# Impact of ultrasound-assisted intermittent hydration during pumpkin seed germination on the structure, nutritional, bioactive, physical and techno-functional properties of flours

Flaviana Coelho Pacheco, Irene Andressa, Ana Flávia Coelho Pacheco, Fábio

Ribeiro Dos Santos, Jeferson Silva Cunha, Nathalia de Andrade Neves,

Raquel Guidetti Vendruscolo, Marcio Schmiele, Paulo Henrique Costa Paiva,

Alline Artigiani Lima Tribst, et al.

► To cite this version:

Flaviana Coelho Pacheco, Irene Andressa, Ana Flávia Coelho Pacheco, Fábio Ribeiro Dos Santos, Jeferson Silva Cunha, et al. Impact of ultrasound-assisted intermittent hydration during pumpkin seed germination on the structure, nutritional, bioactive, physical and techno-functional properties of flours. LWT - Food Science and Technology, 2025, 222, pp.117654. 10.1016/j.lwt.2025.117654. hal-05008449

HAL Id: hal-05008449

https://hal.science/hal-05008449v1

Submitted on 27 Mar 2025

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# Impact of ultrasound-assisted intermittent hydration during pumpkin seed germination on the structure, nutritional, bioactive, physical and techno-functional properties of flours

Flaviana Coelho Pacheco<sup>a</sup>, Irene Andressa<sup>a</sup>, Ana Flávia Coelho Pacheco<sup>b</sup>, Fábio Ribeiro dos Santos<sup>a</sup>, Jeferson Silva Cunha<sup>a</sup>, Nathalia de Andrade Neves<sup>c</sup>, Raquel Guidetti Vendruscolo<sup>c</sup>, Marcio Schmiele<sup>c</sup>, Paulo Henrique Costa Paiva<sup>b</sup>, Aline Artigiani Lima Tribst<sup>d</sup>, Pedro Esteves Duarte Augusto<sup>e</sup>, Bruno Ricardo de Castro Leite Júnior<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

- Emerging technologies

- Clean-label ingredient

- Sustainability

- Pumpkin seeds flour

- Agro-industrial by-products

- Valorization

## ABSTRACT

This study investigated the ultrasound (US)-assisted intermittent hydration of pumpkin seeds (2 cycles of 10 min, at 25 or 45 °C, 38 W/L, 25 kHz), followed by germination (3 days, 25 °C), evaluating the structure, nutritional, bioactive, color, and techno-functional properties of the obtained flours. US-assisted hydration produced flours that were more porous and lighter in color. Germination reduced starch crystallinity (≤14.3 %) and altered the flour's aliphatic hydrocarbons, amino acids and carbohydrates, particularly in the C-H and C-O bonds, as evidenced by Fourier transform infrared (FTIR) spectroscopy. US treatment did not affect the flours' mineral nor fatty acid profiles. On the other hand, sonication effectively increased the content of gamma-aminobutyric acid (GABA) (15.6 % and 13.7 %) and the antioxidant activities, measured both by 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (29.4 % and 9.7 %) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (40.8 % and 2.4 %) methods compared to non-sonicated flours at 25 °C and 45 °C, respectively. US increased the water retention capacity, solubility index, foam capacity and foam stability by up to 49 %, 68 %, 143 % and 208 %, respectively. The enhanced nutritional and techno-functional properties suggest that germinated pumpkin seed flour hydrated under US, especially at 25 °C, is a promising and sustainable ingredient that can be used in products such as bread, cakes, cookies, and beverages.

## 1. Introduction

The food industry produces substantial quantities of waste during vegetable processing (Massironi et al., 2023). Sustainable practices aim to minimize waste and maximize resource efficiency, promoting a circular economy, such as valorizing by-products by converting them into valuable products (Santos et al., 2024).

Pumpkin seed, a by-product from pumpkin (*Curcubita mochata*)

processing, is a potential functional ingredient for the food industry (Syed et al., 2019) due to its nutritional and techno-functional properties (Massironi et al., 2022). This seed is rich in phenolic compounds, antioxidant compounds, proteins (~25 g/100 g dry basis), lipids (~36 g/100 g dry basis), and minerals (~4 g/100 g dry basis) (Andjelkovic et al., 2010).

Previous studies have explored the application of pumpkin seeds. For example, bioactive peptides can be produced through the hydrolysis of

<footnote><sup>a</sup> Department of Food Technology, Federal University of Viçosa, Avenue Peter Henry 10 Rolfs, s / n, University Campus, 36570-900, Viçosa, MG, Brazil</footnote>

<footnote><sup>b</sup> Instituto de Laticínios Cândido Tostes, Empresa Agropecuária de Minas Gerais (EPAMIG), Tenente Luiz de Freitas, 116, 36045-560, Juiz de Fora, MG, Brazil</footnote>

<footnote><sup>c</sup> Institute of Science and Technology, Federal University of Jequitinhonha and Mucuri Valleys (UFVJM), 39100-000, Diamantina, MG, Brazil</footnote>

<footnote><sup>d</sup> Núcleo de Estudos e Pesquisas em Alimentação (NEPA). Coordenadoria Interdisciplinar de Centros e Núcleos (COCEN). Universidade Estadual de Campinas (UNICAMP), Albert Einstein, 291, 13083-852, Campinas, SP, Brazil</footnote>

<footnote><sup>e</sup> Université Paris-Saclay, CentraleSupélec, Laboratoire de Génie des Procédés et Matériaux, Centre Européen de Biotechnologie et Bioéconomie (CEBB), 3 rue des Rouges Terres 51110, Pomacle, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: bruno.leitejr@ufv.br (B.R.C. Leite Júnior).</footnote>
pumpkin seed proteins (Pacheco et al., 2025). Additionally, pumpkin seed protein complexes can stabilize bioactive compounds (Lelis et al., 2025), while oil-protein complexes contribute to the formation of stable emulsions (Fu et al., 2025). The use of pumpkin seed flour in baking and in the development of other food products has also been proposed (Bialek et al., 2016; Serdaroğlu et al., 2018). However, no study has yet evaluated the impact of germination, combined with ultrasound (US), on the functional characteristics of the final flour.

Germination is a bioprocessing method that can improve the nutritional, physical and chemical quality of seeds, potentially converting them into functional clean-label ingredients (Andressa et al., 2024; Miyahira et al., 2021). Those ingredients, if incorporated into food, can reduce the need for additives and improve the food techno-functional properties, meeting the growing demand for clean label (Avezum et al., 2023). Germination is a natural process with low environmental impact, as it does not require the use of chemical additives, in line with sustainable practices (Reed et al., 2022).

Despite these advantages, germination presents several challenges, including long hydration and germination periods (Kalita et al., 2021), making the process slow, costly, and increasing the risk of microbial contamination (Ding & Feng, 2019). To overcome these challenges, new strategies are being developed, including the use of emerging technologies, such as ultrasound (US), to accelerate and improve both the hydration and germination steps (Pacheco et al., 2024a; Kheto et al., 2024).

Ultrasound technology can promote changes in the seed microstructure, inducing the formation of micropores and microcracks in the cell wall through cavitation, sponge effect, and microjet generation (Miano et al., 2019). These effects can enhance seed permeability to water, improving hydration (Kumar et al., 2023). Previous studies demonstrated the efficacy of US in increasing the water absorption of various seeds, such as Andean lupine (Miano et al., 2019), durum wheat (Yildirim et al., 2022), and rice (Aires et al., 2023), with positive impacts on the germination of mung bean (Miano et al., 2016), Pinus tabuliformis (Zhang et al., 2023), rice seeds (Huang et al., 2024), barley malt (Carvalho et al., 2024), sunflower (Liu et al., 2024), chickpeas (Bender et al., 2024), and guar seeds (Kheto et al., 2024).

A previous study evaluated how the US-assisted intermittent hydration process (i.e., applying ultrasound technology during the hydration process ("US-assisted") in cycles ("intermittent")) affects the water uptake, germination and proximate composition of pumpkin seeds (Pacheco et al., 2024a). This study assessed different sonication cycles (up to four cycles of 10 min) and temperatures (25 and 45 °C). Based on the results, processing conditions of 2 cycles of 10 min at 25 and 45 °C were selected, and the present study focused on the evaluation of the flours obtained under these conditions.

Therefore, the obtained flours were evaluated for structural modifications (scanning electron microscopy (SEM), fluorescence spectroscopy, FTIR, X-ray diffraction (XRD)). Additionally, their nutritional and bioactive properties, including fatty acid profile, minerals content, gamma-aminobutyric acid levels, antioxidant activity, were analyzed. Techno-functional properties, such as water and oil holding capacities, foaming capacity and stability, swelling and solubility indices, and color parameters, were also assessed.

This in-depth analysis is essential to understand the impact of sonication on the structural modifications of germinated pumpkin seed flour and to identify the best industrial applications for this product. Consequently, this study is aligned with the development of a sustainable, clean-label, and high-added-value ingredients for the food industry.

