Effect of Alkaline Treatment on Characteristics of Bio-Calcium and Hydroxyapatite Powders Derived from Salmon Bone - MDPI
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
applied
sciences
Article
Effect of Alkaline Treatment on Characteristics of
Bio-Calcium and Hydroxyapatite Powders Derived
from Salmon Bone
Anthony Temitope Idowu 1 , Soottawat Benjakul 1 , Sittichoke Sinthusamran 1 ,
Thanasak Sae-leaw 1 , Nobuo Suzuki 2 , Yoichiro Kitani 2 and Pornsatit Sookchoo 3, *
1 Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai,
Songkhla 90112, Thailand; tonitop17@yahoo.com (A.T.I.); soottawat.b@psu.ac.th (S.B.);
sinthusamran@hotmail.com (S.S.); thanasaki_am@hotmail.com (T.S.-l.)
2 Noto Marine Laboratory, Institute of Nature and Environmental Technology, Kanazawa University, Ogi,
Noto-cho, Ishikawa 927-0553, Japan; nobuos@staff.kanazawa-u.ac.jp (N.S.); yki@se.kanazawa-u.ac.jp (Y.K.)
3 Department of Material Product Technology, Faculty of Agro-Industry, Prince of Songkla University Hat Yai,
Songkhla 90112, Thailand
* Correspondence: pornsatit.s@psu.ac.th; Tel.: +66-742-863-51
Received: 11 April 2020; Accepted: 11 June 2020; Published: 16 June 2020
Abstract: Alkaline treatment has been extensively implemented in the extraction process of
hydroxyapatite (HAp) extraction from various kinds of bio-materials, such as animal bone and
scales. The main purpose of such treatment is to remove proteinaceous substances from raw materials.
The influence of the alkaline treatment that could alter not only the organic contents but also chemical
composition—specifically the Ca/P mole ratios of bio-calcium, HAp, and the biphasic apatite powders
derived from salmon bone, a by-product from the salmon industry—was investigated. Both HAp
and biphasic apatite powders were obtained from the calcination of bio-calcium powders with and
without alkaline treatment, respectively. An X-ray diffraction analysis confirmed the presence of
hydroxyapatite and β-tricalcium phosphate (β-TCP) in the calcined bone powder without alkaline
treatment while only a single phase of hydroxyapatite was observed in the alkaline-treated sample.
Calcium and phosphorus contents were measured by an inductively coupled plasma optical emission
spectrometer (ICP-OES). A variation of Ca/P ratios was observed among all samples, depending on
the chemical and heat treatment conditions. Organic molecules, such as protein, fat, hydroxyproline,
and TBARS, were significantly lowered in bio-calcium powders with the alkaline treatment. This work
represents important research on chemical treatment prior to the raw material conversion process,
which significantly influences chemical and phase compositions of the bio-calcium and hydroxyapatite
powder derived from salmon bone waste.
Keywords: bio-calcium powder; calcined powder; hydroxyapatite; salmon bone
1. Introduction
Fish consumption has increased tremendously, and its demand by 2050 is estimated to be
9.8 billion tons [1]. During the processing of fish, more than 60% of fish mass is generated as leftovers,
including viscera (liver, kidney, and roe), frames, trimmings (containing muscle, bone, and skin), heads
(containing the gills), and mince [2]. Salmon (Salmo salar) constitutes a large portion of the fish globally
served due to its high market demand. It is usually sold as a fillet or as whole, which often leads
to the generation of frames attached with remaining meat. Consequently, a large amount of waste
is generated [3]. The hydrolysis of salmon frames prepared in the mince and chunk form has been
studied [3]. After hydrolysis, a high amount of fish bones remains as residues, particularly when chunk
Appl. Sci. 2020, 10, 4141; doi:10.3390/app10124141 www.mdpi.com/journal/applsciAppl. Sci. 2020, 10, 4141 2 of 12
form is used. These residual bones could be used as a starting material for the production of bio-calcium
for calcium supplement. Calcium is an essential element that is abundantly found in the structure of
human bones and teeth. It is also involved in the numerous physio-logical activities of the human
system, including maintaining nerve impulse transfer and heart rate, facilitating blood flow within
capillaries, participating in blood coagulation, and modulating muscle function [4]. Deficiency of
calcium is a general problem associated with reduced bone mass and osteoporosis [5]. This is due to
inadequate calcium in most regular meals consumed by people. Therefore, an alternative source of
calcium in necessitated.
In general, fish bones are rich in calcium and phosphorus as well as other trace elements such as
Na, Mg, Fe, etc. [6]. Calcination at high temperature has been used to produce the hydroxyapatite
(HAp: Ca10 (PO4 )6 (OH)2 ), Ca:P = 1.67) compound from natural bio-mass, such as animal bones
and shells [7]. This compound has been widely used in dental and bio-medical applications [7–9],
as well as in catalysis, ion exchange, and heavy-metal removal sorbents [10]. HAp produced from
different fish bone species resulted in a variation of chemical composition and a deviation of Ca/P
ratios from 1.67. Several studies have reported the formation of other compounds in addition to HAp
such as CaO and β-tricalcium phosphate (β-TCP) when Ca/P ratios are higher and lower than 1.67,
respectively [10]. The HAp/β-TCP biphasic compound has been extensively studied due to its ability
to promote osteoconductive property [11].
Extraction of HAp from fish and other animal bones usually involves the elimination of organic
matters during the pre-treatment process. Alkaline treatment has been documented to be one of
the effective means to remove proteinaceous substance from the bone matrix [3,4]. Nevertheless,
no information exists regarding whether there is an influence of alkaline treatment on the Ca/P
ratios of treated bones, which could determine the formation of HAp and β-TCP upon calcination.
Therefore, this work aims at elucidating the effect of alkaline treatment used in the extraction process
of bio-calcium and HAp from salmon bone on their characteristics, including chemical compositions,
Ca/P ratios, and phase morphology.
2. Materials and Methods
2.1. Chemicals
Hydrogen peroxide, sodium hydroxide, and sodium hypochlorite were supplied from
QReC (Auckland, New Zealand). Hexane was procured from LabScan (Bangkok, Thailand).
In addition, 1,1,3,3-tetramethoxypropane, trichloroacetic acid, and hydrochloric acid were bought from
Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), while 2-Thiobarbituric acid was purchased from
Fluka (Buchs, Switzerland).
