Development of Fruit-Based Carbohydrate Gel for Endurance Athletes

Abstract

The aim of this study was to produce a carbohydrate gel based on genipap and banana and analyze its physico-chemical, rheological, and sensory quality, as well as its proximate composition and antioxidant activity. Three gel samples were formulated containing different concentrations of genipap and clarified banana juice. The formulated samples followed the minimum parameters required and were subjected to analyses of their pH, soluble solids, titratable acidity, moisture, ash, lipids, proteins, glucose, fructose, sucrose, polyphenols, antioxidant activity, and rheology. Commercial carbohydrate gel was used as a control sample. It can be concluded that the gel formulations were formulated following the minimum parameters required, with a moderate sensory acceptance. The physico-chemical parameters and proximate composition the developed gels were similar to the commercial gel, while their glucose, sucrose, fructose, polyphenol, and antioxidant activity contents were higher and their rheological properties were within the expected range for this category of commonly marketed products. In the two blocks of analysis mentioned above, data variability was mostly explained by PC1–PC3 at almost 100%. Rheologically, the commercial gel is considered to be a Newtonian fluid, and the developed formulations can be considered as pseudoplastic fluids due to the insoluble solids still present.

1. Introduction

Sports supplements originated in the 20th century and have since been used by different populations over the years to improve the performance of athletes in training and sports competitions [1].

In North America, dietary supplements, in general, are regulated and monitored only after entering the market by the Food and Drug Administration (FDA) [2]. In Europe, supplements with nutritional or physiological purposes must be licensed in advance by the Ministry of Agriculture and Food Sovereignty before their production and sale [3]. In Brazil, regarding the creation and production of sports supplements, there is a designated classification with composition and labeling requirements for foods intended for athletes, established by the resolution of the National Health Surveillance Agency (ANVISA), RDC No. 18, of 27 April 2010 [4].

It is known that carbohydrate replacement energy supplements (drinks, gels, and energy bars) are generally preferred over natural food sources (e.g., lentils, bananas, oats, honey, raisins, potatoes, rice, and pasta) due to their greater transportability and ease of use during physical exercise [5]. Furthermore, it is a fact that cold beverages are generally more palatable, and flavored sports drinks are more accepted by athletes compared to water [6].

However, regardless of the form in which they are administered, newly created sports drink products primarily focus on optimizing hydration and energy replenishment [7].

Endurance sports athletes usually replace carbohydrates with energy in the form of liquid, semi-solid, or solid mixtures, with the latter two being the most common during exercise [8].

Energy replacement in gel form is common among runners, and its formulation is most often composed of maltodextrin, water, fructose, sodium citrate, medium-chain triglycerides, sea salt, potassium citrate, citric acid, calcium carbonate, gellan gum, L-isoleucine, L-leucine, L-valine, sodium benzoate, potassium sorbate, and natural citrus flavoring [9], thus making it a complex food.

Supplementary Table S1 shows the main published data on the possibilities for formulating energy-replenishing gels for endurance athletes. Fruit-based energy gels also have a similar effect to the gels sold in food supplement stores, by increasing glucose levels in the body [10]. In addition, they also have a potential protective effect on the human body, due to the high levels of bioactive compounds present in fruit, favoring antioxidant, anticoagulant, and antithrombotic activity [11].

The fruits genipap (Genipa americana L.) of the Rubiaceae family and banana prata (Musa spp.) of the Musaceae family can be highlighted as potential fruits for energy replacement in athletes. G. americana L. is popularly known as genipap tree or genipap, and its species is distributed from Mexico and the Antilles to northern Argentina [12].

G. americana L. is one of the species listed in the Food and Agriculture Organization’s (FAO) Plants for a Future project, a database of information on over 8000 tropical and subtropical edible and useful plants. This project has a global geographic focus, with the primary goal of improving the knowledge base and increasing awareness of the value of local crop diversity [13].

Additionally, the publication “Native Species of the Brazilian Flora of Current or Potential Economic Value—Plants for the Future—North Region”, developed by the Brazilian Ministry of Environment with the collaboration of renowned experts, included the G. Americana L., among other native species from the North region, exploring the potential economic value of the sustainable production of products and by-products of socio-economic interest, such as medicines, foods, and flavorings [14].

The Musa spp. is one of the most widely consumed banana species in the world and is very present in international trade [15]. Regarding its nutritional value, the macronutrients present in the fruit are carbohydrates, such as sucrose and glucose, with lower levels of proteins and fats, and concerning micronutrients, sodium, potassium, and magnesium are present in its composition [10].

Hence, adding these two fruits in the production of a carbohydrate gel provides an alternative to be consumed before and during endurance training. The variety of micronutrients present in genipap [16,17] and banana [10] provide nutritional support for this audience.

Therefore, this study aimed to develop a carbohydrate gel based on genipap and banana, analyze the physico-chemical, rheological, and sensory quality of different formulations, present the profiles of its proximate composition, antioxidant activity, and polyphenols, and demonstrate the applicability of this energy replenisher in endurance-type physical exercise practitioners.

2. Materials and Methods

2.1. Preparing the Carbohydrate Gel

2.1.1. Ethical Aspects and Patent Deposit

The project was submitted to and approved by the Research Ethics Committee of the State University of Ceara (UECE), under CAAE number 47593721.8.0000.5534, with opinion number 4.866.034. All the individuals involved signed an Informed Consent Form (ICF) to participate in the focus group and affective descriptive sensory analysis.

