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Sequential extraction protocol for bio-calcium from saltwater crocodile (Crocodylus porosus) bone: Physicochemical and molecular studies
(2026-06-01)
Yarnpakdee, Suthasinee
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Senphan, Theeraphol
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Benjakul, Soottawat
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Sriket, Chodsana
Saltwater crocodile ( Crocodylus porosus ; SC) farming generates 45% bone waste, creating environmental challenges but also representing an underutilized source of calcium. This study presents the first comprehensive protocol for extracting bio-calcium from SC bone through sequential processing: autoclaving, alkaline soaking, ethanol extraction, and hydrogen peroxide bleaching. The extraction process yielded 16.94% bio-calcium with a high mineral density (26.37% Ca and 13.83% P) and an average particle size of 6.77 μm. XRD confirmed the preserved hydroxyapatite structure, while FTIR revealed the complete elimination of the organic matrix in the resulting bio-calcium. SEM-EDS demonstrated uniform elemental distribution with no detectable heavy metals. Remarkably, in vitro calcium bioavailability reached 15.24%, representing a 78% enhancement over fish bone bio-calcium and doubling synthetic supplement performance. Preserved collagen-derived amino acids facilitate calcium-peptide chelation, explaining the superior absorption. The developed valorization process converts aquaculture waste into high-value ingredients with potential applications in food and pharmaceuticals.
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Improving calcium bioavailability from fish bone waste: the role of sodium hydroxide in bio‑calcium extraction from tilapia (Oreochromis niloticus) bones
(2025-10-01)
Senphan, Theeraphol
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Mungmueang, Natthapong
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Choommongkol, Vachira
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Sriket, Chodsana
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Kishimura, Hideki
Calcium deficiency is a global health issue motivating the search for sustainable calcium sources from aquaculture waste. This study investigated sodium hydroxide (NaOH) concentration effects on bio‑calcium quality and bioavailability extracted from tilapia (Oreochromis niloticus) bones. Tilapia bones were treated with 0.5 M, 1 M, and 2 M NaOH solutions for 30 min compared to untreated controls. Increasing NaOH concentrations decreased yield while improving brightness, ash content, and calcium bioavailability. The 2 M NaOH treatment produced bio‑calcium with highest calcium bioavailability (8.57 %), surpassing both control (7.26 %) and commercial calcium carbonate (0.72 %) by 12-fold. Higher NaOH concentrations reduced moisture, protein, and fat contents while increasing hydroxyproline and decreasing lipid oxidation. SEM showed smoother surfaces with homogeneous pores in 2 M NaOH-treated samples. ATR-FTIR and EDS confirmed consistent hydroxyapatite structure. This optimization provides a sustainable approach for converting fish waste into high-bioavailability calcium supplements, supporting circular economy in aquaculture while addressing global calcium deficiency.
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Synthesis of soluble calcium compound from skipjack tuna bones using edible weak acids
(2022-06-01)
Aenglong, Chakkapat
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Wang, Yu Ming
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Limpawattana, Maruj
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Sukketsiri, Wanida
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Tang, Qing Juan
This study aimed to enhance the water solubility of calcium from tuna (Katsuwonus pelamis) bones (TB). Calcium hydroxyapatite (HA) was obtained by calcination of TB. For mineral profiles of TB and HA, Ca had the highest content followed by P with Ca/P of 3.47 and 1.94. Mg, K, and Na as macro minerals and Cr, Cu, Mn, and Zn as micro minerals were also found. Subsequently, HCl was applied to form structured HA-chloride compound (HA-Cl) then reacted with alkaline to obtain structured HA-hydroxide compound (HA-OH). In order to enhance the water solubility, citric and lactic acid were individually reacted with HA-Cl and HA-OH to obtain structured HA-citrate compounds (HA-Cl-Ci and HA-OH-Ci) and structured HA-lactate compounds (HA-Cl-Lac and HA-OH-Lac). HA-Cl-Ci had the highest water solubility (93.34 ± 0.45%) followed by HA-OH-Lac (90.35 ± 0.23%) with Ca/P ratio of 2.28 and 2.24, respectively. HA-Cl-Ci and HA-OH-Lac demonstrated the similar mineral profiles to those of HA but with different contents. The results of FT-IR indicated the carboxyl group of acid played an important role in binding Ca<sup>2+</sup>. X-ray diffractograms indicated that calcined bone was HA whereas the major composition of HA-Cl-Ci and HA-OH-Lac was related to calcium citrate tetrahydrate and calcium lactate pentahydrate, respectively.