2.2. Collection and Preparation of Bone from Salmon Frame
Frames of salmon (Salmo salar) of about 30–35 cm (in length) were obtained from Kingfisher
Holding Ltd., Songkhla, Thailand. The frames were cut into the length of 4–5 cm using a sawing
machine. The prepared frames (chunks) were subjected to hydrolysis, as detailed by Idowu et al. [3].
Bone residues obtained after hydrolysis were cleaned using a high-pressure water jet cleaner (Model
Andaman 120 bar, Zinsano, Bangkok, Thailand) to remove the meat attached to the bones at a pressure
of 120 bar. After cleaning, the bones were divided into two portions and kept at 4 ◦ C before use.
2.3. Pre-Treatment of Bones
The first portion of prepared bones (50 g) were immersed in 2 M NaOH with a bone/solution
ratio of 1:10 (w/v) at 50 ◦ C up to 120 min. Continuous stirring of the mixture was done at a speed of
150 rpm using an overhead stirrer attached to a propeller (Model RW 20n, IKA-Werke GmbH & CO.KG,
Staufen, Germany). At different times (0, 10, 20, 30, 40, 50, 60, 90, and 120 min), 5 mL of the solution
were taken for the determination of the total soluble protein content by the biuret method [12] andAppl. Sci. 2020, 10, 4141 3 of 12
hydroxyproline [13]. The time used for rendering the solution with the highest soluble protein and
the lowest hydroxyproline content was selected for alkaline treatment. The second portion was not
subjected to alkaline treatment. Both bones were dried separately with a laboratory scale rotary dryer
(air velocity = 1.5 m/s; temp. = 50 ◦ C; time = 2 h). Dried samples were ground using a crushing
mill (Model YCM1.1E, Yor Yong Hah Heng, Bangkok, Thailand) until particle sizes of approximately
3–4 mm were obtained.
2.4. Preparation of Bio-Calcium and Calcined Bone
Both alkaline and non-alkaline treated samples were subjected to lipid removal by soaking them
in hexane with a matter/solution ratio of 1:10 (w/v) at 25 ◦ C and uninterruptedly stirred for 60 min.
Bleaching was done by soaking the samples in 2.5% (v/v) sodium hypochlorite with a matter/solution
ratio of 1:10 (w/v) at room temperature for 30 min with continuous stirring. Thereafter, the samples
were washed with running water for 5 min and then bleached with 2.5% (v/v) hydrogen peroxide using
a matter/solution ratio of 1:10 (w/v) at room temperature (28–30 ◦ C) for 60 min. Grinding into fine
particles was implemented using a planetary ball mill (Model PM 100, Retsch GmbH, Haan, Germany).
The rotation mode in one direction was performed at a speed of 200 rpm for 2.5 h. The obtained powders
were sieved using a sieving machine to collect particles with sizes less than 75 mm. All procedures
were detailed by Benjakul et al. [14].
Bio-calcium powders obtained from alkaline and non-alkaline treated salmon bones were termed
as Bio-cal-A and Bio-cal-H, respectively. Another portion of Bio-cal-A and Bio-cal-H was calcined
using a muffle furnace (Model 320, P Nabertherm, Bremen, Germany) at 900 ◦ C for 6 h and 9 h, and the
resulting powders were named Cal-A (6 h), Cal-A (9 h), Cal-H (6 h), and Cal-H (9 h). All the samples
were ground to obtain fine particles using the same instruments and procedures as described above.
All the samples were subjected to analyses.
2.5. Characterization of Bio-Calcium and Calcined Bone
2.5.1. Chemical Composition
The moisture, protein, fat, and ash contents of all the samples were determined [15]. An inductively
coupled plasma optical emission spectrometer (ICP-OES) (Model Optima 4300 DV, Perkin Elmer,
Shelton, MA, USA) was used for the determination of Ca and P in all the samples as per the method of
Feist and Mikula [16].
2.5.2. Thiobarbituric Acid-Reactive Substances (TBARS)
Thiobarbituric acid-reactive substances (TBARS) of the samples were determined as per the
method of Benjakul et al. [14] to measure the decomposition of hydroperoxides into the secondary
oxidation products. The samples (2.0 g) were homogenized with 10 mL of a solution containing 0.375%
thiobarbituric acid (w/v), 15% trichloroacetic acid (w/v), and 0.25 M HCl. After being heated in a boiling
water bath (95–100 ◦ C) for 10 min to develop a pink color, the mixture was then cooled with running
tap water and centrifuged at 3600× g at 25 ◦ C for 20 min. The absorbance of the supernatant was
measured at 532 nm, and 1,1,3,3-tetramethoxypropane (0–10 ppm) was used for standard preparation.
The TBARS value was calculated and reported as mg malonaldehyde/kg sample.
2.5.3. Color
The color of samples was determined using a Hunter lab colorimeter (Colour Flex, Hunter Lab Inc.,
Reston, VA, USA). Here, the L*, a*, b*, ∆E*, and ∆C* values were recorded.
2.5.4. Mean Particle Size
The mean particle size was determined following the method of Mad-Ali et al. [17] using
a laser particle size analyzer (LPSA) (Model LS 230, Beckman Coulter® , Fullerton, CA, USA).Appl. Sci. 2020, 10, 4141 4 of 12
The powder sample was first dispersed in distilled water. Five successive readings were conducted.
A volume-weighted mean particle diameter (d43) representing the mean diameter of a sphere with the
same volume was recorded.
2.5.5. X-ray Diffraction Analysis
The phase compositions and degree of crystallinity of the samples were determined by X-ray
diffraction (XRD) using an X-ray diffractometer (X’ Pert MPD, PHILIPS, Eindhoven, the Netherlands)
as tailored by Benjakul et al. [14] All powder samples were measured at a 2theta angle ranging from
20 to 60 degrees, with a step size of 0.05 and with X-ray power of 40 kV and 40 mA. A peak profile
matching method was employed to identify the phase compositions of each sample by matching the
measured peak positions to the Joint Committee on Powder Diffraction Standards (JCPDS).
2.5.6. Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM-EDX)
The microstructure, surface morphology, and local elemental analysis of bone powder samples
before and after calcination were observed by SEM-EDX as detailed by Chuaychan et al. [18] using a field
emission scanning electron microscope (FEI-XL30, FEI Company, Hillsboro, OR, USA) equipped with
an electron-dispersive X-ray spectroscope (EDX). After gold coating, the surface of the specimens was
observed with the secondary electron mode using an accelerating voltage of 20 kV and a magnification
of 50,000 while the elemental analysis was performed at a 5 kV.
