Abstract
The use of microencapsulated ferrous-sulfate is among the various options recommended for food fortification, as the protective wall material surrounding the compound can preserve it from undesirable alterations and also protect the food. Microencapsulated iron can be produced using different wall materials and encapsulation methods. Thus, a microparticle was developed through spray chilling, containing ferrous sulfate (FS), as active compound, and a fat mixture as the coating material. The resulting samples analyzed to determine encapsulation efficiency, particle size distribution, and morphology. Furthermore, the oxidative stability and bioaccessibility of FS microparticles were investigated by simulating in vitro digestion. The findings indicated that the encapsulation technique effectively retained FS, resulting in microparticles physically stable at room temperature with typical morphology. The encapsulation efficiency revealed that lower concentrations of FS led to reduced superficial iron content. However, the oxidative stability demonstrated that the presence of iron in the microparticles accelerated the lipid oxidation process. The in vitro digestion test demonstrated that the microparticles with lower iron content exhibited a higher percentage of bioaccessibility, even when compared to non-encapsulated FS. Additionally, the coating material successfully released FS during the simulation of gastrointestinal digestion, resulting in a bioaccessibility of 7.98%.
Supplementary Information
The online version contains supplementary material available at 10.1007/s13197-023-05820-1.
Keywords: Microparticles, Stability, Bioaccessibility, Palm fat, Iron
Introduction
Iron deficiency in the human body is the most common and widespread nutritional disorder worldwide. While certain studies have shown a decrease in the overall occurrence of global anemia, the prevalence rates remain considerably elevated (Blanco-Rojo and Vaquero 2019; WHO 2006).
Iron is a crucial element that plays a vital role in numerous metabolic processes within the bodies of nearly all living organisms. Insufficient levels of iron can have severe implications for human health and the development of children, manifesting in symptoms such as weakness, drowsiness, impaired psychomotor and cognitive development, and an increased risk of premature birth, among other adverse effects (Blanco-Rojo and Vaquero 2019). Therefore, iron deficiency anemia is still a worldwide public health problem, and one of the strategies that have been adopted in many countries, to minimize the lack of this essential element, is food fortification with iron salts (WHO 2006).
The efficiency of fortification programs relies on the vehicle used, as it should cover the largest part of the population as possible. The World Health Organization adopted some strategies to reduce the incidence of anemia by adding iron to wheat flour. Wheat flour is an important component in many consumed products like bread, biscuits and pasta (Hurrell et al. 2010; WHO 2006). In Brazil, the fortification of wheat and corn flours has occurred since 2002, and the Collegiate Board Resolution (RDC 150/2017), allows only the use of the compounds ferrous sulfate, ferrous fumarate and its encapsulated forms (Brasil 2017) for this purpose. Limited information is available regarding the process of encapsulating ferrous sulfate for its application in the food industry.
Although water-soluble iron compounds, such as ferrous sulfate, are considered more bioavailable, they can promote the development of undesirable color, aroma, and flavor, and contribute to lipid oxidation in food (Hurrell et al. 2010). Still, they are associated with other side effects, such as blackening of the teeth, abdominal pain, nausea, diarrhea, and constipation. These side effects can increase the rejection of products fortified with iron by consumers (Contreras et al. 2014).
Scientists have suggested replacing the free ferrous sulfate with its microencapsulated form (Akhtar et al. 2011; Cocato et al. 2007; Majeed et al. 2013). This technique has the advantage of protecting not only the active agent from the processing conditions, but also the food, from undesirable organoleptic changes, preventing lipid oxidation and reducing the interaction with the food matrix. Besides that, the microencapsulated active may have a similar bioavailability to the non-encapsulated compound.
Numerous techniques can be used in order to obtain microencapsulated products, such as spray drying, spray cooling or spray chilling, extrusion, among others; as well as several compounds can compose the coating material (proteins, maltodextrins, gelatins, fats, and others). The technique selection depends on its application. In this regard, it is of utmost importance to obtain not only information on the processes and the physical–chemical characteristics of the microparticles, but also about their bioavailability. This can be assessed by tests that estimate the bioaccessibility of the encapsulated compound during processes that simulate the gastrointestinal digestion.