The process for producing a carbohydrate energy gel for physical exercisers and the carbohydrate energy gel product was filed, with the petition number of the patent process in BR 10 2023 021.438 0.

2.1.2. Sampling

The genipap (G. americana L.) used in the formulations came from family farms and backyards located in the municipality of Cascavel, Ceara, Brazil (−4.14450 latitude and −38.33056 longitude). The other ingredients were purchased in local shops in the city of Fortaleza, Ceara.

The fruits were previously selected and sanitized with 200 ppm sodium hypochlorite for 10 min, then rinsed in running water, pulped, macerated, and stored at freezing temperature (−12 °C to −18 °C).

The pulp of the genipap (G. americana L.) and banana (Musa spp.) fruits was processed in the Food Drying Laboratory, where an enzymatic clarification process took place and, finally, concentration in a rotary vacuum evaporator [18]. The concentrated clarified juice was stored at freezing temperature (−12 °C to −18 °C).

2.1.3. Formulations

The gels were formulated in the dietetics and sensory analysis laboratory. The ingredients were weighed on a Shimadzu semi-analytical balance (model BL3200H—Sao Paulo, Brazil) and homogenized in a food processor for 2 min.

The ingredients used to prepare the carbohydrate gels were demerara sugar, glucose syrup, and agar-agar gum in fixed quantities for all the formulations, and concentrated clarified genipap and banana juice, with variations in their percentage. Supplementary Table S2 shows the different compositions of the gels, related to the variation in fruit concentration and the respective laboratory tests.

To compare the analyses carried out on the genipap and banana gel formulations, we used a commercial gel packaged in a 30 g sachet, guarana- and açaí-flavored, with the ingredients maltodextrin, purified water, fructose, dextrose monohydrate, magnesium bisglycinate, sodium chloride, citric acid acidulant, potassium phosphate acidity regulator, ascorbic acid antioxidant, sodium benzoate, and potassium sorbate preservatives, EDTA sequestrant, and amaranth and brilliant blue artificial colorants.

2.1.4. Focus Group Testing

Six endurance exercisers who consumed carbohydrate gels during races or training sessions were invited to take part in a focus group sensory test. The test was characterized by a discussion about the tasting of six different formulations and a structured questionnaire consisting of questions about their knowledge of the product (overall aspect, texture, and taste), whether they recognized the importance of the preparation, whether they would buy or use it in their workouts, and positive and/or negative points to be adjusted in the preparation [19].

Six different formulations were prepared for the focus group sensory test, and three different formulations were chosen for the affective sensory test. The amount of concentrated clarified genipap juice and banana in the different formulations was combined in inverse proportions, while the other ingredients were fixed in all formulations.

2.2. Evaluation of the Carbohydrate Gel

The physico-chemical and rheological analyses were conducted in triplicate with the fruit and commercial gel, and in duplicate for the three formulations of genipap and banana gel for batch 1 (L1), batch 2 (L2), and batch 3 (L3). The microbiological analysis was conducted in duplicate for batches L1, L2, and L3 of formulations A, B, and C of the affective sensory test. Sensory analysis was conducted only once for formulations A, B, and C.

2.2.1. Physico-Chemical Analysis

The pH, soluble solids (SS), titratable acidity (TA), and soluble solids to titratable acidity ratio (TA/SS) followed standardized methodologies [20].

2.2.2. Rheology: Viscosity

A gel viscosity analysis was conducted using a MARS III rheometer (HAAKE MARS III) (Rheology Solutions Pty Ltd., Victoria, Australia). A 25 °C MTMC (MARS Temperature Module Controller) and geometric plates (P35 Ti L—L11004) with a diameter of 35 mm were used. Certain parameters were followed, such as A-factor, 118,827,000 Pa/Nm; M-factor, 17.497 (1/s)/(rad/s); inertia, 1.547 × 10⁻⁶ kg/m²; damping, 30.00; coefficient of thermal expansion, 1.400 µm/°C; compliance, 0.003900 rad/Nm; torque offset, off; and gap (spacing between plates), 1.000 mm.

For the rotation pre-test, a shear rate of 0.10001/s was used for 1 min until a temperature of 25 °C was reached. The elements were defined as ID 3: Set Temperature; CR; 0.1000 1/s; t 1.00 min; T 25.00 °C > ±1.00 °C; Break -> Goto End of job; ID 2: Rot Ramp (cont); CR; 0.10001/s–100.01/s lin; t 3.00 min; #100; T 25.00 °C.

In the first stage, the rotational shear ramp test took place from 0.1 to 1001/s for 3 min at a temperature of 25 °C, with the following elements: ID 4: Rot Ramp (cont); CR; prev 1/s–0.10001/s lin; t 3.00 min; #100; T 25.00 °C. In the second stage of the rotational test, the shear ramp ran from 100 to 0.11/s for 3 min at a temperature of 25 °C.

