Abstract
The modern food industry requires new analytical methods for high-demand food supplements, personalized diets, or bioactive foods development. One of the main goals of the food industry is to discover new ways of food fortification. This applies to food products or supplements for human and animal diets. In our research, we focused on the solid particles of AproTHEM (dried porcine hemoglobin), which is approved for animal feeding and as a meat product additive, and AproFER 1000 (heme iron polypeptides), which is still being investigated. The study showed the possible application of advanced techniques for the examination of iron-based food additives. We evaluated selected techniques for particle size and morphology examination such as laser diffraction, optical microscopy, as well as scanning electron microscopy, and briefly discussed their usefulness compared with other techniques. On the basis of our results, we proposed a path of microscopic analysis for the study of material homogeneity. The structure of heme iron was evaluated by X-ray diffraction, FT-IR, and Raman spectroscopy supported with thermal behavior analysis (differential scanning calorimeter). Furthermore, a portable colorimeter was applied for L*a*b* color analysis. Our study proved that for new food product development, particle size analysis as well as typically used advanced materials techniques can be successfully applied.
1 Introduction
Iron, an essential mineral, is necessary for organism growth, development, and synthesis of some hormones. As a component of hemoglobin and myoglobin, it plays a role in oxygen transport and storage [1–3]. Iron deficiency is the most frequent nutritional deficiency worldwide and one of the leading contributors to the deterioration of global health [4]. It affects billions of people worldwide, particularly infants, young children, women of reproductive age, and people with low socioeconomic status [5] being the cause of anemia with significant negative impacts on their health [6,7]. Iron deficiency anemia (IDA) is one of the crucial nutritional deficiency disorders in the world. WHO estimates that 42% of children younger than 5 years and 40% of pregnant women worldwide are anemic [8]. The prevalence of anemia is higher in low- and middle-income countries [9]. In high-income countries, however, this problem is increasing due to the growing number of people on a vegan and vegetarian diet. IDA has a negative impact on infants, affecting their growth, cognition, and behavior. Adults suffering from IDA deal with health problems, e.g., fatigue and weakness, trouble concentrating, lack of work productivity, diminished quality of life, and restless legs syndrome [5].
To prevent IDA, iron level has to be maintained within the normal range through intestinal dietary iron absorption. Two types of iron are present in the diet, i.e., heme, found in animal products and characterized by better absorption efficiency, and nonheme, with a lower absorption rate, present mainly in plant-derived products [5].
Designing supplements and fortified food products is an element of the prevention strategy against iron deficiency diseases [10]. There are many available dietary iron supplements. WHO recommends [11,12] several iron compounds for food fortification, i.e. ferrous sulphate, ferrous fumarate, encapsulated ferrous sulphate or fumarate, and electrolytic iron and ferric pyrophosphate (both at double the iron amount as ferrous sulphate).
There are some critical technical barriers specific to the conventional iron fortification of food. They include [13,14] an iron compound bioavailability, a fortified vehicle selection (food/matrix) less sensitive to sensory quality and shelf-life deterioration after iodine fortification, and a balance of inhibitors and enhancers of iron bioavailability in the selected vehicles (staple food selected the most often as iron vehicles, such as cereal-based food, contain potent iron absorption inhibitors). Recommended iron fortifiers differ in their relative absorption, which is associated with solubility [12]. On the basis of the solubility, they are divided into three groups: readily water soluble, poorly water soluble but soluble in dilute acids (gastric juice), and water insoluble but partially soluble in dilute acids. On the other hand, many food products undergo changes in color or flavor caused by iron compound addition. They also can affect the oxidation of food compounds (e.g., unsaturated fatty acids, proteins, vitamins). It has been found that the readily water-soluble (better absorbed) iron compounds were the most reactive with the food matrix and caused the most likely sensory changes in sensitive foods, while compounds only partially soluble in dilute acid were the least likely to cause such effects [13]. These changes limit the amount of these iron compounds that can be added to the food. The same study showed that the particle size of the fortifier could influence the adverse color formation and iron-catalyzed oxidation. The study concerning the double fortification table salt with iodine and iron [15] showed that changing from micronized ground ferric pyrophosphate (particle size <2.5 µm) to regular ferric pyrophosphate (particle size 20 µm) could prevent both the salt yellowing and iron-catalyzed iodine losses in moist table salt. There was no evidence that the larger particle size decreased iron absorption from ferric pyrophosphate.
