increased free radical scavenging activity, as assessed through the DPPH assay. However, the FRAP values increased up to a radiation dose of 2 kGy and decreased beyond that point.

Tocopherols, found abundantly in plant-derived lipids, are the most prevalent natural antioxidants, acting as sacrificial guardians for the lipid components within seeds. In the autoxidation process, they function as chain-breaking antioxidants by capturing free radicals, thus effectively thwarting the initiation and halting the propagation of oxidation. Braunrath et al. [50] observed significant reductions in peroxides and hydroperoxides when δ- and γ-tocopherols were present. Contrastingly, Yun et al. [51] noted that soybeans treated with γ-irradiation at doses of 5.0 and 10.0 kGy displayed a substantial decline in α-tocopherol, with decreases of 24.3% and 35.4%, respectively. However, Dixit et al. [52] discovered that there were no alterations in the overall tocopherol content in soybeans across the entire range of γ-irradiation doses, which included values from 0.5 to 5 kGy. The decrease in tocopherol content in irradiated oil is believed to be associated with the degradation of these antioxidant compounds that take place during the irradiation process.

Minami and co-workers [53] have shown that γ-irradiation, even up to 80 kGy, had no impact on the quantity of unsaturated or saturated fatty acids in soybeans. This phenomenon is attributed to the ample presence of antioxidants, such as tocopherols and carotenoids, in soybeans. These antioxidants effectively scavenge the radicals generated by γ-irradiation. Similarly, in a study by Hafez and colleagues [54], it was noted that no alterations were detected in the fatty acid composition, which included C16:0, C18:0, C18:1, and C18:2, in soybean oil. This observation remained consistent even when exposed to relatively high doses of γ-irradiation, up to 100 kGy. However, a negative correlation was established between the concentration of linolenic acid and the applied radiation dose. Aziz et al. [55] observed that γ-irradiation led to a reduction in the acid values of cereal fats, with the effect becoming more pronounced with the increment in radiation dose within the range of 5–15 kGy. The decrease in acid values can be ascribed to the formation of free radicals during irradiation, which impacts the unsaturated fatty acids in the oil, leading to the production of hydroperoxides and peroxides, as described by Mahrous [56].

Mexis and Kontominas [57] and Bhatti and group [58] found a significant increase in the peroxide value of oils obtained from irradiated almonds and peanuts as the γ-irradiation dose increased. The decrease in the iodine value of oils can be connected to the formation of peroxide compounds and the saturation of double bonds in unsaturated fatty acids, induced by irradiation. Bhatti and co-workers [59] noted a noticeable decline in the iodine value for γ-irradiated peanuts.

