Effect of ultrasound treatment on iron retention and bioaccessibility in fortified rice

Abstract

This study evaluated how ultrasound time and iron source affect Fe retention after cooking and in vitro bioaccessibility in ultrasound-assisted fortified rice. Polished white rice of the Tinajones variety (Sullana, Peru) was selected based on commercial quality criteria, including low broken grain content, absence of chalk and staining, and stable whiteness. A 2×3 factorial design was applied, combining two iron sources — ferrous sulfate (FeSO₄) and iron polymaltose — with three ultrasound exposure times (1, 3, and 5 min) at 40 kHz and 180 W. Moisture content, ash, CIELAB color parameters (L*, a*, b*, ΔE), whiteness (Kett scale), Fe content in raw and cooked rice, Fe retention after cooking, mold count, and relative Fe bioaccessibility by static in vitro digestion (INFOGEST protocol) were determined. Ultrasound time, iron source, and their interaction significantly affected (p < 0.05) moisture, color parameters, Fe content in raw and cooked rice, Fe retention, and bioaccessibility. Whiteness remained stable across treatments, whereas ΔE increased markedly in samples fortified with FeSO₄ at 5 min. Cooking induced differential Fe losses depending on the time–source combination: T3 (FeSO₄, 3 min) achieved the highest Fe content in cooked rice and the greatest retention (99.2%), while T2 (iron polymaltose, 1 min) showed the highest relative Fe bioaccessibility (96.0% w/w). No mold growth was detected in any fortified sample (<10 CFU/g). These results indicate that the nutritional performance of ultrasound-assisted fortification is governed by the interaction between iron source and sonication time, and that process optimization must simultaneously consider post-cooking iron retention, bioaccessible Fe fraction, and color impact.

Keywords:
Acoustic cavitation; Cereal technology; Functional foods; Iron polymaltose; Ferrous sulfate; Cooking stability

HIGHLIGHTS

Ultrasound improved iron incorporation into polished rice

Iron source significantly affected fortification efficiency

Ultrasound-assisted fortification showed industrial potential

1 Introduction

Anemia remains a priority public health problem and disproportionately affects young children and women of reproductive age (Organización Mundial de la Salud, 2025). In Peru, the National Institute of Statistics and Informatics reported that in 2024, 35.3% of children aged 6 to 35 months and 26% of women aged 15 to 49 years presented with anemia, with higher prevalence rates in rural areas (Instituto Nacional de Estadística e Informática, 2025). Given this context, it is crucial to implement dietary strategies aimed at increasing iron intake through commonly consumed foods, complementing pharmacological and public health interventions.

Food fortification consists of incorporating essential micronutrients into widely consumed food products to help prevent or reduce nutritional deficiencies (Bandara et al., 2025). White rice is considered a suitable food vehicle because of its regular consumption and central role in the daily diet of many populations (Saha et al., 2025). In Peru, per capita rice consumption reaches approximately 74 kg/year, reinforcing its potential as a matrix for population-based nutritional interventions (Tuebner, 2025). However, the nutritional value of fortified rice cannot be inferred solely from Fe content in the raw grain. Post-fortification handling and domestic preparation steps (e.g., washing and cooking) can alter the amount of Fe ultimately delivered in the cooked product, while the fraction potentially available for absorption depends on the Fe source and its interactions with the cereal matrix (García-Conde et al., 2025). Therefore, evaluating both post-cooking iron delivery (cooked-rice iron and retention) and in vitro bioaccessibility provides a more meaningful assessment of fortification performance than raw-grain Fe alone.

Multiple rice-fortification technologies have been developed, including extrusion, coating, dusting, soaking/impregnation, and hydrothermal treatments, as well as emerging technologies intended to improve micronutrient incorporation into the grain structure (Bandara et al., 2025; Kumari et al., 2025b). Nevertheless, conventional fortification methods still present important limitations. Surface coating and dusting can result in substantial micronutrient losses during washing and cooking, whereas extrusion requires specialized industrial equipment and may alter the structural characteristics of rice grains. In addition, several studies have reported that iron retention and sensory quality are strongly dependent on processing conditions and fortification methodology, highlighting the need for optimized processes capable of simultaneously preserving nutritional and physicochemical quality attributes (Liu et al., 2025).

In this context, ultrasound has emerged as a promising technology for intensifying mass transfer during soaking and impregnation processes. Its mechanism is primarily associated with acoustic cavitation, a phenomenon involving the formation, growth, and violent collapse of microbubbles in a liquid medium. The collapse of these bubbles generates localized high-energy conditions, including microjets, turbulence, shock waves, and shear forces capable of modifying the surface and internal microstructure of food matrices (Bonto et al., 2024). In rice grains, ultrasonic treatment has been associated with the formation of microchannels, fissures, and microporous regions that increase matrix permeability and facilitate the diffusion of solutes into the endosperm. Consequently, ultrasound may enhance iron incorporation efficiency by improving mass transfer between the fortification solution and the internal structure of the grain.

Additionally, ultrasound-assisted processes may accelerate hydration kinetics and reduce diffusion resistance during impregnation, promoting a more homogeneous distribution of micronutrients throughout the rice kernel. Previous studies have also demonstrated that ultrasound can improve solvent penetration, increase water uptake, and facilitate the transport of bioactive compounds in cereal matrices through the disruption of compact starch-protein structures (Vela et al., 2024). These structural modifications may contribute not only to greater iron retention after washing and cooking, but also to improved release of soluble iron fractions during gastrointestinal digestion. Furthermore, ultrasound has been reported to shorten processing times, reduce the need for high thermal treatments, and enhance the efficiency of impregnation processes while maintaining product quality. However, excessive sonication intensity or exposure time may negatively affect physicochemical attributes such as color, texture, moisture content, and grain integrity, making process optimization essential.