2.6. Statistical Analysis
Experiments were run in triplicate. An analysis of variance (ANOVA) was carried out. Means were
compared using Duncan’s multiple range test. The Statistical Package for Social Science (SPSS 11.0 for
Windows, SPSS Inc, Chicago, IL, USA) was used for statistical analysis.
3. Results and Discussions
3.1. Total Soluble Protein and Hydroxyproline Content of Salmon Bone Leached out during Alkaline Treatment
The total soluble protein content liberated into alkaline solution used for the treatment of salmon
bone, a leftover from the hydrolysis process, was monitored as a function of time (Figure 1a). A sharp
increase in extractable protein from salmon bones was observed up to 50 min of the alkaline treatment.
Thereafter, a slight decrease in protein content was noticeable up to 120 min. Alkali was able to solubilize
the protein attached to the bone that remained after the enzymatic hydrolysis. This could lead to the
removal of proteinaceous substances from the aforementioned bones. With continuous stirring at an
operating temperature of 50 ◦ C, proteins were likely to undergo denaturation or unfolding. This resulted
in an increase in mass diffusivity, which in turn accelerated the mass transfer and solubilization of
denatured proteins from the bone matrix. The result was in line with Kumoro et al. [19], who reported
the positive impact of high temperature on the alkaline extraction of protein from chicken bone waste.
Alkaline solutions were reported to be effective in the removal of non-collagenous proteins from
the starting materials used for collagen or gelatin production [20]. After 50 min, no further increase in
the total soluble protein was found. This could be a result of less availability of soluble proteins in
the bone. A further degradation of peptides to free amino acids or di-peptides could result in a lower
content of proteins detected by the biuret method [21].
The content of hydroxyproline, a distinct amino acid presented in collagen, was determined during
alkaline treatment as shown in Figure 1b. Hydroxyproline in the bone matrix represents collagenous
proteins. Collagenous proteins were released with continuous stirring as a result of softening and
rupturing of the bones induced by the alkaline condition at high temperature. The release of these
collagenous proteins occurred continuously up to 100 min. A decline in hydroxyproline content was
found at 120 min. In the present study, the alkaline treatment for 40 min was selected to remove
non-collagenous proteins in the bone.Appl. Sci.
Appl. Sci. 2020, 10, x4141
2020, 10, FOR PEER REVIEW 55 of
of 13
12
Figure 1. Total soluble protein (a) and hydroxyproline (b) contents as a function of time during the
alkaline treatment of salmon bones. Bars represent the standard deviation (n = 3).
Figure 1. Total soluble protein (a) and hydroxyproline (b) contents as a function of time during the
3.2. Chemical Compositions
alkaline treatment of Bio-Calcium
of salmon bones. Barsand Calcined
represent theBone from deviation
standard Salmon Frame with and without
(n = 3).
Alkaline Treatment
Alkaline
Chemicalsolutions were reported
compositions to be effective
of the bio-calcium andincalcined
the removal
boneofarenon-collagenous
shown in Tableproteins
1. On afromdry
the starting materials used for collagen or gelatin production[20]. After 50 min,
basis, bones that were calcined for 6 h had very low moisture content, of which those calcinedno further increasefor
in
the total soluble
9 h possessed noprotein
moisturewasas found.
a resultThis
of thecould
highbe a result of treatment
temperature less availability
on bonesof soluble proteins in
during calcination,
the bone. A further degradation of peptides to free amino acids or di-peptides
which removed all the organic compounds and water molecules. For bio-calcium, Bio-cal-H could result in a lower
had
content of proteins detected by the biuret method [21].
a higher moisture content than Bio-cal-A (p < 0.05). Fluids or other proteinaceous residues might
bindThe content
or form of hydroxyproline,
a complex with water in a distinct
the boneamino
matrix acid presented by
as indicated in acollagen, was determined
higher moisture content.
during alkaline treatment as shown in Figure 1b. Hydroxyproline in the bone matrix
The ash content of calcined powders was higher than that in their bio-calcium counterparts (p < 0.05). represents
collagenous proteins.
During calcination, allCollagenous proteinswere
organic components werecombusted.
released withIt is continuous stirring as for
noted that calcination a result of
a longer
softening
time yieldedandarupturing
powder withof the bonesash
higher induced
contentbyasthe alkaline
a result of condition at high decomposition
a more complete temperature. The of
release of these collagenous proteins occurred continuously up to 100
the remaining organic matters. Overall, a high ash content of calcined powders (99.55–99.99%) min. A decline in
hydroxyproline
was obtained. content was found at 120 min. In the present study, the alkaline treatment for 40 min
was selected
The Ca/Ptomole
remove non-collagenous
ratios of Bio-cal-A andproteins
Bio-cal-Hinwere
the bone.
1.66 and 1.60, respectively. A slight increase in
the Ca/P ratio of the bone sample with alkaline treatment could be due to the removal of non-collagenous
3.2. Chemical Compositions
phosphoproteins presentedofin
Bio-Calcium and Calcined
the bone matrix Bone from at
[22]. Calcination Salmon Frame with
an elevated and without
temperature for a longer
Alkaline
time alsoTreatment
provided the calcined bone with increases in calcium and phosphorus contents, especially for
the non-alkaline treatment samples.
Chemical compositions The result suggests
of the bio-calcium that the
and calcined removal
bone of non-collagenous
are shown in Table 1. Onprotein
a dry
with alkaline
basis, bones thattreatment could have
were calcined for 6led to a very
h had higherlow ash content content,
moisture of Bio-cal-A. The calcined
of which bones show
those calcined for 9 ha
similar ratio,
possessed no in which Cal-A
moisture (6 h), of
as a result Cal-H (6 h),temperature
the high Cal-A (9 h), and Cal-Hon
treatment (9 h) had during
bones a ratio of 1.66, 1.61,
calcination,
1.66, and 1.63, respectively. Garner and Anderson [23] reported that
which removed all the organic compounds and water molecules. For bio-calcium, Bio-cal-H vertebrate bone contains inorganic
had a
matter moisture
higher in the form of non-stoichiometric
content than Bio-cal-A (p HAp crystals
< 0.05). Fluidsdeposited
or otherinproteinaceous
the matrix of cross-linked
residues might collagen
bind
fibrils.
or formIna general,
complexpure withHAp
waterhas in athe
Ca/P
bonemole ratioas
matrix ofindicated
1.67. In thebypresent
a higherstudy, the mole
moisture ratioThe
content. of Ca/P
ash
from salmon
content bone powders
of calcined (Table 1) was
was higher
relatedthan
closely
thatto inthat
theirofbio-calcium
hydroxyapatite. Based on
counterparts (pAppl. Sci. 2020, 10, 4141 6 of 12
Table 1. Chemical composition of bio-calcium and calcined powders from salmon frame.