Those barriers described earlier encourage the development of new strategies of food vehicle fortification with nutrient-rich food-based fortifiers (food-to-food fortification) [16]. Fortifying staple foods with heme iron has also been suggested. Dietary sources of heme iron are only of animal origin. Heme iron is better absorbed and shows less gastrointestinal discomfort and oxidative stress than nonheme iron [17]. Bovine and porcine types of blood are the reach in readily available heme iron and have been suggested as an iron fortifier. The method of heme iron polypeptide production, i.e., a soluble heme moiety with an attached polypeptide produced by the enzymatic digestion of bovine or porcine hemoglobin, has been designed [18]. The enzymatic hydrolysis of hemoglobin enhances iron absorption by preventing the insoluble heme polymer formation. An example of a commercially available product obtained by this method is Aprofer 1000®.
The nutritional values, health benefits, as well as structure, homogeneity, and color of the food, all may influence customer choices. For new food product development, commonly applied food quality measurement techniques might be insufficient. In the field of food technology and food products, homogeneity as well as particle size (if the ingredients cannot be dissolved) play a crucial role. Here, we propose a selection of advanced techniques typically used for materials characterization in heme iron studies. For the evaluation of particle size and morphology of solid particles, microscopic methods were used (optical microscope and scanning electron microscope (SEM)). For the selected structure analysis, Raman and FT-IR spectroscopy were employed. The last section presented possible applications of heme iron in emulsion systems, which were detailed and characterized in detail previously [19].
2 Materials and methods
2.1 Materials
AproFER 1000 (heme iron polypeptides) and AproTHEM (dried porcine hemoglobin) are commercially available iron supplements. The samples were delivered from a local supplier (JJP Sp. z o.o., Węgorka 20, 60-318 Poznań) and used in the study as delivered, without any processing.
2.2 Methods
2.2.1 Microscopic studies
The morphology of iron supplement particles was evaluated using optical microscopy. For the imaging, two microscopes with different optical light paths were applied. For the lower magnifications stereo microscope ZEISS Stemi 305 (Zeiss, Shanghai, China) was used. For detailed studies, an inverted microscope ZEISS AxioVert.A1 (Zeiss, Shanghai, China) was employed. In both imaging systems, the same color camera Axiocam 208 (Zeiss, China) was applied. For imaging in fluorescent mode, a Colibri 5 system LED light with green LED λ = 555 nm was applied. LD A-Plan 20×/0.35 (air) and LD A-Planx40/0.55 ph1 (air) objectives were used. The solid particles were placed directly on the microscopic glass without additional preparation. For the presentation, the resolution of the images was adjusted using ZEN3.1 software (Zeiss, Jena, Germany).
The samples were analyzed using a digital microscope (DM) Keyence VHX-7000 series with fully integrated objective head VHX-7100. The 3D profiles were obtained using dedicated software.
The microstructure of the samples was characterized by SEM, model Mira 3 FEG SEM (Tescan, Czech Rep.). The electrons were generated by a ZrO/W Schottky field emitter gun (Denka, Japan) and accelerated by a high voltage of 12 kV. For image acquisition, an in-beam SE detector was used.
