Influence of Intercropping on Eugenia dysenterica (Mart.) DC. Fruit Quality

Abstract

Intercropping to integrate cover crops with fruit trees in the Brazilian Cerrado is an innovative strategy for creating a more sustainable food system. This agricultural practice contributes to maintaining soil quality and improves fruits’ chemical and technological properties, such as those of Eugenia dysenterica (Mart.) DC. (cagaita). Given the significant fruit production potential of the Brazilian Cerrado, this study aimed to investigate the impact of an intercropping system involving cagaita trees and various cover crops, specifically Calopogonium mucunoides Desv. (CA), Crotalaria juncea (CR), Lablab purpureus (L.) Sweet (LA), brachiaria (Brachiaria decumbens L.) + nitrogen source (urea) (BRN), and brachiaria (Brachiaria decumbens L.) (BR), on the chemical composition, technological properties, and morphological characteristics of cagaita fruits. Treatments involving leguminous cover crops (CA, LA, and CR) significantly increased nitrogen (N) levels in cagaita fruits, comparable to those observed with the BRN treatment. However, the treatment utilizing BR resulted in the highest levels of macrominerals (Ca, Mg, and K), which are essential for meeting the Recommended Dietary Intake (RDI) and demonstrated a notable positive impact on pulp yield (>78%). Additionally, the antioxidant potential and phenolic content were the highest in the BR, CA, and LA treatments, with the lowest levels recorded for the CR treatment. This study underscores the importance of sustainable agricultural practices in the Brazilian Cerrado, demonstrating their potential to enhance the nutritional quality (both micro and macronutrients), technological properties, and overall development of Eugenia dysenterica DC. fruits, thereby adding value to food and contributing to a more resilient and productive food system.

1. Introduction

The Brazilian Cerrado is considered the richest savanna in the world and the second-largest biome in South America [1]. This biome is an immeasurable heritage of renewable natural resources, sheltering many plant species, including exotic fruit species, unique sensory characteristics, and high concentrations of essential human nutrients [2]. Several Cerrado native species play important economic and nutritional roles in the marketing of their products. One of the fruit species belonging to the Cerrado is Eugenia dysenterica DC., from the Myrtaceae family, popularly known as “cagaiteira” [3].

The cagaiteira trees have a high potential for use in agribusiness production systems, demonstrating high production [4]. The fruits are tasty and rich in nutritious substances such as proteins, lipids, dietary fiber [5], carbohydrates, carotenoids (α-carotene, β-carotene, β-cryptoxanthin, and lycopene), vitamin E (α and β, γ and δ-tocopherol, and tocotrienol), folates (tetrahydrofolate, 5-methyl tetrahydrofolate, and 5-formyl tetrahydrofolate) [3], ascorbic acid (vitamin C) [6], and phenolic compounds [7].

Due to the high nutritional potential of these fruits, it is interesting that commercials use this fruit tree mainly when it can be grown on land, such as in the Cerrado biome. One of the major difficulties in maintaining fruit productivity in the Cerrado biome is the presence of arid and semi-arid soils, which predominate in ancient and weathered soils with low levels of organic matter, making productivity dependent on nutrient cycling systems [8]. Thus, it is essential to incorporate organic compounds, manure, and fertilizers when cultivating these fruits [9].

A sustainable alternative that combines profitability with soil recovery is intercropping. This practice involves planting two or more crops simultaneously in the same area, promoting beneficial interactions between species [10]. The integration of cover crops with native plants has the potential to increase productivity in an economically and environmentally sustainable way, improving the soil’s physical, chemical, and biological properties [11]. These benefits arise through direct mechanisms, such as the release of organic nutrients, nutrient uptake by plants, nitrogen fixation by legumes, and modification of the carbon/nitrogen ratio by incorporating plant residues. Indirectly, cover crops increase soil organic matter, stimulate microbial biomass and eubacterial communities, enhance microbial activity, improve soil structure, porosity, and water conductivity, reduce erosion and nutrient leaching through chelation by root exudates, increase nutrient availability by modifying rhizospheric pH, and assist in more efficient nutrient cycling [11]. In this sense, grasses provide a more stable phytomass (slow decomposition rate), low nitrogen content, and high carbon/nitrogen ratio available in the soil. On the other hand, legumes result in phytomass with a low carbon/nitrogen ratio and a high nitrogen content. This causes legumes to have a mutualistic symbiotic relationship with fungi, contributing to greater nutrient availability for intercropped crops [10].

Under the edaphoclimatic conditions of the Cerrado, cover crops have been increasingly used [11], with grasses such as millet and brachiaria being widely utilized [12]. In intercropping and/or succession systems, particularly in crop-livestock integration (CLI) [13]. Cover crops are a viable and sustainable practice that improves the chemical and physical attributes of the soil, thereby increasing its fertility. This, in turn, directly influences the nutritional content of fruits and enables the sustainable production of alternative products, such as fruits from the Cerrado [14]. Moreover, the rich diversity of native species has significant potential for commercial production and integrated systems. However, there is a lack of studies examining the behavior of these fruit-bearing tree species and cover crops in intercropping systems [13], highlighting the need for further research to characterize the food products obtained from such systems.