The samples (commercial formulation and formulations A, B, and C) were fitted to the Power Law, Newton, Herschel–Bulkley, Bingham, and Casson models, Equations (1)–(5), respectively.

$$\tau =K{\gamma }^n$$

(1)

$$\tau =\mu \gamma$$

(2)

$$\tau ={\tau }_0+K{\gamma }^n$$

(3)

$$\tau ={\tau }_0+{\mathrm{ƞ}}_B\gamma$$

(4)

$${\tau }^{0.5}={\tau }_0+{\mathrm{ƞ}}_C{\gamma }^{0.5}$$

(5)

where ($\tau$) is the shear stress, ${(\tau }_0$) is the residual stress, ($\gamma$) is the shear rate, (n) is the behavior index, ($K$) is the consistency index, ($\mu$) is the apparent viscosity, ${(\mathrm{ƞ}}_B$) is the Bingham plastic viscosity, and ${(\mathrm{ƞ}}_C$) is the Casson plastic viscosity.

2.2.3. Microbiological Analysis

Before the sensory test, the three gel formulations were tested for the presence of Salmonella, molds and yeasts, and enterobacteria. To determine the presence of Salmonella, 25 g of the sample was placed in a flask for homogenization in 225 mL of Buffered Peptone Water. Then, 0.1 mL of the sample was transferred to 10 mL of Rappaport–Vassiliadis (RVS) Soy Broth and 1 mL of the sample to 10 mL of Muller Kauffmann Novobiocin Tetrathionate Broth (MKTTn). From each culture in RVS, a section was streaked on Xylose Lysine Deoxycholate Agar (XLD) and another section on a second culture medium, repeating the procedure with MKTTn broth [21].

For the determination of molds and yeasts, the spread plate method was used, which consists of spreading on a surface with a Drigalsk loop. Using dilutions of up to 10^–3 of the sample, 1 mL was spread on 4 plates (0.3 mL, 0.3 mL, 0.3 mL, and 0.1 mL) of Acidified Potato Dextrose Agar for analysis. The plates were then incubated in Biochemical Oxygen Demand (BOD) at 25 °C for 5 days to count the colonies [22].

To count enterobacteria, the overlay pour plate method was applied, where three dilutions were performed and aliquots of 1 mL of each dilution were inoculated. The number of Colony-Forming Units (CFU/g or mL) was calculated by multiplying the number of typical colonies by the dilution inverse [22].

2.2.4. Sensory Analysis

For the sensory analysis, 100 untrained judges of both sexes were recruited to analyze the gel according to the methodology of Meilgaard et al. (1991) [23]. The evaluators were chosen based on criteria such as age (18 to 59 years), practicing endurance exercise, a habit of consuming carbohydrate gel during training sessions and races, not having diabetes mellitus, and not being pregnant.

The selected participants initially received guidance on the product under development. Before the sensory analysis test, a preliminary investigation was conducted into their knowledge of the existence and consumption of the genipap fruit. The evaluators then indicated how much they liked the taste and texture of the product on a structured 9-point hedonic scale (9 = I liked it very much; 5 = I neither liked it nor disliked it; and 1 = I disliked it very much). To assess purchase intention, another hedonic scale was used, with 5 points (1 = definitely would buy; 3 = maybe yes/maybe no; and 5 = definitely would not buy). The evaluators also indicated their order of preference for each sample [24].

Three samples (formulations A, B, and C) of the gel under development were served to randomized and balanced tasters in transparent plastic cups containing an average of 20 g of the gel, coded with three digits of random numbers [25]. Together with the gel, the evaluators were offered drinking water and a Feddernl cracker, which they were instructed to consume between samples to remove the aftertaste.

To calculate the acceptability index (AI) of each formulation, the following equation was used: AI (%) = [((Average overall acceptance score)/(Maximum score given to formulation))] × 100 [26].

2.3. Characterization of the Carbohydrate Gel

The fresh samples of genipap and banana, the three formulations (A, B, and C) of genipap and banana gel, and the commercial gel were analyzed in triplicate for their proximate composition, carbohydrates, glucose, fructose, sucrose, antioxidant activity, and polyphenols.

2.3.1. Proximate Composition

Moisture, ash, and lipid contents were measured according to the standard proposed by the Adolfo Lutz Institute (2005) [27]. Protein content was obtained following the Association of Official Analytical Chemists (1997) [28] method.

The moisture, ash, and lipid contents were measured [27]. The protein was obtained by the Kjeldahl method [28]. The total carbohydrate content was determined by the difference between 100 and the sum of the moisture, protein, total lipid, and ash contents in percentages. The energy value was calculated from the protein, and the total content of lipids and total carbohydrates was calculated using an Atwater system [29].

2.3.2. Carbohydrates Glucose, Fructose, and Sucrose

The glucose, fructose, and sucrose contents were analyzed using a Shimadzu High-Performance Liquid Chromatography (HPLC) system with a DGU-14A on-line degasser, LC-10 ADVP pump, and CTO-10ASVP column oven. A Kromasil NH2150 × 4.6 mm, 5 μm column was used. The apparatus was equipped with an RID-10A refractive index detector and SCL-10AVP controller. The standards were prepared using 100 mg of the standards dissolved in 10 mL of Milli-Q water. The mobile phase was prepared by dissolving 85% acetonitrile and 15% Milli-Q-grade water, with a run time of 20 min. The oven temperature was 40 °C, using a flow rate of 1.0 mL/min. The samples were extracted using ethanol, filtered using a Buchner funnel and a kitassato, and then roto-evaporated at 75 °C. The samples were then lyophilized, dissolved in Milli-Q-grade water, and filtered through a 0.22-micron syringe filter [30].

