Can biofortification of zinc improve the antioxidant capacity and nutritional quality of beans?

S H O R T C O M M U N I C A T I O N *Corresponding author: Esteban Sánchez, Centro de Investigación en Alimentación y Desarrollo A.C. Unidad Delicias. Av. Cuarta Sur 3820, Fracc. Vencedores del Desierto. Cd. Delicias, Chihuahua. México, C.P. 33089. E-mail: esteban@ciad.mx Received: 05 April 2016; Revised: 29 January 2017; Accepted: 31 January 2017; Published Online: 04 February 2017 Sida-Arreola, et al.: zinc biofortification in bean 238 Emir. J. Food Agric ● Vol 29 ● Issue 3 ● 2017 widespread deficiencies in Fe and Zn that is prevalent in developing countries (Mao et al., 2014). Soil and foliar biofortification with Zn is an effective solution to its deficiency problems in human health and crop production (Cakmak 2008; Prasad et al. 2014). However, there are little information about the way in which Zn biofortification can influence the quality of nutrients in plants and particularly the antioxidant capacity. Thus, this research was focused in the determination of the effect of Zn biofortification with two forms of Zn, on the nutritional status and antioxidant activity of common bean as bioindicator of an efficient Zn biofortification program in bean plants (Phaseolus vulgaris L.). MATERIALS AND METHODS Crop handling Bean seeds were germinated in a substrate mix (peat moss, vermiculite and perlite at ratio 3:1:1) and grown in greenhouse conditions in Delicias, Chihuahua, México. Bean seeds were purchased in Agrow Mexico in Mexico City. This variety of beans is a short cycle (sixty days), and it is grown in soils without saline problems and it is suitable to grown in greenhouse. Greenhouse temperature was at 25°C ± 4°C, humidity of 60-80%, and a light period of 16/8 h (light/darkness). Plants were grown in pots (25 cm diameter) with volume of 8 L, filled with substrate mix. Throughout the growing cycle the bean plants received a growth nutrient solution composed of 6 mM NH4NO3, 1.6 mM K2HPO4, 2.4 mM K2SO4, 4.0 mM CaCl2.2H2O, 1.4 mM MgSO4, 5 μM Fe-EDDHA, 2 μM MnSO4.H2O, 1.0 μM ZnSO4.7H2O, 0.25 μM CuSO4.5H2O, 0.3 μM (NH4) Mo7O24.4H2O and 0.5 μM H3BO3 (pH 5.56.0). Experimental designs and treatments In this research a completely randomized experimental design was used, with two forms of Zn, Zn-DTPA and ZnSO4 at doses of 0, 25, 50 and 100 μM, respectively, according to Sida-Arreola et al. (2015). Sampling and plant analysis Plants were sampled at 60 days of its germination, at complete phenological phase (development and fruit maturity). Organs of each plant were separated (seed, pod, root, stems, leaf). One part of plant material was frozen using liquid nitrogen and stored at -30° C, this material was used for antioxidant capacity assays (DPPH method, Brand-Williams et al., 1995). The other part of plant material was dried at 65° C and used to the determination of the nutritional quality (Karacan and Aslantas, 2008) in bean seed. Determination of the antioxidant capacity (DPPH method) The analysis was performed according to Brand-Williams et al.(1995) methodology. 1 g of seeds was used to macerate it in 5 mL of 80% methanol, and centrifuged at 6000 rpm for 10 min. 0.5 mL of the resulting supernatant extract were taken and added to 2.5 mL of a 0.1 mM DPPH freshly prepared solution, and mixture was incubated for 60 min in dark and cooled. It was measured the absorbance spectrophotometrically at 517 nm. White sample extract consisted of 0.5 mL of methanol. Values obtained by DPPH test were calculated by applying the following formula: g-1 percentage dry weight = (1(samples A517/A517 white) x 100. Determination of nutritional quality (macro and micronutrients) Determination of N, P, K, Ca, Mg, Fe, Mn, Zn and Cu were made according to Wolf (1982). As 0.2 g of dry seeds were mineralized in 12N H2SO4 and 30% H2O2 (v/v) free P at 275-300° C. The result of mineralization was gauged with 50 mL deionized water. The total N concentration was determined based on the Berthelot reaction method with modifications. Total P content was determined using the nitrovanadomolibdate colorimetric method. Total concentration of K, Ca, Mg, Fe, Mn, Cu and Zn were quantified by atomic absorption spectrometry. Statistical analysis It was used a simple ANOVA at 95% confidence, using SAS (SAS Institute Inc., Cary, USA). Means were compared by the Tukey ́s test (P ≤ 0.05). The data shown are mean values ± standard error (SE). RESULTS AND DISCUSSION Antioxidant capacity Antioxidants are compounds found in food and have positive health effects because of its potential to protect humans against reactive oxygen species (Padilla et al., 2008). In our study, we found significant differences in antioxidant capacity (DPPH method) as a result of the application forms and rate of Zn (Fig. 1), where generally the chelate Zn was better to Zn Sulfate to the dose of 25 μM, indicating that this form of Zn improves one of the attributes of bean quality and consumer health. Yuan et al. (2016) studied the result of enrichment of zinc on antioxidant capacity and growth in pea sprouts and found an increase from 10 to 50 μM Zinc improved antioxidant capacity due in turn to increase the Zn content, chlorophylls, phenolic content and amino acids, Zn enrichment could increase the nutritional quality and antioxidant activity as functional foods. Previous studies Sida-Arreola, et al.: zinc biofortification in bean Emir. J. Food Agric ● Vol 29 ● Issue 3 ● 2017 239 showed that the antioxidant capacity may depend on the abundance of metal ions (Zhu et al. 2013). This means that certain metals are required in small quantities by humans and they act as cofactors of enzymes, transcription factors and signaling proteins. These include various enzymes with the function of remove reactive oxygen species, in which the metals are used as cofactors of antioxidant enzymes, like various forms of superoxide dismutase (requires Cu, Mn and/or Zn; Johnson and Giulivi, 2005), catalase (requires Fe) and glutathione peroxidase (requires selenium) (Mates et al., 1999). Ríos et al. (2008) conducted a study with Se biofortification in lettuce and they observed that the treatment with 40 μM selenate was the most adequate for lettuce plants, improving the antioxidant activity and accumulation of selenium, which is important in food and consumer health. Likewise, Blasco et al. (2008) mentions that biofortified lettuce plants with iodine at a dose of 40 μM under the form of Iimproved biomass, antioxidant activity and mineral content of lettuce, ensuring the viability of a program biofortification. Nutritional quality Nutritional quality is one important topic to consider when talking about food, because they determine the functionality of the dietary parameters. The plant foods provide many of the essential compounds, minerals and other compounds that contribute to the enhancement of health. This experiment showed significant increases in nutritional quality of macronutrients and micronutrients as a result of the application of the two forms of zinc (Tables 1 and 2), highlighting the concentration of macronutrients and minerals under the form of sulfate Zn compared to chelate Zn (Tables 1 and 2) enhancing the nutritional quality of the elements as N, K, Mg, Fe, Zn and Ni. The doses and Zn application forms that enhanced the nutritional quality of beans were Zn Sulfate at doses of 25 to 50 μM. There are a little researches analyzing the effect of Zn on the nutritional content of edible plants, after application of this nutrient throughout the growing cycle of the crop. Hermosillo-Cereceres et al. (2013) showed that bean biofortification with selenium showed that the best doses were 40 μM selenite and 20 μM selenate, as they increased the content of Zn and antioxidant activity is, as well as improved in seeds bean, which increased the nutritional value of the crop. Furthermore, Yuan et al. (2016) found that the enrichment of Zn in pea sprouts improved the nutritional quality determined by the content of crude fiber, soluble sugars, protein and Zn content, indicating that biofortification Zn improved nutritional quality, as also occurred in this study.