The sustainable production of fruits for human consumption would enhance the value of conserved areas and improve the quality of fruits obtained through this intercropping system. The nutritional enhancement of cagaita fruits through intercropping can improve local diets, address nutritional deficiencies, enhance food security, and support rural livelihoods and economies in regions where this fruit is grown [15,16]. Promoting the adoption of these intercropping practices can yield significant benefits to human health and sustainable development at the community level. Therefore, it is essential to investigate production methods that enhance food’s nutritional, bioactive, physical, and sensory qualities, thereby stimulating consumer demand while simultaneously addressing sustainability issues. This study investigates the impact of an intercropping system involving cagaita trees (Eugenia dysenterica DC.) and various cover crops, specifically Calopogonium mucunoides Desv. (CA), Crotalaria juncea (CR), Lablab purpureus (L.) Sweet (LA), brachiaria (Brachiaria decumbens L.) + nitrogen source (urea) (BRN), and brachiaria (Brachiaria decumbens L.) (BR), on the chemical composition, technological properties, and morphological characteristics of cagaita fruits. The specific hypotheses include the following: (1) intercropping with legumes is expected to enhance the concentration of essential nutrients, such as nitrogen and potassium, in cagaita fruits due to improved nitrogen availability in the soil. (2) intercropping with legumes is anticipated to promote more efficient nutrient cycling, which may increase the synthesis of antioxidants like phenolic compounds and vitamin C. Additionally, Brachiaria decumbens is expected to enhance the availability of calcium and magnesium, which are essential for the nutritional and sensory quality of the fruits. Finally, (3) intercropping with Brachiaria decumbens is anticipated to significantly increase fruit pulp yield by improving soil conditions, favoring root development and nutrient absorption.

2. Materials and Methods

2.1. Plant Material

The experimental area is located at the Goiano Federal Institute —Campus Rio Verde, southwest of Goiás state, a region with cerrado vegetation (17°48′46″ S and 50°54′ 02″ W and altitude of 693 m). Access to the genetic inheritance for this project has been officially registered in the database of the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (Ministério do Meio Ambiente, Conselho de Gestão do Patrimônio Genético) under the registration code A9DF35D.

The experimental design utilized was a randomized block design (RBD) comprising five treatments that included various cover crops: Calopogonium mucunoides Desv. (CA), Crotalaria juncea (CR), Lablab purpureus (L.) Sweet (LA), Brachiaria decumbens L. + nitrogen source (urea) (BRN), and Brachiaria decumbens L. (BR), all intercropped with Eugenia dysenterica DC. (cagaita). Brachiaria decumbens L. (BR) was designated as the control treatment due to its prevalent application in Cerrado cropping systems, particularly in low-fertility soils, which makes it a suitable benchmark for assessing the effects of other cover crops on the chemical and technological properties of cagaita fruits. Each treatment was replicated four times, resulting in twenty trees per treatment (

Table 1. The intercropping system used to produce cagaita fruits (Eugenia dysenterica DC. Mart.).

Abbreviation	Description
BR	Cagaiteira tree in intercrop with brachiaria (Brachiaria decumbens L.), which was sown only once in October, cut at the beginning of the dry period (July/August) and left on top of the soil for dry matter to decompose.
BRN	Cagaiteira tree in intercrop with brachiaria (Brachiaria decumbens L.) was sown only once in October and received nitrogen fertilizer with 220 g of urea/plant in the crown region (0.5 m diameter). They were cut at the beginning of the dry season (July/August) and left on top of the soil to decompose the dry matter.
CA	Cagaiteira tree in intercrop with Calopogonium mucunoides Desv., which was sown in October (annually), cut at the beginning of the dry period (July/August), and left on top of the soil for dry matter to decompose.
CR	Cagaiteira tree in intercrop with Crotalaria juncea was sown in November (annually), cut at the beginning of the dry season (July/August), and left on top of the soil for dry matter to decompose.
LA	Cagaiteira tree in intercrop with Lablab purpureus (L.) Sweet was sown in November (annually), cut at the beginning of the dry period (July/August), and left on top of the soil for dry matter to decompose.

), with an overall count of 100 trees arranged at 5 × 5 m intervals between rows and/or individual plants (pits measuring 40 × 40 × 40 cm). The soil in the experimental site was classified as Dystrophic Red Latosol [17]. According to the Köppen-Geiger classification, the region is characterized by a tropical climate, with concentrated rainfall in summer and a dry season in winter.

The fruits were harvested 34 days after anthesis, when they exhibited a visually yellow pericarp (ideal ripening stage) over 13 days (the entire harvesting period), twice a day (morning and dusk). The fruits were selected and sanitized (200 ppm sodium hypochlorite for 10 min), and the pulp (CP), epicarp (CE), and seed (CS) were manually separated and weighed for yield calculation. The pulps were packaged in plastic containers, stored at −18 °C until analysis, and then thawed and homogenized before use. The chosen storage temperature was −18 °C because there is effective inhibition of enzymatic and microbiological activities at this temperature.

2.2. Physical Characterization

To perform physical characterization of the cagaita fruits, transverse diameter (TD), longitudinal diameter (LD), and fruit weight (FW) were determined in 50 samples. The measurements were taken using a digital pachymeter or analytical balance, and the results were expressed in mm or g. A Color Flex EZ spectrophotometer (Hunterlab, Reston, VA, USA) was employed to determine the color of 20 fruits (in triplicate) for each treatment and pulp, in agreement with the Commission Internationale de I’Eclairage (CIE) system (standard illuminant D65/10°).

2.3. Chemical Characterization

The reagents used in this work were all analytical standards. DPPH (2,2-Diphenyl-1-picrylhydrazyl), Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), ABTS (2,2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), 2,6-dichlorophenolindophenol, BHT (2,6-ditert-butyl-4-methyl phenol), 2,4,6-tripyridyltriazine (TPTZ), Folin & Ciocalteu phenol reagent, and ferulic acid were all purchased from Sigma Chemical Co. (St. Louis, MO, USA).