Previous studies have demonstrated that ultrasound-assisted fortification can substantially improve Fe incorporation and retention after washing and cooking. For example, Bonto et al. (2024) reported increases in Fe retention from 13.2% to 99.3% under ultrasound-assisted fortification conditions. Similarly, Tiozon Junior et al. (2021) observed that ultrasound treatment enhanced micronutrient penetration into rice grains by promoting structural changes associated with increased porosity and permeability. These findings suggest that ultrasound may represent an effective strategy to improve the nutritional performance of fortified rice while minimizing micronutrient losses during domestic preparation. Nevertheless, the effectiveness of ultrasound is highly dependent on process variables such as sonication time, intensity, frequency, and the physicochemical characteristics of the fortificant solution. Therefore, optimization strategies are necessary to maximize micronutrient incorporation while minimizing undesirable changes in quality attributes.

Furthermore, the Fe source represents a critical factor in fortification performance because it directly influences iron solubility, oxidation stability, interaction with starch components, and release during gastrointestinal digestion (Heinen et al., 2025). Ferrous sulfate (FeSO₄) is one of the most commonly used Fe fortificants because of its high bioavailability and low cost; however, its high reactivity may promote physicochemical instability and losses during processing and cooking (Tiozon Junior et al., 2021). In contrast, polymaltose iron, an iron-polysaccharide complex, presents a different release mechanism and lower chemical reactivity, which may result in distinct diffusion behavior, retention capacity, and bioaccessible fractions after digestion (Ebea-Ugwuanyi et al., 2024).

Despite advances in rice fortification technologies and the growing interest in ultrasound-assisted impregnation processes, important knowledge gaps remain. Most available studies focus primarily on total Fe incorporation or isolated technological responses, without comprehensively evaluating how ultrasound processing and Fe source jointly influence post-cooking iron retention, in vitro bioaccessibility, and physicochemical quality attributes within a single experimental design. In particular, limited information is available regarding the simultaneous evaluation of iron retention after cooking, bioaccessible Fe fractions, moisture and color changes, and the comparative performance of different Fe sources under ultrasound-assisted fortification conditions. Therefore, the aim of this study was to evaluate the effect of ultrasound-assisted Fe fortification on post-cooking iron retention and in vitro bioaccessibility of white rice using FeSO₄ and iron polymaltose as fortificants.

2 Materials and methods

2.1 Selection and characterization of raw materials

Samples of polished white rice from four commercial varieties (Tinajones, Mallares, Puntilla and Valor) were donated by rice processing companies in the cities of Lambayeque and Sullana (Peru).

To select the most suitable variety for the ultrasonic fortification process, 40 g of rice grains were immersed in distilled water at a grain-to-water ratio of 1:1 (w/v) and subjected to ultrasound pre-treatment using an ultrasonic bath (40 kHz, 180 W, AcmeSonic 6L, China) with exposure times of 1, 3, and 5 minutes at 25 °C. The beakers were immersed in the ultrasonic bath with the liquid level inside the beaker adjusted to match the water level in the bath, ensuring efficient transmission of ultrasonic waves.

2.1.1 Grain quality parameters

Samples were taken from different batches using a sampling probe. Authorization requests were sent to various mills, and a general sample of two kg was taken uniformly from the four points of the batch (top, bottom, front, and back). The raw material collection was carried out over one week. Once obtained, the samples were placed in airtight bags and taken to the Emerging Technologies Laboratory of the National University of Frontera, where they were analyzed. The sampling technique was based on the Peruvian Technical Standard ISO 24333-2013, revised in 2018 (Instituto Nacional de Calidad, 2018).

The Tinajones rice variety from Sullana city was chosen for its resistance to ultrasonic treatment without compromising grain integrity, and the selection of the sample for the fortification trials was based on commercial quality criteria, considering: %Q < 10%, absence of chalking and staining, whiteness within the commercial range (38–42) (high whiteness ranges to avoid significant color variation when Fe is incorporated), stable moisture content (11% to 14%), and milling yield ≥ 68% (when available). The results of this characterization were used to identify a representative commercial rice variety that was technologically suitable for the ultrasound-assisted fortification process (Instituto Nacional de Calidad, 2021a; Perú, 2021).

2.2 Fortification process

Fortification was carried out using two sources of iron: ferrous sulfate (FeSO₄) and iron polymaltose (Mobilab – Peru brand). All Fe solutions were prepared at a concentration of 0.5% (w/w) and supplemented with citric acid (0.5%, w/w) (Mobilab – Peru) as a complexing agent.

After selecting a sample of rice based on the characterization of the raw material, a 2×3 factorial design was applied with two sources of iron (FeSO₄ and iron polymaltose) and three sonication times (1, 3 and 5 min), generating six treatments: T1 (FeSO₄ 0.5% –1 min), T2 (iron polymaltose 0.5% – 1 min), T3 (FeSO₄ 0.5% – 3 min), T4 ( iron polymaltose 0.5%–3 min), T5 (FeSO₄ 0.5% – 5 min) and T6 (iron polymaltose 0.5% – 5 min).

For each treatment, 40 g of rice were weighed into beakers and 40 mL of the corresponding fortification solution were added (solid: liquid ratio 1:1, w/v). The samples were homogenized manually for 30 s and then subjected to sonication in an ultrasonic bath (40 kHz, 180 W) for the time defined for each treatment (Bonto et al., 2024; Corrêa Cañizares et al., 2026).

The ultrasonic conditions employed in this study (40 kHz and 180 W) were selected considering their common application in food processing and fortification studies, where acoustic cavitation has been shown to enhance mass transfer phenomena. The application of ultrasound under these conditions may increase the contact between the rice matrix and the iron solution, favoring iron uptake without the need for severe processing conditions. Consequently, these parameters were chosen to assess the feasibility of ultrasound-assisted iron fortification while preserving the technological quality of the rice grains.

