Properties of Tilapia Bone Powder and Its Calcium Bioavailability Based On Transglutaminase Assay
Properties of Tilapia Bone Powder and Its Calcium Bioavailability Based On Transglutaminase Assay
Properties of Tilapia Bone Powder and Its Calcium Bioavailability Based On Transglutaminase Assay
4, July 2013
rpm for 4 min (AM8-Nihonseiki Kaisha, Japan). The bone at the surface of the bone 4. Therefore, it could be removed
powder was kept under vacuum condition at -20 C until almost completely during oven drying. The moisture content
characterization. in the bone powder was found at the low level as the data
shown in Table I. According to the low moisture content, the
C. Fish Bone Characterization
bone powder would be stable even at room temperature. In
Proximate Analysis: Fish bone was analyzed for protein addition, the agglomeration/aggregation to be the cake would
content based on total nitrogen content by Kjeldahl method not occur during the storing bone powder. Besides the
(ISO 5983-1997). Moisture content was performed physicochemical changes, low moisture content also allows
gravimetrically after oven drying at 105 C for 12 h (AOAC, the fish bone powder to be resisted for microbial deterioration.
2000). Crude fat was analyzed using Soxhlet extraction using This is because moisture content at 2% is not sufficient for
petroleum ether as a solvent (AOCS, Ba 3-38). Ash content microbial growth. This stable form provides the easy/safety
was performed using dry ashing by combustion sample at way to use the fish bone powder as the ingredient in many
550C for 16 h (AOAC, 2000). applications.
Color Measurement: Fish bone powder was analyzed for It has been reported that lipid could be filled in the bone,
the color values using colorimeter (CR-10, Minolta Co. Ltd., especially the main bone of fish frame, which contains the
Japan). The color values were reported as the L, a, b. junction of many pieces of bone [5]. In addition, those lipids
Solubility: Tilapia bone powder was mixed with DI-water could not be gotten rid off easily, because those lipids are
at the ratio of powder:water of 1:4 and stirred at room complexes, and hardly remove by just soaking the bone in the
temperature overnight. The mixture was filtered through alkaline solution [6]. According to this information, there is
filter paper (No. 1, Whatman). The retentate was dried before the possibility to observe fat residue in the fish bone powder.
weighing to obtain the insoluble particle. Subsequently, the Indeed, crude fat content in the bone tissue was almost 6%
solubility of bone powder was calculated accordingly. (Table I). The lipid content in fish bone was reported to be in
Ca2+Content: The content of Ca2+ was determined using the range of 1-27% [7]. Lipid content in the bone powder
wet ashing followed by inductively coupled plasma mass from mackerel was approximately 47%, while the lipid
spectrometry (ICP-MS). Tilapia bone powder was digested contents in cod and saithe both were about 1.4% 3. The
with nitric acid (0.1 M) at 90 C for 2 h. The digested sample lipid content in bone is correlated with body fat in each fish
was determined for Ca2+ content using ICP-MS (7500ce, species and large (old) fish normally contains high fat.
Agilent Technology, Tokyo, Japan). Tilapia is considered as a low fat fish species, it contains low
D. Bioavailability of Ca2+ Based on tTGase Assay fat even found in the adult stage. Fish lipid is likely
unsaturated fatty acid. Fatty acid profile in fish bone powder
Crude tTGase Extraction: Tilapia muscle was mixed with
from several species revealed that the content of unsaturated
3 volumes of extraction buffer (10 mM NaCl, 50 mM Tris-Cl,
fatty acids was almost 80% 3. Normally, the unsaturated
2 mM DTT) and homogenized for 2 min. The homogenate
fatty acids are susceptible to oxidative degradation
was centrifuged at 10,000 g for 30 min and supernatant was
(oxidation). However, tilapia bone powder supposes to get
filtered through the filter paper. The filtrate was used as crude
the less risk in autoxidation since there is a little fat content.
tTGase and protein content was assayed using dye binding
Protein content in tilapia bone powder was approximately
method (Bradford).