The fruits collected from each treatment were homogenized in a blender, and the titratable acidity (TA) was determined by titration with 0.1 M NaOH. The pH value and soluble solids (SS) were measured directly using a pH meter (model mPA-210, MS Tecnopon, Piracicaba, Brazil) and digital refractometer (Reichert Brix 35HP, Reichert Inc., Depew, NY, USA), respectively.

Official methods [18] were used to estimate the contents: moisture (method No 967.08), lipid (Bligh & Dyer method), protein (conversion factor = 5.9, method No 988.05), ash (method No 942.05), and carbohydrate (calculated by difference). Caloric values were calculated using Atwater conversion factors (proteins×4, lipids×9, and carbohydrates×4) [19].

For mineral quantification, the samples were dried in an oven at 105 °C and digested in HNO₃ and HClO₄. Regarding minerals, calcium (Ca), magnesium (Mg), copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) were determined by dual-beam atomic absorption spectroscopy (GBC-XPLORAA-2 model, Lakeside, Australia). Potassium (K) was determined by flame photometer (Micronal, Model B-462, Sao Paulo, Brazil) and phosphorus (P) and sulfur (S) were determined by molecular absorption spectrophotometry (Tecnal, SP-1105, São Paulo, Brasil) [20].

2.4. Total Bioactive Compounds and Antioxidant Activity

The total chlorophyll content was determined to be in agreement with that reported by Bruuinsma [21]. One gram of lyophilized pulp was homogenized in 30 mL of acetone: water (80:20, v/v) and filtered through a Whatman no. 4 paper filter. The mixture volume was then adjusted to 50 mL, and the absorbance was measured at 645 and 663 nm. The total chlorophyll content was calculated by Equation (1).

$$\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{c}\mathrm{h}\mathrm{l}\mathrm{o}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{h}\mathrm{y}\mathrm{l}\mathrm{l}=20.2\times A_{645}+8.02\times A_{663}$$

(1)

A₆₄₅ is the absorbance at 645 nm and A₆₆₃ is the absorbance at 663 nm. The results are expressed as mg per 100 g fruit (dry matter).

The total carotenoid content was determined according to Talcott and Howard [22]. Two grams of fresh fruit pulp was homogenized in 25 mL acetone:ethanol (1:1) and 200 mg/L butylated hydroxytoluene (BHT) in the dark at room temperature. Afterward, the extract was washed four times until the residue became colorless. The extract was filtered through the Whatman no. 4 paper filter, and its volume was adjusted to 50 mL. Absorbance was measured at 470 nm. Total carotenoids were calculated in agreement with Gross [23] using Equation (2).

$$\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{c}\mathrm{a}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{i}\mathrm{d}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}=\left(A_{470}\times V\times {10}^6\right)\left(2500\times 100\times \mathrm{g}\right)$$

(2)

where A₄₇₀ is the absorbance at 470 nm, V is the total volume of the extract, and g is the sample weight in grams. The results were expressed as mg per 100 g fruit (dry matter).

The ascorbic acid content was determined using the methodology described by Benassi and Antunes [24], which uses 2% oxalic acid as the extracting solution and 2,6-dichlorophenol-indophenol as the titrant solution. The results are expressed in mg ascorbic acid/100 g fruit (dry matter).

Extraction was carried out as previously described by Larrauri, Rupérez, and Saura-Calixto [25]. Analyses of total flavonoids, phenolic compounds, and antioxidant activities were then conducted. About 40 mL of methanol:water (50:50, v/v) was added to 2 g of pulp and kept at room temperature for 60 min. The mixture was filtered, and the supernatant was mixed with 40 mL acetone:water (70:30, v/v) and kept at room temperature for 60 min. After filtration, the liquids were mixed, and the volume was adjusted to 100 mL with water.

The Folin–Ciocalteu method [26] with modifications as described by Li, Wang, Li, Li and Wang [27] was used to determine the total phenolic content (TPC). Diluted crude extracts (200 mL) were mixed with 1.9 mL of 10-fold freshly diluted Folin-Ciocalteau reagent. The same volume (1.9 mL) of a sodium carbonate solution (60 g/L) was used for neutralizing the mixture. After a 120 min reaction, the absorbance of the mixture was measured at 725 nm in the dark at room temperature. Gallic acid was used as the standard, and the results were expressed as mg gallic acid equivalent (GAE) per 100 g fruit (dry matter).

Antioxidant capacity was measured using DPPH, ABTS, and FRAP. The DPPH method was performed as described by Brand-Williams, Cuvelier, and Berset [28], adapted by Rufino, Alves, de Brito, Pérez-Jiménez, Saura-Calixto and Mancini-Filho [29]. The results are expressed as % inhibition of radical DPPH. For the ABTS method, an ethanol solution was used for calibration, and the results were expressed in mM Trolox/g fruit (dry matter) [30]. The FRAP method, according to the methodology of Pulido, Bravo, and Saura-Calixto [31], with adaptations by Rufino, Alves, de Brito, Pérez-Jiménez, Saura-Calixto, and Mancini-Filho [29]. The reading was obtained at 595 nm, and the results were expressed in mM ferrous sulfate/g fruit (dry matter).

2.5. Identification and Quantification of Phenolic Compounds

The phenolic composition was evaluated using the Thermo Scientific™ UltiMate™ 3000 HPLC System (Thermo Fisher, Waltham, MA, USA) with a reversed-phase HC-C18 (4.6 × 100 mm; 3 μm) (Agilent, Santa Clara, CA, USA), coupled to a high-resolution mass spectrometer (Q Exactive Orbitrap Mass Spectrometer; Thermo Fisher) with source heated-electrospray ionization operating in negative mode, with spray voltage 3.5 kV, sheath gas 30, auxiliary gas 10, capillary temperature 350 °C, auxiliary gas temperature 250 °C, tube lens 55, and mass range 150–700 m/z.

