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In vitro Binding Capacity of Wheat and Barley for Mn, Zn, Cu and Fe

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In vitro binding capacity of organic (wheat bran

and rice bran) and inorganic (perlite) sources for
Mn, Zn, Cu, and Fe

Ali Ghodrat, Akbar Yaghobfar, Yahya Ebrahimnezhad, Habib Aghdam

Shahryar & Abolfazl Ghorbani

To cite this article: Ali Ghodrat, Akbar Yaghobfar, Yahya Ebrahimnezhad, Habib Aghdam
Shahryar & Abolfazl Ghorbani (2015): In vitro binding capacity of organic (wheat bran and
rice bran) and inorganic (perlite) sources for Mn, Zn, Cu, and Fe, Journal of Applied Animal
Research, DOI: 10.1080/09712119.2015.1124338

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 JOURNAL OF APPLIED ANIMAL RESEARCH, 2015
http://dx.doi.org/10.1080/09712119.2015.1124338

In vitro binding capacity of organic (wheat bran and rice bran) and inorganic (perlite)
sources for Mn, Zn, Cu, and Fe
Ali Ghodrata, Akbar Yaghobfarb, Yahya Ebrahimnezhada, Habib Aghdam Shahryara and Abolfazl Ghorbania
a
Department of Animal Science, Shabestar Branch, Islamic Azad University, Shabestar, Iran; bAnimal Science Research Institute, Karaj, Iran

ABSTRACT ARTICLE HISTORY

This study was carried out to determination of in vitro binding capacity of wheat bran, rice bran, and perlite Received 27 August 2014
for Mn, Zn, Cu, and Fe. For this reason, wheat bran and rice bran were chemically analysed for moisture, Accepted 20 November 2015
protein, ash, ether extract, total carbohydrate, neutral detergent fibre, acid detergent fibre, acid detergent
KEYWORDS
lignin, crude fibre, cellulose, hemicelluloses, phytic acid, Non-Fiber Carbohydrate, total dietary fibre, Mineral binding; wheat bran;
soluble dietary fibre, insoluble dietary fibre, DF + lignin, and endogenous minerals. Mineral binding rice bran; perlite; cell wall
capacity of wheat bran, rice bran, and perlite for Mn, Zn, Fe, and Cu under sequential simulated
physiological conditions of the stomach, small intestine, and colon was investigated and compared.
Rice bran showed the highest mineral-binding capacity in small intestine for Mn, Zn, and Cu. Among
the minerals, Mn exhibited the lowest percentage of binding under the simulated physiological
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conditions of the small intestine and the highest percentage of removing occurred in the stomach for
all of the treatments. Cereal bran’s had an affinity for Cu > Zn > Fe > Mn. The amounts of minerals
bound varied among samples. Rice bran showed the highest and perlite had lowest mineral-binding
capacity in small intestine for Mn, Zn, and Cu.

1. Introduction found to significantly affect the in vitro mineral-binding capacity

of DF in numerous previous studies (Camire & Clydesdale 1981;
Recommendation for the increase of dietary fibre (DF) intake has
Elhardallou & Walker 1999; Laszlo 1989; Platt & Clydesdale
raised questions about their possible negative effects on mineral
1987). Neutral detergent fibre (NDF) consists of cellulose,
bioavailability (Idouraine et al. 1995; Idouraine, Khan, Kohlhepp,
lignin, and hemicellulose and is often called cell walls.
et al. 1996). Although this issue has raised interest among
Because NDF represents the total fibre in a feed. Acid detergent
many scientists for the last two decades, conflicting results
fibre (ADF) consists of cellulose, lignin, lignified nitrogen com-
from numerous in vitro (Idouraine et al. 1995; Luccia & Kunkel
pounds (heat-damaged protein), and insoluble ash. ADF does
2002; Persson et al. 1987) and in vivo (Coudray et al. 1997,
not represent the total fibre content in feed, as it does not
2003; Moak et al. 1987; Shah et al. 1990) studies have made it dif-
account for hemicellulose. It is a relatively quick method for
ficult to draw a clear conclusion on this controversial subject.
measuring fibre, often substituting for CF.
By reviewing the adverse effects of DF on mineral bioavailabil-
Non-fibre carbohydrate is made up of different amounts of
ity in previous literatures (Harland 1989; Kelsay 1986; Munoz &
starch, simple sugars, betaglucans, galactans, and pectins
Harland 1993), three postulations on how DF might limit
(soluble fibre) that make up the highly digestible carbohydrate
mineral bioavailability have been suggested. They are (a) short-
fraction in feeds. Non-Fiber Carbohydrate (NFC) is calculated by
ening the transit time of nutrients when DF moves along the
the difference [100 – (%NDF + %CP + %fat + ash)]. The term non-
small intestine and thus reducing the time required for mineral
structural carbohydrate is often used interchangeably with NFC
absorption; (b) directly or indirectly impairing the transportation
but is analytically determined and may be slightly different from
of minerals when DF move across the intestinal mucosa cells; and
Non-Fiber Carbohydrate (NFC). The aim of the study was to deter-
(c) electrostatistic binding and/or trapping of minerals within DF
mine how DF has the ability to bind cations in vitro. However,
particles, leading to the formation of stable, unabsorbable
many components in the diet and the physiological conditions
mineral–fibre complexes and thus reducing the pool of ionized
like colonic bacteria in the gastrointestinal tract will potentially
minerals for absorption. Because the last population is believed
interfere with these binding characteristic.
to be the main factor determining the undesirable effect of DF
on the mineral bioavailability (Laszlo 1989). 2. Material and methods
When DF is ingested, it generally passes through three major
2.1. Materials
gastrointestinal sections including the stomach, small intestine,
and colon (Ji 1998). These three gastrointestinal sections Organic sources of whole fibres were wheat and rice bran (local
possess unique physiological conditions (such as regional pH, produce) and perlite (local source) was the inorganic source in
ionic strength, and average transit time), which had been this study.