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Bioactive alginate/carrageenan/calcium silicate porous scaffolds for bone tissue engineering
(2021-03-01)
Sathain, Ammara
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Monvisade, Pathavuth
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Siriphannon, Punnama
Porous bioactive alginate/carrageenan/calcium silicate scaffolds for bone tissue engineering were fabricated. The scaffolds were prepared by dispersing the synthesized calcium silicate in an aqueous solution of alginate and carrageenan at 90 °C. The scaffolds were shaped by freeze-drying and further crosslinked by 0.5, 1.0 and 1.5 M CaCl<inf>2</inf> for 60 and 120 min. The scaffolds crosslinked by 1.5 M CaCl<inf>2</inf> for 120 min achieved the highest in vitro dimension stability. The formation of hydroxyapatite crystals was observed on the scaffolds surface after soaking in simulated body fluid (SBF) at 37 °C for 7–28 days, indicating in vitro bioactivity of the scaffolds. The presence of calcium silicate could enhance not only the bioactivity, but also the mechanical properties of the scaffolds comparable to the cancellous bone. Moreover, the dimension and mechanical properties of the wet scaffolds could recover to the original after four cycles of mechanical testing at 50 % strain. The scaffolds were nontoxic to human living cells, in which the in vitro drug release behavior of the scaffold using diclofenac as a model drug was suitable for the treatment of acute inflammation after surgery. Therefore, these scaffolds were considered to be the candidate materials for bone replacement.
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In situ ring-opening polymerization of hydroxyapatite/poly(ethylene adipate)-co-(ethylene terephthalate) biomimetic composites
(2013-02-01)
Siriphannon, Punnama
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Monvisade, Pathavuth
Hydroxyapatite/poly(ethylene adipate)-co-poly(ethylene terephthalate) biomaterials (HAp/PEA-co-PET) have been prepared by ring opening polymerization (ROP) of cyclic oligo(ethylene adipate)-co-oligo(ethylene terephthalate) (C-OEA-co-C-OET) in the porous hydroxyapatite (HAp) scaffolds at 250 °C for 24 h under vacuum. The content of ROP-PEA-co-PET in the HAp/PEA-co-PET composite was about 20 wt% with the values of number average molecular weight (Mn) and weight average molecular weight (MW) of 3380 and 7160 g/mol, respectively. Compressive strength and modulus of the HAp/PEA-co-PET composites were about 29 and 246 MPa, respectively. These mechanical properties were higher than those of the porous HAp templates and natural cancellous bone. In vitro bioactivity of the HAp/PEA-co-PET composites was studied by soaking in simulated body fluid (SBF) under the flowing system at the rate of 130 mL/day for 7, 14, 21 and 28 days. The formation of hydroxyapatite nanocrystals was observed on the composite surfaces through the consumption of calcium and phosphorus from the SBF solution, indicating the bioactivity of these HAp/PEA-co-PET composites. These results indicated the competency of HAp/PEA-co-PET composites for biomedical applications. © Indian Academy of Sciences.
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Poly(ethylene terephthalate)/hydroxyapatite biomaterials: Preparation, characterization, and in vitro bioactivity
(2009-02-01)
Siriphannon, Punnama
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Monvisade, Pathavuth
Poly(ethylene terephthalate)/hydroxyapatite (PET/HAp) composites were prepared by mixing HAp powder with a mixture solution of cyclic oligo(ethylene terephthalate) (C-OET) and dibutyl tinoxide catalyst in dichrolomethane, and then shaping the precomposites in cylindrical pellets. The C-OET in the precomposites was ring-opening polymerized (ROP) to PET under vacuum at 250°C for 24 h. The PET/HAp composites were formulated with HAp to PET ratios of 60:40 (H6P4) and 50:50 (H5P5). The ROP-PET in the composites was present as a thin-layer coating on the HAp grains and evenly distributed throughout the samples. Compressive strength of the PET/HAp composites was significantly increased from 8 MPa of the H10P0 to 17 and 29 MPa for H6P4 and H5P5, respectively. In vitro bioactivity of the PET/HAp composites was studied by soaking in simulated body fluid (SBF) at 36.5°C for 7-28 days. After prolonged soaking, the HAp nanocrystals precipitated from the SBF solution and formed as a layer of globular aggregates, coated on the composite surfaces. The H6P4 composite showed faster formation rate of nano-HAp than the H5P5 composite, indicating that the bioactivity of PET/HAp composites depended on the amount of HAp. © 2008 Wiley Periodicals, Inc.
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Preparation and characterization of hydroxyapatite/poly(ethylene adipate) hybrid composites
(2008-06-01)
Siriphannon, Punnama
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Monvisade, Pathavuth
Hydroxyapatite/poly(ethylene adipate) (HAp/PEA) composites were prepared by in situ ring-opening polymerization of cyclic oligo(ethylene adipate) (C-OEA) within the porous HAp templates. HAp was firstly prepared by a co-precipitation method using calcium hydroxide and phosphoric acid and then shaped as a rectangular porous template. PEA precursor was synthesized by bulk polymerization of dimethyl adipate and ethylene glycol in the presence of tetraisopropyl orthotitanate. C-OEA was obtained by cyclo-depolymerization of the PEA precursor under high dilution condition using dibutyl tinoxide as a catalyst. The HAp/PEA composites were prepared by immersing the porous HAp templates in the mixture solution of C-OEA and dibutyl tinoxide catalyst overnight and ring-opening polymerizing at 180, 200 and 220°C for 24 h. The ring-opening polymerized PEA formed as a thin film coating on the surface of porous HAp template. The HAp/PEA composites contained PEA in the range of 20-26 wt%. The weight-average molecular weights of ring-opening polymerized PEA were in the range of 3800-4450 g/mol. Compressive strength of the HAp/PEA composite was significantly increased from 25 MPa in the porous HAp template to 140 MPa in the composite. © 2008 VSP.