2.2.2 Particle size distribution
The Analysette 22 MicroTec plus (Fritsch, Idar-Oberstein, Germany) laser particle sizer was used to determine the particle size distribution of the AproFER 1000 and AproTHEM. The tests were conducted using a predetermined standard operating procedure for full range (0.08–2,000 µm) measurement and theoretical Fraunhofer assumptions. Particle size distributions were created using MaScontrol software (Fritsch, Idar-Oberstein, Germany). This software provides two types of quantities: the sum of the distribution Q(x) and the density of the distribution q(x). The sum of the distribution Q(x) is calculated as a curve that shows a standardized total quantity of all particles measured with size less than or equal to x. Any point of a curve of the sum of the distribution represents the sum of the volume of all particles in a range from x min to x. The curve of the density distribution q(x) is the first derivative of Q(x) by x. Assuming a constant density of tested particles in all size ranges, the volume distributions are also mass distributions.
2.2.3 FT-IR
Spectrum two FT-IR spectrometer equipped with a Universal ATR with a diamond crystal (PerkinElmer, Waltham, MA, USA) was used for the determination of FT-IR spectra in the range of 50–4,000 cm−1. For each sample, the measurements were repeated three times.
2.2.4 Raman spectroscopy
Raman spectra of powder samples of AproFER 1000 and AproTHEM were captured using an inVia Renishaw Raman Microscopy system (Renishaw, Old Town, Wotton-under-Edge, UK). All measurements were acquired at room temperature (RT) (21°C) with 1,800 and 3,000 g·mm−1 grating with 633 nm He/Ne and 514 nm Ar laser, respectively. The laser lights were focused on the sample with a 50x/0.75 microscope objective (LEICA, Wetzlar, Germany). Spectra in the range of 100–2,000 and 100–3,000 cm−1 were obtained with the 514 and 633 nm laser length, respectively. To improve the signal-to-noise ratio, the acquisition time was 20 s. Raman spectra were collected by WiRE™3.3 software linked to the instrument. Peak positions were measured using Lorentz profile with OriginPro 2020b software (Northampton, MA, USA), and all experimental data were normalized to 1.
2.2.5 X-ray analysis
Chemical composition was analyzed using X-ray diffraction (XRD) and energy dispersive X-ray spectrometer (EDS) connected with SEM. The crystallographic structure of iron supplements was evaluated using an Empyrean X-ray diffractometer (Malvern Panalytical, The Netherlands) with CuKα radiation, λ = 1.54178 Å, working at set parameters: voltage 45 kV, and anode current 40 mA. XRD spectra were recorded in the range of 30–120° 2θ angle. The structure and phase composition of the examined samples were analyzed using HighScore Plus software and ICDD- PDF-4+ database, respectively. The EDS analysis was done using an UltimMax 65 detector (Oxford Instruments, England), with a 65 mm2 sensor area.
2.2.6 Differential scanning calorimetry (DSC)
The thermal analysis was done using a DSC, model Q20 (TA Instruments, USA). The measurements started from RT to the end point adjusted at 400°C and followed by cooling to 40°C. The heating and cooling rate was set at 10°C·min−1 and performed under Argon 5.0 gas flow at 50.0 mL·min−1. The powdered samples of 5.2 mg mass were placed into aluminum Tzero pans, and pan was closed with aluminum Tzero lids using a Tzero Sample Press Kit (all from TA Instruments, USA). Pressed pan-lid without powder was used as a reference sample. The obtained DSC data were collected using Advantage/Universal Analysis Software (TA Instruments).·
2.2.7 Color analysis
The color of the samples was analyzed using a PCE-XXM 30 colorimeter (PCE Instruments). For the statistical evaluation, the average values of 10 measurements were presented.
where ΔL*, Δa*, and Δb* are the SD of L*, a*, and b*, respectively, from the series of the measurements of each sample.
2.3 Application of iron supplements into emulsion systems
Solid particles of AproFER 1000 and AproTHEM were used for the principle tests of the emulsion-type food supplement preparation. Oil in water-type emulsions based on hemp seed oil was prepared, with soap nuts extracts as a stabilizer. For all emulsions vitamin D was added as well. Details of the preparation and properties of the emulsions were presented previously [19]. Here, we briefly show possible applications of the AproFER 1000 and AproTHEM.
