Nutritional composition and biological activities (antioxidant and antifungal) of Sesbania virgata (Cav.) Pers. seeds

biological activities stimulate its biotechnological and pharmacological application as exogenous antifungal and antioxidant agent.


INTRODUCTION
For many years, animal-derived nutrients, especially proteins, have been used as functional ingredients in food products. However, this practice is environmentally unsustainable due to the high consumption of natural resources, mainly water, associated with intensive livestock (BURGOS-DÍAZ et al., 2020). Thus, new strategies to provide food sustenance are needed, highlighting legume seeds are used as effective substitutes for animal protein (RUIZ-LÓPEZ et al., 2019). In addition, the progression of diseases caused by oxidative and microbial infections associated with the absence of completely effective treatments encourages the development of research involving the biological applications of legume seeds.
The Fabaceae family (aka Leguminosae) is a widely distributed, economically important group of crops and a staple human food. In the Caatinga, the Brazilian Savanna, Fabaceae is family most diverse (MOHAMMED and QORONFLEH, 2020).
For many years S. virgata is used as green manure specie to improve production of food crops and recover degraded areas (EVANS and ROTAR, 2020).
It is also used as a protein supplement for ruminants, in the tropical regions of Africa and Australia (GUTTERIDGE, 1995), and are edible plants in Argentina, Bangladesh and India (HOSSAIN and BECKER, 2001). However, other uses for S. virgata have not been identified, and to the best of our knowledge, the nutritional and biotechnological potentials of its seeds have been little investigated yet. Therefore, in order to start filling the existing void about the biological applications of S. virgata seeds, this research focusing to investigate on its nutritional composition, and antioxidant and antimicrobial activities. Traditional Knowledge, loose translation) -SisGen n. A1C2041. S. virgata seeds were dried in an environment with air circulation (± 27 °C) and pulverized in an electric mill. The obtained seed meal was homogenized (100 mg) in distilled water (1.0 mL), stirred in vortex-type tube shaker at room temperature and centrifuged at 5000 x g, for 5 minutes. The sediment was discarded and the supernatant, named ASSv, was used in further analyses.

MORPHO-PHYSICOCHEMICAL ANALYSES
Density, hydration (HC) and absorption (AC) capacities, and hydration index (HI) of seeds were determined according to Chavan et al. (1999). The water absorption capacity of seeds (WAC) was determined according to Plhak et al. (1989). Water absorption indexes by seeds (IWAs) and seed flour (IWAf), and seed flour solubility index in water (IFS) were determined according to Okezie;Bello (1988). The morphological standards (length/width) and the form and flattening seeds (thickness/width) were determined according to Silva et al. (2016). The proportions of seed coat + endosperm and cotyledons in the seed were determined by reference to the weight of 100 seeds.

PROXIMATE COMPOSITION AND ENERGY VALUE
The moisture, ash, crude fat and crude proteins contents from S. virgata seeds were determined according to AOAC (2000). Seed flour was dried at 105 °C for 24 hours, with subsequent rest and additional heat treatment. At the end, the moisture content (%) was recorded.

FATTY ACIDS ANALYSIS
The lipid profile and fatty acids methylation were obtained by Folch et al. (1957) and determined according to Hartman;Lago (1973). The identification and quantification of fatty acid esters was carried out by gas chromatography (Varied thioglycol (MOORE et al., 1958). Amino acid analysis was performed in a High-Performance Liquid Chromatograph (VARIAN, Waters 2690, USA) with C18 LUNA 100 Å column (4.6 mm x 250 mm; 5.0 μm particle) (Phenomenex, USA). The amino acids were quantified by comparison to standard (Thermo Scientific, USA).
DL-2-aminobutyric acid was used as an internal standard. The contents of different amino acids are presented as g amino acid per 100g of protein and compared with the FAO/WHO (2007) reference for individuals aged >18 years. The essential amino acid (EAA) score was calculated as: EAA score = (g of EAA in 100 g of protein of S. virgata seed / g of EAA in 100 g of protein in FAO/WHO standard) x 100.

SOLUBLE PROTEIN CONTENT AND SDS-PAGE
Total soluble protein content was measured using BSA as standard and Coomassie Brilliant Blue G-250 as chromogenic reagent (BRADFORD, 1976). The estimation of the relative molecular weight of the proteins was conducted by electrophoresis (SDS-PAGE) in the presence of 1.0% SDS and -mercaptoethanol (LAEMMLI, 1970). The application gel was prepared in the concentration of 3.5% and the separation gel, 12.5%. The gel was fixed in 12.5% trichloroacetic acid and stained with 0.005% Coomassie Brilliant Blue R-250. The weight estimation was obtained by comparison to the relative electrophoretic mobility of the molecular weight standard.