INTRODUCTION
Legumes represent an important group in nutrition in the world, and represent an important source of protein, oil, vitamins, and minerals (Katoch, 2013;Marquez-Quiroz et al., 2015). Common bean is a food crop, which is one of the main protein sources in the developing world and also in minerals, fiber, and phytochemicals with analgesic and neuroprotective properties (Jha et al., 2015). Common bean is a staple crop and also a source for these minerals having more iron (around 55 µg/g) and zinc (around 35 µg/g) than cereals (Beebe et al., 2000). It was showed that there is a correlation between Zn and Fe in bean cultivars (Hoppler et al., 2014).
Zn is an important micronutrient because it plays important roles in crop production and human nutrition (Broadley et al. 2007). Approximately 10 % of human proteins require Zn for maintain their catalytic activity (Andreini et al. 2006). Zn is involved in the biosynthesis of proteins and in scavenging of reactive oxygen species (Cakmak 2000;Broadley et al. 2007). Zinc is deficient in 30 % of soils used for agriculture in the world (Alloway 2008), and WHO reports that about 33 % of the population is affected by Zn deficiency, which represent 450,000 dead children under five years old every year (Black et al., 2008). Increasing Zn levels in crops will lead to more Zn in humans. Zn application was an effective strategy of biofortification to increase Zn concentration in rice and wheat (Cakmak, 2008, Shivay et al., 2008, but information specific to common bean is limited. Micronutrient deficiencies in humans can be decreasing by increasing the mineral quality in edible crops. Biofortification is a promising strategy aimed to improving the mineral content of the staple plant food. Agronomic biofortification can improve the nutritional quality in the plant without it suffers genetic modifications (Storksdieck and Hurrell, 2009). Agronomic biofortification define the process of increase the concentration of essential elements in portions of plants through fertilization. This strategy was developed as a food-based method to help to decrease widespread deficiencies in Fe and Zn that is prevalent in developing countries (Mao et al., 2014).
Soil and foliar biofortification with Zn is an effective solution to its deficiency problems in human health and crop production (Cakmak 2008;Prasad et al. 2014). However, there are little information about the way in which Zn biofortification can influence the quality of nutrients in plants and particularly the antioxidant capacity. Thus, this research was focused in the determination of the effect of Zn biofortification with two forms of Zn, on the nutritional status and antioxidant activity of common bean as bioindicator of an efficient Zn biofortification program in bean plants (Phaseolus vulgaris L.).