After sonication, the samples were drained to remove excess solution and dried in a temperature-controlled dehydrator at 40 °C for 12 h (Cañizares et al., 2026). Each treatment was carried out in three independent batches.

2.2.1 Cooking method

The fortified samples (T1-T6) and the control (unfortified rice) were cooked in a rice cooker using distilled water. Cooking was carried out at 100 °C for 18 min with a rice: water ratio of 1:1 (w/w). After cooking, the samples were allowed to rest for 10 min in the cooker without the keep-warm function and were homogenized before analysis. In all cases, the water was completely absorbed by the grains (without draining).

The effect of sonication time and Fe source on the physicochemical properties and bioaccessibility of Fe in fortified rice was evaluated. An ultrasonic bath (AcmeSonic, China) set to 40 kHz and 180 W, a temperature-controlled dehydrator (Boxa LT-102, Peru), and an Ultraviolet-Visible (UV-Vis) spectrophotometer (Thermofisher Scientific, Genesys 150 model, USA) were used. Thus, in vitro gastrointestinal digestion was performed following the INFOGEST protocol (static digestion), using simulated fluids prepared with analytical grade salts and the enzymes α-amylase, pepsin, and pancreatin, in addition to bile extract and CaCl₂ (Sigma Aldrich, USA).

2.2.2 Analysis of the selected rice

2.2.2.1 Physicochemical

The moisture content of the rice was determined using a gravimetric method (mass loss), in accordance with Peruvian Technical Standard NTP 205.002 (Instituto Nacional de Calidad, 2021b). The samples were oven-dried at 130 °C until constant weight, and the results were expressed as % on a wet basis.

Ash content was determined according to Peruvian Technical Standard NTP 205.004 (Instituto Nacional de Calidad, 2022), by incinerating the samples in a muffle furnace at 550 ± 25 °C until a residue of constant weight was obtained. The results were expressed as % on a dry basis.

2.2.2.2 Whiteness and color

The whiteness of the raw fortified rice was assessed using a Kett C600 whiteness meter (Japan), employing 20 g of sample per determination (Chen et al., 2024). The measurement principle uses blue LED light and a measurement range from 5.0 to 69.9, with the lowest range being the darkest and the highest the whitest color; about 20 g of sample is placed in the measurement box, with a response time of 20 s for the first measurement and less than one second for subsequent measurements.

The color of the cooked fortified rice was measured in the CIELAB color space (L*, a*, b*) using a CR-400 portable colorimeter (Konica Minolta, Japan) under illuminant D65, recording the values on the surface of the homogenized grains. The total color difference (ΔE) was calculated relative to the unfortified cooked control using the formula reported by Feng et al. (2024) (Equation 1).

$\Delta E=\sqrt{{(L^{*}-L_0)}^2+{(a^{*}-a_0)}^2+{\left(b^{*}-b_0\right)}^2}$ (1)

Where: L ∗, a ∗, b ∗ correspond to the coordinates of the fortified cooked samples and L ₀, a ₀, b ₀ to the cooked unfortified control.

2.2.2.3 Iron content

Iron content was determined according to AOAC method 944.02, with adaptations from Zegeye et al. (2025). Samples were incinerated in a muffle furnace (550 °C ± 15 °C, 5 h), and the ash was digested with HNO₃ and HCl. Iron was reduced from Fe³⁺ to Fe²⁺ with hydroxylamine hydrochloride and quantified spectrophotometrically at 510 nm after complexing with 1,10-phenanthroline. Quantification was performed using a calibration curve (0.5 – 5.0 mg/L Fe²⁺), and results were expressed as mg Fe/100 g sample (dry basis).

Iron content was determined in raw and cooked fortified rice for all six treatments, as well as in the unfortified control. All determinations were performed in triplicate and included analytical blanks. The percentage of iron retention after cooking was calculated from the Fe content in raw and cooked rice.

2.2.3 Microbiological evaluation

Mold counts were performed on all raw fortified samples after one month of storage, according to ISO 21527-1:2008 (Instituto Nacional de Calidad, 2008). Potato dextrose agar (PDA) was prepared according to the manufacturer's instructions. After dissolving the medium in distilled water, chloramphenicol was added at a concentration of 100 mg/L to inhibit bacterial growth. The medium was autoclaved at 121 °C for 15 min. Ten grams of fortified rice were weighed under sterile conditions and transferred to a sterile bag containing 90 mL of 0.1% peptone saline solution. The sample was homogenized by manual shaking for 2 min. Serial decimal dilutions of the samples were prepared in 0.1% peptone saline solution, and 1 mL aliquots were inoculated in duplicate onto potato dextrose agar (PDA) supplemented with chloramphenicol (100 mg/L). The plates were incubated inverted at 28 ± 2 °C for 5 days. The results were expressed as colony-forming units per gram (CFU/g) and compared with the limits established by Peruvian technical standard No. 205.11 for rice (Instituto Nacional de Calidad, 2023).

2.2.4 Evaluation of Fe bioaccessibility by in vitro digestion

Bioaccessibility was assessed using the standardized in vitro gastrointestinal digestion protocol INFOGEST (Brodkorb et al., 2019). Previously, the fortified rice samples were cooked according to Section 2.2.1 (rice: water ratio 1:1 (w/w); 100 °C, 18 min; standing 10 min; no draining).