## 2. Materials and methods

### 2.1. Ultrasound-assisted intermittent hydration, germination process and flour production

Pumpkin seeds (Cucurbita moschata) were obtained from the

company "Doces Mirahy" (Miraí, Minas Gerais, Brazil), as a by-product from the pulp. After receiving them, the seeds were dried at 45 °C for 24 h, vacuum packed and stored at 7 °C for up to 24 h.

For ultrasound-assisted intermittent hydration, approximately 40 g of pumpkin seeds (wet basis) were sanitized with 200 mg/L sodium hypochlorite solution for 15 min (Arienzo et al., 2023). After sanitization, US-assisted hydration was performed using 2 cycles of 10 min, interspersed by 50 min of a traditional hydration step, as the best processing conditions determined by Pacheco et al. (2024a).

Ultrasound processing was conducted in an ultrasonic bath (Unique, model USC 2800 A, Brazil), operating at a frequency of 25 kHz, nominal power of 450 W, and volumetric power of 38 W/L. Temperature was controlled at 25 ± 2 °C (optimum temperature for pumpkin seed germination, according to Zehtab-Salmasi (2006)) and 45 ± 2 °C (highest temperature for pumpkin seed germination, according to Ghaderi et al. (2008)) by a thermostatic bath (SSDu 10L, Solidsteel, Piracicaba, Brazil). The hydration was conducted until reaching a moisture of 45 g/100 g (0.82 g of water/g of dry matter) - determined based on the variation in seed water absorption and mass balance. The conventional hydration (i.e., without ultrasound technology) was carried out in a thermostatic bath under the same temperature conditions until reaching the same moisture.

After hydration, the seeds underwent a germination process. For this, 40 g of seeds from each treatment were incubated (BOD, Novaëtica, Rio de Janeiro, Brazil) at a controlled temperature of 25 °C for 72 h in the dark. To maintain optimal relative humidity (80–85 %), the seeds were sprayed with distilled water three times a day.

After germination, the seeds were weighed and dried in an oven (Q819V2, QUIMIS, Diadema, Brazil) at 50 °C for 12 h. After drying, the rootlets were removed from the germinated seeds, and the germinated and non-germinated seeds were ground in an analytical mill (model IKA® A11 B S32, 180 W). Subsequently, the flours were packaged in vacuum-sealed plastic bags and stored at 7 °C for later analysis. Germinated samples, treated or not with ultrasound, were identified in the text as no-US and US-20 min (25 °C and 45 °C). In addition, for comparative purposes, a control flour was produced with non-germinated seeds, and five treatments were evaluated. Fig. 1 shows the flow of obtaining flour and its proximate composition.

### 2.2. Structural properties

#### 2.2.1. Scanning electron microscopy (SEM)

SEM was conducted using a scanning electron microscope (Vega 3 XMU, TESCAN, Brno, Czech Republic) under vacuum, following the method described by Andressa et al. (2024). The microscope was operated at an accelerating voltage of 10 kV, and images were captured at a magnification of × 1000.

#### 2.2.2. Intrinsic fluorescence spectroscopy

The intrinsic fluorescence of the flours was determined using the methodology described by Sosa, Thompson, Chaves, Acevedo, and Avanza (2020) using a SpectraMax M5 fluorescence spectrophotometer (Molecular Devices, San Diego, USA), with modifications. For this analysis, solutions were prepared in 0.1 mol/L phosphate buffer (pH 7.0) at a concentration of 1 g/100 mL (w/v). A 300 µL aliquot of each sample was then introduced into StakMax™ microplate reader. The samples were excited at an excitation wavelength of 290 nm, and the emission spectra was recorded in the range of 300–420 nm with a slit of 2 nm.

#### 2.2.3. Fourier transform infrared (FTIR) spectroscopic analysis

The flours were analyzed using a Fourier transform infrared spectrophotometer, Cary 630 FTIR (Agilent Technologies, California, USA). The absorbance range used was 4000 to 500 cm⁻¹, with a resolution of 4 cm⁻¹ and 8 scans (Gunarathe et al., 2022). Representative wave-numbers (cm⁻¹) of non-germinated pumpkin seed flour were selected
<figure><img src="image_2.png" /><figcaption>Fig. 1. Experimental planning.</figcaption></figure>

Legend: Non-germinated = flour produced from seeds without hydration or germination. No-US = seeds hydrated without ultrasound. US-20 min = ultrasound-assisted intermittent hydration for 20 min 25 or 45 °C = hydration temperatures.

according to the literature. The percentage relative change (PRC) in intensity was calculated as described in Equation (1) (Eq. (1)). A negative value indicates increased intensity (Chinma et al., 2022).

$$PRC = \frac{\text{raw length} - \text{germinated length}}{\text{raw length}} \times 100 \qquad (\text{Eq. 1})$$

### 2.2.4. X-ray diffraction (XRD) analysis

The crystallographic structure of the flour was determined using an X-ray diffractometer (XRD-6000, Shanghai Drawel Scientific Instrument Co., Ltd., Japan) equipped with a divergence slit, using monochromatic CuK radiation (λ = 0.15406 nm - 40 kV and 30 mA) at a scan rate of 2.0 deg.min⁻¹ covering the 2θ range from 10 to 80°. The relative crystallinity (RC) was calculated using the areas of the crystalline and amorphous regions in the X-ray diffractogram (Equation 2 (Eq. (2))). The spacing were calculated using the Bragg's Equation 3 (Eq. (3)).

$$RC = \frac{\text{crystalline area}}{\text{total area}} \qquad (\text{Eq. 2})$$

$$n\lambda = 2*d*\sin\theta \qquad (\text{Eq. 3})$$

where n is an integer; λ is the wavelength of incident wavelength; d is the spacing between the planes in the atomic lattice, θ is the Bragg's angle in radian (peak center).

### 2.3. Composition, bioactive and physical-chemical properties

#### 2.3.1. Fatty acid profile

The flour lipid fraction was extracted from 1 g of sample using a mixture of methanol, chloroform, and water, according to the method described by Bligh and Dyer (1959). Subsequently, the lipids were derivatized using the method of Hartman and Lago (1973). Then, the fatty acid methyl esters dissolved in hexane were analyzed on a gas chromatograph equipped with a flame ionization detector (GC-FID) (Agilent Technologies, model 7820A, USA). The results were expressed as a percentage of the total area of the chromatograms, considering the FID correction factors and the conversion of esters into acids (Visentainer, 2012).
### 2.3.2. Mineral profile

The flour mineral profile was determined by energy-dispersive X-ray fluorescence spectrometry (EDXRF-model EDX 720, Shimadzu®, Kyoto, Japan, with the software PCEDX 1.11). The equipment operated at voltages of 0–40 KeV (Ti-U) and 0–20 KeV (Na-Sc) for 100 s each. The results were expressed in mg/100 g on a dry basis.

### 2.3.3. Gamma-aminobutyric acid (GABA)

The GABA extraction, derivatization, and quantification were obtained as described by Nascimento et al. (2024). GABA quantification was performed on a 1260 Infinity high-performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA, USA), using γ-aminobutyric acid (Merk, purity >99 %) as standard within a linear range of six points (36.8–184 mg/L; area = 29.245 × concentration of γ-aminobutyric acid + 30.702; r² = 0.9994). The HPLC system was equipped with a quaternary pump and a G1314B diode array detector. The mobile phase consisted of 0.05 mol/L sodium phosphate buffer (pH 7.2) and acetonitrile, with a flow rate of 1.0 mL/min, using a linear acetonitrile gradient. A Zorbax Eclipse C18 chromatographic column (4.6 × 100 mm, 3.5 µm) was employed, with a gradient elution program where the acetonitrile concentration increased from 14 % to 20 % between 0 and 7 min, from 20 % to 22 % between 7 and 10 min, and from 22 % to 25 % between 10 and 15 min. The acetonitrile concentration was then held at 85 % for 3 min before being reduced linearly to 14 % between 25 and 26 min. The injection volume was 10 µL, and detection was performed at 254 nm. The retention time for GABA was 6.7 min, and results were expressed as mg of GABA per 100 g of sample (dry basis for flours).

### 2.3.4. Antioxidant activity

The flour antioxidant activity was evaluated by both DPPH and ABTS methods. For this, 1 g of each flour was mixed with 20 mL of 80 % methanol and sonicated (Planatec, São Paulo, Brazil; 40 kHz, 100 W) for 5 min at 25 °C for antioxidant compounds extraction. The samples were centrifuged (Sorvall Legend Micro 17R-Thermoscientific, USA) at 5000g for 5 min, and the supernatant was retained.

The antioxidant activity using the DPPH method followed the procedure described by Bubelová et al. (2018). The antioxidant activity was calculated using a calibration curve obtained with Trolox (standard curve equation: [Trolox] = 153.81 × $Abs_{at485nm}$ - 3.01; r² = 0.9904), and the results were expressed as mg Trolox/100 g pumpkin seed flour (on a dry basis for flours).