Table 1. Method validation for chromatographic analysis of fructose, glucose, and sucrose.

Parameter	Fructose	Glucose	Sucrose
Detection limit [mg·mL−1]	0.01	0.03	0.1
Quantification limit [mg·ml−1]	0.1	0.15	0.25
Linearity	155,614x + 96,710	137,103x − 7198.3	146,832x − 37,666
Correlation coefficient (R2)	0.9997	0.9990	0.9995
Retention time [min]	4.30	5.10	8.50

shows the method validation for the chromatographic analysis of fructose, glucose, and sucrose.

2.3.3. Antioxidant Activity and Total Polyphenols

To assess their antioxidant activity and determine their total polyphenol content, the extracts of the samples were prepared according to the procedure described by Re et al. (1999) [31]. In total, 1 g of each sample (formulations, banana, genipap, and commercial gel) was weighed and mixed with 10 mL of methanol/water (50:50, v/v). After 1 h at room temperature, the samples were centrifuged, and the supernatant was recovered. Next, 10 mL of acetone/water (70:30 v/v) was added to the residue, and the incubation and centrifugation were repeated. Both supernatants were mixed and distilled water was added up to 25 mL.

The analyses were carried out in a low-light environment. Antioxidant activity was assessed by ABTS (2,2’-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) [31], and the results are expressed as TEAC (Trolox Equivalent Antioxidant Activity) in μmol TEAC/100 g fresh weight. The total polyphenol content in the samples was determined using the Folin–Ciocalteau method [32], and the results are expressed in gallic acid equivalents (mg GAE g⁻¹ sample). Absorbance was measured on a Shimadzu UV-1800 (Sao Paulo, Brazil) spectrophotometer.

2.4. Statistical Analysis

Data from the analysis of variance (ANOVA) and Tukey’s mean test were analyzed at a 5% significance level. Samples and analyses were carried out in triplicate, and the results are expressed as mean ± standard deviation. To facilitate visualization of the results, principal component analysis (PCA) and hierarchical clustering analysis (HCA) were conducted and applied to the physico-chemical data and proximate composition, as well as for glucose, fructose, and sucrose, antioxidant activity, and polyphenols. All the multivariate analyses were conducted using the Sensorial XLSTAT software version 2023.

3. Results

3.1. Physico-Chemical Analysis and Proximate Composition

Figure 1 and

Table 2. Physico-chemical analysis and proximate composition (wet basis) of the fresh material and carbohydrate gel formulations.

	pH	Titratable Acidity (TA)	Soluble Solids (SS)	TA/SS Ratio	Moisture (mg/100 g Sample)	Ash (mg/100 g Sample)	Protein (mg/100 g Sample)	Lipid (mg/100 g Sample)	Carbohydrate (mg/100 g Sample)	Energy Value (Kcal)
Formulation A	3.55 ± 0.01 a	0.07 ± 0.00 a	69.67 ± 0.58 a	966.07 ± 7.23 a	33.10 ± 1.39 a	0.73 ± 0.02 a	0.40 ± 0.04 a	0.53 ± 0.03 a	64.96 ± 1.33 a	266.27 ± 5.35 a
Formulation B	3.57 ± 0.01 a	0.09 ± 0.01 a	69.50 ± 0.00 a	810.23 ± 19.51 b	38.79 ± 1.51 a	0.75 ± 0.06 a	0.35 ± 0.02 a	0.24 ± 0.02 b	59.80 ± 1.38 b	242.80 ± 5.49 b
Formulation C	3.47 ± 0.04 a	0.10 ± 0.00 c	69.83 ± 0.58 a	694.76 ± 26.54 c	37.56 ± 1.30 a	0.98 ± 0.07 a	0.31 ± 0.01 a	0.38 ± 0.05 a	60.76 ± 0.00 bc	247.73 ± 0.00 bc
Commercial gel	3.72 ± 0.08 b	0.06 ± 0.00 ae	64.33 ± 0.57 b	1125.14 ± 29.48 d	36.15 ± 1.89 a	0.67 ± 0.16 a	0.34 ± 0.02 a	0.26 ± 0.04 b	62.58 ± 0.00 c	254.02 ± 0.00 c
Genipap	3.44 ± 0.08 a	0.12 ± 0.00 d	25.00 ± 0.00 c	203.05 ± 14.67 e	76.71 ± 0.32 b	1.22 ± 0.12 a	0.47 ± 0.00 a	0.15 ± 0.06 c	21.45 ± 0.00 d	89.03 ± 0.00 d
Banana	4.33 ± 0.05 c	0.05 ± 0.00 e	24.33 ± 0.58 c	483.90 ± 37.96 f	76.29 ± 0.32 b	0.83 ± 0.14 a	0.76 ± 0.02 b	0.10 ± 0.01 c	22.02 ± 0.00 d	92.02 ± 0.00 d

All tests were performed in triplicate (n = 3) and the results were expressed as mean ± standard deviation by ANOVA and Tukey test. Values with different superscript letters are significantly different (p < 0.05).

show the multivariate and univariate analyses of the physico-chemical tests and proximate composition of the samples analyzed. Figure 1A shows that the variability of the data (moisture, ash, protein, lipid, carbohydrate, pH, acidity, brix, and TA/SS) is mostly explained by PC1–PC3 (96.4%). There is a significant correlation of all the compounds explained in PC1 (60.9%) and PC2 (30.4%).