Chemical Composition Bio-Cal-A Bio-Cal-H Cal-A (6 h) Cal-H (6 h) Cal-A (9 h) Cal-H (9 h)
Moisture (%) 4.82 ± 0.07 d 7.81 ± 0.04 e 0.27 ± 0.00b 0.45 ± 0.01 c 0.00 ± 0.00 a 0.00 ± 0.00 a
Protein (%) * 12.07 ± 0.18 b 20.90 ± 0.06 c 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a
Fat (%) * 0.33 ± 0.01 b 1.70 ± 0.01 c 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a
Ash (%) * 82.78 ± 0.25 b 69.59 ± 0.57 a 99.73 ± 0.05 d 99.55 ± 0.32 c 99.99 ± 0.00 f 99.97 ± 0.00 e
Hydroxyproline (mg/g) * 5.07 ± 0.01 b 14.79 ± 0.02 c 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a
TBARS (mg malonaldehyde/kg sample) * 0.95 ± 0.00 b 3.34 ± 0.01 c 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a
Calcium (%) * 30.88 ± 0.33 b 27.32 ± 0.17 a 36.01 ± 0.26 e 31.54 ± 0.20 c 38.84 ± 0.55 f 34.24 ± 0.40 d
Phosphorus (%) * 14.40 ± 0.39 b 13.22 ± 0.48 a 16.79 ± 0.25 e 15.16 ± 0.32 c 18.11 ± 0.35 f 16.27 ± 0.45 d
Mole ratio Ca/P 1.66 1.60 1.66 1.61 1.66 1.63
Values are presented as mean ± SD (n = 3). Different lowercase letters in the same row indicate significant difference
(p < 0.05). * Dry weight basis.
The lipid oxidation products of bio-calcium samples Bio-cal-A and Bio-cal-H expressed as TBAR
values are also shown in Table 1. Bio-cal-H had a higher TBAR value than Bio-cal-A (Table 1).
The TBARS measurement is used to monitor the degradation of hydroperoxides, which leads to the
formation of secondary oxidation products such as aldehydes [24]. The decrease in the TBARS value of
Bio-cal-A could be as a result of alkaline treatment, which could remove pro-oxidant proteins such as
hemoglobin. The result correlated well with differences in the fat content of Bio-cal-A and Bio-cal-H as
highlighted in Table 1. During alkaline treatment, the bone became soft and possibly led to the leaching
out of some lipids trapped in the bone matrix. Subsequent treatment with hexane could contribute to
the removal of lipid and lipid oxidation products to some extent. Oxidizing agents such as NaOCl and
H2 O2 plausibly disintegrated the lipid oxidation product easily in Bio-cal-A than in Bio-cal-H as a
result of the softness of the bones after the alkaline treatment in the former. This plausibly facilitated
the reduction in TBARS values in Bio-cal-A. In calcined samples, lipids were completely removed,
and the secondary oxidation products were not formed. Hence, TBAR values could not be detected for
all calcined bones. Overall, various pre-treatments and calcination process of fish bones could lower
the lipid oxidation in bio-calcium and calcined powder, respectively.
3.3. Color of Bio-Calcium and Calcined Powders
Bio-cal-A possessed a higher value of lightness (L* value) than Bio-cal-H and the calcined samples
(p < 0.05) (Table 2). The slightly higher creamy whitish color of Bio-cal-A could be a result of the removal
of meat residue as well as the blood during the alkaline treatment of salmon bone. For Bio-cal-A and
Bio-cal-H, during the drying process, carbonyl compound-related products were formed via lipid
oxidation and could undergo a non-enzymatic browning reaction with the proteins, peptides, and amino
group of the free amino acid retained in the bone powders. This resulted in the increased yellow color
obtained in the bio-calcium powders as indicated by the greater positive b* values. Both bio-calcium
powders had low redness (a* value). A similar trend was reported by Benjakul et al. [25] in which
bio-calcium powders from pre-cooked tongol (Thunnus tonggol) and yellowfin (Thunnus albacores) tuna
bone had higher yellowness than their calcined counterpart.
For the calcined samples, an increase in lightness (L*) was observed in both Cal-A and Cal-H when
calcination time increased from 6 to 9 h. Similarly, the increases in *∆C were also observed. It should
be noted that the L* value of Cal-H powders (69.49 ± 0.04) was less than that of Bio-cal-H powder
(98.84 ± 0.08). The darker color could result from the dark grey color of the unburnt carbonaceous
residue that may be trapped inside the porous structure of the fish bones during the calcination process.
The result corresponded with the large quantity of organic constituents present in the Bio-cal-H powder
as shown in Table 1. For a longer calcination period, the aforementioned carbonaceous particles could
be further removed, which resulted in an increment of the L* value to 80.68 ± 0.01. On the other
hand, this observation was less pronounced in Cal-A powders. The result illustrated the impact of
the alkaline pre-treatment and calcination process on the color of both the resulting bio-calcium and
calcined bone powders.Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 of 13
illustrated the impact of the alkaline pre-treatment and calcination process on the color of both the
resulting bio-calcium and calcined bone powders.
Appl. Sci. 2020, 10, 4141 7 of 12
Table 2. Mean particle size and color values of bio-calcium and calcined powders from salmon frame.
Parameters Bio-Cal-A
Table 2. Mean particle size and colorBio-Cal-H Cal-A (6 h) andCal-H
values of bio-calcium (6 h) powders
calcined Cal-Afrom
(9 h)salmonCal-H (9 h)
frame.