Antioxidant activity using ABTS was performed according to Re et al. (1999), with minor modifications. An ABTS + stock solution was prepared by mixing a 7 mM ABTS solution in 2.45 mM potassium persulfate in a 1:1 ratio. After this, the mixture was left to rest in the dark, at room temperature, for 16 h before use. Then, the ABTS + stock solution was diluted with ethanol until its absorbance reached approximately 0.70 ± 0.05. The mixture consisted of 50 µL of sample and 3.5 mL of ABTS + stock solution and was incubated in the dark for 60 min, and absorbance was subsequently measured at 734 nm. Antioxidant activity was determined using the same calibration curve obtained with Trolox, and the results were expressed in mg Trolox/100 g of pumpkin seed flour (on a dry basis for flours).

### 2.3.5. Color parameters

The flour instrumental color was measured using a Konica Minolta CR10 colorimeter (Minolta, Chiyoda, Japan), with results expressed as luminosity (L*) and chromaticity parameters (a*, red/green coordinate; b*, yellow/blue coordinate). The total color difference (ΔE, Equation (4) (Eq. (4))) was used in relation to the non-germinated flour sample as reference.

$$ \Delta E = \sqrt{(\Delta L^*)^2 + (\Delta a^*)^2 + (\Delta b^*)^2} \qquad (\text{Eq. 4}) $$

## 2.4. Techno-functional properties

### 2.4.1. Water and oil holding capacities

To determine the water holding capacity (WHC), 2 g of flour and 30 mL of distilled water were mixed in a 50 mL centrifuge tube. After 2 min, samples were centrifuged at 3500 g for 30 min at 25 °C, drained and the WHC was determined as the grams of water retained per gram of flour on a dry basis (Alfaro-Diaz et al., 2021).

To determine oil holding capacity (OHC), 10 mL of refined soybean oil was added to 2 g of each flour in a 50 mL centrifuge tube and mixed for 2 min. Then, the mixture was rested at 28 °C for 30 min, centrifuged at 15.000 g for 20 min, drained and the OHC was determined as the grams of oil retained per gram of the sample on a dry basis (Alfaro-Diaz et al., 2021).

### 2.4.2. Foaming capacity (FC) and stability (FS)

To analyze the foam's capacity and stability of the flours, 0.5 g of each flour was mixed with 25 mL of distilled water, and the suspension was mixed using a high shear disperser at 15,000 rpm for 2 min at 23 °C. The contents were immediately transferred to a 100 mL graduated cylinder, and the foam volume was recorded (Elkhalifa & Bernhardt, 2018). Foaming capacity (FC) was expressed as a percentage increase in the volume of the mixture after processing. The samples were then allowed to stand for 60 min, and the foam volume was recorded again. Foam stability (FS) was calculated as the percentage ratio between the volume of the mixture after 60 and the initial volume (0 min).

### 2.4.3. Swelling index and solubility index

The swelling and solubility indices were determined according to the method described by Wani et al. (2013). Each sample (0.6 g of flour) was mixed with 20 mL of distilled water in tubes, heated at 90 °C for 30 min in a water bath, and then centrifuged for 15 min at 3500 g at 4 °C. The swelling index was determined as the weight gained by the samples after draining the supernatant. The supernatant containing soluble matter was dried in a static oven, and the solubility index was calculated as the percentage ratio between the residue mass and the initial mass of the sample.

## 2.5. Experimental design and statistical analysis

The processes and experiments were carried out in three replications, and each experimental unit was carried out in triplicate. The results were expressed as mean ± standard deviation. Data were analyzed using one-way ANOVA, followed by Tukey's post-hoc test, to compare the effects among different treatments with a 95 % confidence level using Statistica software, version 9.2 (StatiSoft INC, USA).

# 3. Results and discussion

## 3.1. Structural properties

### 3.1.1. Scanning electron microscopy (SEM)

The effect of germination and ultrasound processing on the microstructure of pumpkin seed flour analyzed by SEM revealed distinct structural modifications (Fig. 2). As expected, flour from non-hydrated and non-germinated seeds exhibited a compact and smooth surface with no ruptures. Structural changes emerged in flours obtained after seed germination at 25 °C and 45 °C, with the appearance of small ruptures (red arrow in Fig. 2). More pronounced modifications were observed when the hydration of seeds was performed with 2 cycles of sonication (US-20 min) at 25 °C and 45 °C. In these conditions, the flours surface became more porous and showed greater structural breakage (blue arrow in Fig. 2).

These structural changes, promoted by ultrasound-assisted intermittent hydration and germination, could impact the functional properties of the obtained flour, which may improve its overall quality for
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Scanning electron microscopy of flours obtained from pumpkin seed non-germinated or hydrated at 25 °C and 45 °C (assisted or not by 2-cycles of intermittent ultrasound) and germinated. White bars represent 30 µm. Red and blue arrows represent structural changes discussed on section 3.1.1. Legend: Control = flour produced from seeds without hydration or germination. Non-US = seeds hydrated without ultrasound. US-20 min = ultrasound-assisted intermittent hydration for 20 min 25 or 45 °C = hydration temperatures.</figcaption></figure>

food applications, particularly in baked products (Bhinder et al., 2022; Dogruer et al., 2023; Gani et al., 2022). To further investigate these effects, the techno-functional properties of the flours were evaluated in this study.

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Intrinsic Fluorescence of flours obtained from non-germinated pumpkin seed or seeds hydrated at 25 °C and 45 °C (with or without 2 cycles of intermittent ultrasound) and subsequently germinated. Legend: non-germinated = flour produced from seeds without hydration or germination. no-US = seeds hydrated without ultrasound. US-20 min = ultrasound-assisted intermittent hydration for 20 min 25 or 45 °C = hydration temperatures.</figcaption></figure>
### 3.1.2. Intrinsic fluorescence spectroscopy

The fluorescence spectrum, influenced by the polarity of the environment and interactions of tryptophan and tyrosine residues, is a sensitive tool for characterizing proteins and their conformations (Pallarès et al., 2004). As shown in Fig. 3, the intrinsic fluorescence spectrum of flour produced from germinated samples exhibited an increase of 38–40 % in fluorescence intensity at the peak, when compared to flour from non-germinated seeds (p < 0.05). This suggests a possible exposure of proteins’ hydrophobic groups, such as tryptophan, tyrosine, and phenylalanine, induced by enzymatic hydrolysis during germination, which is aligned with the increase in soluble protein concentration previously reported (Pacheco et al., 2024a). Notably, the combination of ultrasound and higher temperature (45 °C) during seed hydration led to an additional increase (52 %) in fluorescence intensity (p < 0.05). This finding indicates that protein unfolding, with exposure of tryptophan residues to the polar microenvironment (Cunha et al., 2024), occurred only when ultrasound-assisted intermittent was carried out at high temperature (45 °C).

Another relevant observation in the fluorescence emission spectra was a slight red shift in the peak wavelength of the samples subjected to sonication (US-20 min, 25 °C and 45 °C), with a $\lambda_{max}$ of 360 nm compared to 358 nm in the non-germinated sample (Fig. 3). This shift suggests a subtle change in the microenvironment around aromatic amino acids, such as tryptophan, which may become more exposed to a water-affine environment. This exposure could potentially enhance the techno-functional properties of the flour (Chen et al., 2013). While the 2 nm shift is relatively small, it may still indicate modifications in the protein’s microenvironment that could influence the availability of hydrophobic amino acid residues. A similar shift was observed by Viana and English (2022), where germination and soaking of yellow-eyed beans also resulted in a red shift compared to other treatments. It is important to note that such small shifts should be considered as indicative of minor changes and require further investigation to fully assess their significance.

### 3.1.3. Fourier transform infrared (FTIR) spectroscopic analysis

The flours' FTIR spectroscopy revealed noticeable changes in various bands at different wavenumbers due to germination and ultrasound-assisted intermittent hydration of pumpkin seeds (Fig. 4). The percentage relative change (PRC) in intensity (Table 1) was used to clarify these changes; a negative PRC indicates an increase in intensity relative to the bands in non-germinated pumpkin seeds, highlighting the impact of germination on functional group intensity (Chinma et al., 2022).

The 1100–900 cm⁻¹ region is associated with carbohydrate-related

transitions, specifically C–C stretching vibrations (1200–1103 cm⁻¹) and C–O bending vibrations (1047–994 cm⁻¹) (Vela et al., 2021). The C–O bond elongation peak at 1040.8 cm⁻¹ increased with germination and US-assisted hydration (p < 0.05 – Table 1). According to Vela et al. (2021), the bands at 1022 cm⁻¹ and 1047 cm⁻¹ are attributed to the amorphous and crystalline structures of starch, respectively, suggesting a possible shift in these structures to the peak at 1040.8 cm⁻¹ in the flours from germinated seeds, independent on sonication during hydration. The absorbance ratio of 1047/1022 cm⁻¹ was highest in the flour from non-germinated seeds (0.993) compared to the other flours (<0.973). These results indicate that germination and sonication during seeds hydration promote the transition from highly ordered crystals to disorganized amorphous molecules, a trend also observed by Kaur and Gill (2019). The starch region exhibited evident changes during germination, with a decreasing trend in the 1047/1022 cm⁻¹ ratio in germinated flours compared to the control sample (Kaur & Gill, 2019).

