Study of germination and antioxidant activity of iron-fortified soybean (Glycine max) germs

Iron deficiency in human diet is a serious problem leading, among others, to the development of iron-dependent anaemia, which affects 1.8 billion people worldwide. Biofortification of crop plants, including edible sprouts, is a promising strategy for combating dietary deficiencies. The aim of the present study is the evaluation of the effect of seeds imbibition in FeCl2 solution with Fe at the concentrations of 100 and 500 mgL−1 on the seeds germination, growth of the seedlings, their antioxidant activity and the level of total phenolics and flavonoids. The results show that pre-treatment of seeds with Fe leads to increased accumulation of this metal in the germinated seedlings. In addition, it affects germs antioxidant activity and the level of flavonoids. On the other hand, seeds imbibition in FeCl2 had no effect on the germination rate, growth of seedlings or total phenolic content (TPC). The results indicate that seeds imbibition in Fe solution is a promising strategy for obtaining Fe-enriched soybean sprouts.


Introduction
Iron is an essential metal required for the proper functioning of organisms. This metal is redox active and constitutes a crucial element of the electron transport chains. Thus, proper Fe levels are indispensable for efficient respiration and photosynthesis. In addition, as a cofactor of numerous enzymes, Fe is involved, e.g. in antioxidant response, chlorophyll biosynthesis and hormone and nucleic acids metabolism. In plants, Fe deficiency manifests by chlorosis, decreased photosynthesis efficiency and reduced growth (Balk and Schaedler 2014;Krohling et al. 2016). The uptake of Fe from the soil is dependent on various factors, wherein low soil pH plays crucial role. Plants can improve Fe absorption by its reduction to more bioavailable Fe 2+ form and by exclusion of chelating agents including malic, citric and phenolic compounds (Veléz-Bermudéz and Shmidt 2021, Krohling et al. 2016). The phenolics can play multiply roles under Fe deficiency and/or excess due to their chelating as well as antioxidant/prooxidant functions (Moran et al. 1997).
Importantly, low Fe levels in crop plants adversely affect human nutrition. The most prominent symptom of Fe deficient diet is the development of anaemia, which according to the World Health Organization (WHO) affects over 40% of children under the age of five and over 30% of woman in the reproductive age worldwide (WHO 2021). One of the promising strategies for combating nutrient deficiencies is the introduction of biofortified crops. Biofortification aims to increase the levels of nutrients in crops through the introduction of new varieties by the means of conventional breeding and genome editing and through agronomical approaches (Stangoulis and Knez 2022). The studied approaches include also enrichment of the edible seedlings with essential minerals, e.g. through imbibition in appropriate solutions. This study describes the impact of seeds imbibition in FeCl 2 solution on the germination, growth and chosen biochemical parameters of soybean (cv Nawiko) germinated seedlings. The described results are important for understanding of plants response to the Fe treatment and for possible biofortification of soybean seedlings/sprouts with this micronutrient.

Germination and treatment procedures
Soybean seeds (cv Nawiko) were sterilised through imbibition for 5 min in 75% ethanol and for 10 min in 1% hypochlorite solution. The seeds were washed under running water for 30 min and imbibed for 2 h in 30 ml of following solutions: distilled water (control) or FeCl 2 with Fe at the concentration of 100 or 500 mgL −1 . Then, the seeds were washed under running water, transferred to glass Petri dishes 30 cm in diameter lined with two layers of lignin and one layer of blotting paper, watered with 30 ml of distilled water and transferred to a germination chamber with temperature of 21-22 °C. The percentage of germinated seedlings has been measured after 24, 48 and 72 h and the roots length after 72 h. The measurements of the germination rate and seedlings growth were carried out in three experimental repetitions. For future assessment of antioxidant activity and the level of total phenolic compounds and flavonoids, the germs of approx. 350 mgs were frozen in − 80 °C.