ANTINUTRITIONAL COMPOUNDS
Lectins were detected by hemagglutination assays (DEBRAY et al., 1981), using Oryctolagus cuniculus 3.0% erythrocyte (Ethics Committee for the Use of Animals, CEUA/UFPB, n. 178/2015). The presence of hemagglutination was determined in triplicate, by serial dilution and direct visualization of clots. The results were expressed as the inverse of the title of the highest dilution that still showed visible hemagglutination. For the detection of trypsin inhibitors (XAVIER-FILHO et al., 1989), bovine trypsin (0.3 mg/mL) was used as the standard enzyme and DL-BAρNA, as its chromogenic substrate. The inhibitor unit (IU) was defined as the amount of inhibitor that can decrease by 0.01 nm the absorbance value in the trypsin inhibitor assay, and since specific activity was considered, the relationship between IU and amount of protein used in the assay.

Antioxidant activity
The antioxidant activity was determined by ABTS (RE et al., 1999)  (100 mg mL -1 ) were transferred to test tubes containing 3.0 mL of the ABTS •+ or DPPH • radicals and the percentage of radical scavenging was determined, using Trolox 1.0 mM as positive control and ultrapure water as negative control.

Antimicrobial activity
Bacterial strains were housed in NA, stored at 4 °C and used to determine antibacterial activity. The assays were performed in BHI broth, with chloramphenicol (100 µg mL -1 ) as negative control. Yeast and filamentous fungi were housed in SDA, stored at 37 -35 °C and used to determine antifungal activity.

STATISTICAL ANALYSES
Data are expressed as mean ± standard deviation of three repetitions. Analysis of variance (ANOVA) was performed for data analyses using GraphPad Prism® version 6.01 (GraphPad Software, USA), with Tukey's post-test. A value of p<0.05 was considered statistically significant.

MORPHO-PHYSICOCHEMICAL CHARACTERIZATION
S. virgata seeds showed coloring (light brown tones) and density (0.9 ± 0.01 g mL -1 ), similar to other Sesbania species (HOSSAIN and BECKER, 2001). Its individual weight and weight of 100 seeds were 0.1 ± 0.01 g seed -1 and 7.6 ± 0.01 g 100 seeds -1 , respectively. The seeds dimensions were 0.4 ± 0.00 cm thick, 0.4 ± 0.00 cm width and 0.6 ± 0.01 cm long, thus being the full and elliptical type, according to Silva et al. (2016). The dry mass of S. virgata pods was 0.9 ± 0.00 g pod -1 and each pod houses 6 seeds. The pods showed 0.6 ± 0.00 cm thick, 0.9 ± 0.01 cm wide and 6.1 ± 0.00 cm long. The small size of pods with few seeds is interesting because S. virgata can firmly fix its fruitlessness on peduncle, reducing the chances of the peduncle bending and breaking. In addition, the light weight of pods prevents the branches from touching the soil.
Página | 3655 seed shells, with consequent low hydration and water absorption; the amount of water that is absorbed by the starch granules from flour; and the degree of degradation that water causes in the fibers. Thus, it stands out that the morphophysicochemical characteristics of S. virgata seeds express its quality in different processing, in addition to corresponding to a proper percent of hydrophilic molecules, according to Hodge;Osman (1976) criteria.