Experimental designs and treatments
In this research a completely randomized experimental design was used, with two forms of Zn, Zn-DTPA and ZnSO 4 at doses of 0, 25, 50 and 100 µM, respectively, according to Sida-Arreola et al. (2015).

Sampling and plant analysis
Plants were sampled at 60 days of its germination, at complete phenological phase (development and fruit maturity). Organs of each plant were separated (seed, pod, root, stems, leaf). One part of plant material was frozen using liquid nitrogen and stored at -30° C, this material was used for antioxidant capacity assays (DPPH method, Brand-Williams et al., 1995). The other part of plant material was dried at 65° C and used to the determination of the nutritional quality (Karacan and Aslantas, 2008) in bean seed.

Determination of the antioxidant capacity (DPPH method)
The analysis was performed according to Brand-Williams et al.(1995) methodology. 1 g of seeds was used to macerate it in 5 mL of 80% methanol, and centrifuged at 6000 rpm for 10 min. 0.5 mL of the resulting supernatant extract were taken and added to 2.5 mL of a 0.1 mM DPPH freshly prepared solution, and mixture was incubated for 60 min in dark and cooled. It was measured the absorbance spectrophotometrically at 517 nm. White sample extract consisted of 0.5 mL of methanol. Values obtained by DPPH test were calculated by applying the following formula: g -1 percentage dry weight = (1-(samples A517/A517 white) x 100.

Determination of nutritional quality (macro and micronutrients)
Determination of N, P, K, Ca, Mg, Fe, Mn, Zn and Cu were made according to Wolf (1982). As 0.2 g of dry seeds were mineralized in 12N H 2 SO 4 and 30% H 2 O 2 (v/v) free P at 275-300° C. The result of mineralization was gauged with 50 mL deionized water. The total N concentration was determined based on the Berthelot reaction method with modifications. Total P content was determined using the nitrovanadomolibdate colorimetric method. Total concentration of K, Ca, Mg, Fe, Mn, Cu and Zn were quantified by atomic absorption spectrometry.