Mean particle
26.53 ± 3.49
Bio-Cal-A 24.05 ±Bio-Cal-H
f 3.14 d 25.36Cal-A
± 2.78(6e h) 22.21Cal-H
± 2.84(6ah) 22.39 ± 2.64
(9 h) 23.12
b ± 2.94 c
size (dParameters
43, µm) Cal-A Cal-H (9 h)
L* size (d43 , µm)
Mean particle 0.08 f± 3.49 f94.8424.05
95.29 ± 26.53 ± 0.08 d d93.61 ± 0.01
25.36 c
± 2.78 e 69.49 ± 0.04
22.21 a a 95.01
± 2.84 ± 0.01 e b 80.68
23.12 ± 2.94b c
± 0.01
± 3.14 22.39 ± 2.64
c e a −2.63 a e −0.36
a* L* 0.02 c± 0.08 f−0.3194.84
−0.60 ± 95.29 ± 0.07± 0.08
d d
−1.5993.61 ± 0.01
± 0.01 b 69.49
−0.23 ± 0.04
± 0.04 95.01 ± 0.01
± 0.01 80.68±± 0.01d b
0.03
c d b e a
b* A* −0.60
7.13 ± 0.02 d ± 0.02 −0.31 ± 0.07 −1.59 ± 0.01 −0.23 ± 0.04 −2.63 ± 0.01
6.86 ± 0.14 c c −0.19 ± 0.02 b b −0.10 ± 0.07 b b −1.67 ± 0.01 a a −0.22 ± 0.01 −0.36 ± 0.03b d
B* 7.13d ± 0.02 d 6.86 ± 0.14 −0.19 ± 0.02 −0.10 ± 0.07 −1.67 ± 0.01 −0.22 ± 0.01e b
ΔE* 6.94 ± 0.12 6.60 ± 0.08 c 0.87 ± 0.14 a 24.15 ± 0.15 f 3.00 ± 0.14 b 12.97 ± 0.09 e
f b
∆E* 6.94 ± 0.12 d 6.60 ± 0.08 c 0.87 ± 0.14 a 24.15 ± 0.15 3.00 ± 0.14 12.97 ± 0.09
ΔC* ∆C* 7.15 ± 0.03 f 6.87 ±
7.15 ± 0.03 f 0.14
6.87 ± 0.14 e 1.60 1.60
e ± 0.01
± 0.01 c 0.260.26
c ± 0.06
± 0.06 a 3.11
a ± 0.00
3.11 ± 0.00 d 0.42
d ±±
0.42 0.01 b
0.01 b
Values are
Values are presented
presented as mean
as mean ± SD (n±SD
= 3). (n = 3). lowercase
Different Differentletters
lowercase letters
in the same rowin the same
indicate row difference
significant indicate
(p < 0.05). difference (p < 0.05).
significant
3.4. Mean
Mean Particle
Particle Size
Size of Bio-Calcium and Calcine Bones
All samples
samples showed
showeddifferences
differencesininthethe mean
mean diameter
diameter (d43(d),43which
), which ranged
ranged fromfrom 22.21
22.21 to 27.53
to 27.53 µm.
µm. Bio-cal-A
Bio-cal-A showedshowed
largerlarger
particleparticle
size thansize
Bio-cal-H (p < 0.05)(p(Table
than Bio-cal-H < 0.05) 2).(Table 2). For
For Cal-A, the Cal-A, the higher
higher calcination
calcination
time resultedtime
in resulted in the
the decrease indecrease in particle
particle size. On the size. Onhand,
other the other hand,higher
a slightly a slightlysizehigher size was
was found for
Cal-H when calcination time was increased (p < 0.05). Bio-cal-A showed a bi-modal distribution because
found for Cal-H when calcination time was increased (p < 0.05). Bio-cal-A showed a bi-modal
distribution because of
of non-homogenous non-homogenous
particles as indicatedparticles as indicated
by the peak with theby the peakwhile
shoulder, with Bio-cal-H
the shoulder, whilea
showed
Bio-cal-H
mono-modal showed a mono-modal
distribution, distribution,
indicating the presence indicating the presence
of homogeneous of homogeneous
particles (Figure 2). Theparticles
surface
(Figure
moisture,2).protein,
The surface
and moisture, protein,are
fat concentration andthe
fatfactors
concentration
affectingare thethe factors affecting
stickiness the stickiness
and agglomeration of
and agglomeration
particles, of particles,
thus influencing thus size
the particle influencing the particle
distribution [26]. Duringsize alkaline
distribution[26].
treatment, During alkaline
the collagen in
treatment, the collagen
the bone matrix, whichinisthe bonelinked
tightly matrix,with
whichHAp,is tightly
mightlinked with HAp,
be removed. Thismight
couldbebring
removed.
aboutThis
the
could bringmatrix,
weakened about and
the weakened matrix,
the size could and the with
be reduced size could
ease. be reduced with ease.
Figure 2. Particle size distribution of bio-calcium and calcined bone powders from salmon bones.
Figure 2. Particle size distribution of bio-calcium and calcined bone powders from salmon bones.
For calcined samples, Cal-A and Cal-H had a more ordered and compact form with uniform
particle size distribution as shown by mono-modal distribution. In addition, the removal of organic
compounds possibly resulted in the lower particle diameters due to more compactness of HAp.Appl. Sci. 2020, 10, x FOR PEER REVIEW 9 of 13
For calcined samples, Cal-A and Cal-H had a more ordered and compact form with uniform
particle
Appl. size10,
Sci. 2020, distribution
4141 as shown by mono-modal distribution. In addition, the removal of organic
8 of 12
compounds possibly resulted in the lower particle diameters due to more compactness of HAp.
Therefore, the particle size distribution was greatly affected by the alkaline pre-treatment and
Therefore, the particle size distribution was greatly affected by the alkaline pre-treatment and
calcination temperatures.
calcination temperatures.
3.5. X-ray
3.5. X-RayDiffraction
Diffraction(XRD)
(XRD)Patterns
PatternsofofBio-Calcium
Bio-Calciumand
andCalcine
CalcineBone
BonePowders
Powders
Diffraction patterns
Diffraction patterns of
of both
both the
the bio-calcium
bio-calcium andand bone
bone powders
powders calcined
calcined atat 900
900 ◦°CC atat 66 and
and 99 hh
showed that the HAp (Ca 10(PO4)6(OH)2) phase (JCPDS:01-074-4172) was the dominant phase in all the
showed that the HAp (Ca10 (PO4 )6 (OH)2 ) phase (JCPDS:01-074-4172) was the dominant phase in all
samples
the samples(Figure 3). When
(Figure calcination
3). When was implemented,
calcination was implemented, the removal of organic
the removal matters
of organic and water
matters and
occurred along with the agglomeration of HAp nano-crystals [27]. As a result,
water occurred along with the agglomeration of HAp nano-crystals [27]. As a result, the diffraction the diffraction peaks
became
peaks more more
became pronounced
pronouncedfor theforcalcined powders
the calcined powdersthanthan
in bio-calcium
in bio-calcium powders
powders (Figure
(Figure 3a,b).
