JOURNAL OF

FOOD COMPOSITION
AND ANALYSIS
www.elsevier.com/locate/jfca
Journal of Food Composition and Analysis 17 (2004) 321329
Original Article
In vitro bioavailability of calcium from chicken bone extract
powder and its fortied products
Sirirat Sittikulwitit, Prapaisri P Sirichakwal*, Prapasri Puwastien,
Visith Chavasit, Pongtorn Sungpuag
Institute of Nutrition, Mahidol University, Phutthamonthon 4, Salaya, Nakorn Pathom 73170, Thailand
Received 3 September 2003; received in revised form 1 March 2004; accepted 15 March 2004
Abstract
Chicken bone extract powder (BEP) is an alternative inexpensive calcium source. It is rich in calcium
(30 g/100 g) and contains a Ca : P ratio of 2 : 1, but the bioavailability of its calcium is not known. The
objective of this study was to determine calcium bioavailability of BEP and BEP-fortied products using an
in vitro equilibrium dialysis method. The effects of phytate and dietary ber from food products on the
calcium bioavailability of BEP were evaluated. The results showed that BEP exhibited excellent calcium
bioavailability. It showed higher bioavailable calcium than milk and several calcium forticants. Although
phytate and dietary ber had a negative effect on calcium bioavailability BEP showed the lowest effect
among all calcium sources. The BEP-fortied bakery products were well accepted and not signicantly
different in appearance, taste and texture from non-fortied products.
r 2004 Elsevier Ltd. All rights reserved.
Keywords: Bioavailability; Calcium; Chicken bone extract powder; Fortication
1. Introduction
Calcium is a mineral required by the body for a variety of physiological functions and the
maintenance of bone tissues through life (Broadus, 1996). As a structural component, calcium
combines with phosphorus to comprise the mineral portion of bone and teeth. As a metabolic
component, calcium is important in a variety of biochemical and physiological processes including
neuromuscular excitability, blood coagulation, transfer of inorganic ions across membranes,
*Corresponding author. Tel.: +66-28893947; fax: +66-2441-9344.
E-mail address: nupsr@mahidol.ac.th (P.P. Sirichakwal).
0889-1575/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfca.2004.03.023
hormone secretion, release of cellular enzymes, signal transductions, and reproductive functions
such as sperm motility and fertilization of the ovum (Lobaugh, 1995; Weaver and Heaney, 1999).
Chronic inadequate intake of calcium from the diet is one factor in the etiology of several
disorders (Weaver and Heaney, 1999). Adequate calcium intake during growth is critical to the
achievement of peak bone mass that may reduce the risk of osteoporosis (NIH, 1994; Kanis, 1996;
Martin et al., 1997; Heaney et al., 2000). Furthermore, adequate calcium intake has been
associated with reducing the risk of hypertension (Bucher et al., 1996; McCarron, 1996; Williams,
1987) and colon cancer (Garland et al., 1991).
Osteoporosis has become an important degenerative disease in the world especially in Asia.
Osteoporosis-related fractures can occur in any of the bones but the main fractures occur at the
hip, vertebral spinal column, and the wrist (NIH, 1991). Hip fracture is a major public health
problem in Asia. It has been projected that by the next century, 50% of all hip fractures in the
world will occur in Asia (Cooper et al., 1992). In Thailand, an epidemiological survey has
indicated that the incidence of hip fracture is 162/100 000 population over the age of 50 (Lau et al.,
2001). Nutrition interventions to increase calcium include increasing the consumption of high
calcium foods and the use of calcium supplements.
Chicken bone contains high calcium and is available as a by-product of chicken slaughtering
plants. An alkaline treatment was found to be the best and most inexpensive method to prepare
bone extract powder (BEP) with high quality and a proper calcium and phosphorus ratio
(Kettawan et al., 2002). BEP is a new calcium source that was well accepted when fortied into
shrimp chips and chilli-paste (Kettawan et al., 2002). However, its calcium availability has not yet
been studied. The objective of this study was, therefore, to evaluate the bioavailability of calcium
in chicken BEP and compare it to that of ve common commercial calcium forticants (i.e.,
calcium carbonate (CaC), tricalcium phosphate (CaP), calcium lactate (CaL), calcium citrate
(CaCi), and calcium lactogluconate (CaLG)). The effects of phytate and dietary ber on calcium
dialyzability in BEP and in the three best dialyzable calcium salts were then evaluated. Calcium
bioavailability in BEP-fortied white bread, butter cookies, whole meal bread and oat cookies was
also evaluated. The method used for the determination of calcium bioavailability was the in vitro
equilibrium dialysis method of Miller et al. (1981). The acceptability of BEP-fortied products
was also evaluated.