14%. This value for the bone powder from cod, saithe, blue
Determination of tTGase Activity: The activity was tested
whiting, salmon, trout, herring, mackerel were found in the
based on incorporation of MDC into DMC according to the
method described by Takagi (1986) with slight modification. range of 26-41% 3. Therefore, the protein content in tilapia
bone powder was much lower than in other fish species. This
The reaction contained 2 mg DMC, 15 L MDC, 70 mM
might be due to the different preparation procedures. In the
Tris-Cl, pH 7.5, 3 mM DTT, and 100 L of crude tTGase
previous study, fish meat was removed from fish bone by
(0.42 mg protein). The presence of standard Ca2+ and soluble
only boiling in the hot water, while this study was done by the
Ca2+ from fish bone were tested at 10 M along with negative hot alkaline solution. Therefore, alkaline solution would be
control (without Ca2+). The reaction was done at 37 C for 10 more effective to solubilize and leach out more meat tissue
min. Then, the incorporation of MDC into DMC was and proteins from the bone. However, the alkaline solution
estimated by fluorescent spectrophotometer (RF-1501, was not effective enough to get rid of protein completely
Shimadzu, Kyoto, Japan). The excitation and emission since some of protein still left in the bone powder. Normally,
wavelengths were at 350 and 480 nm, respectively. The unit proteins participated in the bone are categorized into stroma
of enzyme activity was calculated as the amount of enzyme protein 8. This stroma protein is resistant to both acid and
that catalyzes the incorporation of MDC (1nmol) into alkaline solutions. Collagen associated amino acids, glycine,
described amount of DMC within 1 min at 37 C. proline, and hydroxyproline, were found in the bone powder
from blue whiting, herring, and mackerel 3. Protein content
in the bone is increased as fishes grow up.
III. RESULTS AND DISCUSSION The major component in the bone powder was ash content,
A. Proximate Analysis which was found to be 75%. The ash in bone powder from
several fish species could be found up to 40% 3. The ash in
Tilapia bone powder was analyzed and showed the small
tilapia bone powder was much higher than that reported
amount of moisture content. This suggested that the drying
previously. This suggested that the bone preparation was
process is performed perfectly. It has been reported that water
important to get the high purity of bone component.
molecules are not included into bone tissue but bound weakly
Application of alkaline solution was the practical method to
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International Journal of Bioscience, Biochemistry and Bioinformatics, Vol. 3, No. 4, July 2013
remove organic materials particularly protein. The ratio of structures. The cellular bone from fish species in the family
ash/protein is the important criteria to indicate the bone of Salmonadae is less strained than in acellular fish bone.
mineralization, associated with hardness of the bone. This This is because of the lower surface to volume ratio in
value for salmon bone was lower than 1.00 3. The cellular fish bone 11. Thus, Ca2+ from acellar bone could be
ash/protein ratio of tilapia bone powder was 5.35, which is more available when compared to the cellular bone fish. The
much higher than that reported previously. This suggested availability of Ca2+ from tilapia bone powder was expected
that not only the bone mineralization but also the preparation because this fish is classified into the acellular bone.
method could affect this value. In addition, this value could Although the soluble ash was observed at the low level, the
be used to indicate the purity of bone powder. Therefore, Ca2+ content in solution was also observed (Table III). The
alkaline aid solubilization could be the practical method to Ca2+ is considered as the macro mineral in the bone.
produce fish bone powder with high purity. Normally, it complexifies with phosphorous in the form of
hydroxyappatite (Ca5(PO4)OH2) 7. Therefore, bone tissue is
TABLE I: MAJOR COMPONENT OF TILAPIA BONE POWDER
the important depot for storage of calcium and phosphates
Proximate parameter Content (%)
and also is essential in the regulation of plasma
Moisture 2.46 0.03
Crude fat 5.82 0.04
concentrations of these minerals 12. It would be
Protein 14.81 0.33 hypothesized that Ca2+ in the tilapia bone could be
Ash 75.83 0.12 solubilized to be the soluble Ca2+. However, the utilization of
Mean SE was estimated from 3 replications. soluble Ca2+ is also an interesting point. There are several
documents reporting the availability of Ca2+ residue in fish
bone powder. Those studies have been tested with the
B. Color
complicated system, like the rat and human body model 13.
