Development and optimization of the demineralization process for the extraction of chitin from Omani Portunidae segnis

Background: Chitin is an organic polymer and it is rich marine natural polysaccharide after the cellulose. The main natural sources of chitin are exoskeletons of insects, mollusks, the cell walls of certain fungi and crustaceans such as crabs, shrimps and lobsters. The waste of these marine exoskeletons are pollutant for the environment continuously, but this raw material could be used for the production of commercial chitin. The chitin is an important raw material used for water treatment, agricultural, biomedicinal, biotechnological purposes, food and paper industry and cosmetics. Based on the variety of importance, the present targets of this study is to i. optimize the demineralization process for the removal of calcium and phosphorus from the waste of Portunidae segnis ( P. segnis ) by using acid at ambient temperature ii. characterize the isolated demineralized sample as well as the percentage of remaining calcium and phosphorus by using Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Results: The coarse powder samples of P. segnis were demineralized with seven different concentrations of hydrochloric acid at ambient temperature for 1 hour. All the demineralization samples at different concentrations were analysed by using sensitive ICP-OES. The results based on ICP-OES showed that among the seven different concentrations wused in the demineralization process for the isolation of chitin, the best was 2M of HCl concentration for the production of chitin. Conclusion: The develop optimized demineralization process could be used commercially for the isolation of chitin for the preparation of agriculture, biomedicine, biotechnological purposes.


Introduction
Since ancient time, the scientists/researchers have been trying to minimize the amount of marine waste material and turn it into commercial sustainable biologically and agricultural products. Chitin is one of the abundant polysaccharide polymers. It is a well-known polysaccharide in nature which is normally transfer to cellulose. As a structural component of animals, chitin is used mainly for exoskeletons of animals like insects and crustaceans such as shrimps, crabs, and lobsters. Previous reports showed that more than 1011 tons of chitin is produced from sea products worldwide. Marine waste is pollutant for the global environment and nowadays it is a burden for the communities. important due to their use in various sectors. However, marine waste could be used as raw material for the production of chitin and chitosan commercially. Therefore, the purpose of this study is to use marine waste for the production/isolation of biologically active products such as chitin and chitosan biopolymeric compounds and their derivatives. Recently, the policy has been developed by the Government of the Sultanate of Oman is to save the environment and nations to protect or use all kinds of marine waste. The main sources of marine waste are crabs, shrimps, squilla and fish waste.
Marine waste contains several biologically active compounds with significant commercial values. Most of the marine waste such as crab, shrimp, squilla and fish waste contains approximately 25-30% chitin, 25% protein, 40-50% calcium carbonate (Marguerite, 2007). Chitin is commercially used in the medical and agriculture sectors. Marine waste contains a significant percentage of chitin. There are three forms of chitin such as α, β, γ-chitin available in the nature and as solid crystalline. Among the three forms, α-chitin is the most commonly found in nature. It can be isolated commercially from the marine waste like shrimp and crab shells (Panariello et al., 2019). It is white colour, hard, inelastic, nitrogenous polysaccharide polymer (  (Panariello et al., 2019). Due to its structural characteristics, the chitin is strong and has other favorable properties. Therefore, it can be used in the industry for making a suitable alternative for plastics. The chitin and its derivative polysaccharides are widely used for medical applications due to their biocompatibility and antimicrobial potency (Brasselet et al., 2019). It is fixed with a protein matrix of a crustacean shell. Recently, commercial chitin is commonly produced by the marine industry from crab and shrimp shells which are cast-off as massive wastes. The physiochemical and toxicological properties of chitin includes solubility, solution, viscosity, polyelectrolyte behavior, polyoxy salt formation, ability to form films, metal chelation, optical, and structural characteristics (Mottari et al., 2018; Achilonu et al., 2018). It has plenty of applications in biomedical, agricultural, biotechnological, wastewater treatment, food, paper and cosmetics industries (Arabia, 2013). However, the major drawback of using chitin in the clinical field is the insolubility of chitin in most of the organic solvents (Ahyat et al., 2017). Previous reports showed that the extraction of chitin and chitosan with significant amount from P. pelagicus. P. segnis is a most common species available in Oman. It occurs in sandy and sandy-muddy areas including mangroves, sea grass and algal beds. The literature showed that there is not enough research available on the phytochemicals and pharmacological study of P. segnis. The authors used plenty of methods for the extraction of chitin from marine wastes but these methods were not standardized.. in the coastal areas polluting the environment. Therefore, huge amount of these marine wastes can be used as a raw material for different industries to produce different valuable life-saving, and agricultural drugs. In addition, other components from the marine waste like protein, calcium, potassium, calcium carbonate could also be isolated. Throughout the decades, many methods have been developed for the production of chitin. However, the chemical methodis the most effective method found in the literature for the production of industrial chitin. The literature research showed that therehas not been such research conducted in Oman Therefore, the present targets of this study are to i. optimize the demineralization process for the removal of calcium and phosphoruss from the waste of P. segnis by using acidic medium at ambient temperature ii. characterize the isolated demineralized samples as well as the percentage of remaining calcium and phosphorus minerals by using Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES).