Statistical analysis
It was used a simple ANOVA at 95% confidence, using SAS (SAS Institute Inc., Cary, USA). Means were compared by the Tukey´s test (P ≤ 0.05). The data shown are mean values ± standard error (SE).

Antioxidant capacity
Antioxidants are compounds found in food and have positive health effects because of its potential to protect humans against reactive oxygen species (Padilla et al., 2008). In our study, we found significant differences in antioxidant capacity (DPPH method) as a result of the application forms and rate of Zn (Fig. 1), where generally the chelate Zn was better to Zn Sulfate to the dose of 25 µM, indicating that this form of Zn improves one of the attributes of bean quality and consumer health. Yuan et al. (2016) studied the result of enrichment of zinc on antioxidant capacity and growth in pea sprouts and found an increase from 10 to 50 µM Zinc improved antioxidant capacity due in turn to increase the Zn content, chlorophylls, phenolic content and amino acids, Zn enrichment could increase the nutritional quality and antioxidant activity as functional foods. Previous studies showed that the antioxidant capacity may depend on the abundance of metal ions (Zhu et al. 2013). This means that certain metals are required in small quantities by humans and they act as cofactors of enzymes, transcription factors and signaling proteins. These include various enzymes with the function of remove reactive oxygen species, in which the metals are used as cofactors of antioxidant enzymes, like various forms of superoxide dismutase (requires Cu, Mn and/or Zn; Johnson and Giulivi, 2005), catalase (requires Fe) and glutathione peroxidase (requires selenium) (Mates et al., 1999). Ríos et al. (2008) conducted a study with Se biofortification in lettuce and they observed that the treatment with 40 µM selenate was the most adequate for lettuce plants, improving the antioxidant activity and accumulation of selenium, which is important in food and consumer health. Likewise, Blasco et al. (2008) mentions that biofortified lettuce plants with iodine at a dose of 40 µM under the form of I-improved biomass, antioxidant activity and mineral content of lettuce, ensuring the viability of a program biofortification.

Nutritional quality
Nutritional quality is one important topic to consider when talking about food, because they determine the functionality of the dietary parameters. The plant foods provide many of the essential compounds, minerals and other compounds that contribute to the enhancement of health. This experiment showed significant increases in nutritional quality of macronutrients and micronutrients as a result of the application of the two forms of zinc (Tables 1 and 2), highlighting the concentration of macronutrients and minerals under the form of sulfate Zn compared to chelate Zn (Tables 1 and 2) enhancing the nutritional quality of the elements as N, K, Mg, Fe, Zn and Ni.
The doses and Zn application forms that enhanced the nutritional quality of beans were Zn Sulfate at doses of 25 to 50 µM. There are a little researches analyzing the effect of Zn on the nutritional content of edible plants, after application of this nutrient throughout the growing cycle of the crop. Hermosillo-Cereceres et al. (2013) showed that bean biofortification with selenium showed that the best doses were 40 µM selenite and 20 µM selenate, as they increased the content of Zn and antioxidant activity is, as well as improved in seeds bean, which increased the nutritional value of the crop. Furthermore, Yuan et al. (2016) found that the enrichment of Zn in pea sprouts improved the nutritional quality determined by the content of crude fiber, soluble sugars, protein and Zn content, indicating that biofortification Zn improved nutritional quality, as also occurred in this study.

CONCLUSIONS
Biofortification with Zinc improves antioxidant activity and nutritional quality of beans, considered the main attributes bean quality and consumer health. The highest antioxidant capacity was observed with the dose of 25 µM Zn chelate compared to the form of sulfate; while in the nutritional value of beans both forms of Zn chelate and sulfate content had similar doses of 25 to 50 µM. Finally, comment that it is feasible to implement a program biofortification  Zn bean, as this nutrient is more concentrated in the seeds (edible part of the plant), allowing improve its quality and antioxidant capacity.

Author's contributions
Authors that contributed in this work are presented next.