3 Results and discussion
3.1 Particle size and morphology
Particle size determination plays a crucial role for the development of food products and supplements. In the previous article [20], selected food particle size and morphology evaluation techniques were discussed. It should be highlighted that AproTHEM, as opposed to AproFER, has good solubility. This limits the usage of AproFER as an additive in food products. Current European Union as well as the United States regulators recommend detailed studies of each new component that might be used in food product development. Furthermore, there are several agencies working on new regulations for nanocomponents for food products. On the basis of the “Guidance on technical requirements for regulated food and feed product applications to establish the presence of small particles including nanoparticles” [21], we decided to compare the size and the structure of the perspective heme iron supplements.
The difference in particle size of the two iron supplements is visible to the eye. Microscopic examinations with a stereoscopic microscope confirmed the high polydispersity of the particles. Single particles as well as aggregates can be distinguished under magnification ×4 (Figure 1a and e). This encouraged us to perform a more detailed analysis with an inverted microscope. In both samples, the signal in the fluorescent mode was detected, which correlated with the heme structure present in analyzed particles. Figure 1c presents a typical image of the AproFER1000 sample merged view from the halogen light channel and fluorescence channel. Optical microscopic imaging confirmed differences in particle sizes in both iron supplements. It should be noted that the detailed examination of particle morphology allowed to distinguish large particles up to several micrometers from aggregates of smaller particles in the same range (Figure 1b–d and f–h). Figure 1d and h present images in the 2.5D mode where single particles and huge agglomerates were observed.
Optical microscopy images of (a)–(d) AproFER 1000 ((a) stereoscopic microscop image, (b)–(d) inverted optical microscope), and (e)–(h) AproTHEM ((e) stereoscopic microscop image, (f)–(h) inverted optical microscope).
New opportunities for food product studies come from the development of light microscopy, such as confocal laser scanning microscopy or hyperspectral imaging techniques. Pu et al. [22] reported principles and possible applications of different kinds of hyperspectral microscope imaging (HMI) techniques in various modes, such as fluorescence HMI, visible/near-infrared HMI, Raman HMI, and infrared HMI. These techniques are mostly referred to the samples where the size is around a few micrometers. The problems for detailed studies occur in the systems where the particle size ranges from dozens to several hundred micrometers, as presented here. Furthermore, for more than two-dimensional (2D) studies, the good method is missing. One of the possibilities is using the manual extended depth of focus approach proposed by Zeiss. However, some artifacts might occur in the systems that are not stable or flow as shown previously [23]. Due to these concerns, we decided to verify possible applications of DM. This system offers the presentation of the results of solid particle imaging in 3D and automatic 3D sample profile presentation. Figures 2 and 3 present a sample image of the powdered samples taken with a DM. First, what can be observed is that the sample volume can be easily adjusted with the comparison of the traditional optical microscope. Furthermore, with DM, the 3D structure is almost automatically registered and presented with the topography (i.e., Figures 2c and 3c and e). Auty [24] highlighted that unlike traditional materials, food might contain moisture, fat or sugar, and other ingredients such as preserving agents, which limits the usage of electron microscopy. Furthermore, this “contaminations” limit also the application of atomic force microscopy (AFM) for the topography studies (AFM). Akhlaghi et al. [25] stated that microscopic assay is the best way to study nanosystem morphology and showed sample results of AFM, SEM, and TEM of smart stimuli-responsive herbal drug-encapsulated nanoniosome particles. The authors pointed that spherical shapes verified by microscopic examinations correspond to the results obtained by DLS.
AproTHEM powders studied with DM: (a) 2D image, (b) 3D image, (c) 3D image with topography, (d) 2D image with higher magnification (scale bar 50 μm), (e) 2D image with higher magnification (scale bar 50 μm), and (f) 3D image overview of the sample.
