Microwave-Assisted Hydrolysis of Chitosan from Shrimp Shell Waste for Glucosammine Hydrochlorid Production

Chitin is the most widespread renewable natural sources following cellulose as the main source of chitosan. Chitin is isolated from crustacean waste and shrimp shells. Chitosan is derived from chitin throuhgt demineralisation, deproteination, decolorisation and deacetylation process using chemicals such as sodium hydroxide, hydrogen chloride and acetone. Glucosamine hydrochloride (GlcN-Cl) can be produced by hydrolysis of chitosan by using hydrogen chloride. During deacetylation and hydrolysis the solution is heated by hotplate or furnace. In this paper we use microwave instead of hotplate for production chitosan and GlcN-Cl. The research investigates effect of microwaves to amount of rendemen and their property. The chitosan was characterized its moisture content, solubility, and degree of deacetylation (DDA). Whereas the glucosammine hydrochloride characterized its functional groups using FTIR and crystallization by using X-Ray Difraction (XRD). The experimental results show that the use of microwave energy on deacetilation of chitosan and hydrolisis processes can decrease time consuming and reactant concentration during production. the DDA value obtained was very high from 70 to 85%. The results also show that microwaves meet chitosan and GlcN-Cl standards.


Introduction
Osteoarthritis (OA) is one of the leading causes of disability (disability) in the elderly population in the world. OA is the fifth leading cause of disability in the population of developed countries, and the ninth highest cause in developing countries [1]. The main cause of disability (disability) in Australia and Asia is cancer. After cancer, many disability caused by lower back pain and OA with a prevalence of about 15% of the total cases. The results show in the world 9.6% of men and 18.0% of women aged over 60 have symptoms of OA. 80% of them due to OA causing limitation of movement, and 25% can not perform major activities of daily living [2]. WHO also estimates that in 2020 OA will be one of the causes of physical disability in humans after rheumatoid arthritis, osteoporosis and lower back pain.
The prevalence of OA continues to increase along with the increase in population, age, and risk factors such as obesity [1]. Indonesia is the 4th with the number of elderly people (elderly) the most after China, India and the United States [3]. Approximately 56.7% of patients in the outpatient clinic of the Department of Rheumatology (Cipto Mangunkusumo Hospital) has been diagnosed is one one variant OA. Osteoarthritis patients is very haunted by the pain caused by degradation and joint inflammation that occurs [4], so the treatment is focused on reducing pain by administration of nonsteroidal antiinfla-mmatory drug (NSAID) in the long term. However, this drug side effects if used long term. The use of NSAIDs for a long time, especially in older people reportedly turned out to cause a lot of side effects, such as gastrointestinal disorders, impaired liver function, impaired kidney function and so on [4]. Dietary supplements glucosamine and chondroitin sulfate has been recommended as a safe and effective option for preventing and treating symptoms of OA [5]. Glucosamine is a natural compound found in the human body is composed of glucose and amino acid glutamine, other than that glucosamine is a key element of glycosamino-glycans (GAGs) in the cartilage of cartilage and synovial fluid. The function of glucosamine in the body is to produce synovial fluid that serves as a lubricant to the cartilage, so that the movement of the bones to be good. Shortage of synovial fluid in the body will cause joint disorders, such as stiff joints so that movement will result in OA disease. Giving oral glucosamine can help the production of synovial fluid so as to prevent and cure of disease [5]. Until now, glucosamine has not been produced in Indonesia that are imported from other countries with a fairly high. Research to produce glucosamine have been done by some researchers. Islam et al. (2011) have managed to hydrolyze glucosamine hydrochloride amounted to 63.45% of the chitin shells of shrimp [6]. Afridiana (2011) also managed to extract glucosamine hydrochloride from chitin shells of shrimp through chemical hydrolysis method with a concentration of 45.64%. Hydrolysis is safe for human consumption [5].  use of microwave energy to heat material or to speed up chemical reaction as a substitute of conventional heating has been reported. Some peculiar results were found such faster densification of ceramics [7][8][9], improve microstructure and mechanical of ceramics [10][11][12][13][14], drying and chemical reaction [15][16][17], synthesizing organic materials [18][19][20]. In this study, we applied this microwave energy on deacetylation and hydrolysis. The experimental results were then compared to the results of conventional ones.

