Food Hydrocolloids 142 (2023) 108808

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Thermal stability and in vitro digestion of alginate–starch–iron beads for

oral delivery of iron
A. Mihaly Cozmuta a, *, M.A.K. Purbayanto b, A. Jastrzębska b, A. Peter a, C. Nicula a,
A. Uivarasan a, L. Mihaly Cozmuta a
a
Technical University of Cluj Napoca, Department of Chemistry-Biology, Baia Mare, Romania
b
Warsaw University of Technology, Faculty of Materials Science and Engineering, Warsaw, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: Ferrous bisglycinate can be used as an oral therapy for iron deficiency. However, the low thermal stability of
Ferrous bisglycinate encapsulation ferrous bisglycinate powder to oxidation of ferrous ions hinders its application to fortification of foods that are
Ferrous bisglycinate thermal treatment heated before consumption. In this study, ferrous bisglycinate powder and beads prepared from alginate, starch,
Thermal treatment of beads
ferrous bisglycinate powder, and water in a ratio of 2:1:2:100 (w:w:w:v) were subjected to thermal treatment at
In vitro digestion
90 ◦ C and 180 ◦ C for 90 min. Heating changed the geometry of the beads. Large fractures appeared on the surface
Ferrous ion thermal stability
of the heated beads, while multiple pores formed in their cores. The beads increased the oxidative stability of Fe
(II) at 180 ◦ C, with 83.53% Fe(II) and 16.47% Fe(III) after heating, compared with 65.08% and 34.92% for
ferrous bisglycinate powder alone. In a temperature-dependent manner, the amounts of bioaccessible Fe(II) and
Fe(III) released from bisglycinate powder under in vitro gastric conditions ranged from 153.31 to 118.42 mg and
10.81–74.75 mg, respectively, compared to 32.90–42.49 mg and 7.94–14.51 mg released from the beads. At the
end of the simulated oral-gastric-intestinal digestion, the beads heated to 180 ◦ C released 1.22-fold more bio­
accessible ferrous ions (147.85 mg) than were released from the corresponding bisglycinate powder (121.57 mg).
These experimental results indicate that alginate–starch–ferrous bisglycinate beads thermally treated at 180 ◦ C
are promising carriers for oral delivery of iron.

1. Introduction
Iron is a microelement that is required for various metabolic pro­
cesses, including DNA synthesis, oxygen transport, and electron trans­
port (Abbaspour, Hurrell, & Kelishadi, 2014). Iron deficiency anemia is
a common condition that can be mitigated or treated by oral adminis­
tration of iron salts such as ferrous sulfate, ferrous fumarate, ferrous
bisglycinate, ferrous lactate, or ferrous gluconate (Leonard, Chalmers,
Collins, & Patterson, 2014). Ferrous bisglycinate has been suggested to
have potential use as a carrier of ferrous ions in food because it contains
ferrous ions chelated between two glycine molecules. Its structure im­
proves iron absorption and storage and increases hemoglobin levels
more effectively than the more commonly used ferrous sulfate
(Bovell-Benjamin, Viteri, & Allen, 2000). The administration of ferrous
bisglycinate, similarly to other iron salts, can trigger side effects. Un­
pleasant taste and gastrointestinal disturbances such as diarrhea, con­
stipation, nausea, stomach pain, or black-stained stools were the most
commonly reported, although they occurred less frequently and less
severely than with other iron salts (AbuHashim, 2017). Poor thermal
stability hinders the use of ferrous bisglycinate powder as a carrier for
oral iron delivery. Ferrous bisglycinate is heat-stable at temperatures
above 220 ◦ C (EFSA, 2006); however, because of the citric acid added
during the manufacturing process, chelate bonds break upon heating,
and NOx and CO2 are generated. As a result, the Fe(II) is released from
the heterocyclic rings and oxidized by oxygen and water, with the
amount of oxidation increasing with increasing temperature. This
behavior of ferrous bisglycinate makes it unsuitable for fortification of
foods processed at temperatures above 153 ◦ C (FAO, 2003). This limi­
tation can be overcome by encapsulating ferrous bisglycinate before it is
added to food. A coating over a ferrous bisglycinate core prevents in­
teractions with the food matrix and improves bioaccessibility, mitigates
the unpleasant sensory characteristics, prevents oxidation of ferrous ions
to ferric ions, and allows the release of ferrous ions into the gastroin­
testinal tract. Divalent metal cation transporter 1 (DMT1), a protein in

* Corresponding author.
E-mail address: ancamihalycozmuta@gmail.com (A. Mihaly Cozmuta).

https://doi.org/10.1016/j.foodhyd.2023.108808
Received 9 February 2023; Received in revised form 18 April 2023; Accepted 21 April 2023
Available online 29 April 2023
0268-005X/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
A. Mihaly Cozmuta et al. Food Hydrocolloids 142 (2023) 108808