The FTIR region from 1100 to 1700 cm⁻¹ is associated with proteins, including amide I, amide II, and amide III regions (Yakubu et al., 2022). A decrease in the intensity at 1236.6 cm⁻¹, associated with C–F stretching of alkyl halides, was observed in the samples hydrated at 45 °C (p < 0.05), regardless of sonication. Additionally, an increase in intensity was recorded at 1379.0 cm⁻¹ (C–H deformation vibrations) for the samples not treated with US at 25 °C (p < 0.05). Changes at this peak suggest an increase in C–H stretching, possibly related to the formation of long-chain linear aliphatic structures (Gunarathe et al., 2022). Higher peaks related to proteins, such as those at 1540.6 cm⁻¹ and 1636.1 cm⁻¹, were not affected by germination, ultrasound treatment, nor increased hydration temperature (p > 0.05).

The flours' FTIR spectra also reveal other distinct vibrational characteristics. The observed vibrations include O–H stretching from 3700 to 3020 cm⁻¹ (up to 3275.8 cm⁻¹) in soluble and non-soluble carbohydrates, C–H stretching from 3020 to 2744 cm⁻¹, and C=O carbonyl group vibrations in lipids prominently observed at 1733 cm⁻¹ (Fang et al., 2020). These peaks were also unaffected by germination, ultrasound treatment, and increased hydration temperature. In general, peaks related to O–H stretching, C–H stretching and C=O vibrations remain unchanged during germination or sonication because they are associated with stable covalent bonds in carbohydrates, lipids and proteins (Rostamabadi et al., 2023). This structural stability may be advantageous for specific food applications, such as bakery products, by ensuring consistency in food texture, as well as for emulsified systems (Cai et al., 2023).

Overall, our results suggest that both treatments reduce the compaction of starch granules and aliphatic hydrocarbons, enhancing

<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Fourier transfer infrared spectroscopy of flours obtained from non-germinated pumpkin seed or seeds hydrated at 25 °C and 45 °C (with or without 2 cycles of intermittent ultrasound) and subsequently germinated. Legend: non-germinated = flour produced from seeds without hydration or germination. no-US = seeds hydrated without ultrasound. US-20 min = ultrasound-assisted intermittent hydration for 20 min 25 or 45 °C = hydration temperatures.</figcaption></figure>
<caption>**Table 1**</caption>

Description and relative change in intensity of FTIR functional groups of flours obtained from non-germinated pumpkin seed or seeds hydrated at 25 °C and 45 °C (with or without 2 cycles of intermittent ultrasound) and subsequently germinated.

<table><thead><tr><th rowspan="2">Representative wave numbers (cm<sup>-1</sup>)<br>Reference: non-germinated Sample</th><th rowspan="2">Functional group</th><th rowspan="2">Probable compound</th><th colspan="5">Relative percentage change in intensity</th></tr><tr><th>non-germinated</th><th>no-US/25 °C</th><th>US - 20 min/ 25 °C</th><th>no-US/45 °C</th><th>US - 20 min/ 45 °C</th></tr></thead><tbody><tr><td>3275.8</td><td>O - H stretching of alcohol</td><td>Soluble and non-soluble carbohydrates</td><td>0.00 ± 0.04<sup>a</sup></td><td>0.05 ± 0.03<sup>a</sup></td><td>0.00 ± 0.06<sup>a</sup></td><td>-0.04 ± 0.08<sup>a</sup></td><td>-0.01 ± 0.05<sup>a</sup></td></tr><tr><td>2922.5</td><td>C - H stretching of alkane</td><td>Carboxyl group of triglycerides</td><td>0.00 ± 0.03<sup>a</sup></td><td>0.00 ± 0.03<sup>a</sup></td><td>-0.01 ± 0.03<sup>a</sup></td><td>0.01 ± 0.02<sup>a</sup></td><td>0.01 ± 0.02<sup>a</sup></td></tr><tr><td>2853.1</td><td>C - H stretching of alkane</td><td>Carboxyl group of triglycerides</td><td>0.00 ± 0.02<sup>a</sup></td><td>0.00 ± 0.03<sup>a</sup></td><td>-0.01 ± 0.02<sup>a</sup></td><td>0.02 ± 0.03<sup>a</sup></td><td>0.01 ± 0.01<sup>a</sup></td></tr><tr><td>1743.3</td><td>C=O Stretching</td><td>Lipids</td><td>0.00 ± 0.03<sup>a</sup></td><td>-0.01 ± 0.07<sup>a</sup></td><td>-0.03 ± 0.02<sup>a</sup></td><td>-0.01 ± 0.05<sup>a</sup></td><td>0.01 ± 0.04<sup>a</sup></td></tr><tr><td>1636.1</td><td>Group C = N/amine stretch/C = O of oxalic acid</td><td>Protein</td><td>0.00 ± 0.03<sup>a</sup></td><td>0.01 ± 0.03<sup>a</sup></td><td>-0.01 ± 0.02<sup>a</sup></td><td>0.01 ± 0.04<sup>a</sup></td><td>-0.01 ± 0.04<sup>a</sup></td></tr><tr><td>1540.6</td><td>N - O stretching of nitro compounds</td><td>Protein</td><td>0.00 ± 0.04<sup>a</sup></td><td>0.00 ± 0.04<sup>a</sup></td><td>-0.03 ± 0.03<sup>a</sup></td><td>0.01 ± 0.04<sup>a</sup></td><td>-0.01 ± 0.03<sup>a</sup></td></tr><tr><td>1379.0</td><td>C - H deformation vibrations</td><td>Aliphatic hydrocarbons</td><td>0.00 ± 0.33<sup>b</sup></td><td>-1.20 ± 0.02<sup>a</sup></td><td>-0.62 ± 0.56<sup>ab</sup></td><td>-0.31 ± 0.65<sup>b</sup></td><td>-0.41 ± 0.40<sup>b</sup></td></tr><tr><td>1236.6</td><td>C - F stretching of alkyl halide</td><td>Amino acids</td><td>0.04 ± 0.08<sup>b</sup></td><td>0.10 ± 0.05<sup>ab</sup></td><td>0.12 ± 0.04<sup>ab</sup></td><td>0.19 ± 0.08<sup>a</sup></td><td>0.16 ± 0.03<sup>a</sup></td></tr><tr><td>1157.4</td><td>C - O - C elongation of antisymmetric bridge</td><td>Amino acids</td><td>0.00 ± 0.08<sup>a</sup></td><td>0.13 ± 0.13<sup>a</sup></td><td>0.18 ± 0.16<sup>a</sup></td><td>0.08 ± 0.11<sup>a</sup></td><td>0.15 ± 0.10<sup>a</sup></td></tr><tr><td>1040.8</td><td>C-O bending vibrations</td><td>Carbohydrates</td><td>0.00 ± 1.04<sup>b</sup></td><td>-1.13 ± 0.04<sup>a</sup></td><td>-1.12 ± 0.07<sup>a</sup></td><td>-1.21 ± 0.11<sup>a</sup></td><td>-1.14 ± 0.05<sup>a</sup></td></tr></tbody></table>

Different lowercase letters (line) indicate significant differences between different treatments by Tukey's test at 5 % probability (p < 0.05). References of associated functional groups and probable compounds: Vela et al. (2021); Kaur & Gill (2021); Gunarathne et al. (2022). Legend: non-germinated = flour produced from seeds without hydration or germination. no-US = seeds hydrated without ultrasound. US-20 min = ultrasound-assisted intermittent hydration for 20 min 25 or 45 °C = hydration temperatures.

water penetration into the starch matrix, similarly as described by Fang et al. (2020).

### 3.1.4. X-ray diffraction (XRD)

X-ray diffraction was used to assess the crystallinity of compounds, especially starch, present in the flours. The peak intensity reflects the ordered crystalline regions (Fig. 5). The sharp peaks represent the crystalline regions, and the diffuse peaks represent the amorphous region. The flours from germinated and non-germinated seeds showed different diffraction peaks (p > 0.05), suggesting a shift in the type of crystalline structure due to germination, ultrasound treatment, and temperature of hydration.

As shown in Table 2, the crystallinity index, diffraction peaks, peak intensity, and D-spacing of all five flour samples ranged from 66.3 to 77.4 %, 20.2–20.9 %, 487.3–875.5 and 4.25–4.40 nm, respectively. The crystallinity index of the flour from non-germinated seeds was higher

(77.4 ± 0.73) than that obtained from germinated and sonicated seeds (p < 0.05). The relative crystallinity of the flour decreased by up to 14.3 %, reflecting changes in the complex organization of starch. Similar results were reported for quinoa and chickpea (Sofi et al., 2023; Xing et al., 2021).

In addition, a shift in the positions of the diffraction peaks was observed between flours from non-germinated (20.9°) and germinated seeds (ranging from 20.2° to 20.4°) (p < 0.05). The flour from non-germinated seeds also exhibited a smaller d-spacing (4.25 nm) compared to the germinated samples (4.38 and 4.40 nm) (p < 0.05). These results suggest that germination altered the crystalline structure of the compounds, particularly starch, slightly increasing the distance between crystalline planes and reducing the crystallinity (Gutiérrez-Cortez et al., 2024).