Digestion was performed in three sequential phases (oral, gastric, and intestinal). In the oral phase, 5 g of cooked rice were mixed with 3.5 mL of salivary fluid (pH 7.0), 0.5 mL of α-amylase (1500 U/ mL), 25 µL of CaCl₂ (0.3 M), and 975 µL of distilled water, and incubated at 37 °C for 2 min with agitation. The added volumes were adjusted according to INFOGEST to achieve the final conditions recommended in the digestate. For the control sample, 3.55 mL of salivary fluid and 1.5 mL of water were added. In the gastric phase, the oral digest was combined with 3.75 mL of simulated gastric fluid (pH 2.0), stock solutions of 0.8 mL of pepsin (25,000 U/ mL) and 10 µL of CaCl₂ (0.3 M), adjusting the pH with 0.2 mL of HCl (1 M) and 0.35 mL of distilled water, and incubated with shaking at 37 °C for 2 h. For the control, 3.75 mL of salivary fluid and 1.16 mL of distilled water were used. In the intestinal phase, 5.5 mL of simulated intestinal fluid and 5 mL of stock solutions of pancreatin (800 U/mL), 1.25 mL of bile extract (160 mM), and 20 µL of CaCl₂ (0.3 M) were added; the pH was adjusted to 7.0 with 20 µL of NaOH (1 M) and incubated with shaking at 37 °C for 2 h. For the control, 5.5 mL of simulated intestinal fluid, 0.73 mL of NaOH, and 3.77 mL of distilled water were used.

At the end of the intestinal phase, the digestates were centrifuged (3000 rpm, 10 min, 4 °C; rotor-dependent RCF) and the supernatant was recovered as the soluble fraction. Soluble Fe (Fe post-digestion) was quantified in the supernatant using the colorimetric method with reduction to Fe²⁺ and complexation with 1,10-phenanthroline, following the procedure described in Section 2.2.2.3, omitting the incineration step, and taking the reading at 510 nm.

Bioaccessible iron content was defined as the amount of soluble Fe recovered in the intestinal supernatant after in vitro digestion. Relative Fe bioaccessibility was calculated as the mass fraction of bioaccessible Fe with respect to the total Fe present in the corresponding cooked rice sample, following the approach described by Arnal et al. (2025). The relative bioaccessibility was calculated according to Equation 2:

$Relativebioaccessibility\left(\%w/w\right)=\frac{mbioaccessibleFe\text{}}{mtotalFeincookedrice}x100$ (2)

Where m bioaccessible Fe represents the mass of soluble Fe recovered in the intestinal supernatant after digestion, and m total Fe in cooked rice represents the total mass of Fe present in the corresponding cooked rice sample before digestion. Both Fe masses were referred to the same initial mass of cooked rice. Results were expressed as % (w/w). All digestions were performed in triplicate and included reagent blanks.

2.2.5 Statistical analysis

Experimental data were analyzed using STATISTICA version 10.0 (Statsoft Inc., Tulsa, OK, USA). For continuous variables, the assumptions of normality and homoscedasticity were verified using the Shapiro-Wilk and Levene tests, respectively.

Factorial analysis of variance (ANOVA) was applied to evaluate the main effects of ultrasound time, iron source, and their interaction on moisture, ash, color, Fe retention, and Fe bioaccessibility. Multiple comparisons between means were performed using Tukey's HSD test. Treatment results were compared with the control using Dunnett's test.

Microbiological data were analyzed descriptively and compared with the limits established in current Peruvian health regulations. In all cases, a significance level of α = 0.05 was considered, and the results are expressed as mean ± standard deviation.

3 Results and discussion

3.1 Commercial quality of white rice and sample selection

Variability was observed in the commercial quality of white rice according to origin (Table 1), with differences concentrated in broken grains (%Q), moisture (%H), and milling yield (%Rp). Samples from Lambayeque (1-3) showed constant moisture (12%), low %Q (3%), but with chalk (6%-7%), staining (0.5%), and a whiteness close to 40. In Sullana (5-8), moisture varied between 11%-14%, and %Q was the most discriminating attribute, with lots of high broken grains (25%-26%) and lots of low broken grains (5.5%-6.5%); in this origin, chalk and staining were absent (0%), and a whiteness was 36-38. %Rp also varied, being higher in Lambayeque (75%-78.8%) than in Sullana for the samples with available data (69%-71.5%).

Table 1
Commercial quality control parameters for white rice.

Samples	Origin	Variety	% Q	% Chalk	% Stained	% Whiteness	% H	% Rp
1	Lambayeque	Mallares	3.0 ± 1.4	7.0 ± 1.4	0.5 ± 0	40.5 ± 3.5	12 ± 0	75.0 ± 0.0
2	Lambayeque	Puntilla	3.0 ± 1.4	7.0 ± 1.4	0.5 ± 0	40.5 ± 3.5	12 ± 0	78.8 ± 0.4
3	Lambayeque	Valor	3.0 ± 1.4	7.0 ± 1.4	0.5 ± 0	40.5 ± 3.5	12 ± 0	77.0 ± 0.0
4	Sullana	Tinajones	5.0 ± 0.7	NE	NE	38 ± 0	11 ± 0	69.0 ± 1.4

• %Q: percentage of broken grains; %Chalk: percentage of grains with chalk center; %Stained; B°: % of whiteness; %H: moisture; %Rp: pile yield and NE: not found. The values ​​are expressed as mean ± standard deviation. Three repetitions were performed for each variety.

Based on these criteria, sample 4 (Sullana, Tinajones) was selected for further analysis because it combined low breakage (5.0% ± 0.7%), zero chalking and staining (NE), low moisture (11%), stable whiteness (38%), and quantified %Rp (69% ± 1.4%). These values fell within the tolerable ranges for extra-grade rice established in NTP 205.11 (Instituto Nacional de Calidad, 2023), thus minimizing the influence of grain defects on the variables evaluated during fortification and in vitro digestion.