Each lyophilized pulp (2 mg) was solubilized in 1 mL of deionized water, filtered through a polyester membrane filter (0.45 μm), and 10 μL of the extract was injected into a C18 column (4.6 × 100 mm; 3 μm) (Agilent), and the analysis was performed by Santos, Sousa, Santana, Almeida, Silva, and Egea [32]. We used a stock solution of standard phenolic compounds in methanol at a concentration of 1 mg/mL to identify phenolic compounds. Data were processed using Xcalibur 2.1.0 software (Thermo Fisher Scientific).

2.6. Infrared Absorption Spectroscopy

Fourier Transform Infrared Absorption Spectroscopy (FTIR) analyses were performed with the freeze-dried sample on a spectroscopy (Frontier-FTIR/NIR Spectrometer, PerkinElmer, MA, USA) following the following operating conditions: 650–4000 cm⁻¹ region, with 8 scans and 2 cm⁻¹ resolution.

2.7. Statistical Analysis

Data were subjected to analysis of variance (ANOVA) to detect significant differences among treatments, and means were compared using Tukey’s test using GraphPad 8.0. The t-test was used to compare treatments with BR treatment since this coverage is normally associated with livestock pastures, and our objective was to demonstrate that cagaita can be used in crop−livestock systems. Differences were considered significant at p < 0.05. All experiments were performed in triplicate.

3. Results and Discussions

3.1. Physical Parameters and Proximal and Mineral Composition

In the present work, different intercropping systems resulted in fruits yielding >70%, indicating that they can produce good use of fruits for processing in the food industry. The BR treatment was highlighted, presenting an average yield value higher than 78%, statistically differing from the CR treatment, which resulted in the lowest average value (72%) (p < 0.05).

Figure 1 presents the results obtained for the morphometric properties (transverse diameter, TD; longitudinal diameter of the fruits, LD; and fruit weight, FW) of cagaita fruits from different intercropping systems. The lowest coefficient of variation was obtained for LD, TD, and FM in the LA treatment, while the CR treatment obtained the highest coefficient of variation (Figure 1). The high coefficient of variation between measurements is related to the lack of standardization in the physical characteristics of the fruits collected in each treatment, which is representative of the species since no genetic selection has yet been carried out.

Although this occurred, the results of the present work were similar to what had been reported by Bueno, Guedes, de Souza, Madeira, Garcia, Taroco, and Melo [33] (LD between 1.92 and 3.29 cm, TD between 2.21 and 4.34 cm, and FM 6.66 to 36.36 g) who characterized cagaita fruits from the tropical highland region. Furthermore, several factors influence physical parameters, such as harvest time, sun exposure, climate, maturity stage, soil fertility, and the location of fruits on the plant [34,35]. Alves et al. [36] evaluated the physiological performance of the species Hancornia speciosa (mangaba fruit) during different seasons of the year, grown in full sun and in consortium systems to recover degraded areas. The researchers observed that H. speciosa intercropped with Syagrus oleracea favored more robust and healthy growth of mangabeira fruits and a significant increase in productivity compared to monoculture cultivation.

Intercropping with C. ochroleuca and the grass U. ruziziensis resulted in fruits with higher average mass compared to intercropping with other cultivars (U. decumbens and Canavalia ensiformis) in a ‘Pera’ orange orchard [37].

Table 2. Colorimetric analysis of the fruits and pulp and the physicochemical and chemical composition of the cagaita pulp grown in intercropping systems with Calopogonium mucunoides Desv. (CA), Crotalaria juncea (CR), Brachiaria decumbens L. (BR), Brachiaria decumbens L. + nitrogen fertilization (BRN), and Lablab purpureus (L.) Sweet (LA).

