TECNOCIENCIA CHIHUAHUA, Vol. XX (2026): enero-diciembre, e2089

https://revistascientificas.uach.mx/index.php/tecnociencia

ISSN-e: 2683-3360

Scientific Article

Development of a supplement containing cocoa,

mesquite, agavins, pea protein, vitamins and

minerals: effects on bone density and body

composition

Desarrollo de suplemento con cocoa, mezquite, agavinas, proteína de

chícharo, vitaminas y minerales: efectos en densidad ósea y

composición corporal

*Correspondencia: Correo electrónico: kherrera@itdurango.edu.mx (Karen Marlenne Herrera-Rocha)

DOI: https://doi.org/10.54167/tch.v20i1.2089

Recibido: 21 de septiembre de 2025; Aceptado: 04 de diciembre de 2025

Publicado por la Universidad Autónoma de Chihuahua, a través de la Dirección de Investigación y Posgrado.

Editora de Sección: Dra. Jazmín Leticia Tobías-Espinoza

Abstract

The development of supplements is a strategy to promote population health. This study aimed to

develop a powdered supplement containing mesquite flour, pea protein, cocoa, agavins, xanthan

gum, vitamins, and minerals; characterize its phytochemical and nutritional composition; and

evaluate its glycemic index, glycemic load, and effects on bone mineral density and body

composition. The impact of particle size and mixing time on wettability, solubility, fluidity, and foam

stability was assessed through physicochemical analysis; formulations with 600 µm particles

exhibited greater stability, water absorption, solubility, and fluidity. The nutritional profile was

established in accordance with NOM-051-SCFI/SSA1-2010, yielding an energy value of 366.03

kcal/100 g, with 29.53 g of carbohydrates, 58.59 g of proteins, and 1.15 g of lipids. The phytochemical

Karen Marlenne Herrera-Rocha¹*, Nuria Elizabeth Rocha-Guzmán¹, Martha Rocío Moreno-

Jiménez¹, José Alberto Gallegos-Infante¹, Silvia Marina González-Herrera¹, Daniela de Jesús-

Sánchez¹, Julieta Nataly Durán-González²

1 Laboratorio Nacional CONAHCYT de Apoyo a la Evaluación de Productos Bióticos (LaNAEPBi), Unidad

de Servicio, Tecnológico Nacional de México/Instituto Tecnológico de Durango, Felipe Pescador 1830 Ote.,

34080 Durango, Dgo., México.

2 Facultad de Medicina y Nutrición, Universidad Juárez del Estado de Durango, Avenida Universidad s/n,

Los Ángeles, 34076 Durango, Dgo., México.

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profile identified by LC-MS/MS revealed a polyphenol content of 6608.57 µg/100 g, highlighting

hydroxycinnamic acids (2597.12 µg/100 g), flavanols (1606.77 µg/100 g), and flavonols (1526.47

µg/100 g). In an adult intervention study (n=32, 18–40 years, 30 g/day for two months), the

supplement showed a glycemic index of 11.18 and a glycemic load of 5.59. Consumption improved

bone health in both sexes, particularly in the radial and femoral regions, and reduced body fat,

especially visceral fat.

Keywords: supplement, functional food, bone health, body composition, health

Resumen

El desarrollo de suplementos es una estrategia para favorecer la salud poblacional. Este estudio

buscó elaborar un suplemento en polvo con harina de mezquite, proteína de chícharo, cocoa,

agavinas, goma xantana, vitaminas y minerales; caracterizar su composición fitoquímica y

nutricional; y evaluar índice glucémico, carga glucémica y efecto sobre densidad mineral ósea y

composición corporal. Se analizó fisicoquímicamente el impacto del tamaño de partícula y tiempo

de agitación sobre humectabilidad, solubilidad, fluidez y estabilidad de la espuma; las

formulaciones con partículas de 600 µm mostraron mayor estabilidad, absorción de agua,

solubilidad y fluidez. El perfil nutricional se estableció conforme a la NOM-051-SCFI/SSA1-2010,

obteniéndose un valor energético de 366.03 kcal/100 g, con 29.53 g de carbohidratos, 58.59 g de

proteínas y 1.15 g de lípidos. El perfil fitoquímico identificado por LC-MS/MS mostró un contenido

de 6608.57 µg/100 g, destacando ácidos hidroxicinámicos (2597.12 µg/100 g), flavanoles (1606.77

µg/100 g) y flavonoles (1526.47 µg/100 g). En el modelo de intervención en adultos (n=32, 18-40 años,

dosis de 30 g/día por dos meses), el suplemento presentó un índice glucémico de 11.18 y carga

glucémica de 5.59. Su consumo mejoró la salud ósea en ambos sexos, especialmente en regiones

radial y femoral, y redujo grasa corporal sobre todo visceral.

