Guazuma ulmifolia Lam . )-physical , physicochemical and antioxidant characterization

Guazuma ulmifolia Lam., popularly known as mutamba, mutambo and mucungo, is widespread throughout Latin America. Studies on the physicochemical characteristics and the technological application of its constituents can support its use as raw material for enrichment and formulation of food products. This study aimed at to identify substances of food interest Research, Society and Development, v. 9, n. 7, e176973680, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i7.3680 3 derived from mutamba fruit, through its physical, physicochemical, proximate, mineral, bioactive compounds and antioxidant activity characterization in two maturation stages. The incomplete and full physiological maturation had, on average, mass of 5.52 and 3.54 g, respectively, and acid pH of 4.84 on average. Soluble solids content increased with maturity reaching 32.9 °Brix. Regarding proximate composition, it was observed an increase in protein, lipid, crude fiber and ash; and reduction of 80% in moisture. The most concentrated minerals were K, N, Ca, P and Mg. The content of anthocyanins, polyphenols, yellow flavonoids and the antioxidant activity increased with maturation, while vitamin C remained at 1500 mg.100 g. This study is innovative as mutamba fruits with incomplete physiological maturation were characterized for the first time. That mutamba fruits, no matter the physiological maturation, have potential of exploitation in the food industry, due to their high content of soluble solids, as well as in the enrichment of other food products as source of vitamin C and minerals.


Introduction
Guazuma ulmifolia Lam., popularly known as mutamba, mutambo and mucungo, is widespread throughout Latin America. It comes from a perennial tree plant, with a height ranging from 7 to 30 meters. The leaves are oval and hairy, with small flowers that show long light yellow hue filiform appendages measuring from 5 to 10 mm long, and slightly perfumed (Brandão et al., 2002;Carvalho, 2010). The fruit is a subglobose, dry, warty capsule (Gómez-Gurrola et al., 2014), of green color when immature and black when ripe, with small whitish seeds; it is hard and varies in length from 1.5 to 4.0 cm, containing, on average, 87 seeds (Pereira et al., 2019).
Leaves and stem leverage researches related to antiretroviral action (HIV -human immunodeficiency virus and herpesvirus) and against androgenic alopecia, being used to treat numerous other diseases such as diarrhea, asthma, bronchitis, fever, elephantiasis, syphilis, obesity, leprosy, dysentery, among others, in the form of herbal infusion or essential oil (Lopes et al., 2009). Regarding the mutamba fruits, whether ripe or green, studies are still taking place very timidly, but it is already possible to find some works on the proximate and phytochemical composition of maturity mutamba fruit (Pereira et al., 2020), as well as its application in the form of flour for the production of tea, bread and popsicles (Assis et al., 2019;Tene et al., 2007).
The aim of this work was to identify substances of food interest derived from mutamba fruit, through the physical, physicochemical, centesimal, mineral, bioactive compounds and antioxidant activity characterization of fruits in two maturity stages Fruit with Incomplete Physiological Maturity and Fruit with Full Physiological Maturity. Development, v. 9, n. 7, e176973680, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i7.3680 5

Materials and Methods
This work corresponds to a basic research of experimental nature and quantitative approach. This research can be classified as explanatory in terms of objectives, since it seeks to verify cause and effect relationships from data collected in the laboratory (Cervo et al., 2007;Pereira et al., 2018). Its purpose is to identify in mutamba fruits substances of food interest, through the physical, physical-chemical, centesimal, minerals, bioactive compounds and antioxidant activity characterization of fruits in two maturity stages, by obtaining numerical data and statistical evaluation.

Chemicals
All standards and reagents were of analytical grade. The solutions were also prepared with reagents of analytical grade.

Plant material
The Fruits with Incomplete Physiological Maturity (FIPM), which are green, and the Fruits with Full Physiological Maturity (FFPM), which are black (Figure 1), were manually collected from mutamba trees located on rural properties of Limoeiro do Norte, Ceará, a tropical climate city with dry season (AW), located at 05º 08' 44" South, 38º 05' 53" West, with an altitude of 30 meters in relation to sea level, showing average annual temperature and rainfall of 27.6 °C and 762 mm, respectively. Research, Society and Development, v. 9, n. 7, e176973680, 2020 (CC BY 4. Fruits with incomplete physiological maturation: green (a) and fruits with full physiological maturation: black (b). Source: authors.