Parameters	BR	BRN	CA	CR	LA
Fruit colorimetric analysis (epicarp) (n = 20)
L*	54.60 ± 6.92 a	53.91 ± 6.44 ab	51.30 ± 6.03 b#	52.85 ± 6.80 ab	54.57 ± 4.76 a
a*	3.84 ± 6.46 a	1.11 ± 2.85 bc#	1.31 ± 3.23 ab#	−1.37 ± 3.42 c#	−0.04 ± 7.67 bc#
b*	47.44 ± 8.70 a	46.75 ± 7.98 a	45.73 ± 7.41 a	43.77 ± 7.91 a#	45.52 ± 6.77 a
C*	48.26 ± 7.24 a	46.85 ± 7.96 a	45.87 ± 7.36 a	43.93 ± 7.88 a#	46.22 ± 6.26 a
°h	83.17 ± 10.22 b	86.98 ± 2.49 a#	86.82 ± 3.38 a#	85.97 ± 4.41 a	84.37 ± 8.67 ab
Pulp colorimetric analysis (mesocarp)
L*	43.10 ± 1.25 a	39.98 ± 1.93 bc#	38.93 ± 1.91 c#	40.20 ± 1.02 bc#	41.63 ± 1.39 ab#
a*	−0.95 ± 0.26 d	2.52 ± 0.20 a#	0.11 ± 0.15 b#	−0.95 ± 0.15 d	−0.33 ± 0.27 c#
b*	33.00 ± 1.24 a	30.38 ± 2.33 b#	31.19 ± 1.71 ab#	30.49 ± 1.94 b#	32.99 ± 1.69 a
C*	33.01 ± 1.24 a	30.48 ± 2.32 b#	31.19 ± 1.70 ab#	30.48 ± 1.93 b#	32.98 ± 1.68 a
°h	91.64 ± 0.41 a	85.22 ± 0.62 c#	90.07 ± 0.93 b#	91.79 ± 0.21 a	90.65 ± 0.32 b#
Physicochemical and chemical composition
pH	3.05 ± 0.01 c	3.14 ± 0.01 b#	3.15 ± 0.01 b#	3.16 ± 0.01 b#	3.19 ± 0.01 a#
Titratable acidity (g citric acid/100 g)	0.86 ± 0.01 b	0.97 ± 0.01 c#	0.89 ± 0.01 d#	0.86 ± 0.01 b	1.06 ± 0.02 a#
Soluble solids (°Brix)	8.30 ± 0.10 c	9.53 ± 0.15 a#	8.60 ± 0.0 bc#	8.23 ± 0.1 c	8.90 ± 0.10 b#
Moisture (g/100 g)	91.87 ± 0.12 ab	91.17 ± 0.09 bc#	92.32 ± 0.14 a#	92.16 ± 0.05 a#	91.00 ± 0.62 c
Lipid (g/100 g)	4.48 ± 2.92 a	4.05 ± 2.87 a	4.66 ± 2.77 a	3.37 ± 2.21 a	3.61 ± 1.87 a
Ash (g/100 g)	3.74 ± 0.09 a	2.68 ± 0.24 c#	3.12 ± 0.03 b#	2.87 ± 0.1 bc#	2.71 ± 0.17 c#
Protein (g/100 g)	5.76 ± 0.42 b	12.24 ± 2.47 a#	11.93 ± 0.53 a#	11.97 ± 1.34 a#	14.91 ± 3.93 a#
Carbohydrate (g/100 g)	6.99 ± 0.24 ab	7.15 ± 0.17 a	6.16 ± 0.25 c#	6.41 ± 0.28 bc	7.08 ± 0.17 a
Energy (Kcal)	33.13 ± 1.19 b	36.17 ± 1.34 a#	31.54 ± 1.09 b	31.77 ± 0.87 b	36.72 ± 0.84 a#
Macrominerals
Phosphor (mg/100 g)	172.70 ± 0.01 a	127.82 ± 0.01 a	163.15 ± 17.75 a	150.32 ± 35.43 a	161.22 ± 17.54 a
Potassium (mg/100 g)	1579.01 ± 0.01 a	1150.38 ± 36.15 b#	1305.23 ± 0.01 ab	1252.64 ± 70.86 b#	1190.52 ± 140.30 b
Sulfur (mg/100 g)	12.34± 0.01 a	12.78 ± 0.01 a	18.82 ± 8.87 a	18.79 ± 8.86 a	12.40 ± 0.00 a
Calcium (mg/100 g)	1437.15± 218.07 a	287.59 ± 63.27 b#	301.21 ± 70.99 b#	225.47 ± 17.71 b#	241.82 ± 8.77 b#
Magnesium (mg/100 g)	376.25 ± 8.72 a	102.26 ± 36.15 b#	125.50 ± 17.75 b#	106.47 ± 26.57 b#	99.21 ± 17.54 b#
Microminerals
Copper (mg/100 g)	0.99 ± 0.17 a	0.89 ± 0.18 a	1.00 ± 0.18 a	1.13 ± 0.35 a	1.24 ± 0.53 a
Iron (mg/100 g)	9.07 ±1.66 b	8.56 ± 0.18 b	13.87 ± 0.44 a	11.27 ± 1.77 ab	11.47 ± 0.44 ab
Manganese (mg/100 g)	1.60 ± 0.35 a	2.75 ± 0.63 a	2.00 ± 0.18 a	2.07 ± 0.26 a	3.84 ± 1.05 a
Zinc (mg/100 g)	1.48 ± 0.35 a	1.79 ± 0.9 a	1.82 ± 0.27 a	2.69 ±1.15 a	2.54 ± 1.31 a
Boron (mg/100 g)	0.31 ±0.09 a	0.32 ± 0.09 a	0.69 ± 0.27 a	0.31 ± 0.09 a	0.31 ± 0.09 a

Average followed by standard deviation. Means with different letters show a statistical difference in the same line by Tukey’s test (p > 0.05). Means with symbol ^# demonstrate a significant difference with the BR treatment by Student’s t-test (p > 0.05).

presents the colorimetric analysis of the fruits and pulp and the physicochemical and chemical composition of the cagaita pulp grown in intercropping systems. Regarding the a* parameter of the colorimetric analysis of the fruit epicarp, the CR and LA treatments showed a slightly greenish color (negative values). In contrast, CA, BR, and BRN showed a slightly reddish color (positive values). The increase in the red levels may indicate advancement in fruit ripening [38]. Regarding the b* parameter, all treatments presented values in the yellow range (positive values), with no statistical difference between them. When the b* parameter is negative, the cagaita fruits appear bluish, indicating degradation of chlorophyll and synthesis of carotenoids, that is, very ripe fruits [39]. In addition to being related to the degree of ripeness of the fruit, color is a quality attribute that influences consumption and acceptability [40].

All fruits and their pulp demonstrated a color between orange and red in terms of tone, with medium saturation and luminosity (Figure 2). The L* parameter represents the luminosity and varies on a scale from 0 to 100, with 0 (completely dark) and 100 (completely clear) [41]. The L* values presented by the fruits were approximately 50, and therefore, a color of average brightness.