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Preparation and characterization of hydroxyapatite/poly(ethylene glutarate) biomaterials
(2007-05-01)
Siriphannon, Punnama
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Monvisade, Pathavuth
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Jinawath, Supatra
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Hemachandra, Khemchai
Hydroxyapatite/poly(ethylene glutarate) (HAp/PEG) biomaterial composites were prepared by ring-opening polymerization (ROP) of cyclic oligo(ethylene glutarate) (C-PEG) in porous HAp scaffolds. The HAp/C-PEG pre-composites were prepared by immersing the porous HAp scaffolds in the mixture solution of C-PEG and dibutyl tin-oxide catalyst overnight and polymerizing at 200°C for 24, 48, and 72 h under vacuum. The successful ROP of C-PEG in the porous HAp scaffolds was corroborated by the signals of hydroxyl end-group of PEG shown in the <sup>1</sup>H NMR spectrum of the ROP-products extracted from the composites. PEG in the composites was present as a thin layer coating on the HAp grains and was evenly distributed throughout the samples. The PEG content was about 13-16 wt % and decreased with increasing polymerization time. Its molecular weight (M̄<inf>w</inf>, weight average) measured by gel permeation chromatography was in the range of 4300-6800 g/mol. Compressive strength of the HAp/PEG composites was significantly increased from 3 MPa of the porous HAp scaffold to 11-15 MPa, depending on the PEG content in the composites. In vitro bioactivity of the HAp/ PEG composites was studied by soaking in simulated body fluid (SBF) at 36.5°C for 7-28 days. After prolonged soaking, the HAp nanocrystals precipitated from the SBF solution and formed as a layer of globular aggregates, coated on the composite surfaces. This result suggested that the HAp/PEG composite was a bioactive material. © 2006 Wiley Periodicals, Inc.
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Comparative study of the formation of hydroxyapatite in simulated body fluid under static and flowing systems
(2002-01-01)
Siriphannon, Punnama
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Kameshima, Yoshikazu
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Yasumori, Atsuo
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Okada, Kiyoshi
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Hayashi, Shigeo
α-CaSiO<inf>3</inf> ceramics of nominal composition CaO 46.0, SiO<inf>2</inf> 54.0, and Na<inf>2</inf>O 0.4 mass% were soaked in simulated body fluid (SBF). The soaking systems were maintained under both static and flowing conditions to study their effect on the formation of hydroxyapatite (HAp). Two different flowing systems were designed for soaking, namely, a closed system using a fast flow rate of about 2.8 mL/s (circulating system) and an open system using a slow flow rate of about 40 mL/day (slow flowing system). The HAp layer in all samples initially formed as a rough layer of ball-like particles. Under a fast flow of SBF solution, silica gel particles peeled from silica-rich interlayer during the first soaking period. The silica gel particles then reattached to the product HAp layer and induced the formation of new HAp particles of smaller size. In the slow flowing system, the rough HAp layer initially formed on the ceramic surfaces became gradually smoother after prolonged soaking. The formation rate and thickness of the HAp layer decreased with increasing flow rate of the SBF solution. These results indicate that flowing SBF solution gives rise to differences in the formation rates, formation behavior, and microstructure of the HAp layer. © 2002 John Wiley & Sons, Inc.
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Hydroxyapatite formation on CaSiO3 ceramics in protein containing system
(2001-01-01)
Okada, Kiyoshi
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Siriphannon, Punnama
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Kameshima, Yoshikazu
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Yasumori, Atsuo
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Hayashi, Shigeo
α-CaSiO<inf>3</inf> ceramics with a nominal composition of CaO 46.0, SiO<inf>2</inf> 54.0, and Na<inf>2</inf>O 0.4 wt% were soaked in two different solutions, simulated body fluid (SBF) and α-minimum essential medium (α-MEM), to study the effect of protein on the formation of hydroxyapatite (HA). The SBF solution contained the same inorganic salts as in the human blood plasma, while the α-MEM solution contained both inorganic salts and various proteins. Both soaking systems were maintained at 36.5°C and a flow rate of about 40 ml/day for various periods of times. A complete coating of HA formed on the surface of the α-CaSiO<inf>3</inf> ceramics in both solutions, but with different formation behavior and microstructure. The HA formed in the α-MEM system had ball-like particle morphology with a smaller particle size than the material formed in the SBF system. Cristobalite and/or unknown silicate phase coprecipitate with the HA in the α-MEM system but not in SBF. The HA layer formed on the ceramic surface achieved a thickness of about 80 and 93 μm after 20 days soaking in SBF and α-MEM solutions, respectively. The presence of protein in the α-MEM solution was considered to effect the microstructure of HA and possibly enhanced HA precipitation on the α-CaSiO<inf>3</inf> ceramic. © 2002 Trans Tech Publications, Switzerland.

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