the structure of the apical membrane of enterocytes, transports ferrous Table 1

ions across the membrane into the cell. More bioaccessible ferrous iron Encapsulation parameters.
implies that more will be available for cell metabolism. Various mate­ Composition (w: Total iron Encapsulation The amount of dried
rials have been reported to encapsulate iron compounds. w:w:v) content in the efficiency, EE% beads containing the
Jiménez-Alvarado, Beristain, Medina-Torres, Román-Guerrero, and dried beads, iron amount equivalent
mg/g to iron amount in 1 g of
Vernon-Carter (2009) incorporated a ferrous bisglycinate solution into
ferrous bisglycinate
the inner phase of water-in-oil-in-water multiple emulsions stabilized powder, g bead/1 g
with protein:polysaccharide complexes. Furthermore, the effect of the powder
relative concentrations of the biopolymers was determined, along with Alginate:Starch: - - -
the pH at which electrostatic complexation was maximized. Ferrous Ferrous
glycinate nanoliposomes made from egg phosphatidylcholine by the bisglycinate:
reverse-phase evaporation method have been shown to be fit for oral Water
1:1:0:100
administration of ferrous bisglycinate because they had acceptable sta­
(control beads)
bility in simulated gastrointestinal juice at 37 ◦ C for 5 h (Ding et al., Alginate:Starch: 26.47 ± 0.92 59.60 ± 2.53(e) 8.43 ± 0.29(a)
2011). Beads prepared by encapsulation of heme (bovine erythrocytes) Ferrous (d)
and non-heme (ferrous sulfate) iron in a maltodextrin matrix resulted in bisglycinate:
the high bioavailability of iron released under in vitro gastrointestinal Water
1:1:1:100
conditions (Churio & Valenzuela, 2018a). Sodium alginate has been Alginate:Starch: 27.82 ± 0.81 68.30 ± 2.47(d) 8.02 ± 0.21(a)
widely used to produce beads specifically to release bioactive com­ Ferrous (d)
pounds in a controlled manner. Alginate beads have poor functionality; bisglycinate:
their major limitations are deficiencies in their wall structure, high Water
1:2:1:100
porosity, and poor mechanical properties, which cause their deteriora­
Alginate:Starch: 33.38 ± 1.36 74.53 ± 2.12(c) 6.69 ± 0.13(bc)
tion and early diffusion of the ferrous core from the beads during gastric Ferrous (c)
digestion. In addition, low efficiency of compound incorporation into bisglycinate:
the alginate matrix has also been reported (Zhang, Liu, Huang, Wang, & Water
Wang, 2015). 2:0:1:100
Alginate:Starch: 78.14 ± 1.23 77.18 ± 0.84(c) 2.86 ± 0.08(d)
These challenges can be overcome by blending alginate with other
Ferrous (b)
compatible components to reduce the porosity, reinforce the wall, and bisglycinate:
enhance the encapsulation efficiency of the beads. Starch has been re­ Water
ported to reinforce the alginate matrix and increase encapsulation effi­ 2:0:2:100
Alginate:Starch: 33.17 ± 1.15 78.97 ± 0.98(bc) 6.73 ± 0.21(b)
ciency by providing additional hydrogen bonding sites between the
Ferrous (c)
amylose and amylopectin and the hydrophilic groups of the encapsu­ bisglycinate:
lated compounds (Córdoba, Deladino, & Martino, 2013; Mihaly Coz­ Water
muta et al., 2021; Ziar, Gerard, & Riazi, 2012). Although alginate-based 2:1:1:100
gels have been intensely studied, the influence of thermal treatment on Alginate:Starch: 87.71 ± 3.40 82.30 ± 0.78 2.55 ± 0.05(d)
Ferrous (a) (ab)
their properties or their performance during heat exposure is not well
bisglycinate:
understood (Abbaszadeh, Gandomi, Misaghi, Bokaei, & Noori, 2014; Water
Kim, Jeong, Cho, & Kim, 2019; Saqib et al., 2022). This paper reports the 2:1:2:100
development of alginate–starch–iron beads that could be used as iron Alginate:Starch: 35.92 ± 0.95 83.96 ± 1.57(a) 6.22 ± 0.13(c)
Ferrous (c)
carriers in food subjected to thermal treatment such as bread, bakery
bisglycinate:
products, and canned products. Firstly, different types of alginate–­ Water
starch–ferrous bisglycinate beads were prepared by varying the ratios of 2:2:1:100
the raw materials, and the encapsulation efficiency of each formulation
Samples 2:2:2:100, 1:1:2:100 and 1:2:2:100 were not considered due to the high
was calculated; the bead type with the most suitable encapsulation pa­
viscosity of gels which not allowed the proper extrudation
rameters was selected for further investigations. The selected beads and Samples 1:0:1:100 was not considered because the gelation did not occurred
ferrous bisglycinate powder were heated to 90 ◦ C or 180 ◦ C for 90 min, properly, and beads with no well defined form resulted.
and the bead morphology and ferrous and ferric ion content were Results are expressed as average ± standard deviation SD (n = 3).
characterized. Finally, in vitro digestion studies were conducted with In each column, mean values with different letters are significantly different at P
ferrous bisglycinate powder and alginate–starch–ferrous bisglycinate < 0.05.
beads to assess the bioaccessibility of the iron species.
left in the bath for 15 min to harden, and, after filtration, were rinsed
2. Materials and methods with ultrapure water to remove excess calcium ions and ferrous bis­
glycinate. The beads were dried at 20 ◦ C in the dark (to prevent Fe(II)
2.1. Bead preparation oxidation) until they reached a constant weight. Control beads free of
ferrous bisglycinate were also prepared to quantify the contribution of
The external gelation method was used to prepare iron-containing the raw materials to the Fe(II) and Fe(III) content of the ferrous bis­
beads (Table 1) by blending different amounts of ferrous bisglycinate glycinate beads.
powder (Biosynth Carbosynth, USA), sodium alginate with a viscosity of
25.7 cps at 25 ◦ C and a mannuronic/guluronic ratio of 35/65 (data
supplied by the manufacturer, Sigma-Aldrich, USA), and soluble potato 2.2. Determination of total iron content and encapsulation efficiency
starch (Sigma-Aldrich, USA), in 100 mL of ultrapure water (Thermo
Fisher Scientific Barnstead™, USA). After stirring for 24 h at 25 ◦ C (MR The total iron content of the ferrous bisglycinate beads, control
Hei-End, Heidolph Instruments, Germany), the solution was fed through beads, and ferrous bisglycinate powder was determined through acid
a peristaltic pump (PM05 AES Laboratoire, France) at 50 rpm and digestion (Berghof Method), followed by atomic absorption spectrom­
extruded through a capillary tube (diameter 0.5 mm) into a bath con­ etry. Around 300 mg of the sample was dried to constant weight and
taining 500 mL of 0.5 mol/L CaCl2 (Merck, Germany). The beads were accurately weighed, placed in 6 mL of 65% HNO3 and 1 mL of 2 mol/L