2. Materials and methods
Preparation of chicken BEP: Chicken bone was kindly provided by Leamthong Co., Ltd
(Thailand). Chicken BEP was prepared by an alkaline treatment method using 3% NaOH and
neutralized bone was dried at 100

C and ground (Kettawan et al., 2002). This was to make sure

that BEP was treated appropriately to get rid of all the organic materials as well as any microbes
before using as a calcium forticant.
Commercial calcium forticants: Five common commercial calcium forticants used in this
experiment included calcium carbonate (CaC) (Thai food and Chemical, Co., Ltd.), tricalcium
phosphate (CaP) (Tinkorn Sale and Promotion, Co., Ltd.), calcium citrate (CaCi) (Thai food and
Chemical, Co., Ltd.), calcium lactate (CaL) (Tinkorn Sale and Promotion, Co., Ltd.), and calcium
lactogluconate (CaLG) (Nutrition, Co., Ltd.). All were dry powder of food grade.
S. Sittikulwitit et al. / Journal of Food Composition and Analysis 17 (2004) 321329 322
Preparation of bakery products fortied with BEP: Four bakery products: white bread, whole
meal bread, butter cookies and oat cookies were selected for BEP-fortied products. BEP, as the
source of calcium, was added to the bakery products in amounts sufcient to provide a nal
calcium content of 240 mg per serving of bread (50 g) and cookies (30 g). Whole meal our and
oatmeal were used as natural sources of phytate and dietary ber. White bread and butter cookies
were the control products to study the effect of phytate and dietary ber on calcium
bioavailability. The bread and cookies recipes are shown in Table 1. The most accepted levels
of whole meal our and oatmeal used to replace the wheat our in single serving recipes, obtained
from the preliminary study, was 40% and 60% of initial amount of wheat our, respectively.
In vitro calcium bioavailability study: The bioavailability of calcium in all samples was
determined by the in vitro equilibrium dialysis method of Miller (Miller et al., 1981). This analysis
is based on the simulation of a gastrointestinal digestion of food with pepsin-HCl during the
gastric stage and pancreatin-biliary salts during the intestinal stage. The fraction of diffused
element through a semi-permeable membrane during the intestinal stage is measured to predict
the elements dialyzability. Whole milk powder (Nestle Thailand, Co., Ltd.) was used as a control
sample in each set of determinations throughout the study. The amount of each sample was
adjusted to have a calcium content of 240 mg, which approximates the calcium level in 200 mL of
cow milk. The dialyzability of calcium in each product was determined in three independent
Table 1
Ingredients of bread (1 loaf) and cookies (about 31 pieces)
Ingredients Weight of ingredients per serving
White bread Whole meal Butter cookies Oat cookies 60%
bread 40% whole oatmeal
meal our substitute substitute
g g/50 g g g/50 g g g/30 g g g/30 g
Hard wheat our 250 28.90 150 17.14
Whole meal our 100 11.43
All purpose our 80 13.81 32 5.52
Oatmeal 48 8.29
Butter 33.5 5.79 33.5 5.79
Shortening 19 2.20 19 2.17 17 2.93 17 2.93
Sugar 21 2.43 21 2.40 23 3.97 23 3.97
Salt 4 0.47 4 0.46 0.3 0.05 0.3 0.05
Water 135 15.60 140 16
Yeast 3.5 0.40 3.5 0.4
Whole milk 19 3.28 19 3.28
Baking powder 1 0.17 1 0.17
Total 432.5 50 437.5 50 173.8 30 173.8 30
BEP fortied products
BEP 6.65 0.77 6.74 0.77 4.36 0.75 4.36 0.75
S. Sittikulwitit et al. / Journal of Food Composition and Analysis 17 (2004) 321329 323
replicates. The experiment was conducted on a timed schedule, so that all samples were incubated
for the same period of time. The calcium content in the diluted dialyzates and in the original
samples was analyzed by Atomic Absorption Spectrometry (AAS). The relative availability of
calcium in various samples after the simulated gastrointestinal digestion was calculated directly
from the amount of the element that passed through the dialysis membrane in proportion to the
total elemental content of the original food sample. The dialyzed calcium in each sample was then
expressed as a percentage of the total amount of calcium present in the sample and reported as
mean7SD.