Other studies have suggested the use of microencapsulated iron for wheat flour fortification (Akhtar et al. 2011; Hurrell et al. 2010). However, detailed studies that involve the encapsulation techniques and coating materials to encapsulate ferrous sulfate, are still scarce in the literature. In this study, we aimed to create a microparticle that incorporates ferrous sulfate by using a fat mixture as a coating material and employing the spray chilling technique. We still analyze the efficiency of encapsulation, particle size distribution and the morphology of the generated samples. In addition, we evaluated the oxidative stability and iron bioaccessibility through the in vitro digestion simulation to investigate gastric resistance and enteric release of ferrous sulfate microparticles.
Material and methods
Material and equipments
Ferrous sulfate monohydrate (M. Cassab, Brasil), palm oil totally hydrogenated (AA Óleos Vegetais, Brasil), and vegetable fat LEVIA + E (Cargill, Brazil) were used for the particles production. Three batches were carried out to obtain the ferrous sulfate microparticles with different concentrations.
Standard solutions of iron (1000 mg/L) (Sigma Chemical Co., St. Louis, USA); nitric acid, analytical grade (Merck, Darmstadt, Germany); hydrogen peroxide (Merck, Darmstadt, Germany); purified water (Sartorious System, Germany); qualitative filter paper (Nalgon, Brazil) were used to the samples mineralization in digestion block (model 4025, Marconi, Brazil).
For INFOGEST 2.0 protocol: porcine pepsin (P6887, 3100 U/mg), bovine bile (B3883), pancreatin (P7545, 6U) were used; and the other reagents used to prepare the digestion fluids (simulated salivary fluid, simulated gastric fluid and simulated intestinal fluid), and enzymes followed the protocol specifications. All reagents used were from Sigma Chemical Co., St. Louis (USA).
For mineral quantification: an atomic absorption spectrophotometer with flame (model AAnalyst 200, Perkin Elmer, USA); hollow cathode lamps for determination of iron (248.3 nm, Perkin Elmer, USA); standard solutions of Fe (1000 mg/L) (Sigma Chemical Co., St. Louis, USA); nitric acid 65% v/v, analytical grade (Merck, Darmstadt, Germany); hydrogen peroxide 30% v/v (Merck, Darmstadt, Germany); purified water (Sartorious, Germany); qualitative filter paper (Nalgon, Brazil); acetylene gas (Messes Gases, Brazil); metabolic bath (Dubnoff MA093, Marconi, Brazil); centrifuge (SL 706, Solab, Brazil); and air-circulation oven (model 520, Fanem, Brazil) were used.
Methods
Microparticles production
The coating material consisted of 60% hydrogenated palm fat (AA Óleos Vegetais, São Paulo, SP) and 40% LEVIA + E vegetable fat (Cargill, São Paulo, SP) (approximate melting point of 54 °C, according to Fadini et al. (2019)). The lipid materials were weighed in a glass beaker, melted in a microwave oven, at intervals of 30 s, and the final temperature of the mixture did not exceed 70 °C, the mixture was kept in a heating plate until use. Ferrous sulfate was added to the lipid mixture and homogenized by mechanical stirring (mechanical stirrer, IKA-Werke, RW 11 Lab egg-stirrer, Staufen, Germany) on a heating plate.
The final mixture was subjected to an ultrasonic bath (Mod. 750A, MaxiClean Unique, Indaiatuba, São Paulo, Brazil) (70 °C/1 min), to better disperse the suspension of ferrous sulfate in the melted fat, and transferred to a heated sample reservoir (70 °C) of the spray chilling for subsequent formation of the microparticles. The equipment used was a mini spray dryer laboratory scale B-290 (Büchi, Flawil, Switzerland) containing a cooling module (Dehumidifier B-296) for use as a spray chiller. The spraying occurred through a double fluid atomizer nozzle with a diameter of 0.7 mm. The chamber temperature was maintained at 5 ± 2 °C. The equipment's operating conditions were: air pressure of 5 bar; aspiration of 70% of the power of the vacuum cleaner and air flow of 500 L/h, as described by Alvim et al. (2016).