2
A. Mihaly Cozmuta et al.
HCl, and digested in a microwave digestion system (MWS-2, Germany).
Digestion proceeded in three steps at a temperature of up to 200 ◦ C and a
total digestion time of 30 min. The resulting solution was diluted with
ultrapure water in a 100 mL volumetric flask, and the total iron content
was determined using an AAnalyst 800 spectrometer (PerkinElmer,
USA) at 248.3 nm. The result was calculated as an average of three
replicates and expressed as mg total iron/g of sample.
The encapsulation yield was calculated according to Equation (1):
EE (%) = FeB × 100/FeFeBg (Eq. 1)
where EE (%) is the ferrous bisglycinate encapsulation efficiency, %; FeB
is the total iron content in the dried beads, mg iron/g of beads; and
FeFeBg is the total iron content in the added dried ferrous bisglycinate
powder, mg iron/g of ferrous bisglycinate powder.
The ferrous bisglycinate beads with the best iron encapsulation pa­
rameters were selected for further investigation.
2.3. Thermal treatment of ferrous bisglycinate powder and selected beads
Samples of the ferrous bisglycinate powder (FeBP) and selected
beads (FeBB) were subjected to thermal treatments similar to those
applied to dough during bread-making. They were tested at the tem­
peratures reached during baking, either in the core or at the crust of the
bread. The samples were placed in a proofing chamber (Matina D 6040,
Bucharest, Romania) at 32 ◦ C and 75% relative humidity, and after 1 h,
they were heated for 90 min at 90 ◦ C (temperature in the core of bread)
or 180 ◦ C (temperature of the bread crust). Steam was introduced to the
chamber for the first 10 min of baking. Untreated (20 ◦ C) and thermally
treated (90 ◦ C and 180 ◦ C) samples were physically and chemically
characterized. The samples were labeled according to the composition
and intensity of thermal treatment they received: FeBP-20, unheated
ferrous bisglycinate powder; FeBP-90, 90 min of thermal treatment at
90 ◦ C; and FeBP-180, 90 min of thermal treatment at 180 ◦ C. FeBB-20,
FeBB-90, and FeBB-180 are the corresponding ferrous bisglycinate
beads.
2.4. Physical characterization of selected beads
A Bresser LCD micro 5 MP (Bresser GmbH, Germany) microscope
was used to collect digital photographs of the FeBPs and FeBBs. An
S–3500N scanning electron microscope (SEM; Hitachi, Japan) was used
to visualize the changes in the surface morphology and core of the beads
during thermal treatment and in vitro digestion, respectively. Because of
the non-conductive nature of the beads, they were placed onto
conductive carbon tape and sputtered with a conductive gold nano­
particle layer before they were imaged in the SEM.
The shape of 100 beads of each type was quantified using a digital
image processing technique (ImageJ, National Institutes of Health and
the Laboratory for Optical and Computational Instrumentation LOCI,
University of Wisconsin). The equivalent diameter, length, width,
maximum and minimum Feret diameters, spherical factor, circularity,
and elongation were obtained (details are given in section 2.4.SM of
Supplementary Material).
2.5. Oxidative stability of ferrous iron in ferrous bisglycinate powder and
selected beads
The level of iron oxidation in the ferrous bisglycinate and beads was
assessed by measuring Fe(II) and Fe(III) content using the method re­
ported by Niedzielski et al. (2014) and adapted to our study. Samples
were dried at 50 ◦ C until they reached a constant weight, ground, and
sieved. An accurate weight of 2 g of powder with a diameter of less than
0.1 mm was extracted with 20 mL of 2 mol/L HCl at 25 ◦ C for 1 h, and at
80 ◦ C for 1 h under a reflux condenser. The solution was filtered through
a 0.45 μm filter and diluted with ultrapure water to 25 mL in a
3
Food Hydrocolloids 142 (2023) 108808
volumetric flask. An aliquot of 1 mL of solution was transferred into a
25-mL volumetric flask and diluted with ultrapure water. This solution
was used to measure the Fe(II) and Fe(III) contents. The analysis was
conducted in three steps. Firstly, the initial amount of Fe(III) was
measured, then the Fe(II) in the solution was oxidized to Fe(III), and
finally the amount of Fe(III) was measured again. The initial Fe(III) was
measured by reaction with ammonium thiocyanate (Niedzielski et al.,
2014). A mixture of 10 mL 1 mol/L ammonium thiocyanate solution and
10 mL iron solution was stirred for 10 min, and the absorbance of the
stable red color was measured at 490 nm (Perkin-Elmer Lambda 35
UV-VIS Spectrophotometer). The total iron content was determined by
oxidizing Fe(II) in the solution with 30% H2O2 (3:1 v:v) (Fenton reac­
tion) and allowing it to react for 10 min. The solution was degassed by
boiling for 10 min, cooled, and diluted with ultrapure water to 25 mL in
a volumetric flask. The total iron amount was measured using the re­
action with ammonium thiocyanate, as indicated above. A calibration
curve was constructed by plotting known concentrations of iron(III)
chloride (Merck, Germany) dissolved in ultrapure water against absor­
bance at 490 nm.
The Fe(II) content was calculated as the difference between the final
and initial Fe(III) measurements. The preparation and measurements
were repeated three times for each sample. The results were expressed as
Fe(II) or Fe(III) from the total iron (%), average values ± standard
deviation.
The total iron amount was also measured by atomic absorption
spectrometry (described in section 2.2) to confirm the accuracy of the
results obtained using the ammonium thiocyanate method.
2.6. Speciation of Fe(II) and Fe(III) in the in vitro digestion samples
Thermally treated and untreated samples of FeBP and FeBB were
submitted to in vitro digestion to assess the bioaccessibility of Fe(II) and
Fe(III). In vitro digestion of 1 g of FeBP-20, FeBP-90, and FeBP-180 and a
total iron equivalent (2.55 g) of FeBB-20, FeBB-90, and FeBB-180 (de­
tails are given in section 4.1) was performed in triplicate, following the
INFOGEST protocol (Brodkorb et al., 2019) which simulates the oral,
oral-gastric, and oral-gastric-intestinal stages of digestion. Samples were
taken after the oral (2 min), oral-gastric (2 h), and oral-gastric-intestinal
stages (4 h). To ensure reproducibility of the results, the digestion was
started with individual tubes for each phase and time point. Samples
taken at different time points received heat shock treatment on ice for 5
min to ensure enzyme inactivation. The oral, gastric, and intestinal
digestates were centrifuged at 6000×g for 30 min to separate the su­
pernatant containing the bioaccessible fraction. The Fe(II) and Fe(III)
contents of an accurately measured 20-mL portion of the supernatant
were analyzed. To exclude the contribution of iron from the simulated
digestion fluids, procedural blank digestions were run, and the iron
concentration in the resultant fluids was measured at the same time
points as the sample digestates. The bioaccessible iron in the superna­
tant was expressed as the amount of Fe(II), Fe(III), and total iron in the
supernatant (mg), and the bioaccessible fractions of Fe(II), Fe(III), and
total iron calculated with Eq. (2):
Bioaccessible fraction (%) = (FeS − FeB) × 100/FeU, (Eq. 2)
where FeS and FeB are the amounts of Fe(II), Fe(III), or total iron in the
supernatant of the digested sample and the blank digestions, respec­
tively, mg/100 g, and FeU is the total amount of Fe(II), Fe(III), or total
iron in the undigested sample, mg/100 g.
Details regarding the composition of simulated bodily fluids and the
digestion procedure are provided in the Supplementary Material.
2.7. Statistical analysis
Three independent measurements were conducted for each analysis.
100 beads of each type were measured to quantify the shape parameters.
A. Mihaly Cozmuta et al. Food Hydrocolloids 142 (2023) 108808

The results are presented as mean ± standard deviation (SD). One-way
analysis of variance (ANOVA) was performed using IBM SPSS Statis­
tics 24 to assess the difference between the means, where the signifi­
cance was defined as P < 0.05.
3. Results and discussion
3.1. Appearance of beads and selection of the optimal formula
Fig. 1 shows the prepared beads. With some exceptions (alginate,

Fig. 1. Digital photographs showing overall appearance of freshly prepared and dried alginate-starch-ferrous bisglycinate beads (alginate:starch:ferrous bisglycinate:
water w:w:w:v).

4
A. Mihaly Cozmuta et al. Food Hydrocolloids 142 (2023) 108808

starch, ferrous bisglycinate powder, and water in a ratio of 1:1:2:100,
1:2:2:100, 1:0:1:100, and 2:2:2:100), the beads were well-formed and
had no tendency to aggregate. When fresh, the beads appeared glossy
with a color from light to dark green depending on the ratio between the
white (alginate and starch) and the red-brown (ferrous bisglycinate
powder) raw materials. Upon drying, they turned brown. When the raw
materials were mixed in ratios of 1:1:2:100, 1:2:2:100, and 2:2:2:100,
highly viscous solutions were generated that could not be extruded
(Fig. 2.SM). The lower amount of alginate in those formulas resulted in a
low concentration of the carboxyl groups that cross-link the calcium ions

Fig. 2. Digital photographs and SEM micrographs of ferrous bisglycinate powder and beads:
Digital photographs of ferrous bisglycinate powder:
A1. Unheated ferrous bisglycinate powder;
A2. Ferrous bisglycinate powder heated at 90 ◦ C for 90 min;
A3. Ferrous bisglycinate powder heated at 180 ◦ C for 90 min;
B. Unheated ferrous bisglycinate beads:
B1, B2 – digital photographs; B3, B4 – outer and core SEM micrographs;
C. Ferrous bisglycinate beads heated at 90oC for 90 min:
C1, C2 – digital photographs; C3, C4 – outer and core SEM micrographs;
D. Ferrous bisglycinate beads heated at 180oC for 90 min:
D1, D2 – digital photographs; D3, D4 – outer and core SEM micrographs;
The yellow circles indicate the aggregates of ferrous bisglycinate particles.