Palabras clave: suplemento, alimentos funcionales, salud ósea, composición corporal, salud

1. Introduction

The development of nutritional supplements with functional ingredients represents a

promising strategy for supporting metabolic and bone health, thereby contributing to body

recomposition and musculoskeletal integrity in adults. An appropriate combination of

macronutrients (proteins, carbohydrates, and lipids, as primary sources of energy and structural

substrates) and micronutrients (vitamins and minerals, required cofactors for enzymatic catalysis

and regulators of metabolic homeostasis), together with glycemic index and caloric content, can

promote body recomposition and maintenance of musculoskeletal tissue through thermogenesis and

bone regeneration processes (Martiniakova et al., 2022).

Cocoa-derived compounds contribute to the prevention of cardiovascular, neurodegenerative,

metabolic, and bone-related conditions (Fideles et al., 2023). Xanthan gum, employed as a stabilizing

agent in cocoa-based beverages, enhances viscosity and stability without affecting antioxidant

activity (Muhammad et al., 2021). In addition, agave, mesquite, and pea protein provide dietary

fiber, polyphenols, and proteins with anabolic, hypoglycemic, hypolipidemic, and prebiotic

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properties, which promote the balance of the gut microbiota and metabolic homeostasis (Jaimes-

Morales et al., 2022; Medina-Larqué et al., 2022; Wang et al., 2022).

Considering these physiological benefits, it is relevant to assess the use of functional supplements in

clinical settings where body composition alterations (such as visceral and subcutaneous fat

accumulation and decreased bone mineral density) are increasingly observed, even in adults under

45 years old. These conditions may compromise quality of life and predispose to long-term health

difficulties (Herath et al., 2022). Diagnostic assessment is typically performed via dual-energy X-ray

absorptiometry (DXA), the gold-standard imaging modality for estimating T-scores (-1.0 to -2.5) and

accurately measuring fat, lean, and bone mass, as well as body fat distribution.

Based on this evidence, a supplement was formulated with functional ingredients (mesquite flour,

pea protein, non-alkalized cocoa, agavins, xanthan gum as a stabilizer, and a premix of vitamins and

minerals) to characterize its physicochemical and nutritional properties, determine its glycemic

index and glycemic load, and evaluate its effects on bone mineral density and body composition in

a longitudinal study involving adult men and women.

2. Materials and methods

2.1 Materials

2.1.1 Chemical reagents

Catechin, epicatechin, epigallocatechin gallate, procyanidin B1, quercetin, quercetin-3-O-

glucoside, naringenin, apigenin, rutin, hesperidin, taxifolin, phloretin, phloridzin, kaempferol,

quinic acid, protocatechuic acid, syringic acid, chlorogenic acid, caffeic acid, p-coumaric acid,

shikimic acid, gallic acid, salicylic acid, ferulic acid, sinapic acid, trans-cinnamic acid, and benzoic

acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile, formic acid, and

ethyl acetate were obtained from J.T. Baker Inc. (Phillipsburg, NJ, USA). The vitamin and mineral

premix (A, B1 (thiamine), B2 (riboflavin), B5 (pantothenic acid), B6 (pyridoxine), B7 (biotin/H), B9 (folic

acid), B12 (cobalamin), C (ascorbic acid), D3 (cholecalciferol), K, calcium, phosphorus, potassium,

magnesium, iron, zinc, copper, manganese) was purchased from Alimentos América S.A. de C.V.

(Guadalajara, Jalisco, Mexico).

2.1.2 Experimental materials

Mesquite pods were collected in Durango, Dgo., Mexico (24° 02′ N, 104° 40′ W). The pods

were disinfected using a 1 % sodium hypochlorite solution, dried at 32 °C for 36 hours, pulverized,

and homogenized through a 2 mm mesh sieve. The resulting flour was vacuum-sealed and stored at

ambient temperature until use. Pea protein isolate (90 % protein content) was obtained from a local

commercial supplier. Agavins were acquired from Nutriagaves de México (Guadalajara, Jalisco,

Mexico). Cocoa powder and xanthan gum were sourced from a local retail outlet in Durango, Dgo.,

Mexico.

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2.1.3 Formulation and mixing procedure

The supplement was formulated by mixing powders at different proportions, including

hydrolysed pea protein, mesquite flour (Prosopis juliflora), agave fructans, non-alkalized cocoa

powder, xanthan gum, and a premix of vitamins and minerals (Table 1). All powders were

homogenized to ensure a uniform mixture and transferred to a 250 mL beaker, where they were

stirred using an Ika Eurostar Power digi-visc homogeniser (Staufen, Germany) equipped with helical

turbine geometry, at 50 rpm for 0, 180, and 240 seconds.

Table 1. Distribution of powders in the supplement blend

Tabla 1. Distribución de los polvos en la mezcla del suplemento

Ingredients

Proportion (% w/w)

Pea protein

Mesquite powder

Agave fructans

Non-alkalized cocoa powder

Xanthan gum

0.50

Vitamins

0.25

Minerals

0.25

This formulation was used as the foundational matrix for all

assays conducted in this study

2.2 Methods

2.2.1 Physicochemical analysis of powder attributes

2.2.1.1 Bulk and compacted density

Bulk density and compacted density of the powder mixture were evaluated according to the

methodology described by Camargo et al. (2019). To determine Bulk density, 10 g of the sample was

weighed and transferred to a 25 mL graduated glass cylinder (2.5 cm in diameter). The volume

occupied by the powder was recorded directly from the cylinder. Compacted density was obtained

using the same cylinder and sample mass. The cylinder was tapped 30 times against a stainless steel

surface covered with a sheet of ethylene vinyl acetate, and the final volume was recorded. Both

densities were calculated as the ratio of sample mass to the volume occupied in the cylinder (Goula

and Adamopoulos, 2012).