The flour from germinated samples showed higher peak intensity compared to the flour from non-germinated one (p < 0.05). According to Sofi et al. (2023), germination promotes starch hydrolysis by activating amylases, which provide energy for root growth and affect the structure of starch double helices (Li et al., 2017). Enzymes such as α-amylases and β-amylases hydrolyze starch by cleaving α-1,4-glycosidic bonds in amylose and amylopectin, leading to depolymerization and reduced crystallinity (Zheng et al., 2024). Furthermore, germination induces swelling and partial gelatinization of starch granules, which further disrupts their ordered structure (Zheng et al., 2024). In the present study, the modification in the distribution of debranched amylopectin chains may account for the observed reduction in crystallinity (Xing et al., 2021). However, further investigations are necessary to determine whether other factors, such as the extent of branching and molecular weight, also contribute to this decrease (Sun et al., 2015). Additionally, these findings align with the FTIR results, which revealed a shift in the peak at 1040.8 cm⁻¹, suggesting potential changes in the amorphous and crystalline regions of starch in the germinated samples.

<figure><img src="image_6.png" /><figcaption>**Fig. 5.** XRD patterns of flours obtained from non-germinated pumpkin seed or seeds hydrated at 25 °C and 45 °C (with or without 2 cycles of intermittent ultrasound) and subsequently germinated. Legend: non-germinated = flour produced from seeds without hydration or germination. no-US = seeds hydrated without ultrasound. US-20 min = ultrasound-assisted intermittent hydration for 20 min 25 or 45 °C = hydration temperatures.</figcaption></figure>

## 3.2. Composition, bioactive and physical-chemical properties

### 3.2.1. Fatty acid and mineral profile

The flour fatty acid profiles (Table 3) are predominantly composed by polyunsaturated (linoleic, 49.85 g/100 g), monounsaturated (oleic, 21.07 g/100 g), and saturated fatty acids (palmitic and stearic, 17.66 g/
<caption>**Table 2**</caption>

XRD parameters of flours obtained from non-germinated pumpkin seed or seeds hydrated at 25 °C and 45 °C (with or without 2 cycles of intermittent ultrasound) and subsequently germinated.

<table><thead><tr><th>Sample</th><th>Crystallinity index (%)</th><th>Peak diffraction (°)</th><th>Peak intensity</th><th>D-spacing (nm)</th></tr></thead><tbody><tr><td>non-germinated</td><td>77.4 ± 0.73<sup>a</sup></td><td>20.9 ± 0.09<sup>a</sup></td><td>487.3 ± 29.14<sup>b</sup></td><td>4.25 ± 0.02<sup>b</sup></td></tr><tr><td>no-US 25 °C</td><td>68.9 ± 0.71<sup>b</sup></td><td>20.2 ± 0.23<sup>b</sup></td><td>875.5 ± 41.03<sup>a</sup></td><td>4.39 ± 0.05<sup>a</sup></td></tr><tr><td>US - 20 min 25 °C</td><td>66.6 ± 0.70<sup>c</sup></td><td>20.4 ± 0.32<sup>b</sup></td><td>764.0 ± 56.64<sup>a</sup></td><td>4.39 ± 0.04<sup>a</sup></td></tr><tr><td>no-US 45 °C</td><td>66.3 ± 0.79<sup>c</sup></td><td>20.2 ± 0.21<sup>b</sup></td><td>771.3 ± 94.85<sup>a</sup></td><td>4.38 ± 0.04<sup>a</sup></td></tr><tr><td>US - 20 min 45 °C</td><td>66.5 ± 0.69<sup>c</sup></td><td>20.2 ± 0.11<sup>b</sup></td><td>811.3 ± 86.38<sup>a</sup></td><td>4.40 ± 0.02<sup>a</sup></td></tr></tbody></table>

Different lowercase letters (column) indicate significant differences between different treatments by Tukey's test at 5 % probability (p < 0.05). Legend: non-germinated = flour produced from seeds without hydration or germination. no-US = seeds hydrated without ultrasound. US-20 min = ultrasound-assisted intermittent hydration for 20 min 25 or 45 °C = hydration temperatures.

<caption>**Table 3**</caption>

Fatty acid composition of flours obtained from non-germinated pumpkin seed or seeds hydrated at 25 °C and 45 °C (with or without 2 cycles of intermittent ultrasound) and subsequently germinated.

<table><thead><tr><th rowspan="2">Fatty acid (g/100 g)</th><th colspan="5">Sample</th></tr><tr><th>non-germinated</th><th>no-US 25 °C</th><th>US - 20 min 25 °C</th><th>no-US 45 °C</th><th>US - 20 min 45 °C</th></tr></thead><tbody><tr><td>C8:0 - Caprylic Acid</td><td>0.02 ± 0.00</td><td>N.d</td><td>N.d</td><td>N.d</td><td>N.d</td></tr><tr><td>C10:0 - Capric Acid</td><td>0.04 ± 0.00</td><td>0.03 ± 0.00</td><td>0.04 ± 0.00</td><td>N.d</td><td>N.d</td></tr><tr><td>C12:0 - Lauric Acid</td><td>0.33 ± 0.03<sup>a</sup></td><td>0.10 ± 0.00<sup>b</sup></td><td>0.08 ± 0.00<sup>b</sup></td><td>0.06 ± 0.01<sup>b</sup></td><td>0.09 ± 0.01<sup>b</sup></td></tr><tr><td>C14:0 - Myristic Acid</td><td>0.25 ± 0.01<sup>a</sup></td><td>0.14 ± 0.00<sup>b</sup></td><td>0.12 ± 0.01<sup>b</sup></td><td>0.13 ± 0.00<sup>b</sup></td><td>0.14 ± 0.01<sup>b</sup></td></tr><tr><td>C16:0 - Palmitic Acid</td><td>17.66 ± 0.19<sup>a</sup></td><td>17.52 ± 0.06<sup>a</sup></td><td>17.65 ± 0.28<sup>a</sup></td><td>17.28 ± 0.23<sup>a</sup></td><td>17.43 ± 0.39<sup>a</sup></td></tr><tr><td>C16:1 - Palmitoleic Acid</td><td>0.09 ± 0.00<sup>a</sup></td><td>0.09 ± 0.01<sup>ab</sup></td><td>0.08 ± 0.00<sup>b</sup></td><td>0.08 ± 0.00<sup>b</sup></td><td>0.08 ± 0.00<sup>ab</sup></td></tr><tr><td>C17:0 - Margaric Acid</td><td>0.12 ± 0.00<sup>a</sup></td><td>0.13 ± 0.00<sup>a</sup></td><td>0.12 ± 0.00<sup>a</sup></td><td>0.13 ± 0.00<sup>a</sup></td><td>0.12 ± 0.00<sup>a</sup></td></tr><tr><td>C18:0 - Stearic Acid</td><td>9.41 ± 0.18<sup>a</sup></td><td>9.44 ± 0.47<sup>a</sup></td><td>9.57 ± 0.19<sup>a</sup></td><td>9.62 ± 0.59<sup>a</sup></td><td>9.55 ± 0.65<sup>a</sup></td></tr><tr><td>C18:1n9c - Oleic Acid</td><td>21.07 ± 0.12<sup>a</sup></td><td>21.09 ± 0.22<sup>a</sup></td><td>20.75 ± 0.09<sup>a</sup></td><td>21.25 ± 0.49<sup>a</sup></td><td>20.94 ± 0.02<sup>a</sup></td></tr><tr><td>C18:2n6c - Linoleic Acid</td><td>49.85 ± 0.38<sup>a</sup></td><td>49.64 ± 0.76<sup>a</sup></td><td>49.81 ± 0.02<sup>a</sup></td><td>49.60 ± 0.94<sup>a</sup></td><td>49.86 ± 0.24<sup>a</sup></td></tr><tr><td>C18:3n3 - α-linolenic Acid</td><td>0.28 ± 0.01<sup>c</sup></td><td>0.92 ± 0.05<sup>ab</sup></td><td>0.86 ± 0.01<sup>b</sup></td><td>0.94 ± 0.03<sup>a</sup></td><td>0.92 ± 0.00<sup>ab</sup></td></tr><tr><td>C20:0 - Arachidic Acid</td><td>0.48 ± 0.02<sup>b</sup></td><td>0.50 ± 0.04<sup>ab</sup></td><td>0.52 ± 0.02<sup>ab</sup></td><td>0.58 ± 0.03<sup>a</sup></td><td>0.46 ± 0.04<sup>b</sup></td></tr><tr><td>C20:1n9 - cis-11-eicosenoic Acid</td><td>0.10 ± 0.00<sup>a</sup></td><td>0.10 ± 0.00<sup>a</sup></td><td>0.10 ± 0.00<sup>a</sup></td><td>0.11 ± 0.01<sup>a</sup></td><td>0.11 ± 0.00<sup>a</sup></td></tr><tr><td>C22:0 - Behenic Acid</td><td>0.18 ± 0.01<sup>a</sup></td><td>0.18 ± 0.01<sup>a</sup></td><td>0.19 ± 0.01<sup>a</sup></td><td>0.17 ± 0.02<sup>a</sup></td><td>0.20 ± 0.02<sup>a</sup></td></tr><tr><td>C20:5n3 - Eicosapentaenoic Acid</td><td>0.11 ± 0.01<sup>ab</sup></td><td>0.12 ± 0.01<sup>a</sup></td><td>0.09 ± 0.00<sup>b</sup></td><td>0.06 ± 0.01<sup>c</sup></td><td>0.09 ± 0.01<sup>b</sup></td></tr></tbody></table>

Different lowercase letters (line) indicate significant differences between different treatments by Tukey's test at 5 % probability (p < 0.05). Legend: N.d = not detected. non-germinated = flour produced from seeds without hydration or germination. no-US = seeds hydrated without ultrasound. US-20 min = ultrasound-assisted intermittent hydration for 20 min 25 or 45 °C = hydration temperatures.

