Optimization of the demineralization process for the extraction of chitin from Omani Portunidae segnis

Abstract

Chitin is an organic polymer and it is the most frequent marine natural polysaccharide after 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, but these waste raw materials could be useful for production of commercial products like chitin. Chitin is an important raw material used for water treatment, agricultural, biomedical, biotechnological purposes, food and paper industry and cosmetics. Based on the variety of importance, the present targets of this study are to optimize the demineralization process for the removal of calcium and phosphate contents from the waste of Portunidae segnis (P. segnis) by using acid at ambient temperature and to characterize the isolated demineralized sample as well as the percentage of remaining calcium and phosphorus contents by using Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). The prepared waste carbs coarse powder samples of P. segnis were demineralized with seven different concentrations of hydrochloric acid at ambient temperature for 1 h. All the demineralization samples by the different concentrations were analyzed by using sensitive ICP-OES. The results based on ICP-OES showed that among the seven different concentrations used in the demineralization process for the isolation of chitin, the best was 2 M of HCl concentration for the production of chitin. The results also showed that the optimized concentration 2 M HCl gave the minimum concentration of calcium and phosphorus compared to other concentrations applied in this experiment. In conclusion, the optimized concentration for demineralization process could be used commercially for the isolation or commercial production of chitin for agricultural, biomedical and biotechnological purposes.

Graphical abstract

Chitin and minerals calcium and phosphorus from the carb waste shell of Portunidae segnis.

Highlights

• •Chitin is an organic polymer widely used in the industry, especially in biomedical and agricultural fields.
• •To optimize the demineralization process as the first step in isolation of chitin from P. segnis waste.
• •To identify the best method of the demineralization process as the first step of the isolation of chitin.
• •To identify the demineralized waste by ICP-OES spectrometry and compare with commercial product.
• •It could be used in the medicine, pharmaceutical, and biotechnology sectors.

1. Introduction

Since ancient time, researchers have been trying to minimize the amount of marine waste material and turn it into commercial agricultural products.

Chitin is one of the abundant polysaccharide polymers. It is a prevalent polysaccharide in nature which is normally transfer to cellulose. As a structural component of animals, chitin can be found mainly in 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. On the other hand, it contains several valuable chemicals like chitin, protein, minerals which are commercially important due to their use in various sectors. Marine waste could be used as raw material for the production of chitin and chitosan. 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, regulations have been introduced by the Government of the Sultanate of Oman is to save the environment and to promote the use of all kinds of marine waste. The main sources of marine waste are crabs, shrimps, squilla and fish waste. It contains several biologically active compounds of significant commercial values. Marine waste contains approximately 25–30% chitin, 25% protein, 40–50% calcium carbonate [1]. Chitin is commercially used in the medical and agriculture sectors. There are three forms of chitin such as α, β, γ-chitin available in the nature. Among the three forms, α-chitin is the most commonly found in nature. It can be isolated commercially from marine waste like shrimp and crab shells [2]. It is a white, hard, inelastic, nitrogenous polysaccharide polymer (Fig. 1). The monosaccharide of chitin is N-acetyl-d-glucosamine (NAG, or GlcNAc) linked with β1α-4 linkages [3,4]. It has an orthorhombic crystal and the chains are antiparallel [2]. Due to its structural characteristics, chitin is strong and has other favorable properties. Therefore, it can be used 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 [5]. 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 [6,7]. It has plenty of applications in biomedical, agricultural, biotechnological, wastewater treatment, food, paper and cosmetics industries [8]. However, the major drawback of using chitin in the clinical field is the insolubility of chitin in most of the organic solvents [9]. Previous reports examined that the extraction of chitin and chitosan from P. pelagicus [6]. P. segnis are among the most common species in Oman. They occur 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 phytochemical and pharmacological use of P. segnis. The authors used numerous methods for the extraction of chitin from marine wastes but these methods were not standardized. All extraction methods for chitin are t similar, they only use different ratio of acid and base, different time and the temperature [2,7]. Some of them described the extraction of chitin by chemical methods and it was synthesized by chemical methods for large scale production [10,11]. Statistical data showed that about 6–8 million tons of crab, shrimp and lobster shell waste is produced globally per year. In Oman, marine waste is a major source of environmental pollution in the coastal areas [1]. According to the fish production report in Oman, about 90,000 tons of marine waste is produced per year [12,13]. This waste, shell and other parts, is dumped in open places in the coastal areas polluting the environment. Therefore, huge amount of these marine wastes is available to 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, phosphorous, calcium chloride could also be isolated. Throughout the decades, many methods have been developed for the production of chitin. However, the chemical method is the most effective method found in the literature for the production of industrial chitin. The literature research showed that there has not been such research conducted in Oman Therefore, the present targets of this study are to optimize the demineralization process for the removal of calcium and phosphorus from the waste of P. segnis by using acidic medium at ambient temperature and to 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).