The amount of ferrous sulfate, used as an active compound, was calculated based on the obtention of three types of microparticles: (LM-F4) Lipid Microparticle with 4 mg Fe/g of particle; (LM-F9) Lipid Microparticle with 9 mg Fe/g of particle, and (LM) Lipid Microparticle without iron. The microparticles obtained in each process were collected and stored under refrigeration for further characterization.
Characterization of microparticles
The microparticles were characterized in terms of ash content (AOAC 1997); humidity (AOAC 1997) and iron content (Fe) (Rebellato et al. 2018). The analyzes were performed in triplicate (n = 3).
Encapsulation efficiency
The Total Encapsulation Efficiency (TEE) was evaluated by the amount of active compound (ferrous sulfate) in the microparticles after processing in relation to the amount of active substance used before the process, multiplied by 100, as described by Alvim et al. (2016).
The Effective Encapsulation Efficiency (EEE) was calculated by subtracting the amount of superficial iron in the total amount of iron present in the microparticles. The obtained value was compared with initial amount of iron added. The ratio was expressed as a percentage representing the EEE value, according to Sartori et al. (2015).
Determination of superficial iron
Superficial iron determination was carried out according to (Morselli Ribeiro et al. 2012), with some modifications. For this purpose, approximately 0.5 g of microparticles were weighed, suspended in 15 mL of 1% (v/v) nitric acid solution and submitted to an ultrasound bath for 5 min. Subsequently, the mixture was filtered using filter paper and then transferred to a 25 mL volumetric flask. The flask was filled with acidified ultrapure water (1%) to reach the desired volume. The iron content was obtained using a flame atomic absorption spectrophotometer (FAAS), in triplicate.
Evaluation of the average diameter and size distribution of the microparticles
The average diameter and size distribution of the microparticles were determined by scattering light (laser diffraction) through the apparatus LA 950 V2 (Horiba, Kyoto, Japão). The microparticles were dispersed in a polysorbate 20 (Tween 20) solution (0.5% w/w) and added to the equipment's reading chamber, containing ultrapure water as a dispersion medium (adapted from Alvim et al. 2016). The average diameter of the microparticles was expressed as the diameter referring to 50% of the accumulated distribution (D50). The polydispersity was obtained by the span index calculated as follows: span = (D90–D10)/D50; the parameters D10, D50 and D90 correspond to the diameters referring to 10, 50 and 90% of the accumulated distributions. The analyses were performed in triplicate.
Morphology
The morphology evaluation was performed by optical microscopy in an Olympus BX40 microscope (Tokyo, Japan), with observation and image capture using a digital camera, as described by Alvim et al. (2016).
Oxidative stability
For the evaluation of oxidative stability, the microparticles LM (Lipid Microparticle without iron) and LM-F9 (Lipid Microparticle with 9 mg Fe) were used. Oxidative stability was conducted according to Firestone (2014), through Rancimat stability, which consists of the measurement of the resistance of oils or fats to oxidation, measured in an induction period, in hours. The equipment conditions were 100 °C and 20 L of air per hour. Approximately, 3 g of each sample were weighed, melted and coupled to the equipment, where they remained in contact with the hot air stream. The analyzes were performed in triplicate.
Static in vitro digestion method (INFOGEST 2.0) to estimate bioavailability of iron.
For this step, in addition to the LM, LM-F4 and LM-F9 microparticles, a sample of non-microencapsulated ferrous sulfate (FS) was also evaluated, for comparative purposes. The bioaccessibility assay was performed according to the protocol INFOGEST 2.0 (Brodkorb et al. 2019), with the following modifications:
Oral phase The salivary amylase enzyme was not used in this phase, due to the composition of the microparticles, mostly based on lipids.