100 g and 9.41 g/100 g, respectively). Overall, the results showed that neither ultrasound-assisted intermittent hydration nor germination changed the flour's fatty acids profile (p > 0.05). Similarly, Ahmed et al.

(2024) observed that the fatty acid composition of germinated pumpkin seeds was very analogous to the non-germinated sample. Generally, bigger changes in the fatty acid profile are only noted at longer germination times (Ahmed et al., 2024).

According to Table 4, ultrasonic treatment during hydration, irrespective of temperature and germination, did not change the mineral composition of the obtained flours, even though some variations were statistically significant (<5 %). Despite this, the action of endogenous enzymes synthesized during germination is expected to enhance the minerals bioavailability due to the hydrolysis of macronutrients, where minerals are typically complexed, as well as the reduction of antinutritional compounds within the matrix (Wu et al., 2023).

Furthermore, pumpkin seeds flours are notable for their high content of iron (118.13 mg/100g in DM), zinc (70.15 mg/100g in DM), and manganese (54.40 mg/100g in DM) (Table 4), which exceed those found in other seeds (both germinated and non-germinated) reported in the literature, such as sunflower seeds (Andressa et al., 2024), green beans (Alkaltham et al., 2020), and green perilla (Bhaswant et al., 2024). It is important to note that extrinsic factors such as soil type, light exposure, harvesting season, and irrigation notably influence the mineral content of seeds (Ghafoor et al., 2022). Therefore, further studies are necessary to assess the bioavailability of these nutrients.

### 3.2.2. Gamma-aminobutyric acid (GABA) and in vitro antioxidant activity

An important substance accumulated during germination is GABA, which plays a relevant physiological role. This compound is a non-protein amino acid primarily produced through the irreversible α-decarboxylation of L-glutamic acid, catalyzed by glutamic acid decarboxylase, and can be a valuable ingredient in functional foods and nutraceuticals (Zhang et al., 2023).

As shown in Fig. 6A, GABA content in flour from germinated seeds was influenced by hydration temperature and sonication. GABA levels increased by 239 % in the non-sonicated samples and by 285 % in the sonicated samples, after hydration at 45 °C followed by germination. Hydration and germination can induce stress conditions in seeds, activating endogenous enzymes, particularly glutamate decarboxylase, and thus increasing GABA concentration (Li et al., 2022). Additionally, ultrasound exposure enhances energy metabolism during seed germination, further promoting GABA accumulation by increasing glutamate decarboxylase activity and supplying more L-glutamate (Wu et al., 2023). The literature indicates an increase in GABA content with ultrasound application during germination. For example, Ding et al. (2018) reported that wheat flour with an initial GABA content of 14.68 mg/100g increased to 49.72 mg/100g after germination, and further to 64.98 mg/100g with sonication.

GABA is naturally present in various seeds, including beans, rice, and sesame (Vann et al., 2020). Wu et al. (2023) monitored GABA content during 96 h of germination in brown glutinous rice (Oryza sativa L.), observing an increase from an initial concentration of 4.16 mg/100g to 5.88 mg/100g. Similarly, Tiansawang et al. (2016) observed an increase in GABA content in bean samples, from 13.25 mg/100g to 80.68 mg/100g after 48 h of germination. In comparison, pumpkin seeds exhibited substantial GABA concentrations that were further enhanced
<caption>**Table 4**</caption>

Mineral profile of flours obtained from non-germinated pumpkin seed or seeds hydrated at 25 °C and 45 °C (with or without 2 cycles of intermittent ultrasound) and subsequently germinated.

<table><thead><tr><th rowspan="2">Mineral (mg/100g in DM)</th><th colspan="5">Sample</th></tr><tr><th>non-germinated</th><th>no-US 25 °C</th><th>US - 20 min 25 °C</th><th>no-US 45 °C</th><th>US - 20 min 45 °C</th></tr></thead><tbody><tr><td>Phosphorus (P)</td><td>0.73 ± 0.01<sup>c</sup></td><td>0.78 ± 0.02<sup>b</sup></td><td>0.83 ± 0.02<sup>a</sup></td><td>0.82 ± 0.01<sup>a</sup></td><td>0.80 ± 0.01<sup>ab</sup></td></tr><tr><td>Potassium (K)</td><td>0.50 ± 0.01<sup>c</sup></td><td>0.50 ± 0.01<sup>cb</sup></td><td>0.51 ± 0.02<sup>abc</sup></td><td>0.53 ± 0.01<sup>ab</sup></td><td>0.54 ± 0.02<sup>a</sup></td></tr><tr><td>Calcium (Ca)</td><td>0.05 ± 0.00<sup>b</sup></td><td>0.06 ± 0.00<sup>ab</sup></td><td>0.06 ± 0.00<sup>a</sup></td><td>0.06 ± 0.00<sup>a</sup></td><td>0.06 ± 0.00<sup>ab</sup></td></tr><tr><td>Magnesium (Mg)</td><td>0.38 ± 0.00<sup>c</sup></td><td>0.43 ± 0.00<sup>b</sup></td><td>0.45 ± 0.01<sup>a</sup></td><td>0.46 ± 0.01<sup>a</sup></td><td>0.46 ± 0.01<sup>a</sup></td></tr><tr><td>Copper (Cu)</td><td>12.21 ± 0.71<sup>a</sup></td><td>10.90 ± 0.44<sup>a</sup></td><td>11.57 ± 0.49<sup>a</sup></td><td>12.13 ± 0.55<sup>a</sup></td><td>11.57 ± 2.13<sup>a</sup></td></tr><tr><td>Iron (Fe)</td><td>118.13 ± 5.84<sup>a</sup></td><td>102.36 ± 3.52<sup>b</sup></td><td>112.56 ± 6.52<sup>ab</sup></td><td>118.21 ± 3.22<sup>a</sup></td><td>115.76 ± 1.52<sup>a</sup></td></tr><tr><td>Zinc (Zn)</td><td>70.15 ± 1.19<sup>b</sup></td><td>78.02 ± 2.77<sup>ab</sup></td><td>81.82 ± 4.16<sup>a</sup></td><td>85.23 ± 4.13<sup>a</sup></td><td>85.68 ± 1.52<sup>a</sup></td></tr><tr><td>Manganese (Mn)</td><td>54.40 ± 0.70<sup>b</sup></td><td>57.81 ± 0.66<sup>b</sup></td><td>62.43 ± 1.10<sup>a</sup></td><td>63.57 ± 2.09<sup>a</sup></td><td>63.77 ± 1.71<sup>a</sup></td></tr><tr><td>Sodium (Na)</td><td>0.01 ± 0.00<sup>c</sup></td><td>0.05 ± 0.00<sup>a</sup></td><td>0.05 ± 0.00<sup>a</sup></td><td>0.04 ± 0.00<sup>b</sup></td><td>0.05 ± 0.00<sup>a</sup></td></tr></tbody></table>

Different lowercase letters (line) indicate significant differences between different treatments by Tukey's test at 5 % probability (p < 0.05). DM: dry matter. Legend: non-germinated = flour produced from seeds without hydration or germination. no-US = seeds hydrated without ultrasound. US-20 min = ultrasound-assisted intermittent hydration for 20 min 25 or 45 °C = hydration temperatures.

by ultrasound and elevated temperature.

Germination is a technological process that can enhance the availability of antioxidants through biochemical transformation (Ti et al., 2014). In this study, germination increased the antioxidant activity against both ABTS and DPPH radicals (Fig. 6B and C) (p < 0.05).

Specifically, the antioxidant capacity for the ABTS radical in flour from samples sonicated during hydration at 25 °C increased by up to 172 %, while the DPPH radical showed an increase of up to 74 %, when compared to flour from non-germinated samples. However, when the hydration temperature was raised from 25 °C to 45 °C, these gains were less pronounced, reaching only 132 % for ABTS and 44 % for DPPH.

The enhancement in antioxidant activity promoted by ultrasound can be attributed to ultrasonic cavitation, which facilitates the decomposition of antioxidant compounds during germination, including phenolic compounds, GABA, chlorophyll, carotenoids, and glucosinolates (Zhang et al., 2020). Conversely, the increase in temperature minimized the rise in antioxidant activity in germinated samples, possibly due to a partial degradation of sensitive antioxidant compounds, such as flavonoids and phenolic acids (Song et al., 2020).