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2.2.1.2 Wettability

Wettability was determined using a previously established method (Ji et al., 2016). A 6 g

portion of each powder sample was carefully sprinkled into a 400 mL beaker (70 mm diameter)

containing 100 mL of distilled water at 25 °C. The time required to achieve complete wetting was

recorded.

2.2.1.3 Solubility

Solubility was determined following the procedure proposed by Liaotrakoon et al. (2022).

One gram of powdered sample was weighed into a centrifuge tube, mixed with 10 mL of distilled

water, and vigorously homogenized. The mixture was incubated at 37 °C for 30 min, followed by

centrifugation at 3000 rpm for 10 min. The insoluble residue was dried in a convection oven at 105 °C

for 3 h. Solubility was expressed as the percentage of soluble solids, calculated using Eq. (1).

  󰇡󰇛󰇜

󰇛󰇜󰇢   Eq. (1)

2.2.1.4 Carr and Hausner indices

The Carr and Hausner indices were calculated using the method proposed by Macías-Cortés

et al. (2022). In summary, the Carr index was determined using Eq. (2) and the Hausner index using

Eq. (3).

  󰇡󰇛󰇜

 󰇢   Eq. (2)

  

 Eq. (3)

2.2.1.5 Foam properties

Foam formation in reconstituted powders was considered a fundamental quality parameter,

closely linked to consumer acceptance. Foams were produced by vigorously shaking a sealed

cylindrical container (130 mL internal volume, graduated to 100 mL in 1 mL increments) filled with

40 mL of solution and 90 mL of air (Marinova et al., 2017). Foam stability was evaluated across three

time points (0, 180, and 600 s) during a 600-second observation. The total volume of foam and

underlying liquid (VF+L, upper limit) and the volume of drained liquid (VDrL, lower limit) were

registered. Calculation of the liquid fraction within the foam (ε) was performed based on Eq. (4), and

the gas fraction (ε⁻¹) was determined using Eq. (5) (Marinova et al., 2017).

  󰇛󰇜

󰇛󰇜 Eq. (4)

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 

 Eq. (5)

2.2.2 Characterization of the supplement

2.2.2.1 Determination of caloric content

The energy content of the supplement was estimated based on its nutritional composition,

in accordance with Mexican Official Standard NOM-051-SCFI/SSA1-2010. Quantification of the total

macronutrient content (proteins, lipids, and carbohydrates) was performed. Subsequently,

Atwater’s general conversion factors were applied, assigning 4 kcal per gram to proteins and

carbohydrates, and 9 kcal per gram to lipids (Sánchez-Peña et al., 2017). Results were represented as

kilocalories per 100 grams of supplement (kcal/100 g).

2.2.2.2 Phytochemical characterization

Triplicate portions of 100 mg were prepared for phytochemical analysis. The samples

dissolved in distilled water at a 1:10 (w/v) ratio and homogenized for 1 minute using an Ultra-Turrax

(Ika Eurostar Power Digi-visc homogenizer, Staufen, Germany) for 1 minute. The homogenates were

centrifuged, and the supernatant was recovered. Subsequently, liquid-liquid microextraction was

performed with ethyl acetate (2:1, v/v). Vortex agitation was applied for 1 minute, and the sample

was then centrifuged to recover the organic phase. The washing was repeated a second time. Both

supernatants were combined in a single tube and concentrated using a Labconco CentriVap

centrifugal vacuum concentrator (Thermo Fisher Scientific, US). The samples were dissolved in 200

µL of methanol and filtered using 0.45 µm PTFE membranes before analysis.

The phytochemical profile was defined according to the methodology described by De Santiago-

Romero et al. (2024). Chromatographic separations were performed using LC-MS/MS, employing

7.5 mM formic acid in Milli-Q water (solvent A) and acetonitrile (solvent B). A concentration gradient

of solvent B was applied at 0, 1.2, 3.8, 11.4, and 13.2 minutes, with concentrations of 3 %, 9 %, 16 %,

50 % and 3 %, respectively. An Acquity BEH C18 column (1.7 µm, 2.1 × 50 mm) was used at 35 °C,

with a constant flow rate of 0.250 mL/min⁻¹ and a stabilisation time of 2.8 minutes at 3 % solvent B.

Electrospray ionisation (ESI) was performed in negative mode, using nitrogen as the nebulising gas.

ESI source included a capillary voltage of 2.25 kV, cone voltage of 30 V, and a collision gas flow of

0.15 mL/min⁻¹. Collision energies were adjusted to 5.0 eV for MS and 20.0 eV for MS/MS. Desolvation

and source temperatures were maintained at 400 °C and 150 °C, respectively. Compounds were

identified and quantified through multiple reaction monitoring (MRM), ensuring selective detection.

