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Effect of adding different thickening agents on the viscosity properties
and in vitro mineral availability of infant formula
Carlos A. González-Bermúdez
⇑
, Carmen Frontela-Saseta, Rubén López-Nicolás, Gaspar Ros-Berruezo,
Carmen Martínez-Graciá
Department of Food Science and Nutrition, Faculty of Veterinary Sciences, Regional Campus of International Excellence ‘‘Campus Mare Nostrum’’, University of Murcia, Spain
article info
Article history:
Received 29 November 2013
Received in revised form 17 February 2014
Accepted 27 February 2014
Available online 12 March 2014
Keywords:
Infant formula
Thickeners
In vitro digestion
Calcium
Mineral availability
Viscosity
Phytate content
abstract
The effect of adding different thickening agents (locust bean gum (LBG), modified corn and rice starches
(MCS, MRS)) to an infant formula on both in vitro mineral availability (Ca, Fe and Zn), quantified by atomic
absorption spectrophotometry (AAS), and formula viscosity, after in vitro gastrointestinal digestion, was
investigated. LBG was the most effective agent to increase formula thickness. However, it showed a neg-
ative effect on Ca, Fe and Zn in vitro solubility and dialysability. MCS and MRS only affected calcium sol-
ubility and dialysability when they were used at P50% of the maximum legal limit. No negative effect
was observed for Fe and Zn when modified starches were added at the different concentrations assessed.
The phytate content in the thickening ingredients was also analysed. Despite finding a considerable
amount of phytic acid in the raw ingredients, its final concentration in the infant formula was insufficient
to decrease in vitro mineral availability.
Ó2014 Elsevier Ltd. All rights reserved.
1. Introduction
Reflux episodes are frequent in infants, although most of them
are mild and brief. However, when this passage of gastric content
into the oesophagus is accompanied by abdominal pain, oesopha-
gitis or inspiratory disorders, as well as others pathological conse-
quences, therapeutic intervention becomes necessary (Horvath,
Dziechciarz, & Szajewska, 2008). In this regard, a variety of
approaches have been proposed, including pharmacological and
non-pharmacological therapies. In the treatment of non-compli-
cated gastroesophageal reflux, thickening of infant formulas has
commonly been recommended (Vandenplas, Hauser, Devreker,
Mahler, Degreef & Veereman-Wauters, 2013). Thickening agents,
such as locust bean gum (LBG) or modified starches, have fre-
quently been added to infant formulas with the aim of increasing
their viscosity. The efficacy of thickening agents depends on their
ability to increase gastric retention time, avoiding a return to the
oesophagus during the first digestion phase, and reducing almost
consistently the frequency and volume of regurgitation (Corvaglia,
Martini, Aceti, Arcuri, Rossini, & Faldella, 2013). Nevertheless, the
number of studies on the effect of thickening agents on rheological
properties of infant formulas is very limited, and those that do
exist have not made reference to the ideal viscosity value that will
lead to a positive effect on children (Bosscher, Van Caillie-Bertrand,
& Deelstra, 2003a; Miyazawa, Tomomasa, Kaneko, Arakawa, &
Morikawa, 2007; Miyazawa, Tomomasa, Kaneko, & Morikawa,
2004; Vanderhooh, Moran, Harris, Merkel, & Orenstein, 2003).
These types of products are commercialised under the name of
antireflux or antiregurgitation (AR) infant formulas, and are
promoted with the claim that they benefit infants who have
gastroesophageal reflux or who spit up regularly (Pina, Llach,
Ariño-Armengol, & Iglesias, 2008; Vandenplas, 2008; Vanderhooh
et al., 2003). The use of AR formulas has been recommended by
the North American and the European Society for Pediatric Gastro-
enterology, Hepatology, and Nutrition (NASPGHAN and ESPGHAN,
respectively) (Vandenplas et al., 2009) as far as it has been demon-
strated that these products significantly reduce regurgitation in
infants with recurrent vomiting. (Agget et al., 2002; Chao &
Vandenplas, 2007; Vandeplas, 2008; Vanderhooh et al., 2003)
Locust bean gum and modified starches, as ingredients for AR
formulas, are legally allowed in Europe, where different maximum
concentrations have been established for each group (European
Parliament and Council, 1995; European Parliament and Council,
2006). According to this legislation, modified starches may be
added to infant formulas up to either 30% of total carbohydrates
or 2 g/100 mL. In the case of LBG, it may be added up to a maximum
level of 10 g/L from birth onwards. These maximum levels are sim-
ilar to those recommended by ESPGHAN in its Global Standard for
the Composition of Infant Formula (Koletzko et al., 2005) and by
http://dx.doi.org/10.1016/j.foodchem.2014.02.168
0308-8146/Ó2014 Elsevier Ltd. All rights reserved.