Assessment of Fe level
For the quantification of Fe soybean seedlings were imbibed for 2 h in distilled water (control) or Fe solutions with Fe at the concentration 100 and 500 mgL −1 , washed thoroughly, germinated for 72 h, dried for three days in 60 °C and send for Fe quantification using ICP-OES to commercial company (Scallad, Poznań, Poland). The Fe level is presented as µg per one gram of dry weight.
The histochemical detection of Fe has been carried out according to Bramborova and Ivanov 2014. Fresh seedlings germinated for 72 h were placed in Eppendorf tubes and incubated at room temperature for 90 min in staining solution containing 4% K 4 Fe(Cn) 6 (Merck, A1684984) and 4% HCl (POCH-BASIC, BA5283115) mixed in a 1:1 proportion. In parallel, the seeds were cut in half and incubated in the staining solution for 24 h. In the case of the whole seedlings, the prolongation of staining was not possible as the staining solution caused their softening and damage. After the incubation, seedlings and/or seeds were washed with distilled water and photographed.

Assessment of antioxidant activity
The antioxidant activity was measured according to the method described by Brand-Williams et al. (1995). Seedlings of soybean were homogenised in 2 ml of 80% methanol (StanLab, Cas: 67-56-1), transferred to Eppendorf tubes, incubated for 2 h at 37 °C and centrifuged 10 min at 12 000 rcf. Then the mixtures containing 100 µl of supernatant, 2000 µl of 80% methanol, and 250 µl of DPPH (2,2-diphenyl-1-picrylhydrazyl) (Sigma-Aldrich) were prepared. Straight after the preparation of the mixtures, their absorbance was measured by λ = 517 nm. The samples were incubated for 10 min at RT in the dark and thereafter their absorbance was measured by the same wavelength. The antioxidant activity was expressed as % differences in DPPH quenching between the first and the second measurement. The measurements were carried out in three independent experimental repetitions.

Measurement of the level of total phenolic compounds (TPC)
The TPC level has been measured on the basis of the method described in Diaz et al. 2005. Germinated seedlings were homogenised in mortar with 4 ml of 80% methanol, transferred to plastic tubes, incubated for 15 min at 70 °C, cooled and centrifuged for 15 min at 5000 rpm. The supernatant was transferred to glass tubes and supplement with 80% methanol up to 5 ml. Thereafter, the reaction mixtures containing 3750 µl of distilled water, 250 µl of Folin-Ciocalteu reagent (Merck, 1090010100) and 250 µl of supernatant were prepared. In the case of the blank, 250 µl of 80% methanol was added to the reaction mixtures instead of the supernatant. After 3 min, 750 µl of 20% Na 2 CO 3 (Polskie Odczynniki Chemiczne, BN69/6191-88) has been added to the reaction mixtures. The mixtures were incubated for 2 h and the absorbance was measured by λ = 760 nm. The standard curve was prepared on the basis of gallic acid solutions at the concentration of 0, 10, 20, 50 and 100 mgL −1 . The measurements were carried out in four independent experimental repetitions. The level of total phenolics is expressed as mgL −1 of gallic acid equivalents in one gram of fresh weight.

Measurement of the flavonoid level
The level of flavonoids was measured as described by Jia et al. (1998). The germinated seedlings were homogenised in a mortar with 2 ml of 80% methanol, transferred to Eppendorf tubes and incubated for 2 h at 37 °C. The samples were mixed every 0.5 h. Thereafter, the samples were centrifuged for 10 min at 12,000 rcf. The mixtures containing 250 µl of supernatant, 1250 µl of distilled water and 75 µl 5% NaNO 3 (POCH, 792690115) were prepared. The samples were mixed on vortex and incubated for 5 min at RT. Then, 150 µl of 10% AlCl 3 (Merck, B469484) has been added to the samples. The samples were mixed on vortex, incubated for another 6 min at RT and supplemented with 500 µl of 1 M NaOH (StanLab, CAS: 1310-73-2). The absorbance of the samples was measured by λ = 510 nm. The blank contained Page 3 of 6 22 250 µl of 80% methanol instead of the supernatant. The standard curve was prepared on the basis of catechin solutions at the concentration of 0, 5, 10, 20 and 25 mgL −1 . The measurements were carried out in three independent experimental repetitions. The level of flavonoids is expressed as mgL −1 of catechin equivalents in one gram of fresh weight.

