Preparation of Nano-hydroxyapatite Obtained from Lates Calcarifer Fish Bone by Alkaline Hydrolysis Method

: Fish bone by-products are considered as abundant and cheap sources of Hydroxyapatite (HAp). The preparation of HAp powders from fish bones not only contributes to improving the added value of by-products but also reduces undesirable impacts on the environment. In this study, nano-HAp was successfully obtained from Lates calcarifer fish bone originated from a seafood export company in Khanh Hoa province. After pretreatment of fish bones for removing organic matters, the bones were under alkaline hydrolysis at 200°C within different time intervals of 30 mins, 1 and 1.5 hours. Results of XRD and SEM analysis showed that the calcium formed was HAP and it possessed an average size of 50-64 nm. The values of the Ca/P molar ratio from 1.896 to 1.921 prove that the nano-HAp powders are B-type biological hydroxyapatites which have been confirmed by FTIR spectrum. In addition, the contents of heavy metals such as As, Pb, Hg, Cd are measured by emission spectrophotometer and detected within safety limits of regulatory requirements of Vietnam regulation and US Pharmacopeia for food and dietary supplement standard. These properties show that nano-HAp from Lates calcarifer fish bone are applicable and to be used as an input material in food and medicine field.


Introduction
Calcium hydroxyapatite, also known as hydroxyapatite (HAp), is a natural calcium phosphate with high biological compatibility with cells and tissues [1]. The chemical formula of HAp is Ca 10 (PO 4 ) 6 (OH) 2 , and it is the main ingredient in human and animal bones and teeth (up to 60-70% by weight in bones [2]; and 97% in teeth [3]). HAp has the Ca/P molar ratio similar to that in bones and teeth (Ca/P=1.67) [4,5]. With the above valuable properties, HAp in the fine powder, super fine, porous, and film form has been studied to expand their applicability. HAp is currently being employed as a calcium supplement [6]; as surgical materials used in bone and tooth implants [4,7,8]; and as an absorbent material [9]; and in regeneration of cranial defects [10].
In recent years, millions of tons of fish are caught each year for human consumption, but only 50-60% of the total catch is used, and the rest is discarded [33]. Accompanying the increase in seafood products contributing to economic development, the seafood processing industry generates a large number of by-products. If not handled carefully, these by-products will be a big challenge for the environment. However, these by-products are the raw materials to extract valuable compounds for human life. Therefore, the bones of many fish species have been used to separate HAp by different methods such as swordfish (Xiphia gladius) and tuna (Thunnus thynnus) [34], cod fish [35], Japanese sea bream [38], salmon [39], grouper [40], tilapia [41], skipjack tuna (Katsuwonus pelamis) [42].
Recently in Vietnam, there is an abundant source to extract HAp: seabass. Seabass is being raised by two main methods: farming in the mud areas or in cages at sea. These fishes are cultured along the coasts of Quang Ninh, Hai Phong, Thua Thien -Hue, Quang Nam, Da Nang, Binh Dinh, Khanh Hoa, Binh Thuan, Ba Ria -Vung Tau and Spratly archipelago (Source of Vietnam Fisheries Newspaper, 2018). The total production is estimated to reach 4382 tons of seabass culture by 2014. After the fillets are being exported to the US, Europe, Taiwan and South Korea (Source of Science and Technology Associations of Khanh Hoa province, 2014), the amount of fish bone by-products that is left is an abundant source for extracting HAp. It has been well-known that the extraction of HAp by the thermal calcination method is a traditional way [8]. Another method, the alkaline hydrolysis, is also a route to obtain nanostructured HAp and carbonated HAp without the milling process [43]. On the basis of these observations, in our paper, we have studied HAp isolation from seabass Lates calcarifer bone through the alkaline hydrolysis method within different time intervals of 30 mins, 1 and 1.5 hours at 200°C. The present study is intended for the preparation of HAp directly from fish bone for various biomedical applications.