⇑
Corresponding author. Tel.: +34 868 88 47 98; fax: +34 868 88 84 97.
E-mail address: cagb1@um.es (C.A. González-Bermúdez).
Food Chemistry 159 (2014) 5–11
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
the European Commission Scientific Committee for Food (1997).
Moreover, several authors have indicated the need to explore
further the effect of these ingredients on the nutrition and health
of infants, as some studies using an in vitro model have suggested
that the bioavailability of calcium, iron and zinc may be affected
by thickening agents and probably also by the presence of certain
antinutrients, such as phytic acid (Bosscher et al., 2000, 2003a;
Vandenplas et al., 2013). In particular, this antinutrient has been
reported as a strong chelator of multivalent metal ions, specifically
iron, zinc and calcium (Frontela, Haro, Ros, & Martinez, 2008).
With this background, the aim of this study was to evaluate the
effect of different concentrations of LBG and pregelatinised corn
and rice starches added to a commercial infant formula on both
viscosity and mineral (calcium, iron and zinc) availability in vitro.
2. Materials and methods
2.1. Samples
Standard infant formula (Hero Baby
Ò
1) was provided by Hero
España SA (Alcantarilla, Murcia, Spain). As thickening agents,
locust bean gum (Grinsted LBG 860, Danisco, Portugal), modified
corn starch (MCS; Multi-Thick
Ò
, Abbott Nutrition, Spain) and
modified rice starch (MRS; Beneo-Remy Industries, Belgium) were
selected. For the infant formula reconstitution, 200 mL of deionised
water were mixed with 30 g of powder according to the manufac-
turer’s instructions.
2.2. Materials and reagents
Deionised water (MilliQ; Millipore, Bedford, MA) was used
throughout the study. Pepsin (P-7000, from porcine stomach muco-
sa), bile salts (B-8756) and pancreatin (P-1750, from porcine pan-
creas) were purchased from Sigma (St. Louis, MO). To simulate
the gastrointestinal conditions of children less than 6 months of
age, pepsin solution was prepared by dissolving 1.6 g of pepsin in
10 mL of 0.1 N HCl. The pancreatin-bile extract solution was pre-
pared by dissolving 0.2 g of pancreatin and 1.25 g of bile in 50 mL
of 0.1 M NaHCO
3
. The working solutions of these enzymes were
prepared immediately before use. For mineral dialysis assays, dial-
ysis membranes with molecular mass cut-offs (MMCO) of
12,000 Da were purchased from Medicell Intl Ltd., London, UK).
Ca, Fe and Zn contents were determined by flame atomic absorption
spectroscopy (AAS) according to the AOAC method (Jorhem 2000).
The glass material was washed with detergent, soaked in concen-
trated nitric acid (SG 1.41) and rinsed three times with distilled
deionised water before use. In the case of calcium determination,
a lanthanum chloride 1% (w/v) solution (Fluka Analytical, Buchs,
Switzerland) was used to suppress phosphate interferences.
2.3. Sample preparation
Different concentrations of each thickening agent were added
to the standard infant formula (Hero Baby
Ò
1), and then samples
were homogenised using a VH-5 high-efficiency mixer (Comecta
SA, Barcelona, Spain). As can be seen in Table 1, for each thickener,
the selected concentrations were 7.5%, 15%, 50% and 100% of their
respective maximum legal limit (European Parliament and Council,
2006, 1995).