Sensory evaluation and chemical analysis: The sensory evaluation of BEP-fortied bakery
products was conducted with 50 panelists. The panelists were faculty, staff and graduate students
at the Institute of Nutrition, Mahidol University (INMU). The test was performed at the Sensory
Science Laboratory, INMU. A nine-point hedonic scale was used to evaluate sensory
characteristics including general appearance, overall acceptability, odor, taste and texture after
tasting. Five categories of just-about-right scale (much too light/ ne=1; just about right=3;
much too dark/rough=5) were used to evaluate sensory characteristics of color and visual texture
before tasting. All samples tested were randomly coded and served in random order. Proximate
composition, ash, calcium, phosphorus, dietary ber and phytate in the BEP-fortied products
were analyzed according to the AOAC Ofcial Methods (AOAC, 2000).
3. Results and discussion
The in vitro equilibrium dialysis method of Miller et al. (1981) was used to evaluate the calcium
bioavailability in all experiments because the technique can be applied to study both calcium
bioavailability and factors affecting calcium bioavailability in various kinds of foods (Benway and
Weaver, 1993; Dyck et al., 1996; Kenneck and Cashman, 2000; Lucarini et al., 1999).
The dialysis rate of calcium in milk powder, 2871.8 (CV=6.3%), which is based on a calcium
content of 240 mg/200 mL, was similar to that reported by Bosscher et al. (1998) who applied the
same in vitro method and by Heaney et al. (1990) who used the digestion in vivo method. The
calcium bioavailability of BEP and ve commercial calcium forticants with the same calcium
content were signicantly higher, 1.42 times, than that of milk powder (Table 2). It has been well
recognized that calcium bioavailability is inuenced by different types of calcium forticants and
other food components (Nicar and Pak, 1985). Among the ve calcium forticants studied,
tricalcium phosphate showed the best calcium dialyzability, about 2 times that of the milk
powder. The phosphorus in BEP provided a Ca : P ratio of 2 : 1. Calcium was more absorbable
from BEP than from commercial salts except tricalcium phosphate. This was in accordance with
previous ndings in total bone extract of bovine origin reported by Miura and Nakano (1998) and
in bone meal reported by Heaney et al. (1990).
With respect to the calcium content, cost and relative calcium bioavailability, three calcium
forticantscalcium carbonate, tricalcium phosphate and calcium lactatewere selected to study
the effect of phytate and dietary ber on calcium bioavailability compared to that of BEP.
Natural sources of phytate and berwhole meal our and oatmealwere selected because they
contain high phytate (867 and 433 mg/100 g, respectively) and considerable amounts of dietary
ber (7.1 g and 7.44 g/100 g, respectively). In addition, the target products must also be taken into
S. Sittikulwitit et al. / Journal of Food Composition and Analysis 17 (2004) 321329 324
consideration. In this study, two bakery products were chosen, bread and cookies, in which whole
meal our and oatmeal are part of the ingredients. Both whole meal our and oatmeal are rough
in texture, and the amount used in the bakery formula must be the one accepted by consumers.
Based on the sensory evaluation in the preliminary study, 11.43 g of whole meal our (containing
99 mg phytate and 0.81 g dietary ber) and 8.29 g of oatmeal (containing 36 mg phytate and 0.62 g
dietary ber), were the accepted amounts to be used in the products. Added whole meal our and
oatmeal signicantly reduced bioavailability of calcium in all calcium sources (Po0.05) (Table 3).
The most pronounced effect of whole meal our and oatmeal was shown in calcium lactate
(29.6% and 22.2% reduction, respectively). The lowest effect of adding whole meal our and
oatmeal was found in BEP (14.1% and 19% reduction, respectively). The mechanism by which
phytate reduced calcium dialyzability is believed to be through the formation of phytatecalcium
complexes (Weaver et al., 1993; Wolter et al., 1993; Dyck et al., 1996; Kenneck and Cashman,
2000). The lower effect found on BEP and tricalcium phosphate might be due to the presence of a
good Ca : P ratio of 2 : 1 which may reduce the effect of inhibitory food components on their
dialyzability. For dietary ber, in vivo studies have found that the addition of cereal ber to
human and animal diets has consistently depressed absorption and retention of calcium (Knox
et al., 1991). The ndings on the effect of phytate and ber on calcium bioavailability in this study
was in agreement with numerous studies both in vivo (Heaney et al., 1991; Weaver et al., 1993;
Hansen et al., 1996; Younes et al., 1996; Levrat-Verny et al., 1999) and in vitro (Benway and
Weaver, 1993; Dyck et al., 1996; Bosscher et al., 1998; Lombardi-Boccia et al., 1998; Lucarini
et al., 1999; Kenneck and Cashman, 2000; Mamiro et al., 2001). Therefore, the presence of food
components high in phytate and dietary ber such as whole meal our and oatmeal can act as
inhibitors on calcium bioavailability.