Fig. 1.

Chemical structure of Chitin.

2. Materials and methods

2.1. Chemicals and reagents

Hydrochloric acid used in this experiment was purchased from BDH, UK. Other chemicals and reagents used during the treatment were analytical grade. The commercial chitin (Purity 98%) was obtained from Sigma-Aldrich Company Limited, Germany.

2.2. Sample collection and process

P. segnis crabs include exoskeletons of the crabs waste were collected from Bahla Fish Market. The samples were collected in Bahla on September 12, 2018 at 11 am. Soon after collection, the samples were taken to the Chemistry Laboratory at the University of Nizwa and kept in the freezer until the necessary extraction process. Then, the collected samples were washed with tap water and dried in an oven overnight at 45 °C. The dried crab waste samples were ground into coarse powder size 0.30–0.35 mm by using a blender machine. The ground coarse powder was kept in a bottle for the production of chitin. The collected carbs samples were properly https://www.google.com/search?source= univ&tbm=isch&q=P. +segnis+ crabs,image&sa=X&ved=2ahUKEwiArJr1k5_ pAhXn UhUIHV JaAYQQsAR6BAgJEAE&biw=1280&bih=881identified by the local people as well as with the help of.

2.3. Inductively coupled plasma optical emission spectrometry (ICP-OES)

The sensitive ICP-OES (Optical Emission Spectroscopy, Optima 8000, PerkinElmer, USA) was used to analyze the demineralized samples. The demineralized samples obtained from the crab shell waste by using chemical treatment with different concentrations of HCl were analyzed by using ICP-OES to detect the concentration of calcium (Ca) and phosphorus (P) after digestion of 0.8 gm of each demineralized sample by ultra-microwave (Single Reaction Champer Microwave Digestion System, Ultrawave, Mileston, USA).

2.4. Demineralization of the crab sample waste

Different chemical treatment methods were used for the extraction of chitin from different marine waste samples that were described by several authors [[14], [15], [16]]. In the present demineralization process at the chemistry laboratory, the chemical procedure was used at different concentrations of HCl at ambient temperature incubation for 1 h.

2.5. Demineralization process

Each crab shell coarse powder sample (5 gm) was treated with seven various concentrations such as 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, and 2.25 M of hydrochloric acid. The powder samples (5 gm) were put in a beaker and various concentration of HCl were added separately. The ratio of samples and acid was 1:15. Then all the treated samples were kept at ambient temperature for 1 h incubation with constant stirring to remove the phosphate and carbonate contents from the crab shell powder [14]. Triplicate samples for each concentration were used. After demineralization, the treated products had a pale pink color and the average mass of each concentration process was measured. Each demineralized sample was digested in ultra-wave digestion system at 230 °C at 120 atmospheric pressure for 15 min. The digested samples were analyzed for calcium and phosphorous by using ICP-OES. The concentration was measured as parts per million (ppm) from 0.8 gm after digestion and dilution in 50 ml of distilled water for each treatment (Fig. 2).

Fig. 2.

Process of carb samples for the measurement of chitin.

3. Results

Chitin is a commercial raw material widely used in food, agriculture and pharmaceutical products as an edible films, as food thickeners and as stabilizers as well. It is also used to reduce blood cholesterol level and body weight. In addition, it is also used to improve kidney function, weakness, appetite loss and insomnia.

The huge amount crab shell of P. segnis include exoskeletons crab waste were available in Oman. The carb sample was collected from the local fish market in Bahla. The collected crab waste samples were washed with water and dried in an oven at 45 °C for 7 days. The dried carb waste shell samples were ground into coarse powder.

3.1. Demineralization process

The carb shell waste samples were demineralized by using chemical methods which were previously described by several authors [[14], [15], [16]]. In this present experiment, the crab shell coarse powder was demineralized by various concentrations of HCl at ambient temperature for 1 h incubation. After incubation, the results of all demineralized samples are presented in Table 1. The highest percentage of demineralization was obtained by using 2 M HCl and the lowest was the result of using 0.75 M HCl.