2.4. Inositol phosphate (IPs) extraction and measurement
Inositol phosphates (IPs), including phytic acid (myo-inositol
hexaphosphoric acid), were extracted from the different samples
with 0.5 N HCl at room temperature for 2 h. Each extract was then
centrifuged and the supernatant frozen overnight, followed by
thawing and centrifugation. An aliquot of supernatant was poured
onto an anion exchange (SAX) column (500 mg; Supelco, Belle-
fonte, PA) connected to a vacuum manifold set at 20 mmHg. The
resin-bound inositol polyphosphates were eluted with 2 mL of
2 M HCl. Eluted samples were evaporated to dryness in vacuo at
40 °C and dissolved in 1 mL of deionised water.
Inositol phosphates were determined by LC–MS (Liu, Villalta, &
Sturla, 2009) using reverse-phase chromatography on an Agilent
1100 series (Agilent Technologies, Santa Clara, CA, USA) HPLC sys-
tem equipped with a thermostated micro-well plate autosampler
and a quaternary pump, and connected to an Agilent Ion Trap
XCT Plus mass spectrometer (Agilent Technologies) using an elec-
trospray interface (ESI).
Samples and standards (40
l
L) were injected into a C18 reverse-
phase HPLC column (Agilent Technologies), thermostated at 40 °C,
and eluted at a flow rate of 200
l
L/min throughout the separation.
Samples were passed through 0.22-
l
m HPLC filters before injec-
tion. The mobile phase consisted of two solvents: solvent A, 0.1%
formic acid in water; and solvent B, 0.1% formic acid in acetonitrile.
Inositol phosphates were eluted as follows: from 10% to 100% Bin
30 min; from 100% to 10% Bin 15 min; an isocratic elution of 10%
was maintained from 45 to 60 min to equilibrate the column under
the initial conditions.
The mass spectrometer was operated in negative ion mode with
a capillary spray voltage of 3500 V, and a scan speed of 2200 amu/s
from m/z 50–750. The nebuliser gas pressure, drying gas flow rate
and drying gas temperature were set at 30 psi, 8 L/min and 350 °C.
Control and data acquisition of the HPLC–MS equipment was per-
formed with Agilent Chemstation Rev B.01.03.SR2. Data were
Table 1
Composition (g of thickening agent/100 g of infant formula) and mineral content (mg/100 g of infant formula) per sample.
Sample Thickening agent (%) LBG (g/100 g) MCS (g/100 g) MRS (g/100 g) Ca (mg/100 g) Fe (mg/100 g) Zn (mg/100 g)
1 0 – – – 398 ± 27.9 4.98 ± 0.27 3.81 ± 0.14
2 7.5 0.5 – – 374 ± 24.4 4.80 ± 0.15 3.81 ± 0.15
3 7.5 – 1 – 294 ± 17.2 4.97 ± 0.28 3.92 ± 0.18
4 7.5 – – 1 303 ± 10.6 5.22 ± 0.11 4.59 ± 0.02
5 15 1 – – 338 ± 6.36 5.34 ± 0.13 4.56 ± 0.03
6 15 – 2 – 296 ± 24.4 4.62 ± 0.12 4.01 ± 0.08
7 15 – – 2 301 ± 5.11 4.76 ± 0.15 4.19 ± 0.14
8 50 3.36 – – 342 ± 6.19 4.85 ± 0.36 3.98 ± 0.07
9 50 – 6.66 – 394 ± 2.37 5.11 ± 0.07 4.04 ± 0.08
10 50 – – 6.66 301 ± 22.0 4.48 ± 0.13 3.99 ± 0.05
11 100 6.67 – – 279 ± 5.37 5.11 ± 0.06 4.22 ± 0.11
12 100 – 13.33 – 381 ± 12.8 4.82 ± 0.04 4.23 ± 0.06
13 100 – – 13.33 362 ± 30.5 5.18 ± 0.11 4.18 ± 0.05
LBG: locust bean gum; MCS: modified corn starch; MRS: modified rice starch.
6C.A. González-Bermúdez et al. / Food Chemistry 159 (2014) 5–11
processed using the data analysis software for LC/MSD Trap ver-
sion 3.3 (BrukerDaltomik, GmbH, Bremen, Germany) provided by
the manufacturer.