3a,b).It
Itshould
shouldbebenoted
notedthat
thatthe
thediffraction
diffractionpatterns
patternsof ofCal-H
Cal-H powders
powders showed
showed aa secondary phase of
secondary phase of another
another
apatite material in addition to the major phase of HAp (Figure 3b). According
apatite material in addition to the major phase of HAp (Figure 3b). According to the peak profile to the peak profile
fitting,this
fitting, thisminor
minorapatite
apatitephase
phasecould
couldbe beidentified
identifiedasasβ-tricalcium
β-tricalciumphosphate
phosphate(β-TCP:
(β-TCP: Ca/PCa/Pmole
moleratio
ratio
= 1.5) (JCPDF: 01-073-4869). The XRD data were in good agreement with
= 1.5) (JCPDF: 01-073-4869). The XRD data were in good agreement with the Ca/P ratios shown in the Ca/P ratios shown in
Table 1, where the β-TCP phase was only observed in non-alkaline-treated
Table 1, where the β-TCP phase was only observed in non-alkaline-treated Cal-H powders which Cal-H powders which
possessedaaCa/P
possessed Ca/Pratio
ratioof
ofabout
about1.61–1.63,
1.61–1.63,less
lessthan
thanthat
thatofofstoichiometric
stoichiometricHAp HAp(Ca:P
(Ca:P==1.67)
1.67)asas earlier
earlier
stated. On the other hand, only a single phase of HAp was observed in alkaline-treated
stated. On the other hand, only a single phase of HAp was observed in alkaline-treated Cal-A samples Cal-A samples
withaaCa/P
with Ca/Pofof1.66.
1.66.The
Theresults
resultsarearesupported
supportedbyby numerous
numerous works
works that
that have
have reported
reported thethe formation
formation of
of β-TCP
β-TCP forfor
thethe calcined
calcined HApHAp thatthat
hashas a Ca/P
a Ca/P molar
molar ratio
ratio lower
lower thanthan
1.671.67 [8,10,28].
[8,10,28].
Figure 3. X-ray diffraction patterns of bio-calcium and 900 ◦ C-calcined bone powders at 6 and 9 h with
Figure 3.treatment
alkaline X-ray diffraction patternsalkaline
(a) and without of bio-calcium and(b).
treatment 900 °C-calcined bone powders at 6 and 9 h with
alkaline treatment (a) and without alkaline treatment (b).
3.6. Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM-EDX)
3.6. Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM-EDX)
Figure 4a–d shows SEM images at 50,000× magnification of bio-calcium and bone powders
Figure
calcined at 900 ◦ C shows
4a–d SEM images in
6 h. Nano-particles at the
50,000×
rangemagnification bio-calcium and
of a few tens of nano-meters that bone powders
agglomerated
calcined
into largeratclusters
900 °C were
6 h. Nano-particles
observed for bothin the range of
Bio-cal-A a few
and tens of
Bio-cal-H nano-meters
(Figure that calcination
4a,b). After agglomerated of
into larger
these clusters
powders, were
larger observed
crystals for both
or grains Bio-cal-A
with the sizesand
of Bio-cal-H (Figure
a few hundred 4a,b). After were
nano-meters calcination
formedof
these powders,
(Figure 4c,d) as alarger
resultcrystals orgrowth
of crystal grains with
at an the sizes of
elevated a few hundred
temperature. nano-meters
The results were inwere formed
agreement
(Figure
with XRD 4c,d)
dataasthat
a result
showedof crystal growth
significant peakatnarrowing
an elevated temperature.
along Theincreasing
with a sharp results were in agreement
in peak intensity
with
of the XRD
calcineddatabone
thatpowders.
showed significant
The crystalpeak
sizes narrowing
of samples along with
with 9-h a sharp increasing
calcination (not showninin peak
this
intensity
article) of the
were calcined
similar boneofpowders.
to that The crystalsamples,
the 6-h calcination sizes of samples with supported
which were 9-h calcination (not nearly
by their shown
in this article)
identical wereatsimilar
full width to that of
half maximum the 6-h of
(FWHM) calcination
diffractionsamples,
patternswhich
shownwere supported
in Figure 3. by their
nearly identical full width at half maximum (FWHM) of diffraction patterns shown in Figure 3.Appl. Sci. 2020, 10, 4141 9 of 12
Appl. Sci. 2020, 10, x FOR PEER REVIEW 10 of 13
◦ C 6 h and
Figure 4. SEM
Figure 4. SEM images
imagesatat50,000×
50,000×magnification
magnificationforfor
(a)(a)
Bio-cal-A, (b)(b)
Bio-cal-A, Bio-cal-H, (c) Cal-A
Bio-cal-H, 900 900
(c) Cal-A °C 6 h
(d) ◦
andCal-H 900 900
(d) Cal-H C 6 °C
h. 6 h.
Nano-particles in the range of a few tens of nano-meters that agglomerated into larger clusters
Nano-particles in the range of a few tens of nano-meters that agglomerated into larger clusters
were observed for both Bio-cal-A and Bio-cal-H (Figure 4a,b). After the calcination of these powders,
were observed for both Bio-cal-A and Bio-cal-H (Figure 4a,b). After the calcination of these powders,
larger crystals or grains with the sizes of a few hundred nano-meters were formed (Figure 4c,d) as a
larger crystals or grains with the sizes of a few hundred nano-meters were formed (Figure 4c,d) as a
result
result of
of crystal
crystalgrowth
growthatatananelevated
elevated temperature.
temperature. TheTheresults were
results in agreement
were in agreement withwith
XRDXRD data data
that
showed significant
that showed peakpeak
significant narrowing alongalong
narrowing with with
sharpsharp
increasing in peak
increasing intensity
in peak of theof
intensity calcined bone
the calcined
powders. The crystal sizes of samples with 9-h calcination (not shown in
bone powders. The crystal sizes of samples with 9-h calcination (not shown in this article) were this article) were similar to
that of 6-h
similar calcination
to that samples, which
of 6-h calcination samples,werewhich
supported by their nearly
were supported identical
by their nearlyFWHM of diffraction
identical FWHM of
patterns shown in Figure 3.
diffraction patterns shown in Figure 3.