2.2.3 Biological methods (human intervention model)

The intervention model was approved by the Research Ethics Committee of Comité de Ética

en Investigación (CEITecNM/I.T. Durango) (registration number CEI-003-2022-0301-003). A non-

probabilistic convenience sampling technique was applied, as the participants were volunteers who

met the inclusion criteria and provided written informed consent. This approach is both ethically

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acceptable and methodologically consistent for exploratory nutritional interventions in healthy

populations. It enabled the determination of the glycemic index and glycemic load of the supplement

through a cross-sectional assessment, in accordance with International Standard ISO 26642:2010(E).

The effect of supplement consumption on body indicators and bone density was also analyzed

through a longitudinal assessment.

Figure 1. Flow of participants through the study stages. (1) recruitment (n = 32); (2) intervention start (17 men,

15 women); (3–4) supplement intake, glycemic index and load assessment, DXA scans (baseline and after 2

months), with dropout of 6 men; (5) final data collection and analysis

Figura 1. Se muestra el flujo de los participantes a través de las etapas de (1) reclutamiento (n = 32); (2) inicio

de intervención (17 hombres, 15 mujeres); (3)-(4) consumo del suplemento, medición de índice y carga

glucémica, escaneos DXA (inicio y 2 meses), registrando diserción por abandono del estudio de 6 hombres; (5)

obtención de resultados y análisis final

A total of 32 participants (15 women and 17 men) were initially recruited. Eligibility required an age

range of 18 to 40 years and the absence of any metabolic disease diagnosis, which was verified

through the clinical record of each participant. Evaluations were conducted at the facilities of the

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Laboratorio Nacional CONAHCYT de Apoyo a la Evaluación de Productos Bióticos (LaNAEPBi),

part of the Tecnológico Nacional de México /I.T. Durango. After signing the informed consent form,

clinical records were initiated, including the recording of vital signs such as blood pressure,

respiratory rate, heart rate, and oxygen saturation. Body composition was assessed via DXA,

providing data on weight (kg), body mass index (kg/m²), fat, lean and bone mass (%), subcutaneous

adipose tissue area (cm³), and bone mineral density in various anatomical regions (radius, femur,

and spine). Participants were directed to consume a daily dose of 30 g of the supplement dissolved

in water, a portion defined in the study design as practical, safe, and consistent with ranges reported

in nutritional interventions (20–40 g/day) and commercial powdered supplements. Following the

initial dose, a glucose tolerance test was performed to determine the glycemic index and glycemic

load of the supplement. Daily supplementation continued for two months. At the end of the

intervention, the DXA scan was repeated to assess post-intervention changes in body composition

(Fig. 1).

2.2.4 Experimental design and data analysis

Experimental data of physicochemical properties were expressed as mean ± standard

deviation, and the data of biological model were expressed as mean ± standard error due to the

nature of the data. A one-way analysis of variance (ANOVA) was performed, followed by Tukey's

post hoc test for multiple comparisons (p < 0.05 was considered statistically significant), using

Statistica v12.1 software (StatSoft, Tulsa, OK, USA). Mixture homogeneity was assessed through

analysis of skewness and kurtosis. T-scores and body composition variables were analyzed using

the relative change method (Fold Change) via the MetaboAnalyst v6.0 platform.

3. Results and discussion

3.1 Physical properties of the supplement

The analyses were performed on the formulated supplement, and all results reflect the

properties of the complete mixture (Table 1). Based on the data presented in Table 2, both mixing

time and particle size had statistically significant effects (p < 0.05) on the specific evaluated responses.

However, the analysis of variance showed that the interaction between mixing time and particle size

did not significantly influence the results (p > 0.05), suggesting that each variable independently

impacts the physical properties of the supplement. Regarding wettability, a higher water absorption

capacity was observed in treatments with 600 µm particles, probably attributable to a lower specific

surface area and increased porosity, potentially facilitating water penetration into the particle matrix.

This property is essential, as powder reconstitution represents the initial stage of the process (Gaudel

et al., 2023). Conversely, the decrease in wettability with increasing mixing time could be due to

physical redistribution of the particles; in this case, the resulting values may represent a weighted

average of the entire mixture (Ong et al., 2020).

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ISSN-e: 2683-3360

Table 2. Rehydration and flow properties of the powders used to obtain the supplement

Tabla 2. Rehidratación y propiedades de fluidez de los polvos utilizados para obtener el suplemento

Time

(s)

Particle size

(mm)

Bulk density

(g/cm3)

Compact

density (g/cm3)

Carr index

Hausner

index

Wettability (s)

Solubility

index (%)

0.600

0.3020.08

0.3970.00

23.901.50

1.310.02

33.00.50

25.460.40

0.250

0.2990.02

0.4230.01

24.221.10

1.420.12

26.50.50

26.830.05

0.145

0.2850.01

0.4010.00

29.051.12

1.410.02

25.50.50

21.131.10

180

0.600

0.2940.00

0.4110.00

28.460.00

1.400.00

35.00.50

26.700.50

180

0.250

0.2950.01

0.4010.00

26.681.25

1.360.02

25.50.50

21.502.70

180

0.145

0.3020.01

0.4010.00

25.682.24

1.310.04

25.50.50

20.301.63

240

0.600

0.2940.00

0.3970.00

25.930.74

1.340.01

36.50.50

24.810.40

240

0.250

0.2950.01

0.4090.01

28.110.20

1.380.00

25.50.50

23.230.60

240

0.145

0.2920.01

0.4060.01

28.200.30

1.380.00

25.50.50

21.530.05

The results are expressed as mean ± standard deviation

In terms of solubility, the 600 µm particle size showed greater solubility compared to smaller sizes (25 % vs. 21 %) (p < 0.05). This behaviour can

be attributed to the presence of mesquite flour, which is rich in carbohydrates that enhance molecular interactions, favouring system solubility.