2.5. In vitro digestion
To measure the solubility and dialysability of iron, calcium, and
zinc, each sample was reconstituted in rapidly stirring and pre-
heated (37 °C) deionised water according to the manufacturer’s
recommendations. After reconstitution, samples were digested
using the widespread in vitro method described by Boato, Wortley,
Liu, and Glahn (2002), with modifications aimed at reducing the
amounts of enzymes used, since the gastrointestinal tract in the
early stages of life is not yet fully developed (Frontela, Ros, &
Martinez, 2009; Frontela et al., 2008). The in vitro digestion pro-
cess, which consists of gastric and intestinal stages, was performed
at 37 °C. At the end of the intestinal stage, aliquots of 20 g of sam-
ple were transferred to 50-mL polypropylene centrifuge tubes
(Costar Corning Europe, Badhoevedorp, Netherlands) and then cen-
trifuged (Eppendorf 5804-R Centrifuge, Hamburg, Germany) at
3500gfor 1 h at 4 °C. The supernatant (soluble fraction) was used
to determine the mineral content. Dialysis comprised the gastric
stage, followed by an intestinal step in which a dialysis bag con-
taining 50 mL of deionised distilled water and an amount of
NaHCO
3
equivalent to the titratable acidity (previously measured)
was placed in flasks containing 20-g aliquots of the pepsin digest.
The iron, calcium and zinc dialysed through the semipermeable
membrane represent the bio-available fraction (expressed as a per-
centage) of the total minerals present in the sample (Etcheverry,
Grusak, & Fleige, 2012; Frontela, Ros, & Martínez, 2011).
2.6. Determination of mineral content
The Fe, Ca and Zn concentrations in samples and the mineral
soluble and dialysable fractions were determined by AAS (Thermo
Scientific AA Spectrometer S Series; Thermo, Waltham, MA). Prior
to analyses, the organic matter was destroyed in an ashing oven
(Nabertherm, Lilienthal, Germany) at 525 °C for 32 h. Three millili-
tres of HNO
3
were then added to the ashes and the samples were
heated to dryness. After cooling, the residue was dissolved with
1 mL of HCl (SG 1.9), and the solution was transferred to a 10-mL
volumetric flask and made to volume with water. The mineral con-
tent in the diluted, acidified samples was determined against Fe, Ca
and Zn standard solutions (Merck, Germany). The calibration
curves obtained were between 1 and 15 ppm for Ca, 0.25 and
5 ppm for Fe and 0.25 and 2 for Zn, and showed an acceptable lin-
earity with correlation coefficients greater than 0.995. Mineral sol-
ubility and dialysability (%) were calculated as follows (Frontela
et al., 2011):
Soluble ð%Þ¼ Soluble mineral content ðmg=100 gÞ
Total mineral content of the sample ðmg=100 gÞ
100
Dialysable ð%Þ¼ Dialysable mineral content ðmg=100 gÞ
Total mineral content of the sample ðmg=100 gÞ
100
2.7. Validation criteria for the AAS technique
The reference material obtained from the International Atomic
Energy Agency (IAEA) 153-Milk Powder (Vienna, Austria) was used
as a control to test the method for accuracy. Fe, Ca and Zn were
analysed in the reference material. The measured mean values
(n= 3) for Fe, Ca and Zn were 2.04, 13125 and 37.98
l
g/g
respectively, which were in accordance with the certified range
of 2.56 ± 1.28
l
g/g for Fe, 12855 ± 445
l
g/g for Ca, and 39.45 ±
2.52
l
g/g for Zn. The precision of the method was calculated from
the results obtained in the analysis of the soluble mineral fraction
from six aliquots of a sample. The values expressed as coefficient of
variation (%), were 1.19 for iron, 0.99 for calcium and 1.00 for zinc.
2.8. Viscosity measurement during in vitro gastric digestion
Viscosity, understood as a quantitative rheological measure-
ment of frictional resistance to shear in a fluid, was measured in
each sample, as well as in the standard formula (Hero Baby
Ò
1).