Table 1.

Percentage of yield of demineralization process using different concentrations.

Conc. HCl (M)	Average demineralized sample yield (gm)	Percentage of yield (%)	Percentage of demineralization (%)	Color
0.75	1.611 ± 0.03	32.22 ± 0.03	67.78 ± 0.03	Slightly pink
1	0.937 ± 0.03	18.74 ± 0.03	81.26 ± 0.03	Slightly pink
1.25	0.976 ± 0.01	19.53 ± 0.01	80.47 ± 0.01	Slightly pink
1.5	0.937 ± 0.01	18.74 ± 0.01	81.26 ± 0.01	Slightly pink
1.75	0.914 ± 0.02	18.28 ± 0.02	81.72 ± 0.02	Slightly pink
2	0.909 ± 0.01	18.18 ± 0.01	81.82 ± 0.01	Slightly pink
2.25	0.967 ± 0.02	19.33 ± 0.02	80.67 ± 0.02	Slightly pink
2.5	1.010 ± 0.01	20.20 ± 0.01	79.80 ± 0.01	Slightly pink

The values of average demineralized sample yield are means ± SD of three trails.

3.2. Determination of calcium and phosphorus in the demineralized samples

The concentration of calcium (Ca) and phosphorus (P) as parts per million (ppm) of the demineralized crab shell waste samples was determined by using the sensitive ICP-OES. The average concentration for calcium (Ca) and phosphorus (P) was calculated using the established equation described by several authors [[14], [15], [16]]. All the obtained results for calcium and phosphorus after demineralization process are presented in Table 2.

Table 2.

The concentration of calcium and phosphorus after the demineralization of carbs samples by applied the seven concentrations.

Conc. HCl	Average Conc. (ppm)		Average Conc. in 0.8 gm/50 ml (ppm)
(M)
	Calcium (Ca)	Phosphorous (P)	Calcium (Ca)	Phosphorous (P)
0.75	0	84.24 ± 5.77	0	5258 ± 5.78
1	69.19 ± 26.13	41.04 ± 1.67	6005 ± 26.14	2562 ± 1.66
1.25	51.78 ± 30.72	35.13 ± 2.99	3232 ± 30.71	2193 ± 2.98
1.5	14.74 ± 8.28	33.11 ± 0.50	920.3 ± 8.29	2067 ± 0.51
1.75	12.6 ± 7.37	25.41 ± 1.50	786.3 ± 7.38	1586 ± 1.49
2	11.62 ± 5.57	24.68 ± 0.69	725.3 ± 5.56	1540 ± 0.70
2.25	21.46 ± 1.23	24.48 ± 0.34	1340 ± 1.24	1528 ± 0.35
2.5	20.10 ± 14.63	26.79 ± 2.04	1254 ± 14.62	1672 ± 2.03

The values of average concentration of Ca and P are means ± SD of three trails.

4. Discussion

Chitin was first detected in mushrooms by Braconnot in 1811. According to the list of natural polymers, chitin is considered as the second abundant natural polymer after cellulose. The annual production of chitin is approximately 10¹⁰–10¹¹ tons [17] globally. Based on the chemical structure, chitin is a derivative of cellulose and there is a difference only at Carbon-2 in the structural formula. In chitin, carbon-2 position contains a hydroxyl group while in cellulose, it has an acetamido group [18]. Chitin is widely abundant in invertebrates, plants and fungi. It is the major biochemical compound in the exoskeletons of arthropods and insects. Nowadays, more than 75% of the total weight of marine wastes are used commercially for the production of chitin and other biomedical compounds [19].

Since the Roman time, chitin is extracted traditionally from various marine wastes by chemical methods which include three simple chemical processes: (i) deproteinisation, (ii) demineralization and (iii) bleaching. Almost all marine waste samples were demineralized by treatment in acidic medium such as HCl, HNO₃, H₂SO₄, CH₃COOH and HCOOH at the temperature range of 90–100 °C [20]. The molecular formula of chitin is poly (β-(1 → 4)-N-acetyl-d-glucosamine) and it is isolated as polymer mainly from living organisms [15]. It is the second most widespread polymer ester cellulose. It is odorless and crystalline solid (Fig. 1). However, there are several marine sources available especially from crab and shrimp shell wastes for chitin. The chitin and its derivatives have several medicinal, agricultural and industrial applications. In the medical sector, it is used widely for wound dressing, monitoring bleedings and in antitumor treatments, etc [14].