3.2 Physicochemical analyses

3.2.1 Moisture and ash content

The final moisture content showed a significant effect of ultrasound time and, furthermore, an interaction between ultrasound time and iron source (p < 0.05). Among the fortified treatments, T1 (FeSO₄, 1 min) exhibited the highest moisture content (9.16%), differing from the other treatments (Tukey, p < 0.05). The other treatments showed lower moisture content (4.57%-5.65%) and were significantly lower than the control (Dunnett, p < 0.05), while T1 did not differ from the control. Increasing the ultrasound time from 1 to 3 and 5 min was associated with a marked reduction in moisture content, but this decrease depended mainly on the iron source in the short time interval; at 1 min, moisture content was high with FeSO₄ (T1) and low with polymaltose iron (T2), demonstrating the reported interaction (Table 2).

Table 2
Moisture content, ash content, Whiteness and color analysis of the fortified treatments.

Treatments	Moisture (%)	Ash (%)	Whiteness (°kett)	L*	a*	b*	Ä E
T1	9.2 ± 0.5 b	0.6 ± 0.0 a	39.1 ± 0.5 a	62.2 ± 0.0 d*	5.2 ± 0.0 c*	0.8 ± 0.0 a*	6.8 ± 0.0 b
T2	5.7 ± 0.4 a*	0.7 ± 0.0 a	40.2 ± 0.3 a	62.9 ± 0.1 e*	2.9 ± 0.0 a*	-1.0 ± 0.0 b*	6.3 ± 0.0 a
T3	5.4 ± 0.3 a*	0.6 ± 0.1 a	40.0 ± 0.4 a	60.5 ± 0.0 a*	5.9 ± 0.0 d*	0.5 ± 0.0 d*	7.9 ± 0.0 c
T4	5.3 ± 0.1 a*	0.6 ± 0.0 a	39.9 ± 0.5 a	60.5 ± 0.0 a*	4.8 ± 0.0 b*	2.1 ± 0.0 e*	6.3 ± 0.0 a
T5	4.6 ± 0.3 a*	0.6 ± 0.0 a	40.0 ± 0.5 a	55.7 ± 0.0 b*	11.3 ± 0.0 f*	0.1 ± 0.0 c*	14.5 ± 0.0 d
T6	4.9 ± 0.2 a*	0.7 ± 0.1 a	40.0 ± 0.3 a	58.4 ± 0.0 c*	7.3 ± 0.0 e*	0.8 ± 0.0 a*	9.7 ± 0.1 e
Control	9.0 ± 0.07 *	0.7 ± 0.0	40.2 ± 0.1	62.8 ± 0.0 *	-0.72 ± 0.0 *	4.2 ± 0.0 *	---

• Different letters indicate differences between fortified treatments (Tukey, p < 0.05). *Indicates a significant difference compared to the control (Dunnett, p < 0.05). The values ​​are expressed as mean ± standard deviation. Three repetitions were performed for each variety.

Ultrasound acts as a mass transfer intensifier through cavitation, which can induce microcracks, internal porosity, and surface rupture in rice grains, reducing diffusion barriers and improving water movement (Zhang et al., 2024). Liu et al. (2024) reported that ultrasonic treatments generate micropores and decrease the contact angle, modifying water-matrix dynamics and accelerating processes associated with hydration and moisture migration. Therefore, the lower moisture content observed at T2-T6 is consistent with a matrix more accessible to transport, where water migration during conditioning and/or subsequent drying occurs more efficiently. This is also relevant because, in dry foods, lower final moisture content is associated with greater stability against microbial spoilage during storage. This behavior aligns with recent evidence linking ultrasound with the formation of internal cracks that facilitate moisture migration and improve drying efficiency in rice (Deng et al., 2022; Rashid et al., 2023, 2025).

The behavior of T1 explains why ultrasound does not have a direct effect on final moisture content. At 1 min, the combination with FeSO₄ did not produce the moisture reduction observed in the other treatments, remaining statistically similar to the control. This supports the idea of an operational sonication threshold (sonication time) beyond which cavitation induces sufficient morphological changes in the matrix or in the water migration kinetics for subsequent drying to result in lower final moisture content.

Ash content showed no significant differences between treatments (p > 0.05) or between the treatment and the control, remaining within a range of 0.60%–0.68%. This is close to the value reported by the Peruvian Food Composition Tables of 0.5% for white rice (Reyes García et al., 2023). This result is expected because ash is a general indicator of mineral content, and the contribution of added iron is usually small compared to the total mineral content of rice. Furthermore, while ultrasound modifies the microstructure, it does not necessarily alter the total mineral fraction at these fortification levels. In line with this, Saha et al. (2025), in a study of fortified rice matrices, reported that fortified products can maintain physicochemical properties similar to those of unfortified rice, even when total iron content increases.

3.2.2 Color analysis

Whiteness did not differ between the fortified treatments (p > 0.05) and remained comparable to the control (Table 2), indicating that ultrasound-assisted fortification did not modify the overall whiteness detected by this index. However, CIELAB analysis showed that color was sensitive to fortification; ANOVA revealed effects of sonication time, iron source, and their interaction on L*, a*, b*, and ΔE (p < 0.05), confirming that ultrasound modulated the chromatic impact in a manner dependent on the type of fortifier.

The L* values varied compared to the control, with a reduction observed with longer sonication times, evidenced by the more pronounced darkening at 5 min (T5_: FeSO₄, 5 min), while a* increased compared to the control, with an increase observed with longer sonication times and for FeSO₄ (T5). On the other hand, for b*, the fortified treatments tended to have lower values compared to the control. ΔE integrated these shifts and confirmed the combined effect of the process: the greatest overall change was observed in T5, followed by T6, while T2 and T4 (iron polymaltose, 1-3 min) showed the lowest ΔE values among the fortified treatments.