CONTACT Akbar Yaghobfar [email protected]

© 2015 The Author(s). Published by Taylor & Francis
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.
 2 A. GHODRAT ET AL.

2.2. Analytical methods small intestinal physiological conditions were further incubated
with 2.0 mM MES buffer solution (pH 5.8; ionic strength = 100
The moisture content of the samples was determined by
mMKCl; 1:100, w/v) in a 250 mL Erlenmeyer flask for 3 h at 37°
heating circa 1 g in an oven, provided with a fan, for 16 h at
C. The resulting mixture was centrifuged at room temperature
105 ± 2°C and 2 h at 130 ± 2°C. The ash content was determined
(2500g, 15 min), and the residue was washed several times
in a muffle furnace by slowly heating 0.5–2.0 g samples (in por-
with ultrapure water until the pH value was approximately 7
celain crucibles) up to 500°C for 16 h. Their protein content was
(samples treated under colonic conditions). All samples
estimated from the quantitative analysis of nitrogen using the
treated under colonic conditions were then freeze-dried, and
Kjeldahl method (N × 6.25) (Egan et al. 1981). It was determined
those samples were retained for mineral analysis.
by a KjeltecTM (Foss, 8400). NDF, ADF, acid detergent lignin
(ADL), and crude fibre were determined according to the Van
Soest (1963) method using FibertecTM (Foss, 2010). Crude fat 2.4. Mineral content determination
was determined according to AOAC (1990). Phytic acid
content was determined according to De Boland et al. (1975). About 0.5 g of the original (endogenous) samples treated under
Also, DF contents of wheat and rice bran were determined by gastric conditions, small intestinal conditions, and colonic con-
using Megazyme assay kits (Megazyme International Ireland ditions were wet-ashed sequentially with nitric acid (65%) and
Ltd, Wicklow, Ireland) according to Approved Methods of the hydrogen peroxide (30%) followed by HCl. Excess HNO3 was
American Association of Cereal Chemists (AACC 2000). evaporated on a hot plate set at 100–120°C. The Mn, Zn, Fe,
and Cu concentrations in the ashed solution were then ana-
lysed by using a flame atomic absorption spectroscopy as
2.3. In vitro mineral-binding capacity
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described by AOAC (1990) (Spectro AA, VARIAN).