Statistical analysis
The results were analysed with a one-way ANOVA test (comparing the means of the control and individual Fe treatments) using XL Miner Analysis ToolPack (Microsoft). Statistically significant differences in relation to the control from the same time point are marked with an asterisk (*). The results are presented as means of independent experimental (biological) repetitions ± standard error (SE).

Results and discussion
Iron deficiency in human diet and the associated development of anaemia is a serious public concern. It is estimated that in 2019 anaemia affected 1.8 billion people, wherein children under the age of ten and woman in the reproductive age were the most susceptible groups (Safiri et al. 2021).
One of the promising strategies to combat nutrient deficiency is the introduction of biofortified crops, including sprouts enriched with Fe (Bouis andSalzman 2017, Vasconcelos et al. 2017). The legume sprouts could be suitable candidates for the biofortification process due to their important dietary role associated with their high protein levels (Joshi-Saha et al. 2022). Introduction of such Fe-enriched sprouts requires detailed insight into Fe effects on legume seedlings. The aim of the present study was to evaluate the impact of Fe-enrichment on the germination rate, growth and antioxidant properties of soybean seedlings.
The results showed that imbibition of seeds in FeCl 2 solutions had no effect on the germination rate or the germs growth expressed as roots length measured after 72 h of germination (Fig. 1A, B). At the same time point, the Fe content quantified using ICP-OES reached approx. 70 µg in g of dry weight (DW) in the control germinated seedlings and increased nearly threefold in the case of germs grown from seeds exposed to Fe at the concentration 100 mgL −1 and over sevenfold in the case of germs grown from the seeds imbibed in Fe solution at the concentration 500 mgL −1 ( Table 1). The Fe accumulation in the germinated seedlings was also assessed histochemically by the incubation in K 4 Fe(Cn) 6 :HCl solution according to Bramborova and Ivanov 2014. Shorter (90 min) staining resulted in the Fig. 1 Germination rate measured after 24, 48 and 72 h (A), root length measured after 72 h (B), Fe accumulation in soybean seedlings (C) and seeds (D) in control soybean seedlings and soybean seedlings previously exposed to FeCl 2 solution with Fe at the concentration of 100 and 500 mgL −1 . The results are presented as means of independent experimental (biological) repetitions ± SE appearance of light blue stains on the roots and seed coat of germinated seedlings grown from Fe-treated seeds (Fig. 1C). In the case of longer (24 h) staining, a few blue stains were noted in the control seeds, even blueish colouring in the seeds treated with lower Fe concentration (100 mgL −1 ) and dark blue colour in seeds imbibed in higher Fe concentration (500 mgL −1 ) (Fig. 1D). The results indicate that imbibition in FeCl 2 leads to concentration-dependent accumulation of Fe within the seeds and in the roots of young seedlings.
In Adzuki beans, the effect of three Fe sources, FeCl 3 , FeSO 4 and Fe-EDTA, has been compared. The results showed that FeCl 3 and FeSO 4 were most effectively taken up by the seedlings. However, application of higher doses resulted in hampered growth and germination rate (Oliveira and Naozuka 2017). Similarly, in broccoli, radish and alfalfa seedlings, Fe-dependent decrease in the fresh weight was observed only in response to the treatment with higher concentrations. On the other hand, mung bean sprouts were unaffected by the exposure to Fe even at the highest applied concentration (36 mgL −1 ) (Przybysz et al. 2016). A study comparing the response of bean and wheat showed that even at high Fe concentrations (up to 1000 mgL −1 ), the beans germination percentage was similar as in the case of the control. In addition, germination in Fe solutions did not affect roots nor shoots length of beans seedlings. In contrast, higher Fe concentrations caused a decrease in wheat germination rate and growth (Rasafi et al. 2016). In general, the described results indicate that legume seedlings show high tolerance to the Fe treatment. Therefore, they could be good candidates for biofortification with this metal. In soybean Fe is stored mainly bound to ferritin and is characterised by relatively good bioavailability. Studies evidenced that the absorption of Fe from ferritin is comparable to absorption form meat products or sulphates. It should be, however, highlighted, that in legume plants high content of phytic acid might negatively affect Fe bioavailability. Thus, various processing techniques are applied to decrease the level of phytates in soybean products, e.g. processing in high temperature, fermentation and soaking (Lӧnnerdal 2009).