2.3 Anti-nutritional factors

Leguminous seeds’ protein content makes up 20% of the total plant-based protein consumption in humans [60]. Nonetheless, specific legumes, particularly soybeans, contain substantial amounts of bioactive compounds that can modify the way nutrients are processed in the body upon consumption. The primary proteins that contribute to the reduction in the nutritional value of unprocessed soybeans are lectin and trypsin inhibitors. However, it’s important to acknowledge that other naturally occurring compounds can also contribute to the observed adverse effects. To mitigate losses during the harvest and storage of these grains, the use of ionizing radiation presents itself as an appealing and health-conscious alternative when compared to conventional chemical treatments. The application of ionizing radiation for the purpose of preserving and sanitizing grains offers a promising strategy to extend shelf life and minimize losses during storage. The anticipated costs and benefits associated with commercial radiation treatment appear to be competitive when compared to other fumigation techniques, as well as thermal and physical treatment methods. Abu-Tarboush [61] reported a 34.9% reduction in trypsin inhibitory activity in soybean flour was assessed after exposure to a γ-radiation dose of 10 kGy. Similarly, Farag [62] found that as radiation doses increased (5, 15, 30, and 60 kGy), with the increment in radiation dose, the degree of inactivation of trypsin inhibitory activity also rose, leading to losses of 41.8%, 56.3%, 62.7%, and 72.5%, respectively. In the research conducted by Villavicencio et al. [63], it was noted that in the Carioca variety, the tannin concentration remained unchanged during soaking and cooking, while in the Macacar variety, it showed an increase. In contrast, Mechi et al. [64] reported a decline in tannin content in black beans when subjected to γ-radiation combined with the cooking process. Amaya et al. [65] proposed that the decrease in tannin content due to cooking is evident and is linked to alterations in chemical and solubility reactivity. This decrease can be attributed to various factors, such as interactions with organic substances, proteins, and modifications in their chemical structure, as suggested by Brigide and Canniatti-Brazaca [66]. Furthermore, it was observed that γ-radiation resulted in a reduction in tannin content as the radiation dose increased, up to a certain threshold. This decrease in tannin content is particularly advantageous, as tannins are recognized as antinutritional factors that can hinder protein digestibility [63, 64]. When the tannin-to-protein ratio reaches 5:1, all the protein tends to precipitate due to the action of tannins, as explained by Pino and Lajolo [67]. Therefore, these findings indicate that, concerning antinutritional factors, their levels decreased with higher radiation doses in cooked and raw samples, with the cooking process itself also contributing to the decrease in these antinutritional factors.

γ-irradiation has been demonstrated to enhance the nutritional quality of soybeans by reducing anti-nutritional components, including phytic acid, trypsin inhibitors, lipoxygenase, oligosaccharides, tannins, urease 3, and haemagglutinating agents, [62, 68, 69]. Currently, there are over 51 countries, China included, have officially approved the use of γ-irradiation in food processing, typically at an average dosage of 10 kGy [70]. A joint study group by FAO/IAEA/WHO has reported that γ-irradiation at doses exceeding 10 kGy remains safe and maintains nutritional adequacy [6]. Therefore, it is plausible that the application of high-dose γ-irradiation could serve as an effective approach to improve the quality and safety of fungus-contaminated soybeans intended for animal feed. Soybean seeds inherently contain three lipoxygenase enzymes, which can impart undesirable beany or grassy flavors. The elimination of these lipoxygenases is a method to enhance the stability and taste of soybean oil and protein products. Additionally, the presence of phytic acid and its mineral derivatives, phytates, creates nutritional and environmental challenges due to their limited digestibility in monogastric organisms. As a result, there has been a substantial rise in endeavors to grow crops with lower phytic acid content. Soybean varieties created through γ-radiation-induced mutations have shown enhanced nutritional qualities owing to their reduced phytate levels along with the reduced lipoxygenase enzyme activity in their seeds [71, 72].

2.4 Allergen

Soybean is a protein-rich food (besides having nutritious fiber, fatty acids, vitamin, minerals etc.) consumed globally. Additionally, it also contains anti-nutritional factors, like agglutinin, protease inhibitors, and allergenic proteins that elicit adverse immune responses, which significantly hamper its nutritional and functional profits. Most of the allergens in soybean are proteins and more than 33 allergens have been characterized so far [73], which influenced around ~0.5% of the general population globally [74]. It affects 0.4% children in USA [75] and its mild symptoms usually appear up to 2 years, however, severe immune responses may persist the whole life [76]. Soyben-induced allergic reactions (allergenicity) have a wide impact on the skin (dermatitis, eczema, urticaria, acne etc) respiratory system (asthma, bronchospasm, dyspnea, edema, Rhinitis wheezing etc.), gastrointestinal tract (colitis, enterocolitis, bowel disease, vomiting etc.) and other prominent symptoms like conjunctivitis, lethargy etc. [76]. Soybean is 8th most common food allergens and become a public health problem globally for its manufacturers as well as consumers [77, 78]. In soyaben allergic patients, its value ranges from 0.0013 to 500 mg, provoking mild to severe immunogenic response [79, 80]. So far, various allergens belonging to the cupin super-family, [81], have been identified from molecular weight ranging from 7 kDa (Gly m1a, Hull protein) to 75 kDa (α’ Subunit of β-conglycinin) [76], provoking immunogenic responses in human [79] as well as various animals [82]. Among them, Glycinin and β-conglycinin are widely studied allergens [82, 83].