The in vitro mineral-binding capacity of the wheat bran, rice
bran, and perlite on Cu, Zn, Fe, and Mn was investigated 2.5. Statistical analysis
under sequential simulated physiological conditions of the gas-
trointestinal tract according to the modified method of Idour- A total of three samples for each treatment were used for the
aine, Khan, and Weber (1996). About 25 g of each sample was present report. SPSS10 was used for statistical analysis. Exper-
gently shaken in a 1% HCl solution (pH: 1.5; ionic strength: imental data, expressed as mean ± SD, were analysed by one
75 mM KCl; 1:20 w/v) in a 600 mL beaker for a period of 1 h at way ANOVA. The differences between means were separated
37°C in order to assess its mineral-binding capacity in the by Duncan’s multiple range tests. Significant differences were
stomach. The slurry was then filtered through a Whatman 541 declared at p < .05.
ashless filter paper, and the residue was washed several times
with ultrapure water until the filtrate tested neutral (pH 7)
3. Results
(samples treated under gastric conditions). All samples treated
under gastric conditions were then freeze-dried, and those Chemical composition as moisture, protein, ash, ether extract,
samples were kept for analysis of minerals. For determining and total carbohydrate content of the wheat and rice bran
the mineral-binding capacity under simulated physiological are presented in Table 1. Other way, amount of NDF, ADF,
conditions of the small intestine, 4 g of each sample was separ- ADL, phytic acid, and crude fibre of the wheat and rice bran
ately mixed in 40 mL of mineral solution (1000 mg/L containing are shown in Table 2. Thus, polysaccharide in the cell wall as
39% Mn (MnSO4·H2O), 38% Zn (ZnSO4·7H2O), 19% Fe hemicelluloses, cellulose, total dietary fibre, soluble dietary
(FeSO4·7H2O), and 4% Cu (CuSO4·5H2O). Then the volume of fibre, insoluble dietary fibre, and DF + lignin content of wheat
the mixture was made up to 400 mL by adding 2.0 mM and rice bran are presented in Table 3.
MES (Merck, Germany) buffer solution (pH 6.8; ionic The endogenous Mn, Zn, Cu, and Fe contents of wheat bran,
strength=100 mMKCl) and incubated at 37°C for 3 h with mod- rice bran, and perlite are presented in Table 4. The endogenous
erate agitation. The slurry was then centrifuged (Hettich Zentri- mineral contents greatly varied among the three samples.
fugen D-78532) at 2500g for 15 min under room temperature. Under the simulated physiological conditions of the stomach,
The supernatant was discarded, and the residue was washed the strong acidic environment removed most of the endogen-
several times with ultrapure water (sample treated under ous Mn from wheat bran, rice bran, and perlite (72.08–91.42%
small intestine conditions). Subsequently, each sample treated removal).
under small intestinal conditions was then freeze-dried, and The mineral-binding behaviour of the samples treated under
those samples were retained for mineral analysis. Finally, to the three gastric conditions under the simulated physiological
assess the mineral-binding capacity under a mimicked physio- conditions of the small intestine is also shown in Table 4.
logical conditions of the colon with slightly acidic pH, portions Among the samples treated under the three gastric conditions,
of the mineral-bound samples (2 g) treated with simulated rice bran exhibited the highest % binding for all of the minerals

Table 1. Proximate analysis of wheat bran and rice bran (%mean ± SD).
Sample Moisture Protein Ash Ether extract CHOa
Wheat bran 9.55 ± 0.47 15.70 ± 0.78 4.60 ± 0.23 1.97 ± 0.09 68.18 ± 3.40
Rice bran 6.48 ± 0.32 9.62 ± 0.48 15.63 ± 0.78 5.37 ± 0.26 62.90 ± 3.14
a
Total carbohydrate (CHO)can be calculated by [100 − (protein + fat + moisture + ash)].
 JOURNAL OF APPLIED ANIMAL RESEARCH 3

Table 2. NDF, ADF, ADL, and phytic acid of wheat bran and rice bran (%mean ± SD).
Sample NDF ADF ADL CF Phytic acid
Wheat bran 45.40 ± 2.27 13.80 ± 0.69 4.20 ± 0.21 11.05 ± 0.55 3.37 ± 0.16
Rice bran 66.60 ± 3.33 44.80 ± 2.24 11.20 ± 0.56 27.86 ± 1.39 4.43 ± 0.22
Note: NDF, neutral detergent fibre; ADF, acid detergent fibre; ADL, acid detergent lignin; CF, crude fibre.

Table 3. Cellulose and hemicellulose of wheat bran and rice bran (%mean ± SD).
Sample Cellulosea Hemicelluloseb NFCc TDF SDF ISDF TNSP
Wheat bran 9.60 ± 0.48 31.60 ± 1.58 32.53 ± 1.62 37.4 ± 1.87 2.90 ± 0.14 35.9 ± 1.79 44.9 ± 2.24
Rice bran 33.60 ± 1.68 21.8 ± 1.09 2.78 ± 0.14 25.5 ± 1.27 0.5 ± 0.02 21.3 ± 1.06 35.07 ± 1.75
Note: TDF, total dietary fibre; SDF, soluble dietary fibre; ISDF, insoluble dietary fibre; TNSP = DF + lignin.
a
Cellulose content was calculated by difference: ADF − ADL.
b
Hemicellulose content was calculated by difference: NDF − ADF.
c
NFC is calculated by the difference [100 − (%NDF + %CP + %fat + ash)].