5
A. Mihaly Cozmuta et al.
and make the beads. Moreover, gelation did not occur properly with low
amounts of ferrous bisglycinate powder (1:0:1:100) and without starch,
resulting in poorly defined beads (Fig. 2.SM). Churio, Pizarro, and
Valenzuela (2018b) also obtained amorphous beads from an alginate
solution containing 0.05% ferrous sulfate, ferrous ammonium sulfate,
and ferric citrate, and a viscous paste at concentrations of 0.1–1%.
Table 1 summarizes the total iron content and the encapsulation
efficiency (EE, %) of the prepared beads. The EE was in the range
59.60–83.96%, corresponding to a total iron content of 26.47–87.71 mg
Fe/g of dried beads. Perez-Moral, Gonzalez, and Parker (2013) reported
the iron content of the beads between 50 and 180 mg iron/g of dried
beads. This results from the incorporation of iron salts of various
oxidation states into the alginate beads. In the presence of Ca2+, the
alginate forms a gel network due to cross-linking between the calcium
ions and the guluronic and mannuronic acid residues in the alginate.
Iron ions can also contribute to the cross-linking of the resulting poly­
meric network, with the calcium and iron ions competing for the elec­
trostatic binding sites within the alginate–starch network. The EE%
values proportionally increased with the amount of alginate, starch and
ferrous bisglycinate powder al in the walls and the core. Chan (2011)
stated that the disproportion between the amounts of core and bead
material reduces the number of binding sites available for calcium ions
and results in less compact gels with collapsed walls and smaller EE%. In
our study, the large amounts of alginate and starch used provided
numerous binding sites in the ß-D-mannuronic and α-L-guluronic
co-monomeric units in the alginate (Durán, Churio, Arias,
Neira-Carrillo, & Valenzuela, 2020) and on the anionic surface of the
starch, for calcium and iron ions, resulting in firm beads with high EE%.
The iron load of the beads increased with the amount of ferrous bis­
glycinate powder added to the gelling bath. Guluronic acid was avail­
able at a larger ratio in the alginate structure as compared to
mannuronic acid (mannuronic/guluronic ratio 35/65) and has a higher
affinity for Fe(II) ions than calcium ions (Perez-Moral et al., 2013). As
will be discussed in section 4.3, the FeBP contains small amounts of Fe
(III) ions, toward which the alginate–starch network exhibits a higher
affinity than for calcium and Fe(II) due to the +3 charge state, which are
better able to compete with the divalent iron and calcium ions for
binding to the active sites (Perez-Moral et al., 2013). The 2:1:2:100 (w:
w:w:v) alginate:starch:ferrous bisglycinate:water beads were selected
for detailed investigation, as they had the highest iron load (87.71
mg/g) and EE (82.30%), and the smallest mass of beads containing the
same iron load as 1 g of bisglycinate powder (2.55 g beads/1 g of
powder). The daily value (DV) for iron is 7 mg/day for children 1–3
years of age and 18 mg/day for adults and children 4 years of age and
older (FDA, 2020). 1 g of the selected beads contains 4.87 times the
adult DV. The ratio of elemental iron in the beads is 8.77%, lower than
values of 33% in ferrous fumarate, 20% in ferrous sulfate and 11.6% in
ferrous gluconate (Saljoughian, 2007).
3.2. Physical characterization of selected beads
After drying, the color of the fresh beads changed from greenish to
red-brown (Fig. 2B1), corresponding to the appearance of dried ferrous
bisglycinate powder (Fig. 2A1). Upon thermal treatment, a visible color
change occurred, which was proportional to the temperature. As shown
in Fig. 2, FeBB-90 turned dark brown (Fig. 2C1), while FeBB-180
became black-brown Fig. 2D1). The color changes are in line with
those that occurred in FeBP-90 and FeBP-180 (Fig. 2A2, 2A3), indicating
increased Fe(III) content (Section 4.3). Fig. 2 shows the SEM micro­
graphs of the outer (Fig. 2B3, 2C3, 2D3) and inner (Fig. 2B4, 2C4, 2D4)
structures of the selected beads. The outer SEM micrograph of FeBB-20
(Fig. 2B3) has a rough and undulating surface, with protrusions sug­
gesting the deswelling of water, characteristic of matrixes prepared from
multiple ingredients (Wang & He, 2002). The spherical geometry in­
dicates hydrophilic interactions between the components (Han, Guenier,
Salmieri, & Lacroix, 2008). Churio et al. (2018b) obtained oblong beads
6
Food Hydrocolloids 142 (2023) 108808
after an alginate-ferrous bisglycinate suspension was dropped into the
gelling solution. Feltre, Silva, Lima, Menegalli, and Dacanal (2018) re­
ported a spherical shape for corn starch-alginate beads, explained by the
improvement in the packaging of the alginate matrix after the starch is
added. In our study, the starch in the alginate–starch–ferrous bisglyci­
nate suspension changed surface tension and viscosity, establishing
additional hydrogen bonds that resulted in a tighter structure. Slow
dehydration during drying and the subsequence shrinkage of droplets
are likely responsible for the appearance of fine cracks on the surface of
the beads. Small depressions were also be observed on the surface of the
beads, but these do not generate internal pores, as shown by the inner
SEM micrograph of the unheated beads (Fig. 2B4). The core is dense,
continuous, smoother than the outer surface, and has rare longitudinal
fractures. FeBP appears as randomly distributed aggregated dark points.
Fig. 2C3 and 2D3 show the changes in the outer structure of the beads
after thermal treatment. The surface damage, which was observed on
the surfaces of FeBB-90 and FeBB-180 beads, suggests that the polymeric
structure has collapsed, reducing the bead wall integrity. Concavities
and fractures that occurred during the extensive release of water vapor
and gases and triggered volume change of the beads, was observed in the
photographs (Fig. 2C2 and 2D2) and SEM micrographs. A less cohesive
structure was observed in the core of the beads (Fig. 2C4, 2D4) resulting
from large fractures and multiple pores connected in labyrinthine pat­
terns. On one hand, this structure may favor heat flow, access of
oxidizing agents to the bead interior, or the premature release of FeBP,
resulting in oxidization of Fe(II) to Fe(III). On the other hand, the pores
may help to release FeBP during digestion. As Gombotz and Wee (1998)
have reported, one mechanism for core material diffusion within algi­
nate beads is the progressive dissolution of the wall material through the
development of pore channels. Table 2 summarizes some characteristic
shape parameters of the beads. According to the sphericity factor (SF),
FeBB-20 can be considered egg-shaped, as only particles with SF < 0.05
are considered spherical (Chan, Lee, Ravindra, & Poncelet, 2009). The
shape of the beads can be tailored by the balance between alginate–­
starch–ferrous bisglycinate solution viscosity and interfacial tension.
The beads lost their initial geometry when exposed to thermal treatment
due to the pressure of the water vapor and gases released at a high rate.
Upon heating to 180 ◦ C, the SF value changed to 0.02, approaching 0.05,
which describes a tear-shaped particle. At 20 ◦ C, the beads had a surface
area of 2.08 μm2, which increased 1.66- and 2.06-fold with temperature,
indicating expansion. These findings were supported by increases in
equivalent diameter, length, width, and Feret’s diameters of the beads,
although they were not significantly different.
3.3. Oxidative stability of ferrous iron state after thermal treatment
No iron was measured in the control beads. The results in Table 3
indicate no significant differences between the total iron amounts
calculated by the ammonium thiocyanate method and by atomic ab­
sorption spectrometry. The Fe(II) in FeBP-20 accounts for 94.82% of the
total iron, significantly higher than Fe(III) at 5.18%. The FeBB-20 beads
contain 90.09% Fe(II) and 9.91% Fe(III). These findings indicate that Fe
(III) ions are present in both FeBP and FeBB. During the thermal treat­
ment, the Fe(II) content of the samples decreased with a corresponding
increase in Fe(III). FeBP had the lowest oxidative stability for Fe(II).
After 90 min at 90 ◦ C, the Fe(II) content reduced 1.082-fold from
94.82% in FeBP-20 to 87.61% in FeBP-90, with the Fe(III) content
correspondingly increased 2.39-fold from 5.18% to 12.40%. A more
pronounced Fe(II) decrease was observed after thermal treatment at
180 ◦ C. The Fe(II) content of FeBP-180 was 65.08%, 1.46-fold lower
than value 94.82% in FeBP-20, while the Fe(III) content increased 6.74-
fold from 5.18% in FeBP-20 to 34.92% in FeBP-180.
Incorporating FeBP into an alginate–starch matrix provided protec­
tion against Fe(II) oxidation, with no significant differences observed
between the amount of Fe(II) in untreated and thermally treated beads.
The percentage of Fe(II) in FeBB-90 (88.05%) and FeBB-180 (83.53%)
A. Mihaly Cozmuta et al. Food Hydrocolloids 142 (2023) 108808