The exploratory principal component (PC) analysis of the evaluated samples (commercial, banana, genipap, and formulations A, B, and C) is shown in Figure 1B. In the PC1 scores, there is a stronger correlation of formulations A and C, given that they are simultaneously in the negative quadrant of the PC1 scores centralized to the PC2 scores. It can also be observed that the commercial sample has the strongest correlation with all the formulations (A, B, and C), as they are grouped in the negative quadrants of the PC1 scores. The correlations of the eigenvalue groups are illustrated by red dashes in Figure 1B. The raw material samples (banana and genipap) are in the positive quadrants of the PC1 scores, but in opposite quadrants of the PC2 scores (positive for the banana sample and negative for the genipap sample).

On the loadings axis (Figure 1B—PC1 loadings vs. PC2 loadings), it is evident that all the samples in negative quadrants in the PC1 loadings, such as all the formulations studied (A, B, and C) and the commercial sample, have higher concentrations of carbohydrates and lipids, as well as higher °Brix values and a higher TA/SS ratio, compared to the raw materials (genipap and banana). The genipap and banana samples have higher concentrations of protein and moisture.

These observations are consistent, as the formulations tend to contain lower concentrations of protein due to the dilution by other compounds, such as demerara sugar and agar-agar gum, which also justifies the formulations having higher concentrations of carbohydrates and high °Brix values. The lower moisture content in the formulations and the commercial gel can be explained by the evaporation process to which they were subjected to concentrate the carbohydrates. All these statements can be confirmed by the univariate analysis (Figure 1 and Supplementary Table S3), given the physico-chemical analyses of the compounds studied (moisture, ash, protein, lipid, carbohydrate, pH, acidity, brix, and TA/SS).

The quality parameters and parallel results of the replicates between batches 1, 2, and 3 of each formulation are shown in Supplementary Table S3, with no statistically significant results.

3.2. Glucose, Fructose, and Sucrose Content, Antioxidant Activity, and Total Polyphenols

Table 3. Glucose, fructose, and sucrose analysis, antioxidant activity, and total polyphenols of the fresh material and the carbohydrate gel formulations.

	Glucose (mg/g)	Fructose (mg/g)	Sucrose (mg/g)	ABTS (μM Trolox/g)	Total Polyphenols (mg Gallic Acid/ 100 g Sample)
Formulation A	153.12 ± 3.87 a	60.14 ± 2.10 a	100.60 ± 2.46 a	6.39 ± 0.38 a	105.29 ± 7.37 a
Formulation B	123.93 ± 3.15 b	42.41 ± 2.32 b	101.22 ± 0.96 a	8.15 ± 0.60 ace	106.01 ± 6.48 a
Formulation C	129.91 ± 4.16 b	46.29 ± 0.71 b	139.71 ± 4.81 b	10.15 ± 1.29 ce	173.74 ± 7.93 b
Commercial gel	4.12 ± 0.54 c	ND c	ND c	ND d	ND c
Genipap	55.97 ± 6.80 d	19.11 ± 0.94 d	29.37 ± 0.86 d	10.88 ± 2.66 e	177.73 ± 15.74 b
Banana	48.37 ± 4.52 d	35.14 ± 1.87 e	ND c	ND d	45.76 ± 1.79 d

All tests were performed in triplicate (n = 3) and the results were expressed as mean ± standard deviation by ANOVA and Tukey test. Values with different superscript letters are significantly different (p < 0.05). ND: Not detected.

and Figure 2A show that the variability of the data (glucose, fructose, sucrose, antioxidant activity, and polyphenols) is mostly explained by PC₁–PC₃ (97.94%). There was a significant correlation of all the compounds explained in PC₁ (74.68%) and PC₂ (19.72%).

The exploratory principal components analysis of the commercial gel, the initial fresh materials (banana and genipap), and the genipap and banana gel formulations (A, B, and C), as shown in Figure 2B, shows that, in the PC1 scores, there is a greater correlation between formulations A and B, as they are simultaneously in the negative quadrant of PC1 scores and PC2 scores. It can also be seen that the commercial gel has a higher correlation with the banana sample, as they are grouped in positive quadrants of the PC1 scores. The correlations of the eigenvalue groups are illustrated by red dashes in Figure 2B.

On the loadings axis (Figure 2B—PC1 loadings vs. PC2 loadings), it is defined that all samples that are in negative quadrants in the PC1 loadings, such as all the formulations studied (A, B, and C), have higher concentrations of glucose, fructose, sucrose, and polyphenols and greater antioxidant activity compared to the commercial sample and the banana and genipap samples. The above statement can be confirmed by the univariate analysis (Figure 2 and Supplementary Table S4), given the concentrations of the compounds and antioxidant activity studied (glucose, fructose, sucrose, polyphenols, and ABTS).

The univariate analyses presented (Figure 2 and Supplementary Table S2) were also decisive in classifying the product as an energy replenisher and muscle restorer for endurance athletes. In addition, the chromatograms of the glucose, fructose, and sucrose analyses are shown in Supplementary Figures S1–S13.