Fish Bone Treatment
Lates calcarifer seabass bone after being filleted and head cut off were purchased at T and H Company Limited (Vinh Phuong ward, Nha Trang City, Khanh Hoa Province, Vietnam) in January 2021. The fish bones were then kept on ice, and transported to the laboratory of the institute of oceanography. Next, the seabass bones are boiled for 1 hour to remove the remaining soft tissue and washed with tap water to remove the remaining muscle, and this process is repeated several times until obtaining white bones. The bones were then dried at room temperature to constant mass.
The washed bones were then boiled with 1.0% NaOH and acetone (the bone: solution=1:50) to remove protein, lipids, oils and organic impurities that still adhered. After being washed continuously with water until the pH was neutral, the bones were ground in a mortar pestle and then dried at 60°C for 24h to obtain powder for the next experiment.

Alkaline Hydrolysis of Fish Bone
The bone powder was treated under alkaline hydrolysis following the protocol that was developed by Venkatesan et al. (2015) with some modifications [39]. Briefly, the bone powder was heated in 2M NaOH (the bone powder: solution=1: 30) for 30 minutes, 1 and 1.5 hours at 200°C. This mixture was then filtered by a pump and rinsed continuously with water until the pH was neutral. The resulting product was dried in an oven at 60°C until constant mass.

Characterisation Techniques
The stretching frequencies of the resulting samples were examined by Fourier transform infrared spectroscopy (FTIR) (Bruker Equinox 55). The phase and crystallinity were evaluated using an x-ray diffractometer (D2 Pharser-Brucker). The resultant XRD spectra were compared with the literature profile from the International Centre for Diffraction Data (ICDD 00-009-0432) to identify the hydroxyapatite compound.
The particle size and morphology of the hydroxyapatite compound were observed by a scanning electron microscope (Hitachi S-4800). The average crystal size is calculated using ImageJ software 1.48V with the scale bar in the image, and this value is expressed as the mean ± SE.
The heavy metals such as Pb, Hg, Cd, As and the calcium and phosphor were measured by emission spectrophotometer (Agilent 7700x-LC-MS).   Figure 1c shows that the powder material before alkaline hydrolysis is fine, yellowish in colour; while calcium hydroxyapatite powder formed at 200°C in different time periods is more yellow in colour, smooth, odourless and tasteless.  IR spectra of samples formed under alkaline hydrolysis at 200°C in different time periods (0.5, 1 and 1.5 hours) are shown in Figure 2 and Table 1. All of these spectra are expressing the similarity of absorption peaks at different functional groups of HAp Ca 10 (PO 4 ) 6 (OH) 2 including PO 3 4-and OH. This result is identical to previous studies on swordfish bones (Xiphia gladius), tuna (Thunnus thynnus) [34] and on salmon bones [39].