Table 2
Morphology of ferrous bisglycinate beads (FeBB) subjected to thermal treatment and in vivo digestion process.
Sample Area Equivalent Length Width MaxFeret MinFeret Sphericity Circularity Elongation
[μm2] diameter [μm] [μm] [μm] [μm] [μm] factor (SF)

Unheated ferrous bisglycinate beads (FeBB-20)

Ferrous bisglycinate beads not 2.08 ± 3.81 ± 1.58(a) 8.53 ± 2.10 ± 5.16 ± 2.42 3.28 ± 1.47 0.22 0.76 ± 0.17 1.51 ± 0.65
subjected to digestion 2.05(a) 2.10(a) 1.23(a) (a) (a) (a) (a)
Ferrous bisglycinate beads 1.94 ± 3.52 ± 1.029a) 7.56 ± 1.95 ± 4.80 ± 1.25 3.21 ± 0.96 0.2 0.77 ± 0.23 1.34 ± 0.35
subjected to oral digestion(2 1.31(a) 1.98(a) 0.63(a) (a) (a) (a) (a)
min)
Ferrous bisglycinate beads 1.9 ± 2.23 ± 1.76(a) 4.23 ± 1.32 ± 3.22 ± 1.17 2.32 ± 0.89 0.16 0.78 ± 0.23 1.29 ± 0.43
subjected to oral- gastric 1.84(a) 1.76(ab) 0.58(a) (a) (a) (a) (a)
digestion (2 h)
Ferrous bisglycinate beads 1.22 ± 1.86 ± 1.01(a) 2.11 ± 0.72 ± 1.25 ± 0.76 0.79 ± 0.11 0.08 0.75 ± 0.12 1.05 ± 0.34
subjected to oral-gastric- 0.93(a) 1.32(b) 0.34(a) (a) (a) (a) (a)
intestinal digestion (4 h)

Ferrous bisglycinate beads subjected to thermal treatment for 90 min at 90 ◦ C (FeBB-90)

Ferrous bisglycinate beads not 3.45 ± 5.27 ± 1.69(a) 9.14 ± 4.62 ± 7.07 ± 2.75 6.01 ± 2.29 0.08 0.68 ± 0.24 1.42 ± 0.48
subjected to digestion 1.48(a) 2.28(a) 2.15(a) (a) (a) (a) (a)
Ferrous bisglycinate beads 3.32 ± 4.93 ± 0.91(a) 8.85 ± 4.46 ± 6.98 ± 1.13 5.35 ± 0.84 0.13 0.59 ± 0.21 1.32 ± 0.42
subjected to oral digestion(2 1.70(a) 1.73(a) 0.55(a) (a) (a) (a) (a)
min)
Ferrous bisglycinate beads 2.86 ± 3.03 ± 0.59(ab) 1.07 ± 3.50 ± 5.02 ± 0.75 4.12 ± 0.54 0.12 0.42 ± 0.19 1.30 ± 0.34
subjected to oral- gastric 0.85(a) 0.94(b) 0.40(a) (ab) (ab) (a) (a)
digestion (2 h)
Ferrous bisglycinate beads 1.05 ± 1.01 ± 0.48(b) 1.35 ± 1.65 ± 2.20 ± 0.64 1.94 ± 0.44 0.21 0.23 ± 0.20 1.34 ± 0.37
subjected to oral-gastric- 0.65(a) 0.98(b) 0.32(a) (b) (b) (a) (a)
intestinal digestion (4 h)

Ferrous bisglycinate beads subjected to thermal treatment for 90 min at 180 ◦ C (FeBB-180)
Ferrous bisglycinate beads not 4.29 ± 6.33 ± 4.09(a) 10.95 ± 5.32 ± 8.44 ± 2.89 8.09 ± 0.02 0.69 ± 0.21 1.45 ± 0.58
subjected to digestion 2.87(a) 3.60(a) 2.75(a) (a) 4.949a) (a) (a)
Ferrous bisglycinate beads 4.11 ± 5.87 ± 0.87(a) 9.82 ± 4.87 ± 7.80 ± 1.08 7.58 ± 0.82 0.11 0.77 ± 0.17 1.45 ± 0.52
subjected to oral digestion (2 1.60(a) 1.48(a) 0.55(a) (a) (a) (a) (a)
min)
Ferrous bisglycinate beads 3.25 ± 4.61 ± 2.01(a) 6.17 ± 2.23 ± 5.79 ± 0.26 5.42 ± 2.34 0.10 0.74 ± 0.18 1.57 ± 0.67
subjected to oral- gastric 1.82(a) 0.55(ab) 0.10(ab) (ab) (a) (a) (a)
digestion (2 h)
Ferrous bisglycinate beads 0.58 ± 2.09 ± 1.72(a) 3.29 ± 1.09 ± 2.78 ± 1.08 1.79 ± 1.70 0.06 0.76 ± 0.17 1.46 ± 0.54
subjected to oral-gastric- 0.47(a) 1.40(b) 0.50(b) (b) (a) (a) (a)
intestinal digestion (4 h)

Results are expressed as average ± standard deviation SD (n = 3). A number of 100 beads were used to quantify the shape parameters.
In each column, mean values with different letters are significantly different at P < 0.05.