A
A relative
relativeabundance
abundanceofof elements
elements in all
in the
all samples was observed
the samples using SEM-EDX.
was observed Elements
using SEM-EDX. such as
Elements
Ca,
suchP,asC,Ca,
O,P,Na, and
C, O, Na,Mgandwere
Mgfound
were foundin both the bio-calcium
in both the bio-calcium and and
calcined
calcinedpowders
powdersas depicted
as depictedin
Figure 5. It was noted that the detection limit of SEM-EDX could vary from
in Figure 5. It was noted that the detection limit of SEM-EDX could vary from 1–10%wt as a result of 1–10%wt as a result of
spectral
spectral resolution
resolution and and difficulty
difficulty in
in detecting
detecting low-Z
low-Z elements [29]. Consequently,
elements[29]. Consequently, other trace elements
other trace elements
could not be detected in the bio-calcium and calcined powders. Despite the
could not be detected in the bio-calcium and calcined powders. Despite the instrumental limitation, instrumental limitation,
the
the results
results show
show that
that the
the alkaline
alkaline treatment
treatment of of fish
fish bones
bones ledled to
to an
an increase
increase in in Ca
Ca and
and P P contents
contents ofof
the bio-calcium powder. This was in line with higher contents of Ca and
the bio-calcium powder. This was in line with higher contents of Ca and P determined by ICP-OES P determined by ICP-OES
(Table
(Table 1).
1). In
In addition,
addition, for
for all
all calcined
calcined samples,
samples, moremore pronounced
pronounced and and sharp
sharp peaks
peaks indicating
indicating aa higher
higher
intensity of Ca and P were observed. Simultaneously, the contents of C were
intensity of Ca and P were observed. Simultaneously, the contents of C were decreased, re-affirming decreased, re-affirming
the
the removal
removal of of organic
organic matters
matters such
such asas lipids
lipids and
and meat
meat proteins
proteinsas asaaresult
resultof ofcalcination.
calcination.Appl. Sci.
Appl. Sci. 2020,
2020, 10,
10, x FOR PEER REVIEW
4141 11 of
10 of 12
13
Figure 5. Elemental profile of bio-calcium and calcined powders from salmon bones as analyzed
Figure 5. Elemental profile of bio-calcium and calcined powders from salmon bones as analyzed by
by SEM-EDX.
SEM-EDX.
4. Conclusions
4. Conclusions
The present study demonstrates the effect of alkaline treatment in the material conversion process
of theThe present
salmon framestudy demonstrates
by-product the effect
to bio-calcium andofHApalkaline treatment
powders. in the showed
The results materialthat
conversion
alkaline
process of the salmon frame by-product to bio-calcium and HAp powders. The results
treatment played an important role on the characteristics of all samples. The amount of bio-molecular showed that
alkaline treatment played an important role on the characteristics of all samples.
compounds such as fat, protein, and collagen was significantly lowered in the bio-calcium powder The amount of bio-
molecular
with alkalinecompounds
treatment.such as fat,
On the protein,
other hand,and collagen
the Ca/P was
molar significantly
ratio was lowerlowered in the bio-calcium
in the bio-calcium sample
powder with alkaline treatment. On the other hand, the Ca/P molar ratio
without the treatment. Consequently, phase compositions of the calcine bone powders was lower in the bio-calcium
could be
samplea without
either the treatment.
single-phase Consequently,
HAp or mixed-phase phase HAp
between compositions
and β-TCPof the calcine bone
depending powders
on their could
Ca/P ratios.
be addition,
In either a single-phase
salmon frameHAp couldorbemixed-phase between
used as a potential HAp
source and β-TCP
to produce highdepending
value-added oncompounds
their Ca/P
ratios. In addition, salmon frame could be used as a potential source to produce
such as bio-calcium for calcium supplements and apatite compounds for bio-medical applications. high value-added
compounds such as bio-calcium for calcium supplements and apatite compounds for bio-medical
applications.Appl. Sci. 2020, 10, 4141 11 of 12
Author Contributions: Conceptualization, S.B.; methodology, A.T.I. and S.B.; software, S.S., T.S.-l., N.S., and Y.K.;
validation, S.S., T.S.-l., N.S., and Y.K.; formal analysis, A.T.I.; investigation, A.T.I.; resources, S.B. and P.S.; data
curation, P.S.; writing—original draft preparation, A.T.I.; writing—review and editing, S.B. and P.S.; visualization,
S.B. and P.S.; supervision, S.B. and P.S.; project administration, S.B. and P.S.; funding acquisition, S.B. and P.S.
All authors have read and agreed to the published version of the manuscript.
Funding: The authors declared that no funding was secured for this project.
Acknowledgments: This research was supported by the Higher Education Research Promotion and Thailand’s
Education Hub for Southern Region of ASEAN Countries Project, the Office of the Higher Education Commission
and the Graduate School, Prince of Songkla University (Grant number AGR6302013N).
Conflicts of Interest: The authors at this moment declared no conflict of interest.
References
1. United Nations. World Population Prospects: The 2017 Revision, Key Fndings and Advance Tables; Working Paper
No. ESA/P/WP/248; United Nations: New York, NY, USA, 2017.
2. See, S.F.; Hoo, L.; Babji, A. Optimization of enzymatic hydrolysis of salmon (Salmo salar) skin by Alcalase.
Int. Food Res. J. 2011, 18, 1359–1365.