Cagaita pulps demonstrated high acidity, indicated by pH < 3.7 [6,42], which was lower for the BR treatment (control), with a statistical difference from the other treatments. These values were close to the fruit pulp of araçá (Psidium guineense Swartz) (3.57), cajá (Spondias mombin L.) (2.94), grape (Vitis sp.) (2.8–3.8), guava (Psidium guajava) (3.0–3.2), and passion fruit (Passiflora alata Dryand) (2.94). The pH value corroborates with the result obtained for titratable acidity that the values found in the present work were higher than values reported for pulps of cagaita (Eugenia dysenterica DC.) (0.64 g of citric acid/100 g) [43], murici (Byrsonima crassifolia L. RICH) (0.47 g citric acid/100 g), genipap (Genipa americana L.) (0.37 g citric acid/100 g) [44], and close to those reported for mangaba (Hancornia speciosa Gomes) pulps (0.80 g citric acid/100 g) [45]. Even with high acidity, it is reported that fruits with citric acid levels between 0.08–1.95% are well-accepted for human consumption [46].

Total soluble solids contents varied between 8.23 (CR) and 9.53 (BRN)°Brix, differing between treatments, in addition to all treatments presenting values higher than the control (BR treatment). The values found in this work were similar to those reported for cagaita pulp (8.00 °Brix), mangaba (Hancornia speciosa Gomes) (10.33 °Brix), yellow cajá (Spondias mombin L.) (11.00 °Brix), araçá (Psidium guineense Swartz) (12.00 °Brix), marolo (Annona crassiflora Mart.) (13.00 °Brix) [43], and sweet passion fruit (Passiflora alata Dryand) (13.33 °Brix) [44]. Singh et al. [47] observed that guavas intercropped with suran, an Indian yam, showed higher TSS levels (9.03–9.68%). The sweetness of the fruits is associated with freshness and maturity, making them more desirable to consumers. This slight increase in the quality of guavas may be associated with additional nutrient uptake by the fruit crop.

As the moisture content differed between the cagaita pulps evaluated, the other macronutrients were calculated on a dry basis to facilitate comparison. Primavesi, Primavesi, and Armelin [48] reported in studies with cover crops that the amount of accumulated nutrients depends on the species, planting time, soil fertility, and phenological stage at desiccation, in addition to the carbon/nitrogen ratio of the species, and this seems to have happened in this work for some macro and micro components.

There was no significant difference in the lipid content of cagaita pulps from the different intercropping systems. The ash content varied between 2.68 (BRN) and 3.74 (BR), differing from each other (p < 0.05), and the BR treatment was superior and distinguished itself from the others (CR, BRN, LA, and CA).

The evaluation of protein content showed values between 5.76 (BR) and 14.91 (LA) g/100 g, with a significant difference between them. Furthermore, all treatments were statistically higher than the BR treatment considered the control in the present study. The addition of nitrogen to the intercropping system (BRN) increased the protein content of the cagaita pulp, as did the treatments with legumes (CR, CA, and LA), which probably occurred due to the greater efficiency of nitrogen absorption and the biological fixation of the element [49,50,51,52].

All cagaita pulps, except BR treatment, demonstrated protein levels higher than those reported for araçá pulp (Psidium guineense Swartz) (0.42 g/100 g), yellow cajá (Spondias mombin L.) (0.75 g/100 g), and cagaita (Eugenia dysenterica DC) (0.77 g/100 g) [43]; murici (Byrsonima crassifolia L. RICH) (0.18 g/100 g), soursop (Annona muricata L.) (0.57 g/100 g), and genipap (Genipa americana L.) (0.21 g/100 g) [44]; and cerrado pear (Eugenia klotzschiana O. Berg.) (0.06 g/100 g) [53].

There was no significant difference between the intercropping system treatments regarding macro and micronutrients, phosphorus, sulfur, copper, manganese, zinc, and boron content. In contrast, the BRN treatment was significantly lower than the BR treatment (control) in terms of phosphorus content. Values varied between 127 (BRN) and 172 (BR) mg/100 g for phosphorus content. Still, these values are higher than those found for fruit pulp from the Cerrado, such as araçá (Psidium guineense Swartz) (9.62 mg/100 g), buriti (Mauritia flexuosa L.) (6.95 mg/100 g), mangaba (Hancornia speciosa Gomes) (9.16 mg/100 g), and similar to cagaita (Eugenia dysenterica DC) (12.75 mg/100 g) [39]. The sulfur content varied between 12.34 (BR) and 1.47 (CA) mg/100 g, with no significant difference between the intercropping system treatments.

The potassium content of the pulp varied between 1150 (BRN) and 1579 (BR) mg/100 g, with a significant difference between the treatments (Table 2). Espindola, Guerra, Almeida, Teixeira, and Urquiaga [54], in their study of different plant covers intercropped with banana trees, observed greater accumulation of potassium when there was cover by grasses, since the decomposition of residues and the release of potassium are slower in the dry season. Potassium is the most abundant cation in plant tissues and is absorbed from the soil solution in significant quantities by the roots in the form of the K⁺ ion. As it is not part of any organic structure or molecule, this micronutrient is a free or adsorbed cation that is easily exchangeable in cells or tissues [55].

Calcium and magnesium contents ranged from 225 (CR) to 1437 (BR) mg/100 g and 99.21 (LA) to 376.25 (BR) mg/100 g, respectively, with a significant difference between treatments. These values were higher than those found for the murici (Byrsonima crassifolia L. Rich) fruit (2 mg/100 g for Ca and 1.8 mg/100 g for Mg) [56] and similar to cagaita (Eugenia dysenterica DC) pulp (22.50 mg/100 g for Ca and 5.38 mg/100 g for Mg). The BR treatment presented higher values than fruit pulps from the Cerrado, such as marolo (Annona crassiflora Mart.) (39.26 mg/100 g), araçá (Psidium guineense Swartz) (42.29 mg/100 g), buriti (Mauritia flexuosa L.) (37.83 mg/100 g) and mangaba (Hancornia speciosa Gomes) (31.01 mg/100 g) [43].