Major allergenic proteins include Gly m 1 to Gly m 8, Gly m Bd 28 K(P28), Gly m Bd 30 K(P34), [84, 85], Gly m Bd 60 K [86], and the Kunitz soybean trypsin inhibitor (KSTI, 20 kDa), [87] were detected in soybean allergic patients. Gly m 1 (~ 8.3 kDa, hydrophobic protein) and Gly m 2 (~ 8 kDa, defense protein) are considered as hull proteins [88] due to their location in soybean hull and accounts for their 75% and 25% allergenicity respectively [89]. Gly m 3 (~ 14 kDa, hydrophilic protein) is from profilin and labile to heat and hydrolysis [90]. Gly m 4 (~17 kDa, hydrophilic, and pathogenesis related protein), is susceptible to heat, acid, proteases, and found as protein isolates [91]. P34, P28 and Gly m 5 (β-conglycinin, ~ 150 to 210 kDa) are derivatives of 7S globulin and constitutes ~30%, Gly m 6 is a hexameric protein originate from 11S globulin, comprises ~40%, [92, 93], Gly m 7 (~ 76.2 kDa, biotinylated protein) is abundant in seed embryo, heat stable, and comprises <0.01% [75], and Gly m 8 (~ 28 kDa) derivative of 2S albumin comprises 10% of the total soybean protein [94]. Furthermore, Gly m Bd, P34 (~ 30 kDa, anchorage vacuole protein) comprises ~1% [85, 95], and Gly m Bd, P28, (~28 kDa, glycosylated protein) comprises <0.5% of total seed protein [95, 96]. Soybean allergenicity is an IgE-mediated immune response, devoid of immunologic and clinical lenience resulting in clinical symptoms and pathological manifestations [97]. In continuation, Gly m 4, Gly m 5 and Gly m 6 provokes robust immune response in soybean-allergic patients [98]. Gly m 5 and Gly m 6 soybean allergens comprising 30% and 40% of the total soybean protein respectively [99] and are easily identifiable in almost every pediatric patient [100]. Schematic representation analyzing impact of low and high doses of y-irradiation on various pathophysiological aspects of soybean allergenicity, nutrients and intrinsic physiochemical properties is summarized in Figure 2.

Figure 2.

Schematic representation of various pathophysiological aspects of soybean allergen, and risks as well as benefits associated with the use of γ-irradiation on intrinsic properties of soybean. Cobalt-60 (⁶⁰Co) and Cesium-137 (¹³⁷Cs) are major radioactive atoms producing gamma radiation, which alters the conformation of soybean allergen protein and, thus, masks the exposed antigenic epitope responsible for its augmented allergenicity contributing to clinical symptoms and pathological manifestations healthwise. The recommended dose of γ-irradiation is 0.5–10 kGy masks the soybean allergenicity, and microbial infection as well as increases the shelf life and nutrient quality. The higher dose of γ-irradiation (10 kGy–400 kGy) induces a change in secondary structure provoking several disadvantages by altering soybean nutrients, constituents, and various intrinsic properties. AAs: Amino acids UFAs: Unsaturated fatty acids, ANFs: Anti-nutritional factors.