Although mixing time did not exert a statistically significant effect on this parameter, its indirect influence may be related to the dispersion of

soluble ingredients. These values were consistent with results reported by Lai et al. (2024) in alfalfa leaf powder mixtures. In particular, the

results for bulk and compact density did not indicate statistically significant differences with respect to mixing time or particle size, indicating

that these properties are more influenced by powder composition than by processing conditions.

To evaluate fluidity, the intermediate mixing time condition was selected to calculate the Carr and Hausner indices. The values obtained were

favorable (Table 2), indicating that the presence of pea protein decreases the cohesion of the particles present in the supplement, promoting its

handling and transport; therefore, the addition of ingredients that could enhance fluidity is not necessary. The Hausner index and the Carr index

are two empirical parameters, closely related to each other, that are used to estimate the flowability of a powder. Despite the above, it is essential

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to mention that the Carr index was developed based on the concept that the more compressed a material is, the less flowable it will be. In contrast,

the Hausner index was conceived as an indicator of the friction conditions between powders (Saker et al., 2019). Therefore, based on the data

shown and the description above, the addition of an ingredient is not necessary.

Table 3. Foaming properties of reconstituted powder mixtures

Tabla 3. Propiedades espumantes de las mezclas de polvos reconstituidos

Time

(s)

Particle size

(mm)

0 s

180 s

600 s

(-1)0 s

(-1)180 s

(-1)600 s

0.600

0.760.02

0.770.02

0.240.02

0.230.02

0.250

0.770.03

0.790.02

0.230.03

0.210.02

0.145

0.800.02

0.810.01

0.810.03

0.200.02

0.190.03

180

0.600

0.810.03

0.810.01

0.190.03

0.190.01

180

0.250

0.780.03

0.780.02

0.220.03

0.220.02

180

0.145

0.800.03

0.200.03

240

0.600

0.800.02

0.790.02

0.800.01

0.200.02

0.210.02

0.200.01

240

0.250

0.770.02

0.780.02

0.770.02

0.230.02

0.220.02

0.230.02

240

0.145

0.760.02

0.780.01

0.240.02

0.220.01

The results are expressed as mean ± standard deviation

The results in Table 3 show the foaming values at different times. In the case of foaming at time zero, it can be seen that mixing time has an

influence, as a higher liquid fraction value (ε) was observed in the powders that were mixed (0.81 vs. 0.76, approximately), regardless of the

foaming time applied. Although this behavior becomes insignificant over time (ε at 0 s vs. ε at 600 s), there is a tendency for foam stability to

improve in treatments with longer mixing times, as the liquid fraction remains constant.

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Regarding particle size, no significant influence was observed on the liquid or gas fraction of the

foam, indicating that this variable does not directly impact the foam formation capacity or stability

under the conditions evaluated. The drainage volume, calculated as the difference between the

liquid fraction at 600 s and at 0 s, showed that the agitation time had a positive influence on foam

stability (Fig. 2). No significant change was recorded in the agitated powders. In contrast, an increase

in drainage volume was observed in the non-agitated powders. The homogenization of the powder

can explain this behavior during agitation, which allows for a more uniform distribution of the

protein in the system, favoring the formation of more stable bubbles (Narsimhan and Xiang, 2018).

In addition, the inclusion of xanthan gum in the mixture may have contributed to the observed

stability, as this polysaccharide supplies structural support to the foam formed by the protein,

preventing premature collapse of the foam matrix (Sheng et al., 2021).

Figure 2. Effect of agitation time on the liquid drainage of the foam in the reconstituted supplement. The bars

indicate the standard deviation, while the dots are the average values

Figura 2. Efecto del tiempo de agitación sobre el drenaje líquido de la espuma en el suplemento reconstituido.

Las barras indican la desviación estándar, mientras que los puntos son los valores medios

3.2 Supplement formulation

The supplement formulation had a functional and phytochemical profile, as detailed in Table 4. The

total caloric content was 366.03 kcal/100 g, with a macronutrient distribution of 29.53 g of

carbohydrates, 58.59 g of protein, and 1.15 g of lipids. This composition primarily consists of a

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protein supplement, which may promote muscle protein synthesis processes and help regulate

energy metabolism (Lim et al., 2024). Micronutrient analysis revealed that the vitamin and mineral

premix contained 430.93 mg/100 g, reinforcing its functional potential to contribute to physiological

functions, including bone mineralization processes and electrolyte balance (Schiefermeier-Mach et

al., 2020).