Chemicals and reagents
Hydrochloric acid used in this experiment were collected from BDH, UK. The other chemicals and reagents used during the process and treatment were analytical grade. The commercial chitin (Purity 98%) was obtained from Sigma-Aldrich Company Limited, Germany. There were three samples for each concentration. After demineralization, the products were pale pink color and the average masses of each concentration process were measured. The demineralized product of each sample was digested in ultra-wave digestion system at 230 °C at 120 atmospheric pressure for 15 minutes than the samples were analyzed by using ICP-OES. The concentration was measured as parts per million (ppm) from 0.8 g after digestion and dilution in 50 ml of distilled water for each treatment (Fig. 2).

Results
The huge amount of P. segnis crabs include exoskeletons of the crab waste were available in Oman.
The sample was collected from the fish market in Bahla. The collected crab waste samples were washed with water and dried in an oven at 45 o C. The dried samples were ground into coarse powder.

Demineralization process
The carb samples were demineralized by using the chemical methods which was previously described  Table 1. The highest percentage of demineralized samples was obtained using 2M HCl and the lowest was when 0.75M HCl was used.

Discussion
One of the major natural polymers is chitin that is detected first in mushrooms by Braconnot in1811.
According to the list of natural polymers, chitin is the second abundant polymer after cellulose. The annual production of chitin is approximately 1010 to 1011 tons (Gooday, 1994). Based on the structure, chitin is a derivative of cellulose only different at carbon-2 in the chemical structure. In chitin structure, carbon-2 contains a hydroxyl group while cellulosehas an acetamido group (Rinaudo M, 2006). Chitin is widely abundant in invertebrates, plants and fungi It is the main chemical compound in the exoskeletons of arthropods and insects. Nowadays more than 75% of the total weight of marine wastes like shrimp, crabs, prawns, lobster, and krill are used commercially for the production of chitin (Kuddus & Ahmad, 2013).
Since the Roman time, chitin is extracted traditionally from various marine wastes by chemical methods which include three simple process viz. (i) deproteinisation, (ii) demineralization and (iii) bleaching. The marine samples were demineralized by reactions in acidic medium such as HCl, HNO 3 , H 2 SO 4 , CH 3 COOH and HCOOH at various temperatures within the range of 90-100 °C (Percot et al., 2003). The molecular formula of chitin is poly (β-(1→4)-N-acetyl-D-glucosamine). The chitin as a polymer is isolated mainly from living organisms (Yadav et al., 2019). It is the second most widespread polimer aster cellulose. It is odorless and it is crystalline solid (Fig. 1) The chitin is extracted from the marine sources by hydrochloric acid treatment to dissolve the calcium carbonate. The calcium carbonate was decomposed and form calcium chloride along with carbon dioxide gas is involved in demineralization as shown: In the present experiment, the crab coarse powder samples were treated with seven different concentrations of HCl for one hour and then the demineralized sample were analyzed ICP-OES (Fig. 2).
In our target through this experiment is to optimize the concentration of HCl in the demineralization process of the crab samples for the production of chitin commercially. Therefore, to achieve this target, after the demineralization of crab samples, the percentage of calcium and phosphorus present in the demineralized samples is measured to reveal the quality of chitin. Therefore, the measurement of the percentage of calcium and phosphorus present in the demineralize samples was conducted by ICP-OES.

Conclusion
The current target is to extract high percentage of chitin with low concentration of Ca and P from the Omani P. segnis marine wastes by using different chemical methods which was collected from Bahla.
Based on the observation of demineralization method, it is concluded that the products obtained from 2M concentration of HCl at room temperature with duration of 1 hour is the best optimized method for the extraction of chitin from Omani P. segnis. In addition, the percentage of yield chitin, and the ICP-OES analysis of the calcium and phosphore content, also concluded that the present optimized demineralization process could be used successfully for the extraction of best quality of chitin from Omani P. segnis waste. Therefore, the optimized demineralization process could be used for the extraction of chitin from crab shells which will be used as raw material for industrial, medical and pharmaceutical industries. However, our future study will be carried out on the selection of best samples/solvent ratio and temperature during the demineralization process it will be also useful to demineralize the minerals from the crab samples for the use in medicine and other sectors.

Consent for publication
Not applicable

Availability of data and materials
All experimental data used and analyzed in this present study are available from the corresponding author.

Competing interests
The authors declare that they do not have any competing financial interests or institutional and personal relationships that could have appeared to influence the work reported in this paper.

Figure 1
Chemical structure of Chitin