γ-irradiation are primarily used for increasing food shelf-life by abolishing the surface pathogens; however, variable doses and combination of other factors later resulted in augmented additional microbes destruction and marked decrease in protein loss during food storage as well as reduced allergenicity [101]. Food exposure to y-irradiation involves production of low energy electron (β-particle) below 5 MeV from radioactive atoms ⁶⁰Co or ¹³⁷Cs, during radioactive decay [9], and modulates protein conformation structurally, resulted in masking of exposed epitopes in allergen protein, thus, reduced adverse immune response or allergenicity [10]. However, factors affecting reduced allergenicity depend on its types, exposure times and doses. ⁶⁰Co involves simultaneous generation of two γ-rays (1.17 and 1.33 MeV) and low energy β-particle (0.3 MeV), while ¹³⁷Se produces γ-rays of energy 0.66 MeV with maximum penetration at same dose [6, 9]. Study suggests no change in allergenicity upon 25 kGy irradiation dose of ⁶⁰Co γ- exposure on raw kidney bean or black gram seeds [102], and on soybean seeds upon multiple exposure ranging from 2.5 to 30 kGy radiation [103]. Same group also found that co-administration of γ-irradiation (25 kGy) and boiling at 121°C for 15 minutes remarkably suppressed the immunogenicity of soluble and insoluble allergens in kidney bean and black gram seeds against untreated control condition [102]. Another study suggests that γ-irradiation <= 5 kGy administration to soybean protein solution exposed the conformational and linear epitopes of allergen proteins, thus, augments its immunogenecity and γ-irradiation > = 10 kGy suppressed the immunogenicity by 91% compared to control by inducing the protein-degradation or aggregation cellular event masking the conformational epitopes responsible for allergenicity [27, 28]. Collectively, the immonosensitivity of soybean allergen proteins depends on the state of its conformational changes induced by γ-irradiation and additional processing factors; however, its clinical execution warrants further in-depth research.

3. Effects of γ-irradiation in reducing microbial contamination load in soybean

Soybean stands as one of the most vital grains globally, yet fungal infections are an inevitable challenge in its cultivation and storage. Pathogens like Fusarium, Alternaria, and Phomopsis inflict substantial damage on the quality and market value of soybeans [104]. Because of the existence of fungi that produce toxins, soybeans afflicted by fungal damage are unfit for human consumption. Interestingly, these fungi-damaged soybeans boast higher protein content and a comparable amino acid composition to their uninfected counterparts [105]. Consequently, certain portions of these fungi-affected soybeans have been repurposed as raw materials for animal feed by blending them with healthy soybeans [104]. Nonetheless, they quite pose risks to animals. Hence, the elimination of fungi and mycotoxins becomes of paramount importance. γ-irradiation has emerged as a secure and efficient physical approach for decontaminating microbes (Figure 1), controlling pests, and enhancing the quality of both unprocessed and processed agricultural products [106]. Research has provided evidence that γ-irradiation at moderate doses, typically ranging from 8 to 10 kGy, can completely eliminate bacteria and fungi from oilseeds while preserving their chemical composition [59, 107]. Moreover, high-dose γ-irradiation (10–30 kGy) has proven effective in reducing mycotoxins, including T-2 toxin, aflatoxins, zearalenone, ochratoxin, and deoxynivalenol, produced by specific fungal strains in food crops [108, 109]. It’s important to note that higher irradiation doses lead to a more comprehensive degradation of mycotoxins. Moreover, γ-irradiation has demonstrated its effectiveness in decreasing the ruminal degradability of soybean protein and enhancing in vitro crude protein digestibility [69].

γ-irradiation has demonstrated its effectiveness for controlling fungal growth and deactivating mycotoxins, which are highly toxic substances. Given the severe health risks associated with mycotoxins, various methods have been employed to eliminate their presence in various food products. The application of ionizing radiation treatment has been well-established as an effective physical method for preserving the quality of food and prolonging the shelf life of agricultural products, including soybeans [110, 111]. Aflatoxins are renowned secondary metabolites generated by fungi including Aspergillus nomius, Aspergillus parasiticus, and Aspergillus flavus [112]. The sources of these fungi have been linked to groundnut meal contaminated with aflatoxin-producing fungi, which is commonly used in animal feed [113]. Given their virulence, carcinogenicity, mutagenicity, and teratogenicity, AFs are regarded as highly hazardous substances that pose risks to animal and human health [114, 115]. Among the 20 different types of aflatoxins, including AFB1, AFB2, AFG1, and AFG2, are frequently detected in various food products [116].