The color of tilapia bone powder was shown visibly as the The absorption and retention of Ca2+ was correlated well with
white powder and the L value was about 98 as shown in Table
a reduction of bone loss14. In contrast, the simple reaction
II. The color of fish bone powder is correlated well with the
to test the bioavailability of Ca2+ would be more convenient
organic residues presenting in the powder. High content of
and should be developed.
organic compounds resulted in the less lightness of the
powder. The white powder was more preferred to fortify into TABLE III: SOLUBILITY AND SOLUBLE CALCIUM ION CONTENT IN TILAPIA
several food products rather the dark powder. Fish bone BONE POWDER
powder was used as supplementary ingredient in the Quality attribute Content
skimmed milk 9. However, the changes in color might be Solubility (%) 9.38 0.07
possible since the bone powder contains amino acids. The Soluble calcium content (mg.L-1) 116.56 0.15
browning reaction occurs through the non-enzymatic reaction. Mean SE was estimated from 2 replications.
Therefore, the suitable storage condition for tilapia bone
powder should be at low temperature and vacuum condition. D. tTGase Activity
TABLE II: COLOR VALUE OF TILAPIA BONE POWDER Ca2+ is one of major minerals, serving as the cofactor for
Color Value several enzymes in the metabolic pathway. The tTGase is one
L 98.08 0.04 of enzyme, requiring Ca2+ for full activation. Ca2+ is needed
a 16.79 0.06 to induce conformational changes of the enzyme molecule to
b 79.48 0.09 be prompt for catalyzing the reaction. The tTGase assay has
Mean SE was estimated from 3 replications. been developed and Ca2+ has to be included for 5 mM 15. It
has been reported that tTGase bound with Ca2+ could remove
C. Solubility and Ca2+ Content the controlling residue, covering the active site. Therefore,
the reaction could be preceded. This fact would be the basic
Usually, the ash is the indicator of mineral content in the
background for developing the bioavailability test of Ca2+
sample. Tilapia bone powder showed high ash, it supposed to
from fish bone. Since the availability of Ca2+ is important for
have high mineral content. However, the mineral uptake of
activation of tTGase, the bioavailability of Ca2+ from fish
human body depends not only on ash content but also on the
bone could be evaluated. It can be seen that tTGase could not
solubility and availability to absorb. Therefore, the soluble
catalyze the incorporation of MDC into DMC in the absence
mineral has been considered as the quality of ash. Meanwhile,
of Ca2+ (Table IV). However, the tTGase activity increased
it is the good indicator of ash for health. Feeding fish with
instantly when soluble Ca2+ was added for 162 nM. This
soluble Ca2+ promoting the mineralization of salmon was
result clearly demonstrated that the presence of Ca2+ from
reported 10. The bone powder of tilapia was not
tilapia bone powder at this level is enough to activate crude
solubilized completely (Table III). This suggested that all
tTGase from tilapia. This was the evidence that soluble Ca2+
mineral components in the powder could not be absorbed.
from tilapia bone powder is also available for biochemical
The solubility of mineral in the bone powder may be
reaction. The activation ability of this soluble Ca2+ was
associated with several factors including fish species and fish
comparable to that of standard Ca2+ (CaCl2) since the
age. If the bone of old fish is mineralized completely, the
comparable activity was found when soluble Ca2+ was
bone would be hard. In contrast, small fish species are often
replaced by standard Ca2+.
eaten as a whole. Then, Ca2+ from fish bone is soluble and is
absorbed readily into human body. Fish species have the E. Effect of Ca2+ on tTGase Activity
different bone structures: cellular and acellular bone It has been reported that the active site of tTGase contains
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