As a complement to the formulation, the intervention study helped determine the glycemic index

and glycemic load, with values of 11.18 and 5.59, respectively. This indicates a low glycemic

response, which is proper for preventing metabolic sensitivity or the risk of insulin resistance (Yu et

al., 2025). These parameters are vital for preventing postprandial glucose and insulin spikes,

especially in preventive contexts and in metabolic disorders such as type 2 diabetes mellitus or

insulin resistance (Peres et al., 2023).

Table 4. Nutritional statement for the dietary supplement developed from mesquite flour, agave

fructans, agavins, cocoa, pea protein, and a blend of vitamins and minerals

Tabla 4. Declaración nutricional del suplemento dietético elaborado a partir de harina de mezquite,

fructanos de agave, agavinas, cacao, proteína de chícharo y una mezcla de vitaminas y minerales

Macronutrients

Amount per 100 g

Energy content

366.03 kcal

Total protein (g)

58.59

Pea protein

42.18

Total lipids (g)

1.51

Total carbohydrates (g)

29.53

Agave fructans

4.72

Micronutrients

Amount per 100 g

Total vitamins (mg/100 g)

430.993

Thiamine (B1)

2.33

Riboflavin (B2)

2.64

Pantothenic acid (B5)

10.88

Retinol (A)

1.55

Folic acid

311.11

Pyridoxine (B6)

3.11

Ascorbic acid (C)

91.33

Cholecalciferol (D3)

7.77

Biotin (H)

0.150

Vitamin K

0.120

Cyanocobalamin (B12)

0.003

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Total minerals (mg/100 g)

1834.68

Calcium

871.27

Phosphorus

566.57

Potassium

244.70

Magnesium

137.25

Iron

5.88

Zinc

5.88

Copper

1.17

Manganese

1.96

Additional information

Amount per 100 g

Non-alkaline cocoa (g/100 g)

5.00

Total polyphenols (µg/100 g)

6608.57

Glycemic index (GI)

11.18

Glycemic load (GL)

5.59

The data correspond to the nutritional profile of the supplement used in the experimental

trials described in this study

Chemical characterization identified 35 polyphenolic compounds, with a total abundance of 6608.57

± 168.60 µg/100 g, predominantly hydroxycinnamic acids (2597.12 ± 106.65 µg/100 g), flavanols

(1606.77 ± 65.85 µg/100 g), and flavonols (1526.47 ± 94.96 µg/100 g), identified for their antioxidant

activity and modulating effects on inflammatory and metabolic pathways (Rudrapal et al., 2024).

Flavanones (9.84 ± 0.73 µg/100 g) and flavones (3.82 ± 1.75 µg/100 g) were found in lower abundance,

completing the bioactive profile of the supplement (Table 5). These compounds, together with the

functional synergy of the base ingredients (pea protein, mesquite flour, agave fructans, cocoa, and

the vitamin and mineral premix), support their potential impact in promoting advances in bone

health and body recomposition (Bogdanis et al., 2023).

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Table 5. Phytochemical profile of the supplement determined by LC-MS/MS

Tabla 5. Perfil fitoquímico del suplemento determinado por LC-MS/MS

No.

Compound

Nominal mass

(g/mol)

Main

transitions

(min)

λmax

Abundance

(µg/100g)

Shikimate

173.18

111.07, 93.06

0.598

297.53

378.14±34.04

Gallic acid

169.15

125.05, 79.07

1.095

269.15

143.58±8.54

Protocatechuic acid

153.15

108.92, 81.09

2.159

293.53

98.82±14.05

p-salicylic acid

137.04

93.05, 65.09

3.221

254.53

20.18±1.06

Syringic acid

197.21

182.05, 153.11

4.152

273.53

4.42±0.60

Salicylic acid

137.04

93.05, 65.09

6.410

254.53

10.05±0.67

Benzoic acid

121.10

77.06

6.453

272.53

21.34±2.56

Quinic acid

191.20

93.06, 85.06

0.580

320.00

974.61±44.35

Neochlorogenic acid*

353.34

191.04, 85.05

2.616

324.53

9.15±1.14

10.

Caffeic acid

179.19

135.08, 89.09

3.975

322.53

12.02±1.08

11.

Chlorogenic acid

353.34

191.04, 85.05

3.565

324.53

1551.77±81.60

12.

Cryptochlorogenic acid*

22.96±2.35

13.

p-coumaric acid

163.24

119.08, 98.07

5.111

308.53

32.69±2.51

14.

Ferulic acid

193.24

178.07, 134.04

5.720

322.53

18.17±0.61

15.

Sinapic acid

223.24

164.06, 149.04

5.785

323.53

6.81±0.88

16.

3,4-dicaffeoylquinic acid*

515.43

353.20, 179.06

6.638

323.53

0.35±0.11

17.

t-cinnamic acid

147.17

103.08, 77.07

8.574

275.53

8.55±2.79

18.

Gallocatechin*

305.33

125.02

2.646

273.53

1.54±0.16

19.