The whiteness measured on the Kett scale remained around 39-40 in all fortified treatments, with no differences compared to the control, and was within the target ranges reported for commercial polished white rice in Peru (approx. 38-40 °Kett in product technical specifications and 39-42 °Kett as an industrial operating range) (Perez, 2023).

Saha et al. (2025) reported fortification-induced color changes in puffed rice attributed to slight chemical modifications, moisture loss, and variations in grain surface structure; this aligns with Wu et al. (2023), who reported the effects of ultrasound promoting morphological changes that favor greater incorporation and distribution of Fe within and on the grain surface.

3.2.3 Iron content and retention after cooking

In fortified raw rice, the Fe content differed significantly between treatments (p < 0.05). Compared to the control, all treatments showed higher Fe (Dunnett, p < 0.05), except for T6_, which did not differ statistically from the control (Table 3). ANOVA revealed effects of ultrasound time, iron source, and their interaction (p < 0.05), confirming that Fe incorporation into the grain depended on the combination of factors. With FeSO₄, Fe in raw rice increased with increasing sonication time, reaching a maximum in T5 (5.6 times the control). In contrast, with polymaltose iron, Fe in raw rice decreased as time increased (T2 _> T4 _> T6), and in T6 the mean was numerically higher than the control, but without statistical significance.

Table 3
Fe content in fortified rice and % retention.

Treatments	Fe in fortified raw rice (mg Fe/100 g)	Fe in cooked fortified rice (mg Fe/100 g)	% Retention after cooking
T1	1.1 ± 0.0 d*	0.7 ± 0.0 b*	65.0 ± 0.4 b*
T2	0.9 ± 0.0 c*	0.8 ± 0.0 b*	94.2 ± 0.3 e*
T3	1.3 ± 0.1 e*	1.3 ± 0.1 c*	99.2 ± 0.1 f*
T4	0.7 ± 0.1 b*	0.5 ± 0.1 a*	72.9 ± 1.9 c*
T5	3.0 ± 0.1 e*	1.2 ± 0.1 c*	42.1 ± 1.2 ª*
T6	0.5 ± 0.0 a	0.4 ± 0.0 a*	77.3 ± 0.3 d*
Control	0.5 ± 0.0 *	0.4 ± 0.0 *	85.1 ± 1.2 *

• Different letters indicate differences between fortified treatments (Tukey, p < 0.05). *Indicates a significant difference compared to the control (Dunnett, p < 0.05). The values are expressed as mean ± standard deviation. Three repetitions were performed for each variety.

This behavior is consistent with the role of ultrasound as a mass transfer enhancer during soaking, due to cavitation: the formation and collapse of bubbles generate local stresses that can produce cracks and microchannels on the grain surface or in suspended structures, favoring the penetration of solutes from the solution into the matrix (Liu et al., 2024). In rice, it has been explicitly described that surface modification of the grain is attributed to cavitation and that micronutrient fortification in sonicated rice can be achieved by soaking, increasing water absorption, and incorporating dissolved micronutrients (Bonto et al., 2024). Likewise, recent reviews on ultrasound in starch matrices document that the treatment induces surface damage (cracks, pores, channels) and that these changes have also been observed in rice grains, supporting the physical plausibility of increased incorporation with longer soaking times when the fortifier is in ionic form and available for diffusion (Vela et al., 2024).

The interaction between sonication time and Fe source suggests that the effect of time was not universal; while increased sonication favored incorporation with FeSO₄, the inverse behavior with polymaltose iron indicates that Fe uptake efficiency was dependent on the chemical form of the fortifier under the process conditions. As a reference for time-dependent behavior in ultrasound-assisted fortification, Purizaca-Santisteban et al. (2023) reported greater Fe incorporation from ferrous sulfate with increased treatment time in a plant matrix, supporting the idea that sonication time can be a determining factor when iron is available in solution.

In the cooked fortified rice, the Fe content differed between treatments (Tukey, p < 0.05) and all differed from the control (Dunnett, p < 0.05) (Table 3). ANOVA showed effects of sonication time, iron source, and their interaction on Fe in cooked rice (p < 0.05), indicating that cooking acted as a discriminating step where the magnitude of losses depended on the combination of factors applied during fortification. With FeSO₄, cooked Fe was higher at 3 min than at 1 min (T1: 0.7; T3: 1.3 mg Fe/100 g) and remained elevated at 5 min (T5_: 1.2 mg Fe/100 g). In contrast, with polymaltose iron, cooked Fe decreased over time (T2_: 0.8; T4_: 0.5; T6: 0.4 mg Fe/100 g). Consequently, the highest Fe contents in cooked rice corresponded to T3 and T5_, while the lowest were observed in T4 and T6_, confirming an interaction between sonication time and Fe source; increasing time favored the final Fe when FeSO₄ was used, but produced the opposite effect with polymaltose iron.

The Fe value of the cooked control was consistent with that reported in the Peruvian Food Composition Tables for cooked rice, supporting the representativeness of the control matrix used (Reyes García et al., 2023). In technological terms, the dependence of the Fe source on sonication time observed in cooked rice is consistent with the fact that cooking reflects the degree of iron integration into the grain; more soluble fortifiers tend to facilitate incorporation during processing, but their final performance depends on the Fe being sufficiently retained to resist losses during cooking. In fortified cereals, recent studies have shown that the selection of soluble salts can improve retention during processing compared to less readily available sources, although the response depends on the matrix and the process applied (Kumari et al., 2025a). In rice treated with ultrasound, it has been reported that cavitation induces microcracks in the endosperm that favor iron diffusion and improve its retention. In particular, a recent study of ultrasonic fortification in rice reported marked increases in Fe under optimized conditions and warned that excessive exposure can be counterproductive (Canizares et al., 2025)

Regarding the percentage of retention, the ANOVA indicated significant effects of sonication time, the Fe source, and their interaction (p < 0.05), confirming that the stability of iron during firing depended on the combination of factors. Compared to the control, only T2 (1 min, polymaltose iron) and T3 (3 min, FeSO₄) showed higher values, while T1_, T4_, T5_, and T6 showed lower retention than the control (p < 0.05) (Table 3). This result demonstrated that increasing the sonication time did not guarantee greater retention and that the response was specific to the iron source (Table 3).