To present a global interpretation of the variables analyzed in the multivariate analysis, Figure 3 shows a hierarchical heatmap cluster analysis (HCA).

The HCA shows that, in an initial dissimilarity index, the raw material samples (genipap and banana) are the first cluster, and the second cluster is made up of the formulations (A, B, and C) and commercial gel sample.

The progression of dissimilarity (linkage distance) shows four clusters (genipap, banana, commercial gel, and formulations—A, B, and C). It should be noted that the grouping of the formulations would only be separated at low dissimilarity indices and the HCA analysis was conducted with all the physico-chemical and chemical responses, making the hierarchical grouping more rigorous. In this way, the multivariate PCA analysis is confirmed by the HCA analysis, while the univariate analysis (Supplementary Table S1 and Table S2) is fundamental for verifying the dissimilarity of the samples and determining significant and non-significant separations between all the physico-chemical and chemical analyses performed.

3.3. Sensory Analysis: Focus Group and Descriptive Sensory Test

Microbiological analysis was a prerequisite for this test and the results regarding Salmonella, molds and yeasts, and enterobacteria were favorable, according to the parameters required by national legislation. As a result of the focus group intervention, three formulations were obtained to be evaluated according to the parameters of the quality, acceptance, and applicability of the product. The formulations differed only in their concentrations of concentrated clarified genipap and banana juices. Thus, formulation A had a 50:50 ratio of genipap and banana, formulation B had a 70:30 ratio of genipap and banana, and formulation C had a 90:10 ratio of genipap and banana.

Table 4. Quantitative results of taste and consistency focus group.

Formulation	Taste (%)					Consistency (%)
	V.Wea	W	I	Str	V.Str	V.Flu	Flu	I	D	V.D
A	0	20	60	20	0	20	80	0	0	0
B	0	0	60	20	20	0	60	40	0	0
C	0	0	40	20	40	20	40	20	20	0
D	0	0	40	40	20	0	0	40	60	0
E	0	0	0	40	60	0	0	60	20	20
F	0	0	0	60	40	0	0	40	0	60

V.Wea: Very weak; Wea: Weak; I: Ideal; Str: Strong; V.Str: Very strong; V. Flu: Very fluid; Flu: Fluid; D: Dense; and V.D: Very dense.

shows the quantitative data from the focus group regarding the taste and consistency of the gels.

According to the data presented in Table 4, the focus group participants rated the taste of formulations A, B, C, and D as ideal, while formulations E and F were rated as having a strong and very strong taste. Regarding consistency, the participants’ point of view found a greater fluidity in samples A, B, and C and a greater density in samples E and F.

The participant group for the sensory analysis consisted of 100 tasters who practiced endurance physical exercise and regularly consumed carbohydrate gels during their training sessions and races, where the majority were familiar with the genipap fruit (76%), but only 50% of them had ever consumed it. The tasters were of both sexes, with the majority being male (71%), aged between 40 and 49 years (36%), and had completed higher education (64%).

It was observed that there was no statistical difference between the attributes (taste and texture) of the three samples, but there was greater acceptance for formulation A compared to the other two (

Table 5. Scores given by the evaluators for the taste and texture parameters and the acceptability index (AI) (mean ± standard deviation).

	Taste		Texture
	Score	AI (%)	Score	AI (%)
Formulation A	7.11 ± 1.77 a	78.98	7.69 ± 1.33 a	85.40
Formulation B	6.65 ± 2.00 a	73.93	7.55 ± 1.39 a	83.88
Formulation C	6.75 ± 2.20 a	75.05	7.45 ± 1.64 a	82.79

Different letters in the same column indicate a difference at p < 0.05.

).

When analyzing intentions to buy, it was possible to see that all the samples were accepted (referring to rates 1 and 2), ranging from 63% (formulation C) and 64% (formulation B) to 66% (formulation A). As for the rejection zone (rates 4 and 5), formulation A was the least rejected (8%), and formulations B and C obtained 17% and 18%, respectively (Supplementary Figure S14). Therefore, it can be seen that formulation A tended to be the most accepted and least rejected among consumers.

3.4. Rheology

The rheological behavior of the formulations and the commercial gel is shown in Figure 4.

Table 6. Mathematical models—Power Law, Newton’s model, Herschel–Bulkley model, Bingham Model, and Casson’s model—for the rheological study of the commercial sample, formulations A, B, and C.

Sample	Power Law
	τ0 (Pa)	K (Pa·sn)	N	R2
Commercial	N/A	2.79	0.9501	0.998
Formulation A 2		18.78	0.5325	0.967
Formulation B 3		19.86	0.5099	0.941
Formulation C		20.70	0.5350	0.969
Sample	Newton’s Model
	µ (mPa.s)	K (Pa·sn)	N	R2
Commercial 1	2.24	N/A	N/A	0.998
Formulation A	2.58			0.823
Formulation B	2.48			0.773
Formulation C	2.88			0.829
Sample	Herschel–Bulkley Model
	τ0 (Pa)	K (Pa·sn)	N	R2
Commercial	5.39	2.135	1.003	0.998
Formulation A	28.21	7.862	0.696	0.969
Formulation B	64.47	1.241	1.047	0.935
Formulation C 4	30.50	8.859	0.695	0.972
Sample	Bingham Model
	τ0 (Pa)	K (Pa·sn)	N	R2
Commercial	5.19	2.163	N/A	0.998
Formulation A	53.52	1.781		0.963
Formulation B	54.69	1.665		0.939
Formulation C	59.15	1.992		0.966
Sample	Casson’s Model
	τ0 (Pa)	K (Pa·sn)	N	R2
Commercial	0.45	2.005	N/A	0.998
Formulation A	24.37	1.024		0.959
Formulation B	25.61	0.9309		0.931
Formulation C	26.57	1.157		0.959

