Development of Fruit-Based Carbohydrate Gel for Endurance Athletes

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 clari ed

Avenue, 2977, Pici Campus, Building 310, Fortaleza 60165-010, Ceara, Brazil; icarogpv@uol.com.br *Correspondence: renata.carmo@uece.br 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 clari ed 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 uid, and the developed formulations can be considered as pseudoplastic uids due to the insoluble solids still present. Keywords:Genipa americanaL.;Musaspp.; glucose; clean label; plant-based; chemometric analysis 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 Processes2024,12, 2304.

Processes2024,12, 2304 2 of 18 Brazil, regarding the creation and production of sports supplements, there is a designated classi cation with composition and labeling requirements for foods intended for ath- letes, 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 avored 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 triglyc- erides, sea salt, potassium citrate, citric acid, calcium carbonate, gellan gum, L-isoleucine, L-leucine, L-valine, sodium benzoate, potassium sorbate, and natural cit- rus avoring [9], thus making it a complex food. Supplementary Table S1 shows the main published data on the possibilities for for- mulating 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 americanaL.) of the Rubiaceae family and banana prata (Musaspp.) of the Musaceae family can be highlighted as potential fruits for energy replacement in athletes.G. americanaL. is popularly known as genipap tree or genipap, and its species is distributed from Mexico and the Antilles to northern Argentina [12]. G.

fruit, favoring antioxidant, anticoagulant, and antithrombotic activity [11]. The fruits genipap (Genipa americanaL.) of the Rubiaceae family and banana prata (Musaspp.) of the Musaceae family can be highlighted as potential fruits for energy replacement in athletes.G. americanaL. is popularly known as genipap tree or genipap, and its species is distributed from Mexico and the Antilles to northern Argentina [12]. G. americanaL. 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 Brazil- ian Ministry of Environment with the collaboration of renowned experts, included the G. AmericanaL., among other native species from the North region, exploring the po- tential economic value of the sustainable production of products and by-products of socio-economic interest, such as medicines, foods, and avorings [14]. TheMusaspp. 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 for- mulations, present the pro les of its proximate composition, antioxidant activity, and polyphenols, and demonstrate the applicability of this energy replenisher in endurance- type physical exercise practitioners.

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

Processes2024,12, 2304 3 of 18 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 led, with the petition number of the patent process in BR 10 2023 021.438 0. 2.1.2. Sampling The genipap (G. americanaL.) 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 hypochlo- rite 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. americanaL.) and banana (Musaspp.) fruits was processed in the Food Drying Laboratory, where an enzymatic clari cation process took place and, nally, concentration in a rotary vacuum evaporator [18]. The concentrated clari ed 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 xed quantities for all the formulations, and concentrated clari ed 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 aça½-

clari ed 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 aça½- avored, with the ingredients maltodextrin, puri ed water, fructose, dextrose monohydrate, magnesium bisglycinate, sodium chloride, citric acid acidulant, potassium phosphate acidity regu- lator, ascorbic acid antioxidant, sodium benzoate, and potassium sorbate preservatives, EDTA sequestrant, and amaranth and brilliant blue arti cial 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 concen- trated clari ed genipap juice and banana in the different formulations was combined in inverse proportions, while the other ingredients were xed in all formulations.

Processes2024,12, 2304 4 of 18 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/m 2 ; damping, 30.00; coefficient of thermal expansion, 1.400 m/ 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 rst 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

(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), re- spectively. t=Kg n (1) t=mg (2) t=t0+Kg n (3) t=t0+ Bg (4) t 0.5 =t0+ Cg 0.5 (5) where (t) is the shear stress,(t 0) is the residual stress, (g) is the shear rate, (n) is the behavior index, (K) is the consistency index, (m) is the apparent viscosity,( B) is the Bingham plastic viscosity, and( 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 ask 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

Processes2024,12, 2304 5 of 18 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 Acidi ed 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 develop- ment. 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 = de nitely would buy; 3 = maybe yes/maybe no; and 5 = de nitely 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

it very much). To assess purchase intention, another hedonic scale was used, with 5 points (1 = de nitely would buy; 3 = maybe yes/maybe no; and 5 = de nitely 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 equa- tion 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 geni- pap 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 Of cial 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 m

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 m

This study develops a fruit-based carbohydrate gel for athletes and evaluates its quality.