The iron content varied between 8.56 (BRN) and 13.87 (CA) mg/100 g, with differences between treatments. These values are higher than those found for fruit pulp, such as buriti (Mauritia flexuosa L.) (0.67 mg/100 g), mangaba (Hancornia speciosa Gomes) (0.50 mg/100 g), marolo (Annona crassiflora Mart.) (0.65 mg/100 g), araçá (Psidium guineense Swartz) (0.18 mg/100 g), yellow cajá (Spondias mombin L.) (0.37 mg/100 g), and cagaita (0.33 mg/100 g) [43].

The macronutrients calcium, magnesium, and potassium were found in higher concentrations in the BR treatment, differing from the other cover crops (BRN, LA, CA, and CR). The BR sample generally contributed the most to the recommended daily intake (RDI) with significant amounts of minerals (Supplementary Table S1).

Martins et al. [57] reported that intercropping citrus with corn, pumpkin, and cassava resulted in higher nitrogen, phosphorus, and potassium use efficiency, with indices ranging from 0.10 to 1.0 after adding these fertilizers to the soil. This improved efficiency leads to more productive and higher-quality crops, reflecting the ability of plants to convert applied nutrients into plant biomass more effectively. Borges et al. [37] reported that intercropping a ‘Pera’ orange orchard with leguminous species (jack bean Canavalia ensiformis and Crotalaria ochroleuca) and grasses (Urochloa decumbens and U. ruziziensis) along with urea (Bahia state, Brazil), resulted in the combination of urea with Crotalaria ochroleuca and grasses (U. decumbens and U. ruziziensis) being able to replace up to 50% of the mineral nitrogen added as fertilizer, indicating its potential as a green manure.

3.2. Bioactive Compounds and Antioxidant Activity

The content of total carotenoids, ascorbic acid, total chlorophyll, and total phenolic compounds in cagaita pulps intercropping with cover crops is shown in Figure 3A, while the antioxidant activity determined by three different methods (ABTS, DPPH, and FRAP) is shown in Figure 3B.

The chlorophyll content varied between 38.05 (BRN) and 108.39 (BR) µg/g, with a significant difference between treatments. The carotenoid content obtained varied between 74.56 (LA) and 185.09 (CA) µg/g, with a significant difference between treatments. Although only the CA treatment demonstrated a significant difference from the BR treatment for total carotenoids, all other treatments demonstrated lower values, which were significantly different from the BR treatment. Fruits from all treatments can be considered ripe, presenting an intense yellow color, mainly due to the degradation of total chlorophyll and synthesis of carotenoids [58].

Regarding ascorbic acid levels, the results varied from 311.56 (LA) to 354.17 (CR) mg/100 g of pulp with no significant difference, except for LA treatment. The values found in this work were close to what had been reported for cagaita pulp (31.95–94 mg/100 g) [43] and Cerrado pear (Eugenia klotzschiana O. Berg.) (0.87 mg/100 g) [53]. Due to its excellent antioxidant properties, ascorbic acid is fundamental in eliminating reactive oxygen species [59]. According to the Institute of Medicine [60], the recommended daily intake of ascorbic acid is 90 mg/day, and the values found in the present study represent more than 30% of the daily consumption of 100 g of cagaita pulp.

The content of total phenolic compounds varied from 36.1 (CR) to 50.33 (BR) mg GAE/100 g fruit (dry matter), with a difference between treatments with excess CA and BR. However, these results were higher than those reported for Cerrado fruits, such as mangaba (Hancornia speciosa Gomes) (46.85 mg GAE/100 g), araçá (Psidium guineense Swartz) (89.14 mg GAE/100 g), buriti (Mauritia flexuosa L.) (110.72 mg GAE/100 g), yellow cajá (Spondias mombin L.) (98.97 mg GAE/100 g), and cagaita (143.44 mg GAE/100 g) [43]. According to the classification reported by Vasco, Ruales, and Kamal-Eldin [61], cagaita pulps have an average phenolic content (100–500 mg GAE/100 g), and these differences found in the results may be associated with agronomic factors (agricultural practices, soil composition, and climatological conditions) [62] (

Table 3. Phenolic compounds of the cagaita pulp grown in intercropping systems with Calopogonium mucunoides Desv. (CA), Crotalaria juncea (CR), Brachiaria decumbens L. (BR), Brachiaria decumbens L. plus nitrogen fertilization (BRN), and Lablab purpureus (L.) Sweet (LA).

Treatments/ Phenolic Compounds	Catequin	Epicatechin	Galic Acid
Mn	289.07214	289.07205	169.01343
BR	X	X	X
CA	X	-	X
CR	X	X	X
BRN	X	X	X
LA	X	X	X

Mn: molecular mass.

).

Cerrado fruits have received significant attention due to their therapeutic and medicinal potential, and cagaita stands out as a rich source of phenolic antioxidants, mainly flavonoids, such as catechin, epicatechin, and gallic acid. These flavonoids may be responsible for numerous biological effects, such as anti-cancer, anti-diabetic, anti-obesity, anti-inflammatory, anti-neuroprotective, memory enhancer, bactericidal, and, hepatoprotective effects, in addition to minimizing oxidative stress and risk factors for metabolic syndrome [63,64,65].