Table 4. Endogenous mineral content and in vitro mineral-binding capacity of the samples under sequential simulated physiological conditions of the stomach (%
removal), small intestine (% binding), and colon (% releasing) (mean ± SD).
Mineral Samples Endogenous (µg/g) Removala Bindingb Releasingc
Mn Wheat bran 163.40 ± 8.17a 89.19 ± 5.86a 13.10 ± 0.40a 11.46 ± 0.57b
Rice bran 128.60 ± 6.43b 72.08 ± 1.17b 13.53 ± 0.67a 11.16 ± 0.55b
Perlite 121.30 ± 0.60b 91.42 ± 1.27a 0.76 ± 0.05b 71.65 ± 2.37a
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Zn Wheat bran 33.90 ± 1.69b 48.82 ± 4.58b 31.08 ± 1.55b 3.00 ± 0.15b
Rice bran 17.01 ± 0.85c 34.92 ± 1.74c 33.89 ± 1.69a 3.05 ± 0.16b
Perlite 95.70 ± 4.30a 94.30 ± 1.44a 1.74 ± 0.14c 81.33 ± 1.32a
Fe Wheat bran 845.45 ± 42.27a 38.41 ± 2.41 14.14 ± 0.70c 20.13 ± 3.03b
Rice bran 599.70 ± 29.98b 36.86 ± 3.46 21.61 ± 1.08b 17.49 ± 0.87b
Perlite 565.65 ± 24.43b 35.73 ± 2.16 83.09 ± 4.80a 70.46 ± 1.88a
Cu Wheat bran 44.05 ± 2.20b 69.86 ± 4.55b 63.49 ± 3.17a 7.57 ± 0.57b
Rice bran 65.18 ± 3.25a 30.73 ± 2.21c 67.51 ± 3.37a 7.39 ± 0.36b
Perlite 19.80 ± 0.20c 86.99 ± 2.48a 1.41 ± 0.21b 60.00 ± 2.96a
Note: Data within a column with different superscripts differ (p < .05).
a
% Removal = ((endogenous mineral bound – gastric mineral bound) × 100%)/endogenous mineral bound.
b
% Binding = ((small intestinal mineral bound – gastric mineral bound) × 100%)/total exogenous mineral added)
c
% Releasing = ((small intestinal mineral bound – colonic mineral bound) × 100%)/small intestinal mineral bound.

Table 5. Endogenous mineral content and in vitro mineral-binding capacity of the samples under sequential simulated physiological conditions of the stomach (%
removal), small intestine (% binding), and colon (% releasing).
Samples Mineral Endogenous (µg/g) Removala Bindingb Releasingc
Perlite Mn 121.30 ± 0.60b 91.42 ± 1.27a 0.76 ± 0.05b 71.65 ± 2.37b
Cu 19.80 ± 0.20d 86.99 ± 2.48b 1.41 ± 0.21b 60.00 ± 2.96c
Zn 95.70 ± 4.30c 94.30 ± 1.44a 1.74 ± 0.14b 81.33 ± 1.32a
Fe 565.65 ± 24.43a 35.73 ± 2.16c 83.09 ± 4.80a 70.46 ± 1.88b
Wheat bran Mn 163.40 ± 8.17b 89.19 ± 5.86a 13.10 ± 0.40c 11.46 ± 0.57b
Cu 44.05 ± 2.20c 69.86 ± 4.55b 63.49 ± 3.17a 7.57 ± 0.57b
Zn 33.90 ± 1.69c 48.82 ± 4.58c 31.08 ± 1.55b 3.00 ± 0.15d
Fe 845.45 ± 42.27a 38.41 ± 2.41d 14.14 ± 0.70c 20.13 ± 3.03a
Rice bran Mn 128.60 ± 6.43b 72.08 ± 1.17a 13.53 ± 0.67d 11.16 ± 0.55b
Cu 65.18 ± 3.25c 30.73 ± 2.21c 67.51 ± 3.37a 7.39 ± 0.36c
Zn 17.01 ± 0.85d 34.92 ± 1.74bc 33.89 ± 1.69b 3.05 ± 0.16d
Fe 599.70 ± 29.98a 36.86 ± 3.46c 21.61 ± 1.08c 17.49 ± 0.87a
Note: Data within a column with different superscripts differ (p < .05).
a
% Removal = ((endogenous mineral bound – gastric mineral bound) × 100%)/endogenous mineral bound.
b
% Binding = ((small intestinal mineral bound – gastric mineral bound) × 100%)/total exogenous mineral added).
c
% Releasing = ((small intestinal mineral bound – colonic mineral bound) × 100%)/small intestinal mineral bound.