Aprofer 1000 powders studied with DM: (a) 2D image, (b) 3D image, (c) 3D image with topography, (d) 2D image with higher magnification (scale bar 250 μm), (e) 3D image with higher magnification and topography (scale bar 250 μm), and (f) 2D image with higher magnification (scale bar 50 μm).
As presented in Figures 2 and 3, DM allows fast imaging in three dimensions, which is important when the samples, i.e., powders, are not homogenous and uniformly size distributed and many “layers” occur. When DM is compared with optical microscopy, it can be observed that DM presents better focus.
Primavera et al. [26] highlighted that DLS as well as laser diffraction are useful for quick and simple monitoring of changes in both the coarse and fine particle fraction during different steps of production. Both methods can be used for controlling processes and modulating industrial production. The techniques offered flexibility for the evaluation of product consistency and homogenity.
Due to good solubility in water, AproTHEM is frequently used in the meat industry, and for that reason, we decided not to use the light scattering technique for the size evaluation. Figure 4 presents the particle size distribution obtained by the laser diffraction technique. It can be seen that for both samples, multimodal graphs were determined. It corresponds with previously performed microscopic evaluation of particle size. As we recommended in our previous studies [27], evaluation of the size should be done using a minimum of two different techniques, where one allows visualization of the sample (i.e., optical or electron microscopy).
Particle size distribution obtained by laser diffraction: (a) AproFER 1000 and (b) AproTHEM.
As shown in Figure 4, a very small population of particles around 1 μm was detected in both iron supplements. Weak and intensive signals were reported for particles sized in the range of 10–100 μm and hundreds of micrometers, respectively. Further food product development can be considered to involve an additional grinding process for AproFER 1000 application.
For that reason, further application in emulsion systems might be beneficial. The additional homogenization process might result in the disruption of aggregates and better dispersion. Moreover, the addition of surfactant may be beneficial by preventing the aggregation of solid particles.
As we discussed earlier, optical microscopy, especially DM, offered an easy way for sample preparation and quite fast analysis, without sample destruction and waist. However, for the detailed morphology analysis, electron microscopy should be the method of choice because it offers simultaneous element content analysis.
Here, detailed structural characterization of the samples was performed using SEM. The morphology of the samples with different magnifications is presented in Figure 5. Heme structure can be recognized for AproTHEM (Figure 5a–c). In both samples, high particle size can be observed as well. SEM imaging corresponds with LPS. Figure 5e–g shows small structures (single micrometers) as well as larger particles (over 100 μm).
SEM images and EDX spectra: (a)–(d) AproTHEM and (e)–(h) AproFER 1000.
During SEM analysis, EDX spectra were recorded (Figure 5d and h). Carbon was identified as an element with a higher concentration in both samples, which corresponds with heme composition. Iron peaks were also identified; however, the concentration was lower than carbon. It should be noted that for some food products, the electron beam might be destructive. Moreover, sample preparation for SEM is more difficult and time consuming than for optical microscopy.
3.2 X-ray
Additional structural analyses were performed using XRD. In food products, XRD can only be applied where some components exhibit the crystal structure. Moreover, XRD analysis might confirm structural changes observed by SEM [28]. Figure 6 shows diffractograms of the heme iron supplement samples. Three weak peaks were detected for AproFER 1000 but almost at the level of noise what makes the method useless for iron detection. It should also be noted that none of the peaks correspond with pure iron for which 2theta (Cu) signals that are registered at 44.676, 65.028, 82.342, 98.955, 116.395, and 137.178. However, other studies, e.g., by Jendrzejewska et al. [29], showed the successful application of XRD for some other dietary iron supplements that contain iron(ii) bis-glycinate.
XRD spectra of AproFER 1000 and AproTHEM.
3.3 Thermal analysis
Figure 7 shows the calorimetric analysis performed by DSC of AproTHEM (Figure 7a) and AproFER 1000 (Figure 7b). The first and second intensive peak maximum were observed for both samples at c.a.100 and 325°C, respectively. For AproTHEM, an additional intensive peak maximum was registered around 210°C. It should be noted that heme as well as blood samples change their volume during the heating process [30]. That fact must be highlighted at the stage of choosing of investigation pan for the samples to prevent uncontrolled decomposition and damage.