Iron retention during cooking is usually determined by the degree of iron integration into the grain matrix beyond its initial incorporation. Since the cooking water was completely absorbed by the grains and no draining step was applied, the decrease in iron concentration observed after cooking cannot be attributed to the removal of iron with discarded cooking water. Instead, these changes may be associated with physicochemical transformations occurring during thermal processing, as well as the redistribution of iron within the grain matrix and its interactions with rice components, which may influence its measured concentration. In ultrasound-treated rice, cavitation has been described as inducing microcracks in the endosperm that promote diffusion inwards and, therefore, can improve iron retention under optimized conditions (Bonto et al., 2024). This is observed in treatments T2 and T3, which outperform the control. Similarly, a recent review on acoustic cavitation in food summarizes that the efficiency of ultrasound depends strongly on the processing conditions and the interaction with the system (matrix/medium), which supports the existence of optima rather than a monotonic improvement over time (Zhu et al., 2024). Likewise, Kumari et al. (2025a) emphasized that Fe retention during cooking is a primary performance criterion and can vary widely depending on the method and formulation of the fortifier.

3.3 Microbiological evaluation

No mold was detected in any sample, with counts < 10 CFU/g (detection limit of the method), and no differences were observed between treatments (1, 3, and 5 min) and the control. These results fell within the criteria established for dry grains according to NTP 205.11 (Instituto Nacional de Calidad, 2023).

The absence of mold (< 10 CFU/g) indicated that ultrasound-assisted fortification did not increase detectable fungal contamination under the evaluated conditions. This behavior was consistent with the low final moisture content achieved in the treatments, which reduces water availability and limits microbial growth during storage. The results obtained support the conclusion that the processing and drying conditions used maintained microbiological stability comparable to that of conventional rice, regardless of the sonication time.

3.4 Evaluation of the bioaccessibility of fortified Fe

Bioaccessible Fe (post-digestion) was affected by sonication time, iron source, and their interaction (p < 0.05), confirming that the fraction released/solubilized after digestion depended on the combination of factors applied during fortification (Table 4). Differences were observed among the fortified treatments (p < 0.05), and compared to the control, T4 and T6 did not differ (Dunnet, p > 0.05). The highest values corresponded to T2 (1 min, iron polymaltose) and T3 (3 min, FeSO₄), while the lowest was observed in T6 (5 min, iron polymaltose), demonstrating a non-monotonic behavior over time and dependent on the fortifier.

Table 4
Fe content post-digestion and % relative bioaccessibility.

Treatments	Post-digestion Fe	% Relative Fe Bioaccessibility
	(mg Fe/100 g cooked rice)	(%w/w)
T1	0.5 ± 0.0 ab*	71.9 ± 0.5 b*
T2	0.8 ± 0.0 c*	96.0 ± 0.2 d*
T3	0.8 ± 0.1 c*	58.5 ± 3.1 a*
T4	0.4 ± 0.1 a	77.2 ± 2.2 b*
T5	0.6 ± 0.1 b*	45.2 ± 2.7 c*
T6	0.2 ± 0.0 d	59.2 ± 0.7 a*
Control	0.4 ± 0.0 *	90.6 ± 0.9 *

• Different letters indicate differences between fortified treatments (Tukey, p < 0.05). *Indicates a significant difference compared to the control (Dunnett, p < 0.05). The values ​​are expressed as mean ± standard deviation. Three repetitions were performed for each variety.

For FeSO₄, the maximum at 3 min (T3) suggests a process condition where sonication favored sufficient incorporation and/or redistribution of Fe towards sites less susceptible to losses and more accessible to solubilization during digestion, without inducing an adverse effect comparable to that observed at 5 min. It has already been mentioned that ultrasound generates microcracks in the endosperm that facilitate iron diffusion and can improve bioaccessible fractions under optimized conditions; at the same time, excessive exposures can introduce physical changes that negatively affect grain properties and/or the distribution of the fortifier (Canizares et al., 2025).

Polymaltose iron, bioaccessible Fe was elevated at 1 min (T2) but decreased at 3 min (T4) and reached its minimum at 5 min (T6), to the point that T4 and T6 did not differ from the control. A critical reading is that the increased time favored conditions that reduced the releasable fraction of this source in the in vitro digestion. This is plausible considering the nature of iron-carbohydrate complexes, recently described as nanoparticles with a ferric core stabilized by a carbohydrate coating, where the stability of the complex and its interactions with the medium determine the iron release kinetics (Cappellini et al., 2022; Krupnik et al., 2024; Nikravesh et al., 2020). In a cereal matrix, an increase in treatment could favor stronger associations of the complex with grain components or microstructure changes that lead to a less solubilizable fraction in the digestion model, which would explain why bioaccessible Fe (mg/100 g) falls at longer times despite being a fortified matrix.