The antioxidant activities obtained by the different methods (ABTS, DPPH, and FRAP) showed agreement between the values (Figure 2B). Ordering in increasing order the results obtained for cagaita treatments according to antioxidant activity by ABTS method, we found CR < BRN < LA < CA < BR, varying between 36.63 and 24.34 mmol TEs/g; using the DPPH method, we found CR < BRN < CA < BR < LA, varying between 52.68 and 32.36% inhibition; and using the FRAP method: CR < BRN < LA < BR < CA, varying between 23.32 and 13.03 mmol ferrous sulfate/g.

The results obtained by the ABTS method for cagaita pulp, regardless of the treatments, were superior to those found for fruit pulp from the Brazilian Cerrado, cagaita (29.32 µmol TE/g), araçá (Psidium guineense Swartz) (10.92 µmol TE/g), buriti (Mauritia flexuosa L.) (6.03 µmol TE/g), mangaba (Hancornia speciosa Gomes) (2.49 µmol TE/g) [43], genipap (Genipa americana L.) (7.31 µmol TE/g), sweet passion fruit (Passiflora alata Dryand) (10.84 µmol TE/g) [46], araticum (Annona crassiflora Mart.) (5.7 µmol/g), jatobá (Hymenaea stigonocarpa Mart.) (5.2 µmol/g), and pequi (Caryocar brasiliense) (1.7 µmol/g) [66].

Different methods have been used to evaluate the antioxidant activity of fruits. These methods are based on the capture of organic radicals (ABTS and DPPH methods) and the reduction of iron in aqueous solutions (FRAP method). Each methodology has its own execution principles, conditions, and reaction mechanisms. As they are different from each other, it is not possible to quantify all antioxidant substances using a single method. Therefore, using two or more methods may allow additional elucidation of the complete profile of antioxidant activity [67,68].

Cagaita fruits are rich in phenolic compounds, which contribute to their high antioxidant capacity [69]. Cultivation techniques have increased these bioactive compounds’ levels and thus may benefit public health [70]. By reducing oxidative stress, many diseases can be prevented, and the greater desirability of these fruits can be promoted with this cultivation.

3.3. Fourier Transform Infrared Absorption Spectroscopy (FTIR) Analyze

Figure 4 presents the Fourier Transform Infrared (FTIR) spectra of cagaita fruit pulps obtained from different intercropping systems. The intense broad bands centered at 3300 cm⁻¹ and 2920 cm⁻¹ are characteristic of the O-H and NH groups [71]. In the region of 2920 cm⁻¹ it was possible to observe bands characteristic of asymmetric stretching vibrations of methylene CH [72].

The intense peaks observed between 900 and 1200 cm⁻¹ are due to the absorption of glycides, which are characteristic of complex monosaccharides and polysaccharides (C–O, C–C, and C–O–C groups) [73,74,75]. Specifically, in the region of 1025 cm⁻¹, the peaks are characteristic of fructose and glucose [76].

Peaks of medium intensity were observed in the region between 1800 and 1500 cm⁻¹, expressing bands characteristic of the presence of proteins, while those observed at 1722 and 1636 cm⁻¹ are bands characteristic of amide-I (elongation of the C=O carbonyl ester) [76] and amide-II, which are due to NH bending vibrations [77].

The bands in the 1500–1200 cm⁻¹ region were attributed to CH₃, CH₂, and CH bending vibrations [74,75]. The band at 1228 cm⁻¹ was attributed to the asymmetric stretching vibrations of phosphodiesters (p = O of PO²⁻) [75,78].

4. Conclusions

This study highlights the potential of intercropping for fruit cultivation using perennial species in combination with various cover crops. This viable, ecological, and cost-effective practice diversifies agricultural systems and promotes the sustainable intensification of cultivation methods. These results indicate that intercropping cagaita trees (Eugenia dysenterica) with cover crops improves nutritional quality, increases the levels of bioactive compounds, and enhances the technological properties of the fruits. Furthermore, this approach supports dietary diversification, strengthens food security, and contributes to the development of sustainable agriculture.

Leguminous cover crops enhance nitrogen availability for plants through biological nitrogen fixation and nutrient cycling, which are essential for plant nutrition. The intercropping of cagaita trees with cover crops positively influenced the levels of bioactive compounds, particularly increasing the ascorbic acid content associated with Crotalaria juncea (CR treatment). The antioxidant potential and phenolic content were higher in treatments involving Brachiaria (BR), Calopogonium mucunoides (CA), and Lablab purpureus (LA), while the lowest levels were observed in the CR treatment. Intercropping with Calopogonium mucunoides (CA) resulted in a higher iron content than the other treatments. The treatment with Brachiaria decumbens (L.) (BR) exhibited the highest levels of macrominerals (Ca, Mg, and K), which are crucial for meeting the Recommended Dietary Intake (RDI) and are essential for human nutrition. This treatment also significantly positively affected the pulp yield of cagaita fruits.

Further research on the long-term impacts of cultivation systems involving Eugenia dysenterica and/or other native species in conjunction with various annual cover crop varieties is essential to deepen our understanding of the interactions between these plants and optimize fruit quality. It is recommended that local farmers adopt intercropping of perennial plants with various annual crops, particularly in degraded areas with low soil fertility, such as the Cerrado, to enhance the nutritional quality of fruits and promote more sustainable agricultural practices. Specifically, intercropping with legumes and grasses such as Calopogonium mucunoides Desv., Crotalaria juncea, and Brachiaria decumbens L. can increase nutrient availability in the soil and improve fruit production. Careful selection of cover crops, appropriate crop rotation, and continuous monitoring are suggested to maximize benefits and strengthens food security and local economies.