BEP-fortied whole meal bread contains 190 mg phytate and 6.4 g dietary ber per 100 g. As
expected, the bioavailability of calcium in BEP-fortied whole meal bread was signicantly
(Po0.05) lower than that of BEP-fortied white bread, which contained a much lower amount of
phytate (41 mg/100 g) and dietary ber (3.6 g/100 g), 20.4% compared to 34.7%, respectively
(Table 4). BEP-fortied oat cookies contain 285 mg phytate and 4.9 g dietary ber per 100 g. A
similar effect of phytate and dietary ber on calcium bioavailability was demonstrated, the
Table 2
In vitro dialyzable calcium in milk, BEP and common commercial calcium forticants
Calcium sources Amount
a
(g) of
sample used
% Dialyzable Ca
b
(mean7SD)
Milk powder 31.44 28.571.8
Chicken BEP 0.80 52.071.2
Calcium carbonate (CaC) 0.68 47.470.2
Tricalcium phosphate (CaP) 0.68 58.772.1
Calcium lactate (CaL) 1.81 45.371.1
Calcium citrate (CaCi) 2.89 47.671.1
Calcium lactogluconate (CaLG) 10.00 39.571.1
a
Each contains 240 mg calcium.
b
Each value represents the mean 7SD of three independent replicates.
S. Sittikulwitit et al. / Journal of Food Composition and Analysis 17 (2004) 321329 325
calcium bioavailability of 17.8% was found in BEP-fortied oat cookies compared to 41.9% in
BEP-fortied butter cookies (Po0.05).
There was no change in the phytate content in oat cookies, whereas it was 25% decreased in
leavened bread. The reduction of phytate in the whole meal bread is likely due to phytase activity
during yeast fermentation resulting in higher calcium dialyzability (Weaver et al., 1991). These
results agreed well with the report of Turk et al. (1996) who found that more than 50% of phytate
in whole meal bread was hydrolyzed. The appearances of the BEP-fortied bakery products were
similar to the non-fortied products. The same range of over-all acceptability scores obtained for
both products conrmed that both BEP-fortied bread and oat cookies were well accepted by the
consumers (Table 5).
It is worth noting that when bone is used as a calcium source, some quarantine period for the
product and certication of its source are required to guarantee optimum safety before it enters
the supplement market.
Table 3
Effect of phytate and dietary ber on calcium dialyzability of BEP and selected calcium forticants
Calcium sources % Dialyzable calcium (mean7SD)
a
Control
b
Plus whole meal our
c
Plus oatmeal
d
BEP 52.071.2a 44.671.3b [14.1%] 42.171.5b [19.0%]
CaC 47.470.2a 35.870.8b [24.6%] 38.070.4c [19.9%]
CaP 58.772.1a 50.171.2b [14.6%] 45.671.0c [22.4%]
CaL 45.371.1a 31.971.0b [29.6%] 35.270.4c [22.2%]
a
Each value represents the mean 7SD of three independent replicates.
b
Value from Table 2 (without inhibitors).
c
Add 11.43 g whole meal our (containing 99 mg phytate and 0.81 g dietary ber).
d
Add oatmeal 8.29 g (containing 36 mg phytate and 0.62 g dietary ber).
Mean within the same row having the same letter indicates no signicant difference at P=0.05. Number in
parentheses shows % reduction of dialyzable calcium when compared with the control.
Table 4
In vitro dialyzable calcium in bakery products fortied with chicken bone extract powder (BEP)
a
BEP fortied products Weight of sample
(g/serving)
Calcium content
(mg/serving)
% Dialyzable Ca
(mean7SD)
b
% Decreased
White bread 50 232 34.770.2a
Whole meal bread 50 237 20.472.6b 41.2
Butter cookies 30 235 41.971.2a
Oat cookies 30 245 17.872.2b 57.5
Milk powder 31.4 240 28.571.8
a
BEP was used as source of calcium forticant to meet 240 mg Ca/serving.
b
Each value represents the mean 7SD of three independent replicates.
Mean within the same column having the same letter indicates no signicant difference at P=0.05.
S. Sittikulwitit et al. / Journal of Food Composition and Analysis 17 (2004) 321329 326
4. Conclusions
The results of this study showed that BEP, as a natural calcium source, exhibited excellent
calcium bioavailability. It exhibits higher bioavailable calcium than milk and other common
calcium forticants, i.e., calcium lactogluconate, calcium lactate, calcium citrate and calcium
carbonate. The natural phytate and dietary ber from whole meal our and oatmeal had negative
effects on calcium bioavailability from all calcium sources including BEP-fortied products.
According to the properties of BEP from this study, it can be concluded that BEP could be a good
alternative calcium forticant. It contains high calcium with high bioavailability and is well
accepted when added to the bakery products.
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