DSC curves of AproTHEM (a) and AproFER 1000 (b) samples.
3.4 Spectroscopic analysis
Additional information was brought by infrared spectra analysis (Figure 8). Both tested products presented similar signals, however different in intensity. The peaks about 3,300 cm−1 as well as the signal at 1,650–1,500 cm−1 [31,32] represent stretching and deforming vibrations of N–H amine bonds. Stretching signals from C–H bonds in saturated and nonsaturated alkyl groups are visible at around 3,000 cm−1. Carboxylic group bonds (C–O and C═O) show characteristic signals about 1,500 and between 1,200 and 1,000 cm−1. Key differences between samples are evident in the area below 1,000 cm−1, i.e., in the “fingerprint area.” Our results correspond with the previous study reported by Damszel et al. [31].
FT-IR spectra of AproFER 1000 (black line) and AproTHEM (red line).
Powder samples of AproFER 1000 and AproTHEM were analyzed with two different laser wavelengths. It was found that the signal strength was maximal for both samples at the Raman shifts at 1,360 and 1,581 cm−1. A comparison between Raman spectra obtained with excitation by 514 and 647 nm lasers is shown in Figure 9. The characteristic peaks from Raman studies of AproTHEM for 633 nm were registered at 131, 168, 214, 1,360, 1,550, 1,764, 1,880, 1,985, 2,096, 2,210, 2,310, 2,417, 2,511, 2,618, 2,738, while for 514 nm at 647,670, 747, 1,002, 1,120, 1,174, 1,230, 1,360, 1,396, 1,493, 1,533, 1,582, and 1,620. For AproFER 1000, two main peaks were recorded for 633 nm at 1,367 and 1,575, and for 514 nm at 1,360 and 1,581.
Raman spectra: (a) obtained by 633 nm laser of powder samples of AproFER 1000 and AproTHEM in the range of 60–3,200 cm−1 and (b) obtained by 514 nm laser of powder samples of AproFER 1000 and AproTHEM in the range of 60–2,000 cm−1.
Raman spectroscopy has been employed in studies of organic samples related with medicine [33,34].
Many studies proved that color of food products and supplements plays a crucial role for consumers [35,36]. Lee et al. [37] showed that visual perception enhanced the selection of fresher and noncontaminated foods. Furthermore, color analysis and changes inform about the changes or stability of the studied samples [38]. Figure 10 shows an L*a*b* color analysis of both investigated samples. It can be observed that AproTHEM is more reddish than AproFER 1000 (a* parameter), and AproFER 1000 is more dark than AproTHEM (L* parameter). The integrated software identifies the colors of AproTHEM as #23120 f (very dark (mostly black) red) and AproFER 1000 as #100c0C (very dark (mostly black) red). The obtained results confirmed also that the color of AproFER 1000 is not “ideally” black as may suggested by eye observation. For each sample,
Color analysis of AproFER 1000 (left side) and AproTHEM (right side).
3.5 Examination path
On the basis of the presented results, we proposed the food structure examination path according to their size and homogeneity (Figure 11). In our opinion at each of the proposed five stages, the examination can be stopped when the desirable information is achieved. It is obvious that none of the mentioned imaging methods can be applied to food samples (due to their behavior, i.e., fat content limits SEM, TEM, and AFM). Furthermore, in recent days, the costs of the analysis increased. It is due to increasing costs of the chemicals (i.e., fluorescent dyes for CLSM), examination and supportive tolls (i.e., cuvettes, grids for TEM, scanning tips for AFM), or needs of additional processing for samples preparation (i.e., freezing for cryo-imaging in electron microscopy). The proposed examination path can be also applied for sample “triage” for detailed examination. In this study, we focused only on the sample visualization as well as morphology studies.
Proposed examination path from the easiest (and low cost) to most difficult methods (high costs).