μ, apparent viscosity; K, consistency index; n, behavior index; τ, shear stress; τ₀, residual stress; ɣ, shear rate; N/A: not applicable; 1, 2, 3, and 4: model that best fits the commercial sample, formulation A, B, and C, respectively.

shows the values for the rheological models and adjustments to the “n” values, where all the samples were subjected to the Power Law. Fluids with “n” above 0.95 were adjusted to Newton’s Law (only the commercial formulation), and fluids with “n” below 0.95 were adjusted to the Herschel–Bulkley, Bingham, and Casson models, as they showed pseudoplastic fluid behavior.

4. Discussion

The gel formulations exhibited acidic characteristics with low pH values, similar to the commercial gel analyzed, which favored microbiological control, since low levels of water and pH are important to prevent the rapid food deterioration [33]. In addition, when pH values are close to 3.2, crystallization of the saccharide can be prevented, enhancing the gel’s equilibrium, while raising this parameter can destabilize it [34], which can lead to sensory rejection of the product by the consumer. Thus, from the results presented, it can be seen that the data closely align with this recommendation, showing the stability of the gel formulations produced.

The soluble solids analysis detected high levels of total sugars in the formulations. Titratable acidity can be determined by the amount of acid that interacts with a base of a specific concentration [35]. Given the above, the gel formulations, which were fruit-based, had a titratable acidity above 6% with a high classification in terms of acidity. Therefore, genipap has a high titratable acidity content compared to banana. Therefore, the formulations that used higher concentrations of genipap had a higher acidity. Foods that have an inherent acidic nature prevent deterioration, as well as influence the taste and stability of products, contributing to the preservation of such gels [35].

The proximate composition showed a high concentration of carbohydrates and energy value in the genipap and banana gel formulations. The data showed relevance, since a high carbohydrate intake is necessary during intense physical exercise to maintain muscle glycogen and delay fatigue. In addition, adequate energy replenishment prevents damage caused by exercise, maintaining an athlete’s performance during and after physical activity [36].

From the results shown in Supplementary Table S1, there were different values among the formulations in terms of their carbohydrate content and energy value. It is also important to note that only the gel made with maltodextrin [37] was similar to the genipap and banana gel (Table 2). This may have been due to a high moisture content, which was only shown in the study by Morgado et al. (2016) [38], where the moisture value of a gel made from beet was double that of a gel made from genipap and banana (Table 2). On the other hand, studies carried out with different types of ingredients have shown lower carbohydrate and energy values [39,40].

Regarding soluble solids, the genipap and banana gel exhibited higher values than those produced in the other studies shown in Supplementary Table S1, leading to the conclusion that these products did not follow the minimum parameters for classifying food as an energy gel. To fit the definition and be considered as a jelly or similar food product, the formulations adhered to the recommendations of at least 45% fruit extract and at least 65ºBrix soluble solids [41].

In the genipap and banana gel formulations, it was observed that glucose content was prominent in all the samples, followed by sucrose and then fructose, thereby classifying them as foods with a high carbohydrate content. This category of food is considered to promote an improved performance in endurance athletes, since water and electrolytes alone are not effective in restoring endurance capacity during strenuous exercise [42].

Periodizing carbohydrate intake before, during, and after training and competitions can further enhance the likelihood of positively impacting an individual’s athletic performance. In addition, the wide range of rapidly oxidizable carbohydrate sources, including glucose and its combinations (glucose–galactose or glucose–fructose), can provide a greater flexibility in intake options. The combination of glucose–fructose may lead to the synthesis of liver and muscle glycogen simultaneously [43], which was favored in the formulations developed in this study.

Some studies have shown that carbohydrate intake and overall energy intake do not meet sports nutrition recommendations [44,45]. For this reason, nutritional guidance can be beneficial for an adequate nutrient intake for performance, recovery, health, and injury prevention [46].

Accordingly, an athlete’s anabolism is positively influenced by an adequate macronutrient intake, which supports sports performance and rehabilitation. However, it is essential to monitor the consumption of these nutrients to avoid supplementation beyond recommended levels [47]. In addition, to increase their levels of antioxidants, a substance present in the formulation of a gel under development for physical activity practitioners should follow an adequate diet with diverse foods [48], which should be encouraged by nutritionists and physical education professionals who work with athletes [44].

Concerning the specific glucose, fructose, and sucrose contents of the commercial gel, it is important to note that, although it had 62% total carbohydrates, the analysis results showed only slight glucose levels. This may have been due to the degree of polymerization of the maltodextrin present in the commercial carbohydrate gel, being thirty times higher than that of free glucose, which has a 100% equivalence with dextrin, while maltodextrin has less than a 20% equivalence [49], making it more difficult to identify the compound.