Intestinal phase At the end of the intestinal phase, the samples were centrifuged (6000 ×g, for 30 min, at 4 °C); the supernatant was the bioaccessible fraction.
The bioaccessible fraction was transferred to digestion tubes, which were taken to the oven at 100 °C for 24 h. After reducing the volume, 6 mL of nitric acid and 2 mL of hydrogen peroxide were added and the tubes were subjected to mineralization, in a digestor block, for 4 h at 110ºC. After cooling, the transfer was carried out to a 25 mL volumetric flask and the volume was made up with ultrapure water. The solution was filtered and the iron concentration in the sample was obtained using a FAAS. When necessary, dilution of the sample was performed with a 4% (v/v) nitric acid solution, so that the concentration and absorbance values remained at the concentrations of the analytical curve. Analytical curve was constructed at 5 equidistant concentrations, with solutions ranging from 0.25 to 3.00 mg/L for Fe. For the control sample, the iron content was below the limit of quantification of the method (0.003 mg/g).
For the evaluation of the total iron content present in the microparticles, used in the bioaccessibility calculation, approximately 0.3 g of each sample were weighed and submitted to the mineralization process, in a digestor block, as reported by Rebellato et al. (2018). Mineralized samples were placed into a nebulizer and mixed with air-acetylene flame (2.5/10 L h−1) at approximately 2000°C. Background radiation correction was performed with a deuterium lamp; and hollow cathode lamp were used for determination of iron (248.3 nm) (PerkinElmer).
Data analysis
Analysis of variance (ANOVA) and Tukey’s test (p < 0.05) were used to analyze results, to compare the means when necessary, using the Statistica 7.0 program (StatSoft, USA).
Results and discussion
Microparticles obtained by spray chilling
Obtaining the lipid microparticles was successful in the expected operating parameters of the equipment, with no problems with the flow or any clogging of the atomizing nozzle. For the different formulations, the overall yields of the process (percentage of material recovered in relation to the initial formulation processed) were 87.5% (LM-F4), 86.7% (LM-F9) and 87.7% (LM), considering the obtainment of the product was acquired in the chamber, in the cyclone and in the sample collection container of the equipment.
Depending on the composition of fatty acids present in the lipid mixture to obtain the microparticles, changes in the melting point can favor the softening of the particles, increasing their adhesion on the equipment surface. Carvalho (2018) and Sartori et al. (2015) reported similar yields of lipid microparticles obtained by the spray chilling method (73–81.8%).
The appearance of the ferrous sulfate microparticles was a coarse powder, firm in consistency, not pasty, stable at room temperature and slightly yellow in color compared to the LM sample (without iron) (Supplementary Material – Fig. 1). The color became more yellow as the ferrous sulfate concentration increased in the formulation, as expected, since the microparticle contained more than twice the amount of active compound (ferrous sulfate).
Fig. 1.
Ferrous sulfate optical microscopy image and Lipid Microparticle without iron
Characterization of microparticles containing ferrous sulfate
Ash content, moisture and total iron content present in the microparticles was consistent with the concentration of active compound (ferrous sulfate) added in each sample, as can be seen in Table 1.
Table 1.
Characterization of microparticles containing ferrous sulfate
| Microparticles | Ashes (%) | Moisture (%) | Fe content (mg/g of microparticle) |
|---|---|---|---|
| LM | 0.07 ± 0.01c | 0.0013 ± 0.000b | nd |
| LM-F4 | 0.71 ± 0.04b | 0.0020 ± 0.000b | 3.45 ± 0.13b |
| LM-F9 | 1.49 ± 0.04a | 0.0777 ± 0.010a | 8.44 ± 0.76a |
| Microparticles | Superficial Fe (mg/g) | TEE (%) | EEE (%) |
|---|---|---|---|
| LM-F4 | 0.31 ± 0.04b | 86.30 ± 3.11a | 78.54 ± 3.11a |
| LM-F9 | 1.92 ± 0.14a | 84.36 ± 7.69a | 65.12 ± 7.69b |
Mean ± standard deviation (n = 3). LM lipid microparticle without iron, LM-F4 lipid microparticle with 4 mg Fe, LM-F9 lipid microparticle with 9 mg Fe, nd non detected, TEE total encapsulation efficiency, EEE effective encapsulation efficiency. Different lowercase letters in each column represent statistically significant difference (p < 0.05)
As anticipated, the ash content rose significantly as the added iron content in the microparticles increased, as LM-F4 contains less active compound. Similar behavior was verified for the moisture content, as the ferrous sulfate content increased, the higher was the microparticle moisture content.