The relative bioaccessibility (% w/w) was significantly affected by sonication time, iron source, and their interaction (p < 0.05), confirming that the mass fraction of total Fe that became bioaccessible after digestion depended on the combination of factors applied during fortification (Table 4). Differences were observed among fortified treatments (p < 0.05), and all differed from the control (Dunnett, p < 0.05). Only T2 (1 min, iron polymaltose) showed higher relative Fe bioaccessibility (96.0% w/w) than the control. The remaining treatments showed lower values, with a particularly marked progressive decrease for FeSO₄ as time increased. For iron polymaltose, bioaccessibility also decreased with increasing time, going from T2 to lower values in T4 and T6. Overall, the pattern shows an interaction between the variables, where the effect of ultrasound time on bioaccessibility was not uniform and changed according to the fortifier used, with the combination of iron polymaltose + 1 min being the condition that maximized relative Fe bioaccessibility (% w/w).

Considering the recommended dietary intakes of Fe for adults (men 19–50 years: 8 mg/day; women 19–50 years: 18 mg/day) (National Institutes of Health, 2022), and assuming a daily portion of rice of 150 g (within the range reported for Peru; per capita consumption 74 kg/year) (Tuebner, 2025), the treatment with the highest bioaccessible Fe (T2) would provide approximately 1.17 mg Fe/day, equivalent to 15% of the requirement for adult men and 6.5% of that for adult women of childbearing age. This calculation is based on an in vitro digestion model, so it represents a potential bioaccessible contribution and not a direct estimate of absorption; even so, it suggests that optimized fortification can make a significant contribution in diets with a high proportion of rice.

Iron fortification improves the nutritional value of rice by transforming it from a naturally poor source of iron into an effective vehicle for increasing dietary iron intake. Depending on the fortification level and iron retention after cooking, fortified rice can provide more than 30% of the recommended daily iron intake for women of reproductive age and an even greater proportion for adult men. In addition, intervention studies have demonstrated that regular consumption of fortified rice improves iron status and reduces the prevalence of iron deficiency and iron-deficiency anemia (Peña-Rosas et al., 2019).

Iron bioaccessibility was evaluated using the standardized INFOGEST in vitro digestion model, which estimates the fraction of iron released during gastrointestinal digestion and potentially available for intestinal absorption. Although this method is widely accepted and provides high reproducibility for comparing treatments, it does not fully reproduce the complex physiological processes involved in intestinal uptake, transport, and systemic iron metabolism. Therefore, the bioaccessibility values reported in this study should be interpreted as indicators of potential iron availability rather than actual bioavailability. Future studies employing intestinal cell culture models or in vivo approaches are needed to confirm the nutritional efficacy of ultrasound-assisted iron fortification in rice.

A further limitation of this study is that only a single combination of ultrasonic frequency and power was evaluated. Although these processing conditions were selected based on previous evidence, different combinations of frequency, power density, and treatment time may influence iron incorporation and retention. Consequently, additional optimization studies are warranted to maximize fortification efficiency while preserving the physicochemical and sensory quality of rice grains.

4 Conclusions

The commercial quality assessment of different rice batches allowed the selection of sample Tinajones (Sullana) as a homogeneous matrix for fortification. Ultrasound-assisted fortification showed that the sonication time, the iron source, and their interaction determined the system's response: final moisture decreased in the ultrasound-treated treatments (except FeSO₄, 1 min), the percentage of whiteness (Kett) remained stable, and the CIELAB/ΔE color was sensitive to the process. In nutritional terms, the iron content in cooked rice and the percentage retention depended on the time-source combination, with T3 (FeSO₄, 3 min) standing out for maximizing post-cooking iron and retention. In vitro gastrointestinal digestion showed significant effects on bioaccessible Fe (mg Fe/100 g cooked rice) and relative Fe bioaccessibility (% w/w), identifying T2 (iron polymaltose, 1 min) as the condition with the highest relative bioaccessibility. Overall, ultrasound proved to be a viable strategy for modulating rice fortification; however, optimization must be defined by the interaction between Fe source and sonication time according to the prioritized indicator (dose retained after cooking versus fraction released during digestion), while controlling for the impact on color. Furthermore, the absence of mold (< 10 CFU/g) supported microbiological safety under the evaluated conditions.

From a practical perspective, ultrasound-assisted fortification represents a promising strategy for increasing iron content in polished rice while preserving its commercial quality and cooking characteristics. The technology could be incorporated into existing rice processing lines with relatively low operational complexity, providing an alternative approach to support iron deficiency mitigation programs in rice-consuming populations.

Acknowledgements

The authors acknowledge the Programa Nacional de Investigación Científica y Estudios Avanzados (PROCIENCIA) for the funding provided to carry out this research project under contract No. PE501086279-2024-PROCIENCIA, as well as the authorities of the Universidad Nacional de Frontera.

Data Availability Statement

All data generated or analysed in this study are included in this published article.

Cite as:

Espinoza Delgado, M. P., Rodriguez Rodriguez, F. P., Medina Oviedo, J. J., Torres Becerra, P. M., Chaves, E. S., Quindes Niño, A. J., Vargas Tapia, E., Silva Paz, R. J., & Sócola, Z. (2026). Effect of ultrasound treatment on iron retention and bioaccessibility in fortified rice. Brazilian Journal of Food Technology, 29, e2026029. https://doi.org/10.1590/1981-6723.0292026

Funding:

PROCIENCIA (1 subvención, año 2024).

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*

Corresponding Author: Zury Sócola, Universidad Nacional de Frontera, Grupo de Investigación Tecnologías Emergentes, Investigaciones de Mercado y Comportamiento del Consumidor (TEIMEC), Av. San Hilarion, nro. 101, Villa Peru Canada (Zona Expansion Urbana, Margen Izquierda), 20101, Sullana - Peru, e-mail: [e-mail]

Section Editor:

Mateus Petrarca.

Publication Dates

• Publication in this collection
  06 Nov 2026
• Date of issue
  2026

History

• Received
  13 Mar 2026
• Accepted
  15 Sept 2026