was higher than the percentage in the corresponding powders, FeBP-90
(87.61%) and FeBP-180 (65.08%). Our results agreed with the work of
Rivera, Gallego, Villalba, and Gianotti (2017), who reported that starch
encapsulation protected ferrous ions against oxidation during thermal
processing at 180 ◦ C. Despite cracks and pores occurring on the surface
and in the core of the beads during heating (Fig. 2), the alginate–starch
shell protected the enclosed FeBP in three different ways: (i) diluting the
citric acid present in the powder and thereby preventing damage to
heterocyclic rings; (ii) assuring a lower temperature in the core of the
beads than the external temperature; and (iii) providing resistance to the
inward diffusion of the oxidants, oxygen and water vapor. As a result,
the thermal stability of the ferrous bisglycinate chelate, and therefore Fe
(II), increased. The amount of Fe(II) in 2.55 g of ferrous bisglycinate
beads was calculated (Table 2). After the FeBP and FeBB were treated for
90 min at 90 ◦ C, the amount of Fe(II) in 2.55 g of FeBB-90 (200.41 mg)
was 1.02-fold higher than that in 1 g of FeBP-90 (196.53 mg). The
protective effect of an alginate–starch matrix is stronger at 180 ◦ C: the
amount of Fe(II) in 2.55 g of FeBB-180 (197.96 mg) was 1.31-fold higher
than in 1 g of FeBP-180 (150.72 mg).
3.4. In vitro digestion study
3.4.1. Digital photographys, SEM, and morphology of digested samples
The surface morphology of the beads subjected to in vitro digestion
was characterized using digital photographs (Fig. 3A1-3A12) and SEM
micrographs of the outer surface (Fig. 3B1-3B12) and core (Fig. 3C1-
3C12). The digital photographs of FeBB-20 subjected to oral digestion
for 2 min (Fig. 3A2) did not indicate visible changes on the surface of the
beads. However, the SEM micrographs (Fig. 3B2) revealed the formation
of some pores, explained by the action of salivary α-amylase on the
starch. Due to the short contact time of the beads with the simulated
saliva, the pores did not reach to the core of the beads (Fig. 3C2). At the
end of oral-gastric digestion, more pores of different sizes were visible
on the surface of FeBB-20 (Fig. 3B3, 3B4). No pores were observed in the
core of FeBB-20 after oral-gastric digestion; porosity was only observed
at the end of oral-gastric-intestinal digestion (Fig. 3C2-3C4). The frac­
tures already existing on the surface of the beads subjected to thermal
treatment sensitized the beads and made them more susceptible to the
action of digestive juices, with the beads heated to 180 ◦ C more sus­
ceptible than those heated to 90 ◦ C. After each stage of digestion, un­
dulations appeared on the surface of the beads, porosity and pores sizes
increased, new fractures appeared, existing fractures propagated, and
ultimately, the beads broke into smaller pieces after oral-gastric-
intestinal digestion (Fig. 3A6-3A8). The SEM micrographs (Fig. 3B6-
3B8, 3C6-3C8) indicate the propagation of these changes from the sur­
face to the core of the beads. The disintegration of beads during intes­
tinal digestion could be regarded as an advantage in iron release, as it
exposes the core to the intestinal juice (Churio et al., 2018b; Valenzuela,
Hernandez, Morales, & Pizarro, 2016). In this way, Fe(II) was protected
against oxidation to Fe(III), and its bioavailability increased. As
Fig. 3A5, 3A9, 3B5, 3B9, 3C5, and 3C9 show, both the surface and core
of the beads suggest a morphology-dependent heat response of the
alginate–starch–ferrous bisglycinate matrix. The degradation of the
surface and core was more pronounced in beads heated to 180 ◦ C,

7
A. Mihaly Cozmuta et al. Food Hydrocolloids 142 (2023) 108808

Table 3
Content of total iron, Fe(II) and Fe(III) in the ferrous bisglycinate powder (FeBP) and ferrous bisglycinate beads (FeBB).
Sample Total iron content *Total iron content calculated Fe(II) Fe(III) Fe(II) Fe(III) Fe(II) amount in the
calculated based on based on atomic absorption content, content, from total from total 2.545 g ferrous
ammonium thiocyanate spectrometry method, mg/g mg/g mg/g iron, % iron, % bisglycinate beads, mg/
method, mg/g 2.545 g

Ferrous bisglycinate powder, FeBP

Unheated ferrous 223.24 ± 3.79(a) 221.75 ± 3.66(a) 211.68 ± 11.56 ± 94.82 ± 5.18 ± –
bisglycinate powder 3.37(a) 0.57(c) 0.20(a) 0.20(c)
FeBP-20
Ferrous bisglycinate 224.34 ± 5.74(a) 221.43 ± 2.38(a) 196.53 ± 27.80 ± 87.61 ± 12.40 ± –
powder heated 90 5.19(b) 0.83(b) 0.27(b) 0.27(b)
min at 90 C
◦

FeBP-90
Ferrous bisglycinate 231.52 ± 9.03(a) 228.43 ± 2.73(a) 150.72 ± 80.805 ± 65.08 ± 34.92 ± –
powder heated 90 7.33(c) 2.505(a) 0.92(c) 0.92(a)
min at 180 ◦ C
FeBP-180

Ferrous bisglycinate beads, FeBB

Unheated ferrous 87.70 ± 1.84(a) 86.52 ± 1.25(a) 79.01 ± 8.70 ± 90.09 ± 9.91 ± 201.11 ± 5.767(a)
bisglycinate beads 1.72(a) 0.34(c) 0.35(a) 0.35(c)
FeBB-20
Ferrous bisglycinate 89.16 ± 2.01(a) 89.54 ± 2.07(a) 78.74 ± 10.68 ± 88.05 ± 11.95 ± 200.41 ± 7.00(a)
beads heated 90 min 2.32(a) 0.33(b) 0.62(b) 0.62(b)
at 90 C
◦

FeBB-90
Ferrous bisglycinate 93.11 ± 1.65(a) 92.19 ± 1.57(a) 77.77 ± 15.05 ± 83.53 ± 16.47 ± 197.96 ± 4.71(a)
beads heated 90 min 1.14(a) 0.53(a) 0.29(c) 0.29(a)
at 180 ◦ C
FeBB-180

Results are expressed as average ± standard deviation SD (n = 3).