Previous several studies have been conducted by several authors on isolation or extraction of chitin from available marine sources. Most scientists used the same methods [10,[13], [14], [15], [16]]. The main aim of the demineralization process is to remove minerals, predominantly calcium carbonate and calcium phosphate. To achieve these objectives, several acids such as HCl, HNO₃, H₂SO₄, CH₃COOH and HCOOH were used to demineralize the marine waste samples. Among these methods, most of the authors used diluted hydrochloric acid for demineralization, to remove various minerals from the crab sample. After the demineralization process, the mineral content in the samples varied. These variation mainly depends on the process time, temperature, particle size, acid concentration and samples/solvent ratio. In addition, the acid requires two molecules of HCl to convert calcium carbonate into calcium chloride for complete the demineralization process. The equal or even bigger amount of acid is needed to demineralize equal amount of minerals [21,22]. However, in our present experiment, we used the same acidic method with a slight modification. We have tried to optimize the demineralization process by using various concentrations of HCl at constant temperature.

The chitin is extracted from the marine sources by hydrochloric acid treatment to dissolve the calcium carbonate as shown:

CaCO₃(s) + 2HCl (aq) → CaCl₂(aq) + CO₂(g) + H₂O

In the present experiment, the crab shell coarse powder samples were treated with seven different concentrations of HCl for 1 h and then the demineralized sample were analyzed by ICP-OES (Fig. 2). In our target through this experiment was to optimize the concentration of HCl. Therefore, to achieve this target, after the demineralization of crab shell samples, the percentage of calcium and phosphorus present in the demineralized samples was measured to reveal the quality of chitin by ICP-OES. Table 2 shows that among the applied seven different concentrations of HCl, the best results were obtained for demineralization by the treatment of 2 M HCl. However, the percentage of yield of the demineralized samples was different which could be due to the strength of acid, time of incubation and particles size etc (Table 1). It also showed that the concentration of calcium (Ca) and phosphorus (P) in the crab waste samples was different when different concentrations of acid was applied for the demineralization process. In Table 2, it is clearly shows that the concentration of Ca decreased when the concentration of HCl was increased from 1 to 2 M. However, if the concentration was increased to 2.25 M and above then the concentration of Ca also increased. Similarly, P concentration also decreased as the concentration of HCl was increased from 0.75 to 2 M. Again similar trend for P observed when the concentration of HCl was increased to 2.25 M and above. Therefore, in conclusion, the optimized concentration of HCl is 2.0 M for the demineralization process of the crab shell samples. Several previous studies conducted by several authors globally, however, the optimized acid concentrations for demineralization process are not similar [23]. Their optimized concentration of HCl for demineralization process of the marine waste samples is different from each other' or even from our results, even though, they used the same method for the demineralization process of marine samples and the same process was used for the quantification of minerals in the demineralized crab shell samples by using ICP-OES analysis [14]. The concentration of Ca and P also differs from our results as in our present experiment, the optimized concentration is 2 M of HCl for the extraction of chitin from Omani P. segnis. The variation of results for demineralized samples, Ca and P, it could be due process time, temperature, particle size, acid concentration and samples/solvent ratio.

5. 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 chemical Based on the observation of the demineralization method, it is concluded that the products obtained from 2 M concentration of HCl at room temperature with duration of 1 h is the best optimized method for the extraction of chitin from Omani P. segnis. The chitin content and the concentration of calcium and phosphorous was determined by ICP-OES analysis.

In conclusion, the present optimized demineralization process could be used successfully for the extraction of the best quality of chitin from Omani P. segnis shell waste. Therefore, the optimized demineralization process could be used to produce raw material for industrial, biomedical and pharmaceutical industries. Our future study will be carried out on the selection of best samples/solvent ratio and temperature during the demineralization process that will be also useful to demineralize marine waste for the use in medicine and other sectors.

CRediT authorship contribution statement

Noura Hamed Khalifa Al Shaqsi: Data curation, Investigation. Horiya Ali Said Al Hoqani: Data curation, Investigation. Mohammad Amzad Hossain: Writing - review & editing. Mohammed Abdullah Al Sibani: Conceptualization, Project administration, Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.