Moisture of a dry material is an important indicator, both for chemical and microbiological stability, being acceptable below 5% (Pagliarussi et al. 2006). Despite the low percentage of moisture obtained in the evaluated samples, the analysis was necessary because variations in the relative air moisture during processing or storage, as well as the hygroscopicity of the active compound can influence the characteristics of the microparticles and, consequently, in its protection capacity.
The total Fe content (LM-F4: 3.45 mg/g and LM-F9: 8.44 mg/g) was slightly lower than the theoretical value (LM-F4: 4 mg/g and LM-F9: 9 mg/g), due to the losses that can occur during the process.
Encapsulation efficiency
The TEE is related to the percentage of the active material initially added, that is present in the microparticles after the encapsulation process. The EEE measures how much of this active material was actually retained within the structure of the microparticles, since the content that was only adhered to the surface is not counted (Fadini et al. 2019). The results obtained in relation to the amount of superficial iron, TEE and EEE are shown in Table 1.
We verified that the samples LM-F4 and LM-F9 did not present significant difference (p > 0.05) in relation to the total encapsulation efficiency. However, the LM-F4 sample had the lowest surface iron content and the highest percentage of EEE, indicating better encapsulation and protection of the active. The LM-F9 sample showed a higher content of superficial iron and a lower percentage of EEE, demonstrating that the increase in the amount of the active compound intensified the exposure of the iron on the surface of the microparticle. This result is relevant, considering the iron's pro-oxidant activity, it may be responsible for the oxidation in foods resulting in off-flavor and reduced shelf-life.
In the preparation of microparticles using lipid mixture occurs the formation of molecular arrays with spacing, resulting from the sizes diversity of fatty acid chains, and this causes the formation of a disordered structure with great capacity for incorporating the active compound and the possibility of better encapsulation efficiencies (Hu et al. 2005), as checked for MP-F4.
Encapsulation efficiency values similar to those obtained were reported by Leonel et al. (2010), who encapsulated glucose solution in different concentrations, using fatty acids mixtures (stearic, oleic and hydrogenated vegetable fat) as wall materials, and obtained TEE of 78.3–97.8% and amount of superficial glucose between 2.6 and 19.7%.
Evaluation of the mean size and size distribution of the microparticles
Table 2 shows the results of the average diameter and size distribution of the microparticles. The microparticles samples showed values of average diameter (D50) from 40.4 to 44.7 µm, and the samples containing iron differed significantly from the microparticles without it. The values obtained are above those described by Alvim et al. (2016), who reported mean diameter value to 31.2 µm for lipid microparticles containing ascorbic acid. In that same study, the authors reported that greater amounts of active substance increased the size of the particles. In addition, factors such as atomization pressure, feed rate, carrier material, mixture viscosity and atomizer structure, are largely responsible for the average size of the material to be obtained (Albertini et al. 2008).
Table 2.