Material and Method
The shrimp shells were collected from local fisheries in Kendari, Indonesia. Shrimp shell waste was washed and dried in the sun at room temperature before processing.The composition of the shell parts was determined by Thermogravimetry. The degree of deacetylation (DDA) of the chitin, chitosan, and glucosamine were determined by Fourier Tansform Infrared Spectroscopy (FTIR). Crystallinity was determined by X-Ray Difraction (XRD). All chemicals used in this study was bought from SIGMA-ALDRICH. The procedure of preparation of glucosamine from exoskeleton of shrimp is follow the procedure on Mojarrad and Arifin [21][22].

Demineralisation
50 grams of shrimp shell dissolved in hydrochloric acid of 1.0 M with a ratio of 1:10 (sample: solvent), then stirred with a stirrer for 1 hour at a temperature of 75 o C. After filtered and washed using distilled water until neutral pH, then dried in an oven at 60 o C.

Deproteination
Deproteination (removal of protein) is done by a solution of NaOH 3.5% with a ratio of 1:10 (sample: solvent), then stirred with a stirrer for 2 hours at a temperature of 65 o C. Then the samples were filtered and washed with distilled water until pH neutral and dried at 60 o C

Decolorisation
Decolorisation can be done by solution of 0,315% NaOCl for 5 minute or extracted by acetone for several hours. The resulted then washed with water and dried in oven at 60 o C.

Deacetylation
Deacetylation performed 50 % sodium hydrokhlorid solution in water, after that continue by heating wich microwave for several minute.

Hydrolysis
Two gram chitosan and HCl was prepared for glucosamine production. Hydrolysis performed by two methods, microwave and conventional. By using microwave with varying exposure time from 5 to 20 minutes. Conventional hydrolysis was performed by using hot plate at temperature of 90°C for 90 minutes. Samples were soaked in a solution of hydrochloric acid at a ratio 1: 9 (w / v). HCl solution was varied at 20%, 25%, 32% and 37%. The use of microwave in these process are not only in the deacetylation process but also in the hydrolysis of this produced chitosan.

Results and Discussion
Main exoskeleton of shrimp components are protein, mineral, and chitin. After characterization, we found the composition as shown in Table 1. to remove the proteins and minerals do deproteinization and demineralization .Deproteinization was done by soaking the samples in NaOH 3,5% solution for 2 hours at temperature 90 o C.While demineralization was performed by using HCl 1,0 M at ambient temperature for 1 hour. The use of microwave in the deacetylation of chitin and hydrolysis of chitosan decreasing heating temperature and time significantly.    Fig. 4 and 5. Absorption at wave number 1658.78 cm-1 (amide peak) appear due to chitosan is yet deasetilation overall. Quality of chitosan can be seen also from the large percent degree of deacetylation (DD). The existence of chitin into chitosan supported by data degree of deacetylation of chitosan which is calculated using the Baseline Method according to Khan et.al (2002), is 61.78% [23][24]. According Hargono et al. (2008) [25], standard degree of deacetylation of the chitosan from 60 to 100% so that the chitosan found in this experiment meet the standard. The higher DD means the more pure chitosan.  [26]. From the Fig. 6. indicate that the GlcN-HCl found in this experiment is monomer. Figure 6. FTIR spectrum of Glucosamine hydrochloride(GlcN-Cl) hydrolysis by using microwave Figure 6. show FTIR spectrum of Glucosamine hydrochloride(GlcN-Cl) hydrolysis by using by chemical methods with heated by hot plate for 90 minutes. The spectrum shows similarities with the microwave one for 15 minutes. It indicates that microwave accelerate the process of hydrolysis of GlcN-Cl. In addition with shorter time, we can found the almost same yields than that of result in hydrolisis by using conventional method as shown in Figure 8.. It can be seen that the yield obtained in the variation of microwave heating time 15,11,7,and 3 minutes give yield of 30.14%, 38.25%, 6.8% and 6.35% respectively. While by using conventional for 90 minutes we found yield of 51,01 %.

Conclusion
Application of microwave energy for deacetylation of chitin and hydrolysis of chitosan process for glucosammine hydrochlorid production was successfully performed. Experimental results show that microwave decreased heating temperature and heatingtime significantly. It indicates that microwave accelerates these processes. FTIR results showed that absorption bands of glucosamine hydrochloride meet the standard of GlcN-Cl. These results suggested that microwave is appropriate technology for production of GlcN-Cl.

Acknowledgments
This work has been partially financial supported by the Directorate of Higher Education, at the Ministry of Research Technology and Higher Education, Republic of Indonesia and University of Halu Oleo.