Procyanidin B1

577.44

407.21, 289.18

3.253

278.53

82.26±10.50

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20.

Catechin

289.24

245.12, 123.03

3.608

278.53

664.73±40.39

21.

(epi)- catechin

289.24

245.12, 123.03

4.474

278.53

855.64±25.65

22.

(epi)- catechin gallate

441.27

169.04

6.080

276.53

2.58±0.15

23.

Rutin

609.28

300.24, 271.13

5.940

353.53

117.98±18.24

24.

Miquelianin

477.26

301.10, 151.03

6.059

353.53

1166.73±86.03

25.

Isoquercetin*

463.35

300.42, 271.11

6.086

353.53

78.15±8.57

26.

Astragalin*

447.34

284.24, 255.12

6.667

347.53

10.23±0.86

27.

Quercetin

301.20

179.04, 151.02

8.320

370.53

125.78±5.01

28.

Kaempferol

285.22

185.02, 151.02

9.345

363.53

27.58±8.46

29.

Apigenin

269.27

177.04, 148.58

9.151

335.53

3.82±1.75

30.

Hesperidin

609.40

301.15, 164.02

7.080

352.53

0.17±0.04

31.

Neohesperidin*

609.40

301.15, 164.02

7.080

352.53

0.10±0.01

32.

Naringenin

271.28

151.04, 119.06

9.135

288.53

9.57±0.75

33.

Taxifolin

303.03

285.00, 125.02

6.041

303.00

47.05±8.14

34.

Phloridzin

471.34

435.16, 273.15

7.43

283.53

9.86±1.05

35.

Phloretin

273.23

167.03, 123.08

9.233

284.53

118.69±1.00

Retention time; *Compounds identified based on their major transitions and peak spectra, abundance data are mean ±

standard deviation (n=3)

3.3 The impact of supplement consumption on bone mineral density and body composition

The intervention model began with 32 participants (17 men and 15 women) and concluded with 26 active participants (15 women and

11 men). Despite this decrease, the final sample was statistically valid. The supplement showed different effects in women and men, especially

in bone mineral density and visceral fat redistribution. Both groups maintained vital signs within normal ranges (Table 6).

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Table 6. Health indicators and body composition of participants before and after the intervention

Tabla 6. Indicadores de salud y composición corporal de los participantes antes y después de la intervención

Parameter

Women

Men

Beginning

End

Beginning

End

Age (years)

28.26±1.94

24.45±1.08

Height (m)

1.62±0.01

1.71±0.01

Body weight (kg)

68.81±3.30

68.69±3.26

98.32±5.61

97.75±5.52

Body Mass Index (kg/m2)

25.55±1.03

25.92±1.17

33.51±1.64

33.10±1.64

Heart rate

72.53±2.27

69.00±1.36

Respiratory frequency

17.6±0.17

18.18±0.07

Systolic pressure

111.93±2.20

129.27±2.60

Diastolic pressure

72.33±1.21

77.54±2.37

Oxygen saturation

97.66±0.12

97.63±0.13

Data showed are the mean ± standard error (SE).

DXA analysis showed improvements in body and radial T-scores after two months of intervention. In women, an increase in radial bone density

was observed (−1.81 ± 0.17 to −1.50 ± 0.17), while in men, improvements were observed in radial (from −1.90 ± 0.17 to −1.45 ± 0.16) and femoral

(from −0.07 ± 0.15 to 0.17 ± 0.11) densitometry (Table 7). These changes may be associated with the synergistic action of the supplement's

ingredients. Cocoa has been reported to have effects on the femoral and lumbar regions by increasing levels of insulin-like growth factor 1 (IGF-

I), which reduced bone turnover in rats (Sarmadi et al., 2020). On the other hand, agave fructans improved femoral calcium content and bone

mineral density in calcium-deficient rats (Topolska et al., 2020). Additionally, peas and mesquite provide polyphenols such as quercetin, rutin,

catechin, and epicatechin, and micronutrients such as calcium, phosphorus, zinc, and vitamin D, which help modulate physiological pathways

implicated in osteoblastogenesis and bone remodeling (Shaghaghian et al., 2022; Tomczyk-Warunek et al., 2024).

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With regard to body composition, a reduction in visceral fat was observed in both groups (Table 7). Specifically, in the women's group, visceral

fat decreased from 656.04 ± 96.32 g to 603.07 ± 88.31 g, and in men, from 1310.59 ± 174.11 g to 1253.13 ± 154.55 g. This result can be explained by

the plant proteins, dietary fiber, and antioxidant compounds present in mesquite, peas, and cocoa, which promote lipid oxidation and

thermogenesis processes (Jean-Marie et al., 2021; Oliveira et al., 2021). On the other hand, agave fructans may have contributed to the observed

effect by modulating the gut microbiota and enabling the production of microbial metabolites, such as butyrate, which is involved in bone and

lipid metabolism (Ocampo et al., 2023).