Mean diameter (µm) and size distribution parameters for lipid microparticles containing iron
| Microparticles | Particle size distribution parameters1 | |||
|---|---|---|---|---|
| D10 | D50 | D90 | Span | |
| LM-F4 | 17.75 ± 0.15b | 44.68 ± 0.46a | 106.94 ± 4.38a | 2.00 ± 0.10a |
| LM-F9 | 19.00 ± 0.32a | 44.43 ± 0.75a | 95.87 ± 4.74b | 1.73 ± 0.12b |
| LM | 18.05 ± 0.17b | 40.44 ± 0.61b | 88.57 ± 3.00c | 1.74 ± 0.05b |
Mean ± standard deviation (n = 3). LM-F4: Lipid Microparticle with 4 mg Fe; LM-F9: Lipid Microparticle with 9 mg Fe and LM: Lipid Microparticle without iron.
1D10, D50 e D90 represent diameters for 10, 50 and 90% of the accumulated particle distribution. D50 is considered the mean diameter. Span: calculated polydispersity index: (D90–D10/D50). Different lowercase letters in each column represent statistically significant difference (p < 0.05)
Analyzing the variation of the diameters, we verified greater regularity between the particles with smaller sizes (D10), while the larger diameters (D90) differed between the formulations. The span calculation demonstrates how wide the size distribution was and revealed a typical variation of materials obtained by the spraying technique, with the LM-F4 sample containing the highest polydispersity, while the LM-F9 and LM samples showed similar indices.
The size distribution revealed that the samples exhibited comparable patterns, with a greater occurrence of particles ranging from 50 to 70 µm. Albertini et al. (2008) reported that the spray chilling technique is capable to produce microparticles with extensive variation in size, between 50 and 600 μm. This variability in size is influenced by various factors, including the viscosity of the lipid mixture employed.
Morphology
The structure of the non-encapsulated ferrous sulfate crystals (FS 1 and FS 2), which serve as active compound, and the lipid microparticles (LM 1 and LM 2), without the addition of ferrous sulfate), can be observed in Fig. 1. The microparticles (LM1 and LM2) exhibit a spherical morphology and display a range of sizes, as indicated by the results presented in particle size distribution analysis. Clusters were also observed by Carvalho (2018), who evaluated lipid microparticles produced without active and wall material composed of 60% totally hydrogenated palm oil. According to Oriani et al. (2016), this morphological behavior is linked to the lowest melting point of unsaturated fatty acids, present in the lipid mixture of the wall material, which favors agglomeration during the solidification of particles during the processing.
The morphology of the microparticles containing ferrous sulfate is displayed in Fig. 2 (LM-F4 1 and 2; LM-F9 1 and 2). The predominant shape observed was spherical, the particles had a continuous surface and a wide size variation. A greater agglomeration was observed in the formulation LM-F9, with a higher concentration of the active compound. In the higher magnification images (1000x; LM-F4 and LM-F9, Fig. 2_2), the crystals of the filling material within the microparticles can be observed, with a more centralized distribution in LM-F4 (lower concentration). This indicates the successful incorporation of the compound into the produced microparticles. The EEE of LM-F9 was lower, and the images reveal a greater presence of ferrous sulfate crystals at the edges, suggesting a more superficial distribution.
Fig. 2.
Image of the optical microscopy of the microparticles containing ferrous sulfate
The spherical shape tends to be beneficial for the flow dynamics during the material production. Additionally, the presence of varying sizes of the microparticles promotes the accommodation of smaller particles within larger ones, leading to the formation of agglomerates, a phenomenon commonly observed in the spray chilling process (Oliveira 2014).
The morphology of the microparticles produced in the present study is similar to those reported in the existing literature through several experiments employing spray chilling technique (Alvim et al. 2016; Carvalho 2018; Oliveira 2014).
Oxidative stability
The induction time for the LM sample was higher than 45 h (time of analysis) because there was no inflection in the curves during this period. According to Noello et al. (2016) lipid materials with induction times higher than 6 h can be categorized as possessing a high degree of oxidation stability.
For LM-F9, the induction period (PI) was 0.90 ± 0.02 h, indicating an intense oxidative process for this sample. According to the findings, it is observed that the selected lipid matrix exhibits favorable resistance to oxidation, but the presence of iron (pro-oxidant) in drastic conditions induced by the analysis (high temperature and intense air flow) severely accelerated the fat oxidation.