This was done using a rotational viscometer (Haake Viscotester
VT6L plus, Thermo Electron Corporation, Germany) with a number
1 spindle at 60 rpm and 37 °C. With the aim of characterising the
rheology of each agent added to the standard formula, four mea-
surements were made for each sample during the first stage of
in vitro digestion: immediately after reconstitution (0), just after
pH adjustment and enzyme addition (beginning of digestion) (1),
after 30 min (2), after 60 min (3), and after 120 min (4) of
in vitro gastric digestion.
2.9. Statistical analysis
All experiments were carried out six times, and the results were
reported as means ± SD. After confirming the data normality using
the Kolmogorov–Smirnov test and homoscedasticity by the Levene
test, solubility and dialysability of the samples were compared by
one-way analysis of variance (ANOVA) and a Tukey post-test for
multiple comparisons to determine the significance of the effects
of different thickening agents used at the same legal concentration
level (p< 0.05). For each level, the results were compared with the
standard formula. Pearson’s correlation test was performed to
investigate the relationship between the concentration of each
thickening agent’s viscosity values, phytate content and Ca, Fe
and Zn solubility and dialysability. Values of p< 0.05 (two-tailed)
were considered significant. All statistical analyses were per-
formed with the Statistical Package for the Social Sciences (SPSS
version 14.0; SPSS Inc., Chicago, IL).
3. Results and discussion
3.1. Effect of different concentrations of thickening agents on in vitro
mineral availability in infant formulas
The three measured minerals (Ca, Fe and Zn) assessed in infant
formula were aligned to the levels recommended by infant formula
regulation (Commission Directive, 2006). Differences observed in
mineral content between samples can be considered negligible
and are attributed to the different proportions of thickening agents
used (Table 1). The effect of the concentration of each thickening
agent on Ca, Fe and Zn solubility and dialysability percentages of
infant formulas are shown in Figs. 1 and 2, respectively.
Regarding mineral solubility, calcium was the only mineral neg-
atively affected by the three thickening agents when they were
added in high concentrations (>50% of maximum legal limit) com-
pared with the standard formula (Hero Baby
Ò
1 without thickening
agents). This negative effect was significantly higher (p< 0.05) for
LBG than for modified starches at the same concentration (100%).
This could be explained by the higher amount of IP
6
(myoinositol
hexaphosphoric acid) in LBG (47.3 mg/100 g) compared with
amounts observed in modified starches (19.2 mg/100 g in MCS
and 17.5 mg/100 g in MRS). In this regard, it must also be noted
C.A. González-Bermúdez et al. / Food Chemistry 159 (2014) 5–11 7
that during the gastrointestinal process, optimal conditions for
a
-amylase were achieved (Frontela et al., 2009).
Based on this, total starch loss is probably responsible for break-
ing modified starches into oligosaccharides. Moreover, these con-
ditions might favour endogenous phytase activity, which has an
optimal temperature of around 55 °C and which depends on the
phytate content. When iron solubility was studied, a significant
negative effect was observed when LBG was added in high concen-
trations (>50% of maximum legal limit), whereas the solubility of
zinc was reduced only by LBG at 100% of maximum legal limit).
No negative effect occurred when modified starches were used as
thickeners. Moreover, at the different concentrations of MCS, a sig-
nificant improvement of zinc solubility with respect to standard
formula was found. In this context, an explanation could be found
relating to the content of the hexa (IP
6
) and penta (IP
5
) forms of IP
detected in the thickening agents added to the infant formula
(Hurrell, 2004). However, we observed that the phytate (IP
5
+IP
6
)
concentrations measured in LBG, MCS and MRS provided infant
formula with a phytate/mineral molar ratio that was lower than
values reported to be critical in all the proportions studied
(Table 2). Although the critical phytate/iron molar ratio has not
been well established, according to Hurrell (2004), for an optimal
mineral absorption, it should be reduced to below 0.4:1. In the case
of calcium, it should not be higher than 0.24 (Gibson, Bailey, Gibbs,
& Ferguson, 2010) and in the case of zinc, ratios above 1.5:1 may
inhibit zinc availability (Ma, Li, Jin, Zhai, Kok & Yang, 2007).