3. Sinthusamran, S.; Idowu, A.T.; Benjakul, S.; Prodpran, T.; Yesilsu, A.F.; Kishimura, H. Effect of proteases
and alcohols used for debittering on characteristics and antioxidative activity of protein hydrolysate from
salmon frames. J. Food Sci. Technol. 2019, 57, 473–483. [CrossRef] [PubMed]
4. Benjakul, S.; Mad-Ali, S.; Senphan, T.; Sookchoo, P. Biocalcium powder from precooked skipjack tuna bone:
Production and its characteristics. J. Food Biochem. 2017, 41, e12412. [CrossRef]
5. Cashman, K.D. Calcium intake, calcium bioavailability and bone health. Br. J. Nutr. 2002, 87, 169–177.
[CrossRef]
6. Watanabe, T.; Kiron, V.; Satoh, S. Trace minerals in fish nutrition. Aquaculture 1997, 151, 185–207. [CrossRef]
7. Boutinguiza, M.; Pou, J.; Comesaña, R.; Lusquiños, F.; De Carlos, A.; León, B. Biological hydroxyapatite
obtained from fish bones. Mater. Sci. Eng. C 2012, 32, 478–486. [CrossRef]
8. Piccirillo, C.; Silva, M.; Pullar, R.C.; Da Cruz, I.B.; Jorge, R.; Pintado, M.; Castro, P. Extraction and
characterisation of apatite- and tricalcium phosphate-based materials from cod fish bones. Mater. Sci. Eng. C
2013, 33, 103–110. [CrossRef]
9. Figueiredo, M.M.L.; Fernando, A.; Martins, G.; Freitas, J.; Judas, F.; Figueiredo, H. Effect of the calcination
temperature on the composition and microstructure of hydroxyapatite derived from human and animal
bone. Ceram. Int. 2010, 36, 2383–2393. [CrossRef]
10. Goto, T.; Sasaki, K. Effects of trace elements in fish bones on crystal characteristics of hydroxyapatite obtained
by calcination. Ceram. Int. 2014, 40, 10777–10785. [CrossRef]
11. Cheng, L.; Ye, F.; Yang, R.; Lu, X.; Shi, Y.; Li, L.; Fan, H.; Bu, H. Osteoinduction of hydroxyapatite/β-tricalcium
phosphate bioceramics in mice with a fractured fibula. Acta Biomater. 2010, 6, 1569–1574. [CrossRef]
12. Gornall, A.G.; Bardawill, C.J.; David, M.M. Determination of serum proteins by means of the biuret reaction.
J. Biol. Chem. 1949, 177, 751–766.
13. Bergman, I.; Loxley, R. Two Improved and Simplified Methods for the Spectrophotometric Determination of
Hydroxyproline. Anal. Chem. 1963, 35, 1961–1965. [CrossRef]
14. Benjakul, S.; Mad-Ali, S.; Senphan, T.; Sookchoo, P. Characteristics of Biocalcium from Pre-cooked Skipjack
Tuna Bone as Affected by Different Treatments. Waste Biomass-Valorization 2017, 9, 1369–1377. [CrossRef]
15. Ensminger, L.G. The Association of Official Analytical Chemists. Clin. Toxicol. 1976, 9, 471. [CrossRef]
16. Feist, B.; Mikula, B. Preconcentration of heavy metals on activated carbon and their determination in fruits
by inductively coupled plasma optical emission spectrometry. Food Chem. 2014, 147, 302–306. [CrossRef]
17. Mad-Ali, S.; Benjakul, S.; Prodpran, T.; Maqsood, S. Characteristics and gel properties of gelatin from goat
skin as affected by spray drying. Dry. Technol. 2017, 35, 218–226. [CrossRef]
18. Chuaychan, S.; Benjakul, S.; Kishimura, H. Characteristics of acid- and pepsin-soluble collagens from scale
of seabass (Lates calcarifer). LWT 2015, 63, 71–76. [CrossRef]
19. Kumoro, A.C.; Sofiah, S.; Aini, N.; Retnowati, D.S.; Budiyati, C.S. Effect of Temperature and Particle Size on
the Alkaline Extraction of Protein From Chicken Bone Waste. Reaktor 2010, 13, 124. [CrossRef]Appl. Sci. 2020, 10, 4141 12 of 12
20. Liu, D.; Wei, G.; Li, T.; Hu, J.; Lu, N.; Regenstein, J.M.; Zhou, P. Effects of alkaline pretreatments and acid
extraction conditions on the acid-soluble collagen from grass carp (Ctenopharyngodon idella) skin. Food Chem.
2015, 172, 836–843. [CrossRef]
21. Wu, H.-C.; Chen, H.-M.; Shiau, C.-Y. Free amino acids and peptides as related to antioxidant properties in
protein hydrolysates of mackerel (Scomber austriasicus). Food Res. Int. 2003, 36, 949–957. [CrossRef]
22. Al-Qtaitat, A.I.; Aldalaen, S.M. The Isolation and Characterization of Glycosylated Phosphoproteins From
Herring Fish Bones. J. Biol. Chem. 2010, 285, 36170–36178.
23. Garner, S.; Anderson, J. Skeletal Tissues and Mineralization. Diet Nutr. Bone Health 2011, 33, 49–50.
24. Sae-Leaw, T.; Benjakul, S. Physico-chemical properties and fishy odour of gelatin from seabass (Lates calcarifer)
skin stored in ice. Food Biosci. 2015, 10, 59–68. [CrossRef]
25. Benjakul, S.; Mad-Ali, S.; Sookchoo, P. Characteristics of Biocalcium Powders from Pre-Cooked Tongol
(Thunnus tonggol) and Yellowfin (Thunnus albacores) Tuna Bones. Food Biophys. 2017, 12, 412–421. [CrossRef]
26. Pısecký, J. Handbook of milk powder manufacture. Niro A/S, Copenhagen. Proc. Eng. 1997, 3, 3–9.
27. Londoño-Restrepo, S.M.; Jeronimo-Cruz, R.; Millán-Malo, B.M.; Rivera-Muñoz, E.M.; Rodríguez-García, M.E.
Effect of the Nano Crystal Size on the X-ray Diffraction Patterns of Biogenic Hydroxyapatite from Human,
Bovine, and Porcine Bones. Sci. Rep. 2019, 9, 5915. [CrossRef]
28. Meinke, D.K.; Skinner, H.C.W.; Thomson, K.S. X-ray diffraction of the calcified tissues inPolypterus.
Calcif. Tissue Int. 1979, 28, 37–42. [CrossRef]
29. Choël, M.; Deboudt, K.; Osán, J.; Flament, P.; Van Grieken, R. Quantitative Determination of
Low-ZElements in Single Atmospheric Particles on Boron Substrates by Automated Scanning Electron
Microscopy−Energy-Dispersive X-ray Spectrometry. Anal. Chem. 2005, 77, 5686–5692. [CrossRef]
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).You can also read