The Rancimat test is commonly employed to rapidly evaluate the oxidative stability of oils and fats, and its results serve as parameters for predicting shelf life of various products. However, there are criticisms regarding its application due the accelerated conditions of analysis, which involve high temperature, air flow (related to the amount of exposure to oxygen) and quantity of samples used (which influence the speed of the oxidation reaction). These conditions may lead to an overestimation or underestimation of the oxidative resistance of the tested material, as real production conditions, storage and product usage can significantly differ (Farhoosh 2007).
The chilling spray process aims to produce solid lipid microparticles that stabilize diverse assets, including solid particles of salts. These microparticles shield the encapsulated materials from external factors, such as light and oxygen, thereby preserving their integrit (Alvim et al. 2016). In the rancimat analysis carried out in this study, the solid structure of the encapsulating matrix was disrupted by melting the material at 100ºC and, as a result, contact with iron may have accelerated the oxidative process. The bioaccessibility results reinforce the concept that the integrity of the lipid matrix inhibits the oxidative process, because the results of this analysis (described below) were promising for encapsulated iron.
For more conclusive data, conventional oxidative stability tests, under ambient conditions of storage of lipid microparticles, that do not alter their physical structure essential to their role of protection and inhibition of adverse reactions, are recommended.
Static in vitro digestion method (INFOGEST 2.0) to estimate bioaccessibility of iron
Table 3 shows the results obtained regarding the total, soluble and bioaccessible (%) iron content in microparticles and ferrous sulfate.
Table 3.
Total Fe, soluble and percentage of bioaccessible Fe from the ferrous sulphate microparticles
| Samples | Total Fe content (mg/100 g) |
Soluble Fe (mg/100 g) |
Bioaccessibility (%) |
|---|---|---|---|
| FS | 32,560*a | 2038.57 ± 73.86a | 6.26b |
| LM-F4 | 345.23 ± 12.46c | 27.53 ± 0.83b | 7.98a |
| LM-F9 | 843.60 ± 76.94b | 28.88 ± 2.85b | 3.43c |
Mean ± standard deviation (n = 3). FS ferrous sulphate; LM-F4 lipid microparticle with 4 mg of iron, LM-F9 lipid microparticle with 9 mg of iron.
*Value declared in the ferrous sulphate technical sheet. Different lowercase letters in each column represent statistically significant difference (p < 0.05)
The total iron content present in the ferrous sulfate showed a significant difference in relation to the microparticles produced, and, as expected, the LM-F9 had a higher content than the LM-F4. In the solubility test, it was verified that the non-encapsulated compound (SF) had a higher content than the microparticles, but there was no statistically significant difference (p > 0.05) between the particles of different concentrations.
The bioaccessible percentage is obtained through the ratio of the soluble fraction over the total fraction of the compound evaluated, multiplied by 100. We noticed that the ferrous sulfate presented lower content than LM-F4, and this, showed significant difference (p < 0.05) among the other samples evaluated.
Ferrous sulfate (Fe2+) contains in its composition iron with an oxidation number + 2, being considered more soluble and available for absorption in the body. However, Fe2+ when exposed to the environment undergoes oxidation and changes to the Fe3+ (ferric ion) form, becoming unstable, less soluble and, consequently, less available for absorption (Hurrell et al. 2010; Rebellato et al. 2018). When encapsulating ferrous sulfate, we found that the lowest concentration microparticle (LM-F4) was able to protect the compound (SF) from environmental conditions and still allowed better availability for absorption, as observed in bioaccessibility tests.
The microencapsulation of ferrous sulfate with fat mixture and under the studied conditions shows a potential effective use in fortified foods, since a smaller amount of ferrous sulfate presented satisfactory technological parameters in addition to a higher bioaccessible percentage than the compound without encapsulation.