As can be seen on Table 2, LBG showed the highest amount of
phytate (109 mg/100 g) when compared to MCS and MRS (36.2
and 41.4 mg/100 g, respectively). However, as a result of the legal
limit of addition for each thickening agent (European Parliament
and Council, 2006, 1995), the final content of phytate in infant
formula did not compromise the mineral availability.
Concerning mineral dialysability, calcium was affected by the
addition of LBG at concentration levels of 50% and 100%, and by
MCS/MRS at all the tested concentration levels. When iron and zinc
dialysability were analysed, only formula added with a 50% and
Fig. 1. Effect of different concentration levels for each thickening agent, on Ca, Fe and Zn solubility. Different superscripts (a–c) indicate significant differences (p< 0.05)
between Ca, Fe or Zn solubility within the same concentration level for each thickening agent, locust bean gum (LBG), modified corn starch (MCS) and modified rice starch
(MRS). Data expressed as mean ± SD.
8C.A. González-Bermúdez et al. / Food Chemistry 159 (2014) 5–11
100% concentration of LBG showed a significantly negative effect,
which was more important for iron (2.224% and 1.857% for LBG
concentrations of 50% and 100%, respectively) than for zinc
(7.656% and 7.541% for LBG concentrations of 50% and 100%,
respectively). Meanwhile, MCS and MRS did not seem to affect iron
or zinc dialysability when compared with standard formula. These
results are in agreement with those observed by Bosscher, Van
Caillie-Bertrand, Van Cauwenberg, and Deelstra (2003b), who stud-
ied dairy infant formulas with pregelatinised starches added. They
reported that LBG brought about reduced availability of both Fe
and Zn. Meanwhile, pregelatinised starches used as thickeners
increased mineral availability compared to non-thickened infant
formulas.
Fig. 2. Effect of different concentration levels for each thickening agent, on Ca, Fe and Zn dialysability. Different superscripts (a–c) indicate significant differences (p< 0.05)
between Ca, Fe or Zn dialysability within the same concentration level for each thickening agent, locust bean gum (LBG), modified corn satrach (MCS) and modified rice starch
(MRS). Data expressed as mean ± SD.
Table 2
Inositol and inositol phosphates (IP) content (mg/100 g) in each thickening agent.
LBG MCS MRS
IP
6
47.3 ± 1.05 19.2 ± 0.50 17.5 ± 0.39
IP
5
61.7 ± 1.21 17.0 ± 0.52 23.9 ± 0.91
IP
4
13.2 ± 0.63 12.8 ± 0.34 20.0 ± 0.73
IP
3
77.5 ± 0.95 11.7 ± 0.25 21.3 ± 0.72
IP
2
13.4 ± 0.31 13.5 ± 0.44 18.4 ± 0.50
IP
1
9.98 ± 0.20 4.27 ± 0.52 14.1 ± 0.61
Inositol 3.61 ± 0.43 3.62 ± 0.31 3.53 ± 0.53
LBG: locust bean gum; MCS: modified corn starch; MRS: modified rice starch.
Table 3
Pearson’s correlation coefficients between different concentrations of each thickening
agent and Ca, Fe and Zn solubility and dialysability percentages.
Solubility Dialysability
LBG MCS MRS LBG MCS MRS
Ca 0.909
*
0.793
*
0.866
*
0.953
*
0.733
*
0.861
*
Fe 0.912
*
0.483 0.437 0.912
*
0.632
*
0.837
*
Zn 0.834
*
0.187 0.710 0.783
*
0.016 0.279
LBG: locust bean gum; MCS: modified corn starch; MRS: modified rice starch.
*
p< 0.05.
C.A. González-Bermúdez et al. / Food Chemistry 159 (2014) 5–11 9
To determine a possible linear relationship between the different
concentrations of each thickening agent added to infant formula and
Ca, Fe and Zn solubility and dialysability, a Pearson correlation was
run. The results are shown in Tables 3 and 4. A significant negative
correlation was found for Ca, Fe and Zn solubility (0.909, 0.912
and 0.834, respectively) and dialysability (0.953, 0.912 and
0.783, respectively) when LBG was used as a thickening agent.