AFs exhibit remarkable resistance to heat treatment, as their decomposition temperature exceeds 235°C [117, 118, 119]. As a result, conventional drying methods alone do not lead to a substantial reduction in their concentrations in stored grains. However, prolonged exposure to elevated temperatures seems to have a beneficial impact on decontamination. For instance, subjecting soybeans to heat treatments at 100°C and 150°C for 90 minutes resulted in notable reductions of AFB1 contents by 41.9% and 81.2%, respectively [120]. γ-irradiation has proven to be effective in decreasing both fungal contamination and AFB1 concentration in naturally infected corn kernels. γ-irradiation doses in the range of 1 to 10 kGy have resulted in mycotoxin level reductions ranging from 69.8% to 94.5%, respectively [121, 122]. Additionally, for wheat, rice and corn kernels, doses of 4, 6, and 8 kGy have proven effective in reducing AF levels by 15–56% with increasing doses [123]. In the case of soybeans, doses exceeding 10 kGy have shown utility in AFB1 reduction [124]. Ozone treatment has shown remarkable efficiency in the destruction of AFs, achieving reductions of up to 66–95% of the original toxin levels in cereal flours, grains, soybeans, and peanuts [125, 126, 127]. While Hooshmand and Klopenstein [108] found that γ-irradiation doses up to 20 kGy did not have a notable impact on the AFB1 content in soybeans, corn, and wheat. γ-irradiation reduced the yeast and mold populations in soybeans, with yeast and mold becoming undetectable at doses exceeding 5.0 kGy. The application of a 3.0 kGy dose of γ-irradiation effectively lowered the aerobic bacterial populations in soybeans to a level that met acceptable standards from 5.77 × 10⁵ to 1.2 × 10² CFU/g [51].

Furthermore, in the study by Wilson et al. [104], a positive correlation was noted between the fat content of soybeans and fungal damage, which they attributed to a decrease in seed mass. Dogbevi et al. [128] demonstrated that γ-irradiation at 1.5 kGy led to a reduction in the mold population in dry red kidney beans by a factor of 2 log cycles. Additionally, γ-irradiation at doses of 1.0, 3.0, 5.0, and 10.0 kGy resulted in significantly lower counts of total aerobic bacteria in soybeans compared to the control. The counts of mold and yeast in soybeans were also reduced by irradiation, with no presence of yeast or mold in soybeans exposed to doses of 5.0 and 10.0 kGy.