Previous studies with encapsulated or microencapsulated iron under different conditions have already been described in scientific publications. Wegmüller et al. (2004) produced lipid microparticles by spray cooling (hydrogenated palm oil and 1% lecithin), with iron pyrophosphate as an active ingredient. The authors found that the size of the particles significantly influenced the absorption of iron, both in rats and in humans, with the smallest particle presenting the highest availability of absorption (43%). Cocato et al. (2007) used a dialysis test (in vitro) to verify the absorption of microencapsulated ferrous sulfate with natural polysaccharide. The results showed that bioaccessible iron was 2.2 and 3.4% for pure and microencapsulated ferrous sulfate, respectively.
Experiments performed with alginate nanoparticles, with the purpose of protecting iron against possible pro-oxidant effects were carried out by Katuwavila et al. (2016). The authors observed that the highest percentages of iron release from the alginate nanoparticle were between the pH values of 6.0 and 7.4, and this is the most favorable condition, since absorption occurs predominantly in the duodenum, where the pH is around 6.0.
Buyukkestelli et al. (2019) developed and characterized encapsulated ferric chloride with double emulsion and evaluated the bioaccessible fraction of the mineral by the protocol of Minekus et al. (2014). The authors found variation in the percentages of bioaccessibility (41.17–52.97%) in relation to the proportions of the constituents used in the emulsion for encapsulation. The authors observed that the sample with the highest iron content had the lowest bioaccessible fraction, while the sample with the lowest content had the highest bioaccessible fraction, as also observed in the present study.
Conclusions
The ferrous sulfate microencapsulation using the spray chilling technique with a lipid matrix was successful. The application of a fully hydrogenated palm oil and commercial vegetable fat as a wall material resulted in stable particles, able to retain in their volume the crystals of the active material. The in vitro digestion assay showed that the LM-F4 microparticle presented a higher bioaccessibility percentage, even when compared to non-encapsulated ferrous sulfate, and twice as high as the LM-F9 sample.
The findings indicate that the microparticle with the lowest iron concentration exhibits superior performance for food fortification purposes. This is attributed to its ability to offer enhanced protection for the active compound, improved encapsulation efficiency, and a higher percentage of iron bioaccessibility. Furthermore, this microparticle demonstrates the potential to release the encapsulated material during simulated of gastrointestinal digestion. Future studies could focus on investigating the behavior of this microparticle when incorporated into a food matrix or in the fortification of wheat flour.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
The authors acknowledge the financial support of the Coordination for the Improvement of Higher Education Personnel (CAPES), for the scholarship grant (817163-2015), the National Council for Scientific Development (CNPq), for the scholarship grant (Process Number: 142415/2016-2) and the São Paulo Research Foundation (FAPESP), for the scholarship grant (Process Number: 2019/1362-1).
Author contributions
Conceptualization, A.P.R., J.A.L.P. and C.J.S; methodology, A.P.R., P.P.M.; J.G.S.S.; and I.D.A.; validation, A.P.R., P.P.M. and J.G.S.S.; formal analysis and investigation, A.P.R., P.P.M.; J.G.S.S.; and I.D.A.; data curation, A.P.R., P.P.M. and J.G.S.S.; writing—original draft preparation, A.P.R.; writing—review and editing, A.P.R., P.P.M.; J.G.S.S.; I.D.A.; J.A.L.P. and C.J.S supervision and funding acquisition, C.J.S. All authors have read and agreed to the published version of the manuscript.
Funding
Coordination for the Improvement of Higher Education Personnel (CAPES), for the scholarship grant (817163–2015), the National Council for Scientific Development (CNPq), for the scholarship grant (Process Number: 142415/2016–2) and the São Paulo Research Foundation (FAPESP), for the scholarship grant (Process Number: 2019/1362–1.
Data availability
The data that support the findings of this study are available upon reasonable request.
Code availability
Not applicable.
Declarations
Conflicts of interest
The authors declare no conflict of interest.
Ethics approval
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Consent to participate
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Consent for publication
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Footnotes
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Data Availability Statement
The data that support the findings of this study are available upon reasonable request.
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