These results are in accordance with the negative effect of LBG on
mineral availability observed in the present study. However, this
significant negative effect was only found for Ca when MCS and
MRS were added to the standard infant formula, but not for Fe or
Zn. The explanation for these findings could be that, due to the pres-
ence of ionisable groups, LBG could bind Ca, Fe and Zn, forming
unabsorbable complexes and decreasing mineral solubility and dial-
ysability. In addition, LBG might decrease the availabilities of Ca, Fe
and Zn as a consequence of its gel-forming capacities. This will cre-
ate a viscous environment in the small intestine, and thus impair the
digestion of food components. Related to this, Bosscher et al. (2003b)
suggested that LBG could form strong complexes with metal ions,
rendering them unavailable for absorption.
Regarding modified or pregelatinised starches, MCS and MRS
might be considered digestible carbohydrates. According to this
possibility, during digestion process, bound minerals could be
released to the intestinal lumen, increasing mineral availability in
comparison with LBG. Nevertheless, the in vitro availability of cal-
cium seems to be decreased, which could be due to the formation
of unabsorbable complexes (Agget et al., 2002; Commission
Directive, 2006). The results of similar published in vitro studies
also suggest that the bioavailability of Ca, Fe and Zn in infant
formula may be decreased by thickened formulas with non-
digestible carbohydrates such as LBG, but not by those with
modified starches added (Bosscher et al., 2000, 2003a; Commission
Directive, 2006).
3.2. Effect of thickening agent concentrations on in vitro viscosity
The viscosity values of infant formulas mixed with different con-
centrations of each thickening agent are presented in Table 4. The
results are grouped according to the time of measurement and con-
centration of the thickening agent. Overall, after pH adjustment to 4
with 6 N HCL, the viscosity of formulas increased in relation to their
respective viscosities just after reconstitution, being more evident
at a higher concentration of thickening agents (15% or greater). This
finding has been also described by other authors (Vanderhooh et al.,
2003). Although the viscosity-increasing mechanism does not seem
to be well-known, it might have to do with the physical interaction
between the thickening agents and protein components in the
assayed product.
Comparing viscosity values, standard infant formula with a LBG
concentration of 15% or greater, showed a significantly higher
(p< 0.05) viscosity value than that for the standard infant formula
with MCS and MRS added. Focusing on the viscosity provided by
LBG, it reached values higher than 60 cps and 100 cps for a 50%
and 100% concentration, respectively, under the maximum legal
limit (3.36 g or 6.67 g of LBG per 100 g of infant formula). In con-
trast with these values, MCS and MRS provided viscosity values
higher than 20 cps and 27 cps for a 50% and 100% concentration,
respectively, under the maximum legal limit (6.66 g or 13.33 g of
starches per 100 g of formula).
Due to the lack of clinical trials or in vivo assays, no minimum
viscosity limits for infant regurgitation management in children
can be fixed. Nevertheless, a viscosity higher than 100 cps could ex-
ert a negative effect, since oesophageal clearance of refluxed mate-
rial would be reduced (Infante-Pina, Lara-Villoslada, López-Ginés, &
Morales-Hernández, 2010). This would affect the oesophageal acid
exposure time, increasing the risk of oesophagitis secondary to
gastric content reflux (Corvaglia et al., 2013). Moreover, if the vis-
cosity of infant formula is too high, infants could reject it, since they
may have trouble sucking such a formula through a standard
nipple.
4. Conclusions
LBG, MCS and MRS are frequently used as thickening ingredi-
ents in AR-infant formulas. In the present study, the effect of their
addition to a standard infant formula on both, mineral in vitro
availability (solubility and dialysability percentages measured by
AAS) and viscosity has been determined. Analysing the viscosity
results, it can be concluded that LBG is more effective as a thicken-
ing agent than MCS or MRS when added to infant formula under
the legal limits. However, LBG negatively affects the availability
of calcium, iron and zinc in vitro, decreasing mineral solubility
and dialysability, whereas MCS and MRS only affect calcium solu-
bility and dialysability in a negative way. These findings should be
taken into account when developing an antiregurgitation infant
formula, since the acceptance of foods by infants and mineral
requirements could be potentially compromised. A possible solu-
tion could be the adequate combination of these thickening agents
to minimise the negative effect on mineral availability while max-
imising the effect on formula viscosity.