Fe is redox active and can induce formation of reactive oxygen species (ROS). Indeed increased accumulation of anion radical has been observed in broccoli, radish, alfalfa and mung bean sprouts exposed to Fe. In addition, Fe treatment resulted in elevated levels of hydroxyl radical in broccoli and radish sprouts (Przybysz et al. 2016). Defence mechanisms against oxidative stress include stimulation of antioxidant system. In the present study seeds pre-treatment with Fe resulted in decrease in DPPH radical quenching noted after 72 h of germination (Table 1). Similar results were obtained in other studies, showing that soybean seedlings germination in FeSO 4 solution with Fe at concentrations 15, 20 and 25 mM results in attenuated antioxidant activity (Zielińska-Dawidziak and Siger 2012). Sprouts enrichment with Fe may also modify the activity of antioxidant enzymes. For instance Fe-dependent stimulation of glutathione reductase has been observed in broccoli, radish, alfalfa and mung bean, enhanced ascorbate peroxidase activity in broccoli, radish and alfalfa and elevated activity of catalase in broccoli sprouts (Przybysz et al. 2016).
The antioxidant system consists of enzymatic and nonenzymatic antioxidants. Phenolics form a large and diverse group of non-enzymatic antioxidants. Phenolic compounds, including the abundant group of flavonoids, have vast health benefits. Their protective effects have been described, for instance, in the case of cancer, diabetes and cardiovascular and neurodegenerative diseases (Rahman et al. 2021, Gupta andGuliani 2022). The results of the present study show that the content of total phenolic compounds (TPC) in soybean germinated seedlings was similar after 24, 48 ad 72 h of germination (Table 1). Seeds imbibition in FeCl 2 solutions had no effect on TPC at any of the studied timepoints. On the other hand, the level of flavonoids was lower in germinated seedlings grown from Fe-treated seeds. This effect was observed only after 72 h of germination (Table 1). Other studies on soybean seedlings showed that germination in FeSO 4 containing medium did not affect the level of total phenolics nor flavonoids in a statistically significant way. On the other hand, Fe caused an increase in the level of tocopherols and tocotrienols, which are also considered 91 ± 2 70 ± 10 68 ± 3* FeCl 2 (500 mgL −1 ) 98 ± 3 87 ± 21 60 ± 8* Fe content (µg in g of dry weight)* Control 73 ± 7 FeCl 2 (100 mgL −1 ) 220 ± 45* FeCl 2 (500 mgL −1 ) 549 ± 53* strong antioxidant compounds (Zielińska-Dawidziak and Siger 2012). In radish and alfalfa sprouts, no changes in the content of total phenolics have been observed in response to Fe. However, the Fe treatment increased the level of these compounds in broccoli and mung bean seedlings. In addition, Fe-enriched broccoli and alfalfa sprouts were characterised by significantly higher levels of ascorbic acid (Przybysz et al. 2016). Summarising, the results show that soybean seeds imbibition in FeCl 2 solution is an easy and promising method for obtaining Fe-enriched sprouts. Even short-term (2 h) imbibition in the solution results in significant Fe accumulation as evidenced by histochemical staining. Seeds pre-treatment with Fe did not affect the germination rate or seedlings growth. However, longer germination period (72 h) resulted in a decrease in the antioxidant activity and flavonoids level.
The results indicate that the choice of a proper germination period is important for the preservation of the most favourable nutritional characteristics of Fe-enriched sprouts. In general, it can be concluded that legume plants are good candidates for biofortification with Fe. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.