Based on the obtained results, the antioxidant activity of pumpkin seed flour subjected to germination and ultrasound treatment demonstrated superior values compared to other seeds. For instance, soybean exhibited an antioxidant activity of 13.0 µmol of Trolox equivalents/100g after 5 days of germination (Huang et al., 2014), while mung beans reached 20.0 µmol of Trolox equivalents/100g under similar conditions (Huang et al., 2014). Additionally, rice attained 100.09 µmol of Trolox equivalents/100g after 72 h of germination (Wu et al., 2022). This superior performance underscores the potential of the flour obtained from germinated and sonicated pumpkin seed as a promising source of antioxidant compounds.

Furthermore, a relationship was observed between the increase in GABA content and the enhancement of antioxidant activity. The rise in GABA levels during germination and sonication of the seeds coincides with the activation of metabolic pathways that stimulate the production of bioactive compounds (Kabala & Janicka, 2024). GABA is recognized for its neuroprotective and anti-stress properties (Zhang et al., 2023), while the enhanced antioxidant activity plays a crucial role in mitigating oxidative stress by neutralizing free radicals (Patel et al., 2024). This interplay suggests that the applied processing conditions not only improve the nutritional profile of pumpkin seed flour but may also contribute to its potential functional benefits for food applications.

### 3.2.3. Color parameters

Color is a critical quality attribute in foods and can be influenced by processing (Xia & Li, 2018). The color coordinates (L*, a*, and b*) of the pumpkin seed flour changed after germination (Fig. 7A, B, 7C): luminosity (L*) increased from 60.3 to 64.8, redness (positive a*) decreased from 4.4 to 2.9, and yellowness (positive b*) decreased from 29.4 to

27.6. The increase in luminosity corresponds to a visually lighter color (Fig. 7E). Similar results were reported by Sofi et al. (2023) for germinated chickpea flour. Although statistical differences were noted for the a* parameter, these differences are minimal in practical terms. Moreover, the application of ultrasonic pretreatment during hydration and the hydration temperature did not affect luminosity nor the b* parameter (p > 0.05).

The observed change in luminosity can be attributed to a partial leaching of colored compounds, such as carotenoids (especially β-carotene), during hydration, contributing to the yellow hue of pumpkin seeds (Shen et al., 2020).

The total color difference (ΔE) quantifies the extent of color change in the flours after treatments. According to Xiong et al. (2023), ΔE values less than 1 indicate no discernible difference, values between 1 and 3 indicate small differences, and values above 3 indicate noticeable differences compared to the control. In this study, the ΔE values of the samples from germinated seeds, with or without US processing, exceeded 3 when compared to the flour from non-germinated seeds, suggesting that the color differences could be perceptible to the naked eye, as illustrated in Fig. 7E. However, the ΔE values between the germinated samples, whether with or without US treatment, and at the same hydration temperature, did not differ (p > 0.05), indicating that sonication during hydration did not affect the coloration.

### 3.3. Techno-functional properties

Fig. 8 shows the techno-functional properties of the flours. The flour from non-germinated seeds exhibited values of WHC, OHC, swelling index, solubility index, FC, and FS of 1.47 (g/g), 0.92 (g/g), 3.89 (g/g), 7.79 (g/100g), 18.7 %, and 21.7 %, respectively. All these properties improved with germination by up to 39 %, 27 %, 8 %, 40 %, 57 %, and 124 %, respectively (p < 0.05). These increases can be attributed to the partial hydrolysis of polysaccharides and proteins by metabolic enzymes, as well as structural changes (evidenced by intrinsic fluorescence and FTIR), promoting the generation of polar groups in low molecular weight proteins (Handa et al., 2017).

A similar improvement pattern was observed in previous studies. For instance, Sofi et al. (2023) found that chickpea flour increased WHC from 1.7 g/g to 2.2 g/g due to germination. Similarly, Liu et al. (2018) reported an increase in WHC of bean flour from 2.3 g/g to 3.1 g/g after 72 h of germination. In the case of wheat flour, which is commonly used in baking, the reported WHC values in the literature range from 2.1 g/g in non-germinated flour to 2.8 g/g in germinated flour (Dhillon et al., 2020). Moreover, Romano et al. (2023) reported an increase in OHC from 1.39 g/g to 2.49 g/g in lentil flour, while Mao et al. (2024) noted a rise from 0.70 g/g to 0.75 g/g in chickpea flour. Sofi et al. (2023) documented increases in swelling and solubility indexes for germinated chickpea flour. The results showed that the swelling index increased
<figure><img src="image_7.png" /><figcaption>**Fig. 6.** GABA content (A) and antioxidant activity, measured by ABTS (B) and DPPH (C) radicals, of flours obtained from non-germinated pumpkin seed or seeds hydrated at 25 °C and 45 °C (with or without 2 cycles of intermittent ultrasound) and subsequently germinated. Legend: non-germinated = flour produced from seeds without hydration or germination. no-US = seeds hydrated without ultrasound. US-20 min = ultrasound-assisted intermittent hydration for 20 min 25 or 45 °C = hydration temperatures. Different lowercase letters indicate significant differences between different treatments by the 5 % Tukey test (p < 0.05).</figcaption></figure>

from 7.4 g/g to 8.3 g/g, while the solubility index increased from 18.1 g/100g to 25.1 g/100g. Additionally, Gnanwa et al. (2021) observed that germination improved the foaming capacity and foam stability of germinated bean flour, with an increase in foaming capacity from 22.08 % to 26.74 % and foam stability from 19.23 % to 21.75 %.

Moreover, the positive effects of germination were summed with those induced by US-assisted intermittent hydration and/or hydration carried out at a higher temperature (45 °C). For WHC, a 10 % increase was observed for samples sonicated during the hydration step compared to germinated samples without US (Fig. 4A). The enhanced water absorption capacity may be attributed to structural changes in macronutrients, such as starch, as demonstrated in previous microstructure analyses, which showed increased interaction with water (Dogruer et al., 2023).

<figure><img src="image_8.png" /><figcaption>**Fig. 7.** Color parameters of flours obtained from non-germinated pumpkin seed or seeds hydrated at 25 °C and 45 °C (with or without 2 cycles of intermittent ultrasound) and subsequently germinated. Legend: non-germinated = flour produced from seeds without hydration or germination. no-US = seeds hydrated without ultrasound. US-20 min = ultrasound-assisted intermittent hydration for 20 min 25 or 45 °C = hydration temperatures. Different lowercase letters indicate significant differences between different treatments by the 5 % Tukey test (p < 0.05).</figcaption></figure>

By increasing the hydration temperature, the OHC increased 51 % and 59 % for samples without ultrasound (1.39 g oil/g sample) and sonicated samples (1.47 g/g), respectively, compared to the non-germinated sample (0.92 g/g) (Fig. 8B). Higher temperatures can cause the proteins and polypeptide chains to unfold, exposing hydrophilic groups and hydrophobic side chains (Yu et al., 2023).

Sonication also increased the swelling and solubility indices in pumpkin seed flours. For the swelling index, increases of 8 % and 17 % were observed for non-sonicated and sonicated samples hydrated at 25 °C, respectively, and 12 % and 29 % when hydrated at 45 °C (p < 0.05). This indicates that higher hydration temperatures enhanced the swelling effect. The flour solubility index ranged from 7.79 g/100g for the non-germinated sample to 13.10 g/100g for the sonicated and germinated sample at 45 °C, with higher values observed for samples treated with ultrasound. These US-treated samples exhibited increases of up to 62 % (at 25 °C) and 68 % (at 45 °C) compared to the non-germinated sample. These results may be related to the modifications in the structure of the starch granules (as observed in sections 3.1.3 and 3.2.4), as well as the dissociation of proteins (Kaur & Gill, 2019).
<figure><img src="image_9.png" /><figcaption>**Fig. 8.** Water holding capacity-WHC (A), oil holding capacity-OHC (B), swelling index (C), solubility index (D), foam capacity-FC (E) and foam stability-FS (F) of flours obtained from non-germinated pumpkin seed or seeds hydrated at 25 °C and 45 °C (with or without 2 cycles of intermittent ultrasound) and subsequently germinated. Legend: non-germinated = flour produced from seeds without hydration or germination. no-US = seeds hydrated without ultrasound. US-20 min = ultrasound-assisted intermittent hydration for 20 min or 45 °C = hydration temperatures. Different lowercase letters indicate significant differences between different treatments by the 5 % Tukey test (p < 0.05).</figcaption></figure>

For foaming capacity, maximum increases of 114 % and 143 % were observed for the flour from the sonicated seeds during hydration at 25 °C and 45 °C, respectively, compared to the flour from non-germinated seeds. Regarding foam stability, increases of 208 % and 173 % were noted for flour obtained after seeds sonication during hydration at 25 °C and 45 °C, respectively, compared to the non-germinated sample. FC is influenced by the transport, penetration, and reorganization of molecules at the air/water interface. The cavitation phenomenon likely resulted in the exposure of hydrophobic groups, contributing to enhanced interaction at the air-water interface. Furthermore, it led to a partially unfolded protein structure and improved foaming properties (Gani et al., 2022).