With the aim of characterising the in vivo effects of these ingre-
dients, it would be necessary to conduct clinical trials or in vivo
Table 4
Viscosity of formulas (cps) with different under maximum legal limits (7.5%, 15%, 50% and 100%) of thickening agents during in vitro gastric digestion.
Concentration (%) Thickening agent After reconstitution Beginning of digestion 30 min 60 min 120 min
Time of measurement
0 LBG 1.27 ± 0.140 2.13 ± 0.052 2.46 ± 0.057 1.90 ± 0.086 1.64 ± 0.127
7.5 LBG 2.65 ± 0.174
b
3.42 ± 0.342
b
3.44 ± 0.204
b
3.01 ± 0.158
b
2.97 ± 0.132
b
MCS 3.39 ± 0.169
a
4.08 ± 0.180
a
3.99 ± 0.113
a
3.94 ± 0.082
a
3.7 ± 0.228
a
MRS 3.67 ± 0.126
a
4.24 ± 0.063
a
4.03 ± 0.092
a
3.77 ± 0.152
a
3.64 ± 0.199
a
15 LBG 5.09 ± 0.201
a
6.13 ± 0.123
a
5.90 ± 0.094
a
5.75 ± 0.193
a
5.56 ± 0.144
a
MCS 4.67 ± 0.233
b
6.57 ± 0.281
a
4.96 ± 0.135
b
4.97 ± 0.190
b
4.56 ± 0.239
b
MRS 4.01 ± 0.161
c
5.47 ± 0.247
b
5.00 ± 0.126
b
4.72 ± 0.139
b
4.43 ± 0.160
b
50 LBG 68.61 ± 1.214
a
86.6 ± 1.279
a
74.14 ± 1.862
a
67.57 ± 2.878
a
60.2 ± 1.574
a
MCS 18.36 ± 0.463
b
26.9 ± 0.301
b
25.59 ± 0.506
b
23.23 ± 0.507
b
21.5 ± 0.963
b
MRS 18.59 ± 0.369
b
22.6 ± 0.955
c
22.00 ± 0.104
c
21.98 ± 0.273
b
21.9 ± 0.170
b
100 LBG 109.2 ± 6.337
a
136 ± 4.475
a
130.66 ± 5.376
a
119.96 ± 5.201
a
102 ± 3.252
a
MCS 22.43 ± 0.494
b
29.3 ± 0.274
b
33.18 ± 0.345
b
30.27 ± 0.656
b
27.1 ± 0.158
b
MRS 25.7 ± 0.212
b
30.4 ± 0.455
b
28.06 ± 0.185
b
27.98 ± 0.111
b
27.1 ± 0.222
b
Within the same concentration level, different superscripts (a–c) for the same time of measurement, indicate significant differences (p< 0.05) between thickeners.
LBG: locust bean gum; MCS: modified corn starch; MRS: modified rice starch.
10 C.A. González-Bermúdez et al. / Food Chemistry 159 (2014) 5–11
assays in infants. This would lead not only to a better understand-
ing of the nutritional management of infant regurgitation, but also
to the establishment of an effective thickening agent concentration
which leading to a reduction in regurgitation episodes. In these
types of studies, an interesting and innovative focus will be to ana-
lyse the effect of thickening agents on colonic microbiota.
Acknowledgements
We would like to thank Hero España S.A. (Alcantarilla, Murcia,
Spain) for providing the samples for this study. We would like to
thank also to Dr. Alejandro Torrecillas from CAID (University of
Murcia, Spain) for the performance of the HPLC/MS analysis. This
study was supported by the Seneca Foundation and Science and
Technology Agency (Murcia Region, Spain) through the Project
(11978/PI/09). Carlos A. González Bermúdez was supported by a
predoctoral grant from the University of Murcia.
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