Stretching Frequency of HAp
Specifically, the IR spectrum of sample at 200°C in 0.5 hour (similar to that of samples at 200°C for 1 and 1.5 hours) shows the absorption peaks characteristic of PO 3 4group including three main regions. The first region is represented by the peaks of 1091, 1049 cm −1 corresponding to v3 stretching mode and 961 cm −1 associated to v1 stretching mode. The second zone of phosphate ions exhibits the welldefined peaks at 633, 602 and 571 cm −1 corresponding to v4 bending mode. The third region is observed a weak absorption peak at 473 cm -1 corresponding to v2 bending mode. Hydroxyl stretching mode is observed on all sample spectra at 3571 and 3431 cm −1 with a very low intensity peak. In addition to the absorption peaks of the functional groups present in the structure of HAp, three vibrational bands of carbonate ions are observed: a) peak at 875 cm −1 ; b) peaks from 1418 to 1461 cm −1 cm and c) peak at 2009 cm −1 . The absorption of CO 2 in the atmosphere during the experiment is responsible for the formation of carbonate ions [44]. Finally, the presence of organic material (C-H) is detected as a very low intensity peaks at 2975 cm −1 . Accordingly, when comparing the schematic of samples with the standard pattern of HAp, these calcium samples consisted of HAp phase only with peaks that completely coincided with the standard and in previous studies [34,45,46]. However, regarding crystallinity degree of nano-HAp, these peaks were broad and not sharp, indicating that the nanoparticles were formed in small sizes.    Figures 6,7,8 show the SEM images of nano-HAp powder obtained after alkaline hydrolysis at 200°C in 0.5; 1; 1.5 hours. These SEM images at different positions and different magnifications showed that the calcium crystals are mainly flattened, messy-structured particles, which tend to stick together. Previous studies have shown that the alkaline hydrolysis resulted in HAp with less crystallinity producing small size particles [47]. In this study, image J software showed that the particles have an average size from 45 to 53 nm in length and 12 to 14 nm in width (Table 2), and it has been observed that the alkaline hydrolysis time did not affect the average size of HAp. The reason is that the assemblage of nanoparticles did not occur during alkaline hydrolysis time. Therefore, the alkaline hydrolysis method is the best approach for producing nanostructured HAp.     Table 3 shows the evaluation of heavy metals (As, Pb, Hg, Cd) content (mg/kg) and elemental content of calcium and phosphorus (%) in HAp samples formed at 200°C in 0.5, 1 and 1.5 hours by emission spectroscopy method. Hereby, these samples have the Ca/P molar ratios ranging from 1.896 to 1.921 and these values are higher than that of human bones (1.67). This value is minimum when heated for at least 0.5 h and increases with longer heating (1 and 1.5 hours). The absorption of CO 2 in the atmosphere during the experiment time and the alkaline environment during the process allowed the formation of carbonate ions in samples. Therefore, the longer the heating time was, the more carbonate ions were present and these ions then substituted phosphate sites leading to a B-type HAp. This type of carbonate apatite is a main constituent of the biological apatite [48]. The replacement of phosphate ion by carbonate ion is very important, because carbonated HAp has been found to be easily resorbed by living cells and to possess higher solubility than HAp with Ca/P ratio of 1.67 [49,50]. Other authors have also observed the higher Ca/P ratios in biological HAp obtained from swordfish bones (Xiphia gladius), tuna (Thunnus thynnus) [34] and cow bones [51]. In addition, the presence of carbonate ions of type B HAp in seabass Lates calcarifer bone was confirmed by FTIR spectroscopy (Section 3.2). As described in Table 3, Cd and Hg were not detected in all three HAp powders. However, Pb and As contents in Fish Bone by Alkaline Hydrolysis Method nano-HAp were respectively 0.09-0.170 and 0.08-0.13 mg/kg and these levels met the regulatory requirements of Vietnam regulation and US Pharmacopeia (USP) for food and dietary supplement standard (Table 4). The results of the Ca/P molar ratios and the amount within the safe limits of heavy metals (As, Pb, Hg, and Cd) show that nano-HAp powder from Lates calcarifer fish bone can be used as an input material for medicine and dietary supplements foods. However, evaluation of acute toxicity in mice and subchronic toxicity in rabbits of this product is required and this experiment is in progress in order to investigate the potential toxicity before mass production in large scale.

Conclusion
In our study, nano-HAp powders from Lates calcarifer seabass bone was successfully obtained when heated in 2M NaOH for 30 minutes, 1 and 1.5 hours at 200°C. The average size of particles are from 45 to 53 nm in length and 12 to 14 nm in width, and the heating time did not influence the particle size. The Ca/P ratio ranged from 1.896 to 1.921, indicating that this calcium powder was B-type HAp which was confirmed by FTIR spectrum. In addition, the heavy metal content of the calcium powder is completely within the allowable limits for dietary supplement and medicine.

Conflict of Interest
All the authors do not have any possible conflicts of interest.