3.6 Application of heme iron
Solid particles might be applied in emulsion systems as a stabilizer in the Pickering emulsions type [39]. On the other hand, an emulsion structure might be beneficial for insoluble solid particles to prevent or delay their sedimentation. Figure 12 presents oil in water-type emulsion systems with AproFER 1000 after several months of storage at RT. As an oil phase, hemp seed oil was used. It should be noted that the stabilizer, which was extracted from soap nuts, was applied in different concentrations. As shown in Figure 12, samples 2, 4, and 7 were the most homogenous, and iron supplement was generally quite homogenously dispersed in the whole volume of the samples. The other compositions showed fast phase separation as well as a concentration of iron AproFER 1000 particles in the oil phase. Detailed studies of the prepared emulsion properties were discussed in a previous paper [19]. Primary tests of the emulsion preparation with AproTHEM as an active agent in the same conditions such as AproFER 1000 showed enhanced surface active properties (results not shown). However, during the homogenization process, a high precipitate of solid particles occurred on the surface of the homogenizer shaft. This effect limits the application of AproTHEM at this stage of experiments. The observed foaming effect might be caused by a high concentration of hemoglobin in opposition to AproFER 1000. The volume changes were also observed during heating when DSC analysis was performed. Based on the performed experiments, it can be concluded that the emulsion with AproFER 1000 can be considered as a potential food supplement.
Emulsion prepared with AproFER 1000.
4 Conclusions
Heme iron is not so frequently studied in food products. On the other hand, there is high demand for new iron supplement development due to its deficiency in humans and other animals. Food products’ consistency and homogeneity play a crucial role for consumers. Food solid particles affect the taste of the final dish or food supplement. For that reason, detailed studies of the particle size of food are needed. Here, both AproFER 1000 and AproTHEM samples are inhomogeneus and showed broad particle size distribution registered by various microscopic analyses as well as laser diffraction. Our study showed new possibilities of DM techniques for food studies, especially those focused on solid particle characterization. Moreover, we proposed a microscopic study path that could help to reduce the costs of homogeneity investigation and showed how important is the microscopic verification of particle size obtained by light techniques (DLS, laser diffraction). Finally, spectroscopic analysis supported with color analysis allows us to express the differences between various heme iron supplements (which can be used for new emulsion composition used).
Acknowledgments
The authors are thankful to Inter JJP Sp. z o.o. ul. Węgorka 20, 60-318 Poznań Company for the AproFER 1000 samples used in this study.
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Funding information: This study was financially supported by grant MINIATURA 2021/05/X/NZ9/00384 from National Science Centre, Poland. Publication was co-financed within the framework of the Polish Ministry of Science and Higher Education’s program: “Regional Excellence Initiative” in the years 2019–2023 (No. 005/RID/2018/19),” financing amount 12 000 000,00 PLN.
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Author contributions: Maciej Jarzębski: conceptualization, methodology, software, investigation, resources, data curation, writing – original draft, writing – review and editing, visualization, supervision, project administration and funding acquisition. Marek Wieruszewski: validation, formal analysis, writing – original draft, and writing – review and editing. Mikołaj Kościński: software, formal analysis, investigation, writing – original draft, and visualization. Tomasz Rogoziński: software, formal analysis, investigation, writing – original draft, and visualization. Joanna Kobus-Cisowska: software, resources, formal analysis, investigation, and writing – original draft. Tomasz Szablewski: software, resources, formal analysis, and writing – original draft. Katarzyna Waszkowiak: formal analysis, investigation, writing – original draft, and visualization. Joanna Perła-Kaján: investigation, writing – original draft. Jarosław Jakubowicz: software, formal analysis, investigation, writing – original draft, and visualization.
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Conflict of interest: Maciej Jarzębski, who is the co-author of this article, is a current Editorial Board member of Reviews On Advanced Materials Science. This fact did not affect the peer-review process. The authors declare no other conflict of interest.
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