Table 7. Body composition and mineral density of participants at beggining and at the end of the intervention study

Tabla 7. Composición corporal y densidad mineral de los participantes al inicio y al final del estudio de intervención

Parameter

Women

Men

Beginning

End

Beginning

End

Fat mass (%)

39.66±1.28

40.05±1.35

38.09±1.27

38.35±1.21

Lean mass (%)

57.36±1.19

56.80±1.23

59.23±1.16

58.99±1.10

Bone mass (%)

2.96±0.10

2.95±0.10

2.70±0.11

2.66±0.11

Visceral adipose tissue area (cm3)

135.30±13.13

128.87±12.87

208.40±22.66

203.96±19.99

Visceral adipose tissue mass (g)

656.04±96.32

603.07±88.31

1310.59±174.11

1253.13±154.55

Visceral adipose tissue volume (cm3)

110.26±11.53

119.10±10.79

185.49±17.60

188.25±18.75

Body bone mineral density (T-score)

-1.22±0.15

-1.19±0.15

-1.72±0.07

-1.76±0.06

Radial mineral density (T-score)

-1.81±0.17

-1.50±0.17

-1.9±0.17

-1.45±0.16

Femoral mineral density (T-score)

-0.26±0.17

-0.23±0.15

-0.07±0.15

0.17±0.11

Spinal mineral density (T-score)

0.73±0.14

0.78±0.14

0.59±0.06

0.60±0.05

Data showed are the mean ± standard error (SE).

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Likewise, multivariate Fold Change analysis confirmed and expanded upon the previous findings,

showing significant variations in body and radial bone density, as well as in the redistribution of

adipose tissue, particularly in the visceral area (Fig. 3ab). Positive values show patterns of tissue

redistribution and improvement in bone mineral density, which may reflect the physiological impact

of the supplement.

Figure 3. Fold Change analysis in anthropometric parameters and bone densitometry in women (a) and men

(b). The bars represent the magnitude of change in each variable between comparative groups. The asterisk (*)

indicates the variables with the greatest positive changes.

Figura 3. Análisis del cambio relativo en los parámetros antropométricos y la densitometría ósea en mujeres (a)

y hombres (b). Las barras representan la magnitud del cambio en cada variable entre los grupos comparativos.

El asterisco (*) indica las variables con los mayores cambios positivos.

4. Conclusions

The combination of functional ingredients enabled the development of a powdered

supplement with favorable physical properties, including a particle size of 600 µm that ensured

stability, solubility, and fluidity. Its nutritional composition provided sustained energy intake of

366.03 kcal/100 g, with 29.53 g/100 g of carbohydrates, 1.15 g/100 g of lipids, and a high protein

content of 58.59 g/100 g, accompanied by an optimal glycemic index of 11.18 and a glycemic load of

5.59. The phytochemical profile revealed a polyphenol concentration of 6608.57 µg/100 g, with

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hydroxycinnamic acids (2597.12 µg/100 g), flavonols (1606.77 µg/100 g), and flavanols (1526.47

µg/100 g) as the most abundant subfamilies.

Consumption improved bone mineral density in both sexes. In women, radial bone density increased

from −1.81 to −1.50, while in men improvements were observed both in radial (−1.90 to −1.45) and

femoral regions (−0.07 to 0.17). Supplement intake also promoted body recomposition, as evidenced

by a reduction in visceral fat, with values in women decreasing from 656.04 g to 603.07 g, and in men

from 1310.59 g to 1253.13 g.

Overall, these findings may suggest the potential of the supplement with implications for population

health, particularly in supporting bone health and reducing visceral adiposity. Its innovative

formulation and synergistic effects highlight its relevance for nutritional interventions and future

research.

Contributions of the authors

Conceptualization, J.A.G.I. and K.M.H.R.; methodology, D.D.J.S.; software, K.M.H.R.,

J.A.G.I., N.E.R.G.; validation, K.M.H.R., J.A.G.I., N.E.R.G., M.R.M.J., and S.M.G.H.; formal analysis,

J.A.G.I. and K.M.H.R.; research, D.D.J.S. and J.N.D.G.; resources, J.A.G.I.; data preservation,

K.M.H.R. and J.A.G.I.; writing-drafting of the original manuscript, K.H.M.R., J.A.G.I., and N.E.R.G.;

writing-revision and editing, K.M.H.R., J.A.G.I, N.E.R.G, M.R.M.J. and S.M.G.H; visualization,

K.M.H.R, J.A.G.I and D.D.J.S; supervision, J.A.G.I.; project management, K.M.H.R., J.A.G.I, N.E.R.G,

M.R.M.J., and S.M.G.H.; funding acquisition, J.A.G.I, N.E.R.G, M.R.M.J., S.M.G.H and K.M.H.R. All

authors have read and accepted the published version of the manuscript.

Acknowledgements

The Master's students listed as co-authors gratefully acknowledge the financial support

provided by CONAHCYT (currently SECIHTI). The mesquite samples were kindly donated by Dr.

Julio César Ríos-Saucedo, a researcher at the Instituto Nacional de Investigaciones Forestales,

Agrícolas y Pecuarias, Coyoacán, Ciudad de México. Financial support for the project (ID: 16840.23-

p) was also provided by Tecnológico Nacional de México.

Conflict of interest

The authors declare that they have no conflict of interest related to this research.

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