NUTRITIONAL COMPOSITION
The high protein content (60.8% ± 0.18) stands out in relation to the other macronutrients from S. virgata seeds, and in relation to the protein contents recorded for other legumes (RUIZ-LÓPEZ et al., 2019), including other Sesbania species (HOSSAIN and BECKER, 2001). This promising value encourages the use of S. virgata seeds as a surprising protein nutritional matrix. In the food industry, especially, the use of S. virgata seeds can serve as a food base to overcome protein deficiency. If applied for that purpose, the daily intake of protein be quantified according to the age group.
The moisture content of S. virgata seeds (8.4% ± 0.00) is in accordance with the Brazilian legislation (BRASIL, 2005). This content was even higher than the contents presented by other Sesbania species (HOSSAIN and BECKER, 2001). The ash content of S. virgata seeds (4.8% ± 0.02) is slightly at odds with the maximum content (4.0%) allowed by Brazilian legislation (BRASIL, 1978). However, the ash index does not always characterize the real inorganic richness present in the seed, because some chemical properties can be altered in the determination (AOAC, 2005). Also, this value may still be correlated with the S. virgata seed collection site, a soil abundant in wet matter and constant visitation of cattle, which may have interfered in the ash content of S. virgata seeds.
The crude fat content of S. virgata seeds (7.6% ± 0.19) exceeds the contents of other Sesbania species (HOSSAIN and BECKER, 2001) and can play an important role in the nutritional status of the world population, recognizing its importance as renewable sources of fatty acids. Thus, to monitor lipid bioavailability, Table 1 presents the total fatty acids from S. virgata seeds. Eight fatty acids were detected, four saturated and four unsaturated. The sum of all the unsaturated fatty acids detected in the chromatogram was 68.3%. Among them, the polyunsaturated were the most abundant (45.8%), with two important essential fatty acids, linoleic acid and linolenic acid. This data was promising because polyunsaturated fatty acids are essential to the architecture of cell membranes and play fundamental roles in many cellular processes. The remainder of the chromatogram (31.7%) contemplate saturated fatty acids. Among them, palmitic acid was the one that contributed to the total fatty acid profile, followed by stearic acid.
One of the most attractive features of S. virgata seeds is the high protein content and the good balance of amino acids. Both were shown to maintains the balance of nitrogen in biological system. The deficiency of S. virgata seeds in EAAs could be balanced in association with a diet complemented with cereals. According to Anitha et al. (2020), cereals are naturally rich in methionine and cysteine, and the right combination of legumes seeds and cereals has prospects of enhancing the nutritional value of foods and contributing to a balanced dietary system.

ANTINUTRITIONAL FACTORS
The conditions used in the assay were not sufficient to detect the presence of lectins and trypsin inhibitors in S. virgata seeds. Hossain;Becker (2001) report the presence of these antinutritional factors in S. aculeata, S. rostrata and S. sesban seeds, but its contents are very low. Although, when isolated, these antinutritional factors have biological relevance, its absence in S. virgata seed is an interesting finding, because lectins and trypsin inhibitors generally do not degrade easily in the gastrointestinal tract and can be complexed with digestive enzymes, interfering in the nutrient absorption (SILVESTRINI et al., 2017). Additionally, the acceptability and use of legumes seeds have been affected by the presence of these antinutritional factors, which reduce the bioavailability nutrients (SHARAN et al., 2021). The non-detection may have occurred due to the use of water as the only extraction agent and the absence of vigorous mechanical agitation processes, capable of breaking the cell walls of S. virgata seeds and solubilizing these proteins.

Antioxidant activity
Recognizing the importance of neutralizing the deleterious effects of reactive species and offering new therapeutic possibilities from natural compounds, the  infections, we present the S. virgata seeds as a potential antifungal agent (Table 3).
According to our analyses, the MIC required to promote inhibition of C. albicans and C. tropicalis was 256 μg mL -1 , which represents an optimal activity, according to Houghton et al. (2007) criteria. Although S. sesban (MAREGESI et al., 2008) also presents anti-Candida albicans activity, our results are more promising, because the MIC of S. virgata is four times lower. However, not all Sesbania species exhibit activity against C. albicans, such as S. grandiflora (SRINIVASAN et al., 2001).
In turn, the MIC required to promote inhibition of A. flavus and P. citrinum is criteria. Another record of antifungal activity was found for protein from S. virgata seeds (PRAXEDES et al., 2011). This antifungal protein is toxic to Aspergillus niger, Cladosporium cladospoirides and Colletotrichum gloesporioides, reinforcing that S. virgata seeds are excellent sources of antifungal biomolecules.

CONCLUSIONS
This study presents the nutritional characterization of Sesbania virgata seeds, with important highlights for abundance in protein (60.8% of total protein; 75 -31 kDa, the main protein bands), and essential and non-essential amino acids.
However, the carbohydrate and crude fat contents are also promising, highlighting the abundance in polyunsaturated fatty acids, especially linoleic acid and linolenic acid. These results are also associated with a positive health impact, because S. virgata seeds exhibit antioxidant and antifungal activities. By introducing studies on the discovery of ecologically friendly therapeutic possibilities, we believe that S. virgata seeds can be explored as sources of biomolecules with antibiotic and antioxidant properties, besides highlighting its economic and nutritional potential.
The identification and isolation of these biomolecules are being employed by our group. We suggest investigating the toxic effects of S. virgata seeds flour on humans, animals and the environment to confirm the safety of its application in various sectors of the industry.