4. Risks associated with high dose γ-irradiation on soybean nutrients

So far, we have unraveled numerous beneficial impacts of recommended low dose γ-irradiation (0.5 to 10 kGy) in reducing the soybean microbial contamination, allergenicity, ANFs as well as increasing nutrient composition, flavor, shelf-life, anti-oxidant, and ROS scavenging activities. Impact of various doses of y-irradiation also depends on the soybean state (seed, flour, protein solution, flavor, etc.) and its intended use. Numerous studies suggest a high dose of γ-irradiation (>10 to 400 kGy) exerts significant deleterious effects on soybean nutrients, protein conformation, and various intrinsic properties. Increased γ-irradiation using ⁶⁰Co induce changes in the secondary structure of protein and exposes hydrophobic amino acids, thus, increasing protein insolubility and a simultaneous decrease in its emulsifying ability and reduced vitamin-E level was observed [21, 22]. A similar dose further decreases α-tocopherol (an anti-oxidant) by 35.4% compared to the control, increases ROS (hydroperoxide and peroxide) generation [56], as well as proanthocyanidins [46]. Very high radiation dose (400 to 1200 kGy of γ-irradiation) significantly induces the soluble dietary fiber constituent in soybean [34]. In continuation, a dose-dependent increase in soybean trypsin inhibitor activity has been demonstrated by Farag [62]. While γ-radiation can deactivate trypsin inhibitor (TI), an antinutrient and food allergen in soybeans, but its effectiveness depends on moisture levels. In an aqueous solution, TI is almost completely destroyed at 10 kGy irradiation of Cobalt-60. However, in a dry state or with 50% moisture, significant functional and structural losses occurs at higher doses (100 kGy and 30 kGy, respectively). Surprisingly, TI remains unaffected in other legumes, as well as in soaked and dry soybean seeds, even at substantial irradiation doses (50 kGy and 100 kGy respectively). Hence, TI is stable to direct gamma radiation, and its inactivation at lower doses requires excess moisture. Using gamma radiation to reduce TI in dry or soaked seeds would necessitate high doses that could impact its sensory and functional propertie [129]. Interestingly, the impact of y-irradiation on soybean allergenicity is diverse as demonstrated by increased allergenicity at <5 kGy [102] and decreasing allergenicity at >10 kGy [27, 28] by unmasking and masking of linear epitopes of allergen proteins respectively. Increased γ-irradiation (>10 to 30 kGy) dose is also associated with reduced fat, protein, lipase, and anti-oxidant enzyme activities in soybean as reported in previous sections.

5. Conclusion

Irradiation, a cost-effective and environmentally friendly non-thermal food processing technology, is widely employed in the food industry. It has been found to have various benefits, such as reducing food allergens and effectively eliminating insects, molds, and bacteria. Additionally, it aids in reducing the presence of detrimental compounds such as biogenic amines and substances that hinder nutrient absorption in food. When used at appropriate doses, γ-irradiation does not adversely affect the physical, chemical, nutritional, or sensory qualities of food products. For instance, γ-irradiation doses of up to 10 kGy have been shown to have a minimal adverse impact on the natural physicochemical properties, nutritional value, and flavor of soybean oil. Studies indicate that a gamma irradiation dose of 10.0 kGy is a successful method for inhibiting microbes’ infestation and decontaminating soybean seeds. Moreover, γ-irradiation at these doses increases isoflavone content while reduces tocopherol contents and the compounds responsible for flatulence (RFOs). At moderate radiation doses ranging from 8 to 10 kGy, gamma irradiation can entirely eradicate bacteria and fungi in oilseeds while leaving their composition unaffected. Assessments based on sensory criteria have shown that soybeans subjected to irradiation remain satisfactory even at doses as high as 5.0 kGy. Low-dose γ-irradiation appears to enhance the total phenolic content and increase proanthocyanidins, contributing to improved antioxidant activity. Furthermore, high-dose γ-irradiation (10–30 kGy) has been shown to reduce mycotoxins, including aflatoxins and other toxins produced by fungal infections.

γ-irradiation has the added benefit of enhancing the nutritional quality of soybeans by reducing anti-nutritional components, such as phytic acid, trypsin inhibitors, lipoxygenase, oligosaccharides, tannins, urease 3, and hemagglutinating agents. It’s important to highlight that when it comes to reducing the allergenic properties of soybeans, using gamma irradiation at doses lower than 10 kGy may not achieve the intended outcomes. On the other hand, it has been demonstrated that employing higher doses exceeding 10 kGy can effectively diminish the allergenic properties of soybean protein solutions. High doses of γ-irradiation (> 10 kGy) significantly impact soybean nutrients (reduced fat and protein content) and intrinsic properties (like reduced anti-oxidant, lipase activity, increased UFA, ANFs, etc.). In summary, γ-irradiation is a valuable tool in the soybean food industry, offering multiple benefits such as microbial decontamination, enhanced shelf-life, nutritional quality, flavor, and improved safety, with effectiveness and outcomes depending on the specific dose, duration, and other cotreatments (like temperature, pressure, micropulverization, enzyme catalysis, etc.) applied.

Conflict of interest

The authors declare no conflict of interest.