In the case of columns 2 and 3, the results from each row are compared and mean values with the same letters are not significantly different at P < 0.05; *No sig­
nificance differences appear between the total iron amounts calculated based on ammonium thiocyanate and atomic absorption spectrometry, respectively methods; In
the columns in the range 4–7, the results of each column are compared and mean values with different letters are significantly different at P < 0.05; The differentiation
was made based on the Tukey test post-hoc ANOVA (IBM SPSS Statistics Version 24).

indicating a negative relationship between temperature and the stability
of the matrix. These beads also exhibited a higher sensitivity to digestive
juices, regardless of whether they were exposed for a shorter (Fig. 3A6
and 3A10, 3B6 and 3B10, 3C6 and 3C10) or longer time (Fig. 3A7 to
3A12, 3B7 to 3B12, 3C7 to 3C12).
The evolution of the shape parameters of the beads during digestion
confirms the observations from the digital photographs and the SEM
microphotographs (Table 2). Gastric digestion altered the geometry of
the beads to different extents, depending on the intensity of the thermal
treatment. Shrinkage of the surface areas of FeBB-20, FeBB-90, and
FeBB-180 by 1.094-, 1.206-, and 1.320-fold, respectively, was observed,
although these were not statistically significantly different from one
another. The weight loss under the action of the acidic gastric juice was
responsible for this shrinkage. The gastric juice weakened the electro­
static repulsion between the carboxylic groups in the alginate by pro­
tonation (Pasparakis & Bouropoulos, 2006) and hydrolysis of the α(1–4)
glycosidic bonds in starch and resulted in the formation of cleavage
products. Under intestinal conditions, the disruption of the beads was
the greatest. The area of the beads was reduced 1.704-, 3.285-, and
7.396-fold for FeBB-20, FeBB-90, and FeBB-180, respectively.
3.4.2. In vitro digestion of FeBP and FeBB
No significant increases were observed in the total iron or Fe(II) in
the supernatant collected after artificial oral digestion of FeBP or FeBB,
regardless of the intensity of the thermal treatment. During the 2 min of
oral digestion, the alginate–starch–ferrous bisglycinate beads displayed
strong resistance to the action of salivary amylase. Fig. 4 shows the
bioaccessible fractions of total iron, Fe(II), and Fe(III) resulting from
oral-gastric and oral-gastric-intestinal digestion. Under gastric condi­
tions, FeBB showed greater stability than FeBP, reflected by the lower
bioaccessible fractions of iron species. For FeBB-20, the bioaccessible
fractions of total iron, Fe(II), and Fe(III) were 18.30%, 16.37%, and
35.88%, compared to 73.52%, 72.44%, and 93.47% released from FeBP-
20. The differing stability of powder and beads under strongly acidic
conditions explains these large differences. In the presence of pepsin and
HCl, ferrous bisglycinate is unstable due to the chelate bonds being
weakened by the increased H+ concentration, leading to dissociation
into ferrous chloride and glycine (Ding et al., 2011). The iron leakage
from FeBB-20 during gastric digestion resulted from the behavior of the
matrix components under acidic conditions. The pKa values of man­
nuronic and guluronic acid residues in the alginate matrix are 3.38 and
3.65 (Francis, Hunger, & Donius, 2013), which stabilizes the gel
network by intermolecular hydrogen bonds when pH < pKa (Pawar &
Edgar, 2012). This explains the stability of the alginate under gastric
conditions as well as its instability under intestinal conditions. Several
authors have reported that alginate under acidic pH is converted into an
insoluble and porous structure known as alginic acid (Durán et al., 2020;
Vallée et al., 2009). Regarding the starch, some breaking of α(1–4) or
α(1–6) glycosidic bonds of amylose and amylopectin can occur, resulting
in the development of pores on the surface of beads. The porous struc­
ture (Fig. 3B3) allowed the diffusion of ferrous bisglycinate from the
alginate–starch core into the gastric digestate.
The heated powder and beads released even more bioaccessible iron
species due to the weakened chelate bonds in the ferrous bisglycinate
and disruption of the bead matrix (Fig. 2C1-2C4, 2D1-2D4). In the case
of FeBP-180, the bioaccessible fractions for total iron, Fe(II), and Fe(III)
were 83.43%, 78.57%, 92.51%, and in the case of FeBB-180, 24.05%,
21.47%, and 37.17%. The bioaccessible fraction of total iron of 18.30%
released from FeBB-20 under gastric conditions is within the range of
10–25% reported by Valenzuela et al. (2016) from sodium alginate–­
spray-dried bovine blood cells, and higher than the 2.4–11.1% reported
by Durán et al. (2020) for a matrix made of alginate–whey–ferrous
sulfate–bovine spray-dried blood cells. The speciation of bioaccessible
iron ions resulting from oral-gastric digestion of FeBP-20 and FeBB-20
revealed that both ferric and ferrous ions were present, but the ferrous
ions dominated (Table 4): 153.31 mg from FeBP-20, compared to 32.91

8
A. Mihaly Cozmuta et al. Food Hydrocolloids 142 (2023) 108808

Fig. 3. Digital photographs and SEM micrographs of surface and core of

alginate-starch-ferrous bisglycinate beads upon in vitro digestion process A1-
A12. Digital photographs of beads upon in vitro digestion process
B1-B12. SEM micrographs of outer surface of beads upon in vitro digestion
process
C1-C12. SEM micrographs of core of beads upon in vitro digestion process.

9
A. Mihaly Cozmuta et al.
mg from FeBB-20. The concentration of ferric ions was increased in the
supernatants from the heat-treated powder to a higher level than from
the heat-treated beads, especially for samples heated at 180 ◦ C. The
increase in the amount of bioaccessible ferric ions from FeBP-20 to
FeBP-180 was 6.91-fold, compared with 1.83-fold from the corre­
sponding beads, suggesting a protective effect of the alginate–starch
matrix against the oxidation of Fe(II) to Fe(III). At the end of
oral-gastric-intestinal digestion, the bioaccessible fractions of ferrous
ions were between 74.70% and 80.66%, and there were no significant
differences between samples (Fig. 4d). Bioaccessible ferric ions were not
detected in the supernatant of any of the samples. With the transition
from the gastric to the intestinal stage, the change from acidic pH to
neutral led to the precipitation of ferric ions as ferric hydroxides. This
tendency to precipitate at a pH above 3 is well known. The precipitation
of ferrous ions does not occur upon in vivo digestion. Natural gastric
juice provides both low pH and ligands derived from protein peptic
digestion, which, when combined with ferrous and ferric ions, maintain
them in a soluble form in the neutral environment of the intestine (Ja­
cobs & Miles, 1969), making them available for intestinal absorption.
The absence of such ligands in the simulated gastric juice used in our
experiments explains the precipitation of ferric ions during the intestinal
stage and their absence in the supernatant. The bioaccessible fraction of
total iron was between 52.51% and 72.46%. Significant differences were
observed only between total digestion of FeBP-180 and FeBB-180 and
those treated at 20 ◦ C (FeBP-20, FeBB-20) and 90 ◦ C (FeBP-90,
FeBB-90). Our value for total iron in FeBP-20, 71.50%, is higher than the
value of 57% reported by Perez-Moral et al. (2013), and below the range
of 90–100% reported by Churio et al. (2018b). As Table 4 shows, a
1.22-fold higher amount of bioaccessible Fe(II) was released from
FeBB-180 (147.85 mg) than from FeBP-180 (121.57). It is well known
that iron absorption occurs in the duodenum and proximal jejunum, and
the ferrous ions are more easily absorbed than ferric ions, which require
the ferrireductase to be converted to ferrous ions. Fig. 4a, b, and 4c show
the bioaccessible fraction of iron species released during the intestinal
stage alone, calculated as differences between the bioaccessible frac­
tions at the end of oral-gastric-intestinal digestion (Fig. 4d) and
10
Food Hydrocolloids 142 (2023) 108808
Fig. 4. The bioaccessible fractions for total iron, Fe
(II) and Fe(III) in the oral-gastric, intestinal and oral-
gastric-intestinal, respectively digestates.
a) Bioaccessible fractions of total iron in oral-gastric
and intestinal digestates
b) Bioaccessible fractions of Fe(II) ions in oral-gastric
and intestinal digestates
c) Bioaccessible fractions of Fe(III) ions in oral-gastric
and intestinal digestates
d) Bioaccessible fractions of total iron and Fe(II) ions
at the end of oral-gastric-intestinal digestion.
bioaccessible fractions after oral-gastric digestion. The ferrous ions were
released least from ferrous bisglycinate powder, and thermal treatment
did not result in significant differences between the bioaccessible frac­
tions. In contrast, much larger bioaccessible ferrous fractions were
released from the beads, and thermal treatment did not result in sig­
nificant differences. The release of ferrous bisglycinate powder from the
alginate–starch matrix during intestinal digestion may be attributed to
bile salts, which penetrate the beads through the fractures and pores that
were produced during drying or gastric digestion. Table 4 shows that,
depending on the temperature of the thermal treatment, during the in­
testinal stage alone, between 71.26% and 79.38% of the total amount of
bioaccessible ferrous ions was released from the beads, while only
2.59%–4% was released from ferrous bisglycinate powder.
3.4.3. Safety aspects
Beyond their functionality, the safety of the components in our iron-
based beads should also be discussed to identify vulnerable consumers.
The EFSA (2006) considers ferrous bisglycinate, as a source of iron in
foods intended for the general population, food supplements, and foods
for particular nutritional uses including foods intended for infants and
young children, to be safe if the levels do not exceed the levels currently
provided by programs within the EU. Starch consumption results in an
increase of postprandial blood glucose and insulin response in healthy as
well as diabetic individuals (Lal, Singh, Sharma, Singh, & Kumar, 2021).
The ability of starch to raise glycemic index depends on the starch source
and digestion rate. Potato starch could have a lower effect on blood
glucose compared to rice or wheat starch, due to a larger amount of
slowly digestible starch (Lal et al., 2021). Another advantage of potato
starch is the absence of gluten traces, which may trigger symptoms in
individuals with celiac disease. Even though our beads are made from
potato starch, further study is needed to assess their glycemic index. In
this way, their suitability in the diet of people with diabetes or obesity
can be established. Alginate, an algal polysaccharide found in the cell
walls of certain brown seaweed species, can attenuate the postprandial
glycemic response (Williams et al., 2004). The literature reported rare
cases of allergic reactions to alginate (Wan Ali & Ahmad Tarmidzi,
A. Mihaly Cozmuta et al. Food Hydrocolloids 142 (2023) 108808