Physical and physicochemical analyses
For each maturation stage, 20 fruits were used to determine the average mass, in an analytical balance, and the transversal diameter and longitudinal diameter, with a manual caliper. The shape index was calculated by the ratio between the transversal diameter and the longitudinal diameter. The physicochemical determinations and the subsequent analyses were performed in four replicates in triplicate, totaling 200 fruits for each maturation stage. The material analyzed consisted of a ground mixture (fruit + seed). The pH was measured in 10% (w/v) aqueous solutions (pH meter Quimis Q799-02, Diadema, Brazil). Soluble solids were measured in a digital refractometer and the results expressed as °Brix. The titratable acidity was determined by titration with 0.1 N sodium hydroxide (method 942.15) according to Association of Official Analytical Chemists (2005) and the results were expressed as percentage of citric, malic, tartaric and acetic acid using the conversion factor 0.6404, 0.6704, 0.7504, 0.6005, respectively. Water activity was determined using the Aqualab Lite meter (Decagon Devices Inc., Pullman, USA), at 25 °C.

Minerals
The samples were calcined in a muffle furnace at 550 °C for 16 h. The ashes were then digested according to the method described by Malavolta et al. (1989). Macrominerals (Ca and Mg) and microminerals (Cu, Fe, Mn and Zn) were determined by atomic absorption spectrophotometry (iCE 3300; Thermo Scientifc, USA); Phosphor (P) was quantified by the molybdenum blue colorimetric method and sulfur (S) by the barium chloride turbidimetry.

Bioactive compounds
The vitamin C content was determined by titrimetry (method 985.33; Association of Official Analytical Chemists, 2005) and the results were expressed as mg.100 g -1 .
Anthocyanins and yellow flavonoids were quantified by spectrophotometric techniques with absorbances measurement at 535 and 374 nm, respectively, and the results were expressed as mg.100 g -1 (Francis, 1982).
For total extractable polyphenols (TEP) and antioxidant activity determinations, an extract of each sample was prepared by adding 40 of methyl alcohol 50% (v/v) to 17 g of sample, keeping the mixture at room temperature protected from the light for 60 minutes.
After that, it was centrifuged (Eppendorf 5804) at 5000 rpm for 20 minutes, and the supernatant was filtered to a 100 mL volumetric balloon. The procedure was repeated with the residue by adding acetone 70% (v/v), and the supernatant was filtered and added to the same volumetric balloon, whose total volume was completed with distilled water. The extract was kept refrigerated, protected from the light until the moment of the analysis, up to one month.
The total extractable polyphenols were determined by the method described by Obanda et al. (1997) and Larrauri et al. (1997), using Folin-Ciocalteau reagent and a calibration curve of gallic acid. The blank consisted of the same mixture prepared for the samples, replacing it by water. The absorbances were measured in spectrophotometer FEMTO 600 Plus, at 700 nm, and the results were expressed in mg.g -1 of gallic acid. Research, Society and Development, v. 9, n. 7, e176973680, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i7.3680 8

Antioxidant activity
The antioxidant activity of the mutamba fruits was quantified using the methodologies described by Rufino et al. (2010).
Ferric reducing antioxidant power (FRAP) assay: a calibration curve of ferrous sulphate (FeSO4) was used and the absorbances were measured in spectrophotometer FEMTO 600 Plus, at 595 nm. FRAP reagent was used as blanket and the results were expressed as µM of FeSO4.g -1 of sample.
ABTS + radical scavenging assay: a calibration curve of Trolox was used and the absorbances were measured in spectrophotometer FEMTO 600 Plus, at 734 nm. Ethyl alcohol was used as blanket and the results were expressed as μM of Trolox.g -1 of sample.
DPPH radical scavenging assay: a calibration curve of DPPH radical was used and the absorbances were measured after their stabilization in spectrophotometer FEMTO 600 Plus at 515 nm. Methyl alcohol was used as blanket and the results were expressed as EC50, which means the amount of sample, in g, necessary to reduce to half the initial concentration, also in g, of DPPH free radical (g of sample.g -1 DPPH).

Statistical Analysis
Statistical analyses were performed using the Statistica 7.0 software (Statsoft, 2007) and the null hypothesis between the data was verified with Student's t-test, where a significant difference was considered if p < 0.05. Pearson's correlation matrix was applied to some parameters.