As for polyphenol content, significant concentrations of this compound were detected in the genipap and banana gel formulations. According to Vasco et al. (2008) [50], the content of total polyphenols can be classified according to the following parameters: low polyphenol content (<100 mg GAE/100 g), medium (100–500 mg GAE/100 g), and high (>500 mg GAE/100 g). Therefore, the carbohydrate gel formulations produced in this research had a medium classification and may favor muscle recovery from endurance-related damage.

Although polyphenols are secondary metabolites for plants and do not provide energy to humans, these substances have several benefits in terms of biological activity, such as antioxidant and anti-inflammatory effects, being present in vegetables, fruits, and seeds [51]. In addition, polyphenols have a great relevance due to their preventive effects on metabolic diseases. The intake of polyphenols is increased by the type of food consumed, its frequency, and its quantity in meals. Most fruits contain high levels of active antioxidants, as detected in the genipap analyzed, with the proportion of polyphenols in the fruit depending on genetic, technological, and environmental factors, as well as planting and harvesting methods [52].

Regarding the molecular mechanisms of polyphenols, their antioxidant and anti-inflammatory effects have a restorative function after high-intensity training. Oxidative stress and muscle inflammation can be attenuated by the action of polyphenols in capturing free radicals and other reactive oxygen species [53].

Thus, the use of dietary supplements rich in polyphenols may positively regulate muscle recovery, as they prevent oxidative damage caused by physical exercise [54]. In sports that require a high aerobic capacity, such as running, the use of polyphenols is relevant due to reductions in muscle pain [55].

This benefit occurs by improving the mechanism responsible for pumping blood to the lungs and increasing skeletal muscle metabolism [56].

Thus, due to the variety of micronutrients and bioactive compounds, such as vitamin C and carotenoids, present in bananas and genipap, respectively, carbohydrate gel is a food with important nutrients for the human body [16,57].

Just as important as the requirements for preparation in the composition of the product is its sensory acceptance by consumers. Monteiro (1984) [58] states that a product with an acceptability index equal to or greater than 70% is considered to be well-accepted by consumers. Factors such as taste and nutritional value can have a high degree of importance when choosing carbohydrate energy gels, while texture and price have a moderate influence on the choice of this food [10].

Rheologically, it is possible to observe a decrease in apparent viscosity as the shear rate increases. This characterizes a non-Newtonian behavior, indicating that these drinks have “thinning” characteristics under shear [59,60,61]. According to Lucey (2002) [62], shear forces break weak bonds, reducing electrostatic repulsion and hydrophobic interaction between molecules. This interruption is more intense in the first few minutes of the process, due to the intense action of the hydrodynamic forces. Subsequently, the particles align with the flow, resulting in a reduction in viscosity [63].

For the commercial formulation, there was a decrease from 3668 mPa.s⁻¹ to 2150 mPa.s⁻¹ until the initial viscosity was returned, a reduction of ≈41%. This reduction may be related to the fluid being completely clarified, i.e., showing a significant reduction in the insoluble solids content, unlike formulations A, B, and C, which saw a reduction of over 90% for all three samples; these formulations were not completely clarified due to the fact that they were processed by centrifugation, thus leaving a certain insoluble solids content behind in the formulations. Viana et al. (2021) [61] obtained a similar reduction for microfiltered banana juice (45% reduction), i.e., for a fluid completely free of insoluble solids, and for samples of fresh and enzymatically treated pulp, they obtained a reduction of over 90%, corroborating the results of the present study.

The rheograms in Figure 4 show the hysteresis curves observed in all the formulations studied, including the commercial formulation. This behavior is associated with products that have shear properties, demonstrating thixotropic behavior [59]. The hysteresis areas were practically the same for the formulations, which indicates that, rheologically, there was no difference in the intensity of the thixotropic behavior and there was similarity in the adequacy of the models for formulations A, B, and C, all of which can be considered as pseudoplastic. For the commercial formulation, mild thixotropic behavior was observed, which may have been due to the minimal presence of solids in suspension, resulting in a smaller difference in viscosity, as already mentioned, corroborating the studies of Hernández (1996) [64], who reported that a low viscosity is related to smaller hysteresis areas, demonstrating a greater correlation with the fluid’s thixotropy.

5. Conclusions

It is concluded that the three final formulations of the genipap- and banana-based gel were produced in accordance with the minimum parameters established by The Prevention of Food Adulteration Act and Rules for jellies and similar products.

The three formulations showed the same sensory acceptance profiles regarding taste and texture. The physico-chemical parameters (pH, soluble solids, and titratable acidity) and proximate composition (moisture, ash, protein, lipids, and carbohydrates) of the prepared formulations were comparable to those of the commercial gel analyzed, making them high-quality energy replenishment products.

The sucrose, fructose, polyphenol, and antioxidant activity levels were higher in the genipap and banana gel formulations, whereas they were absent in the commercial gel. This can be attributed to the use of natural raw materials in the formulated gel, which helped to preserve the fruits’ aroma and flavor.

In the two blocks of analysis mentioned above, the variability of the data was mostly explained by PC1–PC3 at almost 100%.

Rheologically, the commercial gel is considered to be a Newtonian fluid, and the formulations developed can be considered as pseudoplastic fluids due to the insoluble solids still present.

6. Patents

The process for producing a carbohydrate energy gel for physical exercisers and the carbohydrate energy gel product was filed under patent application number BR 10 2023 021,438 0.