studied except Fe. Perlite showed the lowest binding capacity Perlite showed the lowest binding capacity while rice bran
while rice bran the highest for Mn, Zn, and Cu. Organic the highest for Mn, Zn, and Cu. Organic sources (wheat bran
sources (wheat bran and rice bran) seem to bind more Mn, and rice bran) seem to bind more Mn, Zn, and Cu than perlite
Zn, and Cu than perlite. (Figure 1).
In Table 5, the column titled ‘% binding’ shows that the
cereal brans had an affinity for Cu > Zn > Fe > Mn and inorganic
4. Discussion
source (perlite) had an affinity for Fe > Zn > Cu > Mn. Among
the minerals, Mn exhibited the lowest % binding under the Among the samples treated under the three gastric conditions,
simulated physiological conditions of the small intestine and rice bran exhibited the highest % binding for all of the minerals
the highest % removal under the simulated physiological con- studied except Fe. These results showed that perlite might
ditions of the stomach for cereal brans. contain the highest number of specific sites for Fe or it might
 4 A. GHODRAT ET AL.

Cellulose is a insoluble linear unbranched polymer of β-(1,4)-

d-glucose residues associated with other cellulose chains by
hydrogen bonding and Van der Waals forces. Cellulose chains
aggregate together to form microfibrils, which are highly crys-
talline and insoluble structures, each one about 3 nm in diam-
eter. Cellulosemicrofibrils comprise the core of the plant cell
wall; one-third of the total mass of wall is cellulose. Themicrofi-
brilar disposition allows the existence of micro spaces between
the microfibrils that are fulfilled by matricial polysaccharides
according to the age and tissue type. Minerals can be trapped
in the cell wall matrix. Rice bran has cellulose more than
wheat bran (Table 3).
Figure 1. Mineral-binding capacity of the samples under sequential simulated Phytate (myo-inositol 1, 2, 3, 4, 5, 6-hexakis dihydrogen phos-
physiological conditions of the small intestine (% binding). phate) is an anionic acid with anti-nutritional properties.
Phytate has been shown to form complexes with trace minerals
and decreases their availability (Davies & Nightingale 1975;
Erdman 1979; Maddaiah et al. 1964; Ravindran et al. 1995;
Vohra et al. 1965).
It has been shown that cations bind to DF in vitro (Camire &
Clydesdale 1981). Fibres from different plant sources have been
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shown to have different chemical structures, and thus different

binding capacities (Slavin 1987; Van Soest & Jones 1988). It is
difficult to draw definite conclusions regarding the relation
between the composition of studied products, and mineral
binding, because plant foods are usually associated with sub-
stances which may also bind minerals. Moreover, the fibre
content and composition of these plant foods differ among
studies, depending on the analytical methods used to
measure DF content (Torre et al. 1991).
Figure 2. Comparison of some components and mineral-binding capacity in wheat
bran, rice bran, and perlite.
Although DF from plant material or isolated components has
been shown to bind essential inorganic nutrients, the mechan-
isms involved are still being investigated.
have the same number of sites but with stronger affinity for Fe
than that of the other two treatments. Rice bran seems to bind
more Fe than wheat bran. 5. Conclusion
Under the simulated physiological conditions of the
Mn, Zn, Cu, and Fe content varied significantly among the
stomach, the strong acidic environment removed most of the
samples of wheat bran, rice bran, and perlite. The amounts of
endogenous minerals from perlite (Mn, 91.42% removal; Zn,
Mn, Zn, Cu, and Fe bound to these sources do not appear to
94.30% removal; Cu, 86.99% removal) with the exception of
be correlated only to the parameters that were determined in
Fe (only 35.73% removal). According to Laszlo (1989) as well
this study. This is probably due to the variation in chemical com-
as Thompson and Weber (1981), the ineffective release of
position and chemical structures of these sources when con-
endogenous Fe under gastric condition might suggest that
sidered as a whole. Other components or more complex
the binding mechanism of this mineral might only be partial
mechanisms might also be involved. Further work is needed
via electrostatistic interactions.
on the total binding of different sources for minerals not only
Binding of iron to cellulose has been observed to be minimal
to understand the mechanism involved but also to predict
under any pH. After reviewing the literature on Fe-cellulose
the degree of binding of a given fibre for a given mineral,
binding, Clydesdale (1983) found that a very weak bond was
which could be useful for consumers.
formed between the mineral and the DF component. Southgate
(1987) reported that there is no evidence of bonding between
iron and cellulose and suggests that some Fe is adsorbed to the Disclosure statement
surface of cellulose.
No potential conflict of interest was reported by the authors.
DF is believed to have a negative impact on mineral bioavail-
ability. A similar result had been observed previously (Caprez &
Fairweather-Tait 1982; Davies & Nightingale 1975; McKenzie &
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