Results and Discussion
The mutamba fruits studied presented average mass of 5.52 ± 0.46 g for FIPM and 3.54 ± 0.53 g for FFPM (Table 1). This result can be justified by the marked water loss that occurred during ripening. Research, Society and Development, v. 9, n. 7, e176973680, 2020 (CC BY 4. The average diameter values of FIPM were 2.32 ± 0.14 cm for the transversal one and 2.08 ± 0.18 cm for longitudinal one; while the FFPM presented a transversal diameter of 2.07 ± 0.23 cm and longitudinal diameter of 2.74 ± 0.28 cm; resulting in an shape index of 1.12 ± 0.13 for FIPM and 0.76 ± 0.10 for FFPM. Shape index other than one indicates that the fruits are oval shaped, being equatorially elongated if shape index is greater than one or longitudinally elongated if shape index is smaller than one. The change of orientation in the shape of the fruits can be justified by the loss of moisture which occurs until the end of the maturation stage (Table 1). Knowledge of this parameter is important for fruits that will undergo selections or mechanized processes (Kill et al., 2010).
The mutamba fruits showed 80% of water loss during the ripening process, from 71.14 g.100 g -1 (FIPM) to 14.11 g.100 g -1 (FFPM) of moisture, and the water activity followed the same behavior, from 0.77 in the FIPM to 0.23 in those FFPM. This low moisture contents can justify the hardness and the dryness in the FFPM fruits. The ash content was 1.51 and 4.56 g.100 g -1 for FIPM and FFPM, respectively (Table 1). This shows that the mutamba fruits, at Research, Society and Development, v. 9, n. 7, e176973680, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i7.3680 both maturation stages, are good sources of minerals compared to other crops such as noni (Morinda citrifolia L.) (Palioto et al., 2015), which presents only 0.75 g.100 g -1 of mineral matter in its composition.
For the crude fiber content, we observed that the FIPM showed a value of 13.49 g.100 g -1 , while this value reached 48.86 g.100 g -1 for FFPM. This feature is of great interest mainly to the dietetic products industry, which exploits high-fiber foods.
Protein values, of 5.41 g.100 g -1 for FIPM and 10.19 g.100 g -1 for FFPM, were similar to those reported in the literature, where levels ranging from 7.95 to 14.91 g.100 g -1 were found in mutambas (Miranda et al., 2008, Rojas-Hernández et al., 2013, Gómez-Gurrola et al., 2014, Rojas-Hernández et al., 2015. The pH of the fruits was shown to be acid in both FIPM and FFPM mutambas, with values of 4.98 and 4.70, respectively (Table 1), the latter being similar to the results found by Pereira et al. (2020). The titratable acidity was expressed as a function of different acids, since there is no record of the predominant organic acid in that fruit. In general, it can be observed that the FFPM had three times the acidity of those FIPM.
As expected, the soluble solids (Table 1) increased during maturation, ranging from 4.70 °Brix in the FIPM to 32.90 °Brix in the FFPM, a value much higher than those reported in the main raw materials that presents high contents: pineapple (14 °Brix), sugarcane (16 °Brix), grape (18-26 °Brix), but smaller than tamarind (39-42 °Brix) (Dellacassa et al., 2017, Amorim et al., 2016, Urcan et al., 2017, Taha et al., 2016. Fruits with this feature are usually exploited in the production of fermented beverages, considering that soluble solids are one of the factors that interfere in the alcoholic fermentation (Casimiro et al., 2000). During must fermentation, soluble solids usually show a marked decrease, stabilizing around 5 °Brix.
The correlation matrix (Table 4) shows that both anthocyanins and yellow flavonoids showed a very strong correlation (p < 0.01) with antioxidant activity in the mutamba fruits, except for the ratio between anthocyanins and antioxidants by the DPPH method (r = 0.3913; p = 0.0586), which was characterized as a strong correlation (0.01 ≤ p < 0.05). This demonstrates the effectiveness of these compounds, present in the fruit, as antioxidant by electrons and hydrogens scavenging and by neutralizing free radicals. There was no significant correlation (p > 0.05) between vitamin C and total phenolics with the antioxidant activity of the mutamba fruits. participations in the proper functioning of enzymes, especially those which act as antioxidants, retarding cellular oxidation. Fe alone acts by forming hemoglobin, and Mn acts in energy production processes. According to the Brazilian resolution which regulates complementary nutritional information (Brasil, 2012), mutambas FIPM and FFPM can be classified as fruits with high content of P, Ca, Mg, Fe and Mn.

Conclusions
The food industry currently seeks innovative and sustainable products. This study characterizes for the first time mutamba fruits with incomplete physiological maturation and shows that regardless the maturity stage, mutamba fruits are an excellent source of vitamin C, besides their soluble solids, fibers, proteins and essential minerals (P, Ca, Mg, Fe and Mn) content, being promising for the enrichment of food products.
The high soluble solids content in the mutamba fruit with full physiological maturation indicates that it has potential for exploitation in the food industry with respect to fermented products.
Toxicological and anti-nutrients tests are suggested to be carried out on mutamba fruit with incomplete physiological maturation before their application in human food.