### 3.4. General discussion, possible applications and importance of this study

The growing demand for healthier, sustainable, and “more natural” plant-based foods increases the interest in germinated flours, due to their nutritional and sensory qualities (Lan et al., 2023). This study aimed to transform pumpkin seeds, which are often considered waste or

by-products, into flours with enhanced quality for food applications. This approach not only reduces the environmental impact associated with underutilized vegetable food sources (Miyahira et al., 2021), but also adds value to agro-industrial by-products, thereby contributing to the development of sustainable food systems (Lan et al., 2023).

As discussed in the previous sections, the application of US-assisted intermittent hydration and germination provided various nutritional and techno-functional advantages, thereby improving the flour's quality and its potential for diverse food applications. The flours produced in this study can be used in a wide range of food products, including breads, cakes, cookies, and beverages, particularly for vegetarian and vegan consumers (Náthia-Neves et al., 2024).

The enhancements in bioactive and nutritional properties, such as increased GABA content and antioxidant activity, make these flours suitable for developing functional foods (Hou et al., 2023). For instance, a previous study demonstrated an increase in GABA levels in germinated red rice-based “yogurt”, with values rising from 2.05 mg/100 g to 21.32 mg/100 g after 32 h of germination. In the present study, the GABA content in the flour increased from 5.6 mg/100 g to 21.6 mg/100 g
following US-assisted hydration and 72 h of germination. This positions the flour as a promising ingredient for developing beverages analogous to dairy products, with enhanced GABA levels.

The improved techno-functional properties of the flour suggest that it can partially substitute other flours in food formulations, enhancing the texture and stability of the final products (Angélis et al., 2024). Saeed et al. (2023) demonstrated that combining germinated chickpea flour with wheat flour resulted in cookies with enhanced techno-functional properties and nutritional characteristics, such as water and oil holding capacities, antioxidant activity, flavonoid, and phenolic compound content. Additionally, Bhinder et al. (2022) showed that germinating Tartary buckwheat flour for 48 h improved its functionality, with notable increases in water holding capacity and foaming capacity, leading to muffins with enhanced rheological behavior and reduced glycemic index. Similarly, the results from this study indicated that pumpkin seed flour, after US-assisted hydration and germination, exhibited improvements in water holding capacity (up to 2.16 g/g), oil holding capacity (up to 1.47 g/g), and foaming capacity (up to 143 %), making it a viable candidate to produce functional cookies and muffins.

In addition, more significant results were observed for the solubility index, FC, and FS, demonstrating that this flour can be particularly beneficial for gluten-free products (Singh et al., 2017). An example of this is the study conducted by Brigagão et al. (2021), in which pumpkin seed flour was included as an ingredient in gluten-free muffins, resulting in greater elasticity and better acceptance by consumers.

Transforming pumpkin seeds, often regarded as a waste, into high-value functional ingredients represents a significant opportunity for the food sector. Furthermore, innovative techniques like ultrasound treatment provide a sustainable method for enhancing food ingredient quality, contributing to a circular economy and minimizing food waste. The findings from this study can stimulate the large-scale utilization of sprouted flours, promoting product diversification within the healthy and sustainable food market.

While germination enhanced nutritional and technological characteristics, ultrasound treatment at elevated temperatures resulted in even more improvements. However, in terms of industrial application, ultrasound-assisted intermittent hydration at 25 °C, although not yielding the best results, offers a more practical approach compared to the treatment at 45 °C. By eliminating the need for heating, it reduces energy consumption, better suiting large-scale implementation.

In fact, even though scale-up and techno-economic viability studies are necessary before actual industrial application, we highlight that the choices made in this study were aimed at proposing viable alternatives. For instance, instead of the continuous application of ultrasound, the intermittent application limits the associated costs and complexity of using this technology at industrial scales, such as energy consumption for ultrasound and cooling, rational use of equipment, and minimizing wear. The same reasoning guided the choice of the ultrasonic reactor applied here. Although ultrasonic tips would transfer higher energy densities to the products, we opted to use ultrasonic bath, which is more suitable for the hydration of grains. For example, large ultrasonic baths are already commercially available for cleaning truck engines, demonstrating that they can be scaled.

Finally, it is important to note that there remains a scarcity of research investigating the performance of these flours in actual food products, particularly as substitutes for wheat flour in baking applications with ultrasound pretreatment. Further studies are needed to explore the potential of these flours in healthy food development and to assess the effectiveness of their functional components during processing and consumption – therefore involving aspects such as properties of real foods, sensorial evaluation, consumer perception and expectations, environmental impact and economic viability. Future research efforts should focus on applying germinated pumpkin seeds to create new products and expand their application in the food industry.

## 4. Conclusion

This study demonstrated that germination, when combined with ultrasound-assisted intermittent hydration, improved most of the quality parameters of pumpkin seed flour. Increases in GABA content (up to 15.6 %) and antioxidant activity (up to 29.4 % in the ABTS assay and 40.8 % in the DPPH assay) were observed. Additionally, there were improvements in water retention capacity (up to 49 %), solubility index (up to 68 %), foaming capacity (up to 143 %), and foam stability (up to 208 %). Therefore, the application of ultrasound technology during seed hydration, in conjunction with germination, offers a promising and sustainable approach for transforming pumpkin seeds into functional flour for food applications. Future research should explore the use of these enhanced flours in the development of healthy food products, assessing their functional components during processing and consumption.

### CRediT authorship contribution statement

**Flaviana Coelho Pacheco:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation, Conceptualization. **Irene Andressa:** Investigation, Formal analysis. **Ana Flávia Coelho Pacheco:** Writing – review & editing, Investigation, Formal analysis, Conceptualization. **Fábio Ribeiro dos Santos:** Investigation, Formal analysis. **Jeferson Silva Cunha:** Investigation, Formal analysis. **Nathalia de Andrade Neves:** Investigation, Formal analysis. **Raquel Guidetti Vendruscolo:** Investigation, Formal analysis. **Marcio Schmiele:** Writing – original draft, Investigation, Conceptualization. **Paulo Henrique Costa Paiva:** Writing – original draft, Investigation, Conceptualization. **Aline Artigiani Lima Tribst:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Formal analysis, Conceptualization. **Pedro Esteves Duarte Augusto:** Writing – review & editing, Writing – original draft, Methodology, Formal analysis. **Bruno Ricardo de Castro Leite Júnior:** Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization.

### Funding

The authors are grateful to the Coordination of Improvement of Higher Education Personnel - Brazil (CAPES) - Financing Code 001, Cândido Tostes Dairy Institute, Minas Gerais Agricultural Company (EPAMIG), and the Minas Gerais Research Foundation (FAPEMIG, Brazil) for funding the project APQ-00388-21/APQ-00785-23/RED-00157-23/PPE-00090-23/PPE-00079-24/PPE-00045-24 and FAPEMIG-CNPq for the productivity grant awarded to B. R. C. Leite Júnior (APQ-06600-24). Communauté Urbaine du Grand Reims, Département de la Marne, Région Grand Est and European Union (FEDER Champagne-Ardenne 2014–2020, FEDER Grand Est 2021–2027) are acknowledged for their financial support to the Chair of Biotechnology of Central-eSupélec and the Center Européen de Biotechnologie et de Bioéconomie (CEBB).

### Declaration of competing interest

The authors confirm that they have no conflicts of interest concerning the work described in the manuscript.

### Nomenclature

<table><tr><td>a*</td><td>red/green coordinate</td></tr><tr><td>ABTS</td><td>2,2'-azino-bis(3-ethyl benzothiazoline-6-sulfonic acid)</td></tr><tr><td>ANOVA</td><td>analysis of variance</td></tr><tr><td>b*</td><td>yellow/blue coordinate</td></tr><tr><td>BOD</td><td>Biochemical Oxygen Demand</td></tr></table>
<table><tr><td>DM</td><td>dry matter</td></tr><tr><td>DPPH</td><td>2,2-diphenyl-1-picrylhydrazyl</td></tr><tr><td>FC</td><td>foaming capacity</td></tr><tr><td>FID</td><td>flame ionization detector</td></tr><tr><td>FS</td><td>foaming stability</td></tr><tr><td>FTIR</td><td>Fourier transform infrared</td></tr><tr><td>GABA</td><td>gamma-aminobutyric acid</td></tr><tr><td>GC</td><td>gas chromatograph</td></tr><tr><td>HPLC</td><td>high-performance liquid chromatography</td></tr><tr><td>L*</td><td>luminosity</td></tr><tr><td>OHC</td><td>oil holding capacity</td></tr><tr><td>PRC</td><td>percentage relative change</td></tr><tr><td>RC</td><td>relative crystallinity</td></tr><tr><td>SEM</td><td>scanning electron microscopy</td></tr><tr><td>US:</td><td>ultrasound</td></tr><tr><td>WHC</td><td>water holding capacity</td></tr><tr><td>XRD</td><td>X-ray diffraction</td></tr><tr><td>ΔE</td><td>total color difference</td></tr></table>

## Data availability

Data will be made available on request.

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