2021). Labelling of allergenic component in the beads composition is an

Ferrous bisglycinate beads heated 90 essential step to ensure the safety of consumers.
min at 180 ◦ C FeBB-180 (2.545g)
4. Conclusions

This study provided insight into the thermal stability and in vitro
digestion of ferrous bisglycinate powder and ferrous bisglycinate beads

147.85 ± 6.04(a)

147.85 ± 6.04(a)
105.36 ± 3.84(c)

105.36 ± 3.84(c)
42.49 ± 1.74(c)
14.51 ± 0.58(c)

56.99 ± 1.87(c) in order to develop an effective carrier for oral delivery of iron in food

In each row, mean values with different letters are significantly different at P < 0.05. The differentiation was made based on the Tukey test post-hoc ANOVA (IBM SPSS Statistics Version 24).
subjected to thermal treatment. Ferrous bisglycinate powder was suc­
cessfully encapsulated as alginate:starch:ferrous bisglycinate powder:
water in a ratio of 2:1:2:100 (w:w:w:v). The geometry of the beads
–

–
changed under thermal treatment at 90 ◦ C and 180 ◦ C for 90 min. Wall
integrity was reduced and pores appeared in the core, which depended
Ferrous bisglycinate beads heated
90 min at 90 ◦ C FeBB-90(2.545g)

on the heating temperature. The encapsulation of ferrous bisglycinate

powder into an alginate–starch shell helped the ferrous ions withstand
oxidation upon heating, which is essential for its successful use as an
iron-fortifying agent in food subjected to thermal treatment. No signif­
icant differences were observed between the concentration of Fe(II) in
119.19 ± 2.00(a)

119.19 ± 2.00(a)

155.31 ± 7.24(a)

155.31 ± 7.24(a)
36.12 ± 1.55(cd)

46.06 ± 2.01(d)
9.93 ± 0.36(de)

beads not heated and those heated at 180 ◦ C, while in the case of ferrous
bisglycinate powder, the ferrous ion concentration was reduced by 1.40-
fold. In vitro digestion demonstrated that, regardless of the temperature
of thermal treatment, the beads released ferrous ions during intestinal
–

–
The amount of bioaccessible Fe(II), Fe(III) and total iron in the supernatant of oral-gastric, intestinal and oral-gastric-intestinal in vitro digestion.

digestion, the main site of iron absorption, whereas the ferrous bisgly­
Unheated ferrous bisglycinate

cinate powder released the largest amount of Fe(II) during gastric

digestion. By developing beads that provide thermal protection to Fe(II)
beadsFeBB-20(2.545g)

ions against oxidation and release them at the main site of absorption in
the gastrointestinal tract, our work suggests a new way to fortify foods
126.68 ± 3.28(a)

126.68 ± 3.28(a)

159.59 ± 5.23(a)

159.59 ± 5.23(a)
32.91 ± 0.90(d)

40.85 ± 1.75(d)
7.94 ± 0.355(e)

that will be subjected to thermal treatment. These findings need to be

validated by fortifying thermally treated foods with alginate-starch-
ferrous bisglycinate beads.
–

Funding sources
Ferrous bisglycinate powder heated

This study did not receive any specific grant from funding agencies in
90 min at 180 ◦ C FeBP-180(1g)

the public, commercial or not-for-profit sectors.

Declaration of competing interest

118.42 ± 4.36(b)

121.57 ± 4.93(b)

121.57 ± 4.93(b)
193.16 ± 3.76(a)

3.16 ± 0.15(d)

3.16 ± 0.15(d)
74.75 ± .96(a)

The authors declare that they have no known competing financial

interests or personal relationships that could have appeared to influence
the work reported in this paper.
–

Data availability
Ferrous bisglycinate powder heated
90 min at 90 ◦ C FeBP-90(1g)

The authors do not have permission to share data.

Results are expressed as average ± standard deviation SD (n = 3).

Acknowledgment
173.44 ± 5.02(b)
148.00 ± 3.04(a)

154.29 ± 6.10(a)

154.29 ± 6.10(a)
25.44 ± 0.33(b)

6.29 ± 0.24(d)

6.29 ± 0.24(d)

The instrumental support provided by the Scientific Research Center

of Environment, Food and Health of Technical University of Cluj Napoca
(Romania) and Warsaw University of Technology, Faculty of Materials
Science and Engineering (Poland) is gratefully acknowledged.
–

–
bisglycinate powder FeBP-20

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.

Oral-gastric-intestinal digestion
164.12 ± 5.76(b)
153.31 ± 4.48(a)

161.76 ± 7.89(a)

161.76 ± 7.89(a)
Unheated ferrous

10.81 ± 0.36(d)

org/10.1016/j.foodhyd.2023.108808.
8.45 ± 0.35(d)

8.45 ± 0.35(d)

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