RESEARCH AND DEVELOPMENT OF RADIATION PROCESSING OF CHITIN AND CHITOSAN IN INDONESIA

Gatot Trimulyadi Rekso

Center for Research and Development of Isotopes and Radiation TechnologiesNational Nuclear Energy Agency, Jakarta, Indonesia

INTRODUCTION

Indonesia is the world's largest archipelago with around 13,000 island, of which 6.000

are inhabited, and occupies a 5,100-km stretch from the Indian to the Pacific Ocean. With

a total land area of almost 195 million hectares, it is the largest member country of the

Association of Southeast Asian Nations (ASEAN) and the most important country in

Asia regarding of ocean and lands those rich of natural polymer material.

Center for Research and Development of Isotopes and Radiation Technologies

(P3TIR) which was established in December 20, 1966 the Pasar Jumat Atomic Energy

Research Complex,

Industrial processing division is one of the several divisions in P3TIR. In this

division has five group activities, radiation polymerization, radiation sterilization and

food preservation, industrial dosimeter and waste recycle for industrial purpose. The

waste recycle for industrial purpose group is one of the new groups, which has

established in 1998. The activity of this group is research and development utilization of

shell seafood waste. For industrial purpose such as chitin and chitosan

Shell seafood waste such as prawn shell, crabs shells are abundantly found in

Indonesia. Utilization of the fishery waste mentioned above to be useful product is

suggested not only to recycle the fishery waste but also to reduce the environmental

pollution and to improve the economic situation such as to preparing of the field

employee occupation, to bring socio-economic benefit..

The remarkable rapid and successful expansion of prawn processing industry in

Indonesia suggests the possibility of utilizing prawn-processing waste as raw material for

the manufacture of many valuable products such as chitin and chitosan.

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The main activity of the research in waste recycle for industrial purpose is to do R & D

to provide data that will enable of shell seafood waste to be used in a commercial

production of chitin, chitosan, its derivative and its modification.

THE RESEARCH HAVE BEEN DONE

1. The isolation of chitin /chitosan from waste of fish frozen industry.

In the beginning of our research work was involved in making on the research

advisability of the natural of resource for chitin and chitosan such as various kind of

prawn and crab shell. The survey was done in side beach of north Java Island. We

found that prawn and crabs shells are easily collected from the frozen prawn

processing plant. Shell was ground using a blender. Then served to obtain particle

size of 40 + 60 mesh.

Isolation of chitosan

Dried prawn shells

Grinding

1 N NaOH Deprotenization

Washing

1 N HCl Demineralization

Washing

Drying

Chitin

Figure 1. Flow diagram of the chitin preparation

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Chitosan can be obtained by treating chitin with 50 percent sodium hydroxide with

liquid solid ratio 20 : 1 at 1000 C for 2 hr, 3 hr and 4 hr . After this process, solids

separated from the alkali layer were extensively washed with distilled water to remove

the traces of alkali. The resulting solids were dried in vacuum oven at 500 C for 24 hr.

The preparation of chitosan can be seen in Figure 2.

Chitin solids

NaOH ,50 % Deacetylation

Drying

Chitosan

Figure2. Flow diagram of the chitosan preparation

Result of the experiment

The average yield of isolation chitin from prawn shell is shown in Table I. The data

obtained shows that the number of extraction affected the yield of chitin . It was reported

that the number of stage of extraction affected the mineral removal significantly.

Table 1. The yield of chitin at various number of extraction

Parameter Number of extraction Single Double Triple

Yield ( %) 28,7 14,8 14,2

Visual appliance - Color Brown Brown-light Brown-light - Odor Weak No No

From the Table it was seen that by single stage extraction . The average yield of

chitin was 28,7 % and the solids obtained were brown and still have a weak odor. This

unexpected color and odor was caused by the presence of some protein mineral and

pigment such as carotenoid. The double one gave an average 14,8 percent and no odor,

the color was brown-light. So it is obvious that the number of extraction affected the

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protein and mineral removal. However for three times extraction yield of the solid was

not giving significantly different compare with double extraction. It was meaning that

double stage extraction was enough for isolation of chitin from prawn shell.

The conversion of chitin to chitosan was achieved by extracting chitin in 50 percent

sodium hydroxide with solid –liquid ratio = 1 : 20 at temperature 1000 C for 120, 240,

and 300 minutes. The data obtained gave an average yield of chitosan from chitin were

72,2 percent. Therefore the overall extraction yield of dried prawn shell in chitin form

was 14,8 percent and chitosan form was 10,6 %. The degree of deacetylation was

determined by means of FTIR spectrophotometer.It is determined by the base line

method. An infrared spectrum was recorded in a range 3500 – 500 cm -1 and absorbency

at 2870 cm-1 ( the C-H ) and 1550 cm-1 (the amide band) were evaluated by base line

method. The deacetylation degree ( D ) is calculated from equation : D ( % ) = 1- (

A1655/ A3450 x 1 /1,33 ) x 100 %, A = log P0 / P

Where A1655 and A3450 are absorbencies at 1655 cm-1 ands 3450 cm-1 , respectively.

Table 2. The degree of deacetylation at various time of reaction

Parameter Time of deacetylation ( minutes)

120 240 300

Yield of chitosan from chitin 74,4 72,6 69,6

Degree of deacetylation 60,4 65,2 73,0

It can be seen in Table 2. That the deacetylation time gave significant effect of

deacetylation. After 120 minutes degree of deacetylation reaches 60 4 % , it is gradually

increased as the time increased. The highest degree of the acetylation was achieved for

300 minutes.

Sehchi Mima and coworkers reported deacetylation proceed to about 70 % within the

first 1 hr of alkali treatment in 50 % NaOH solution at 1000 C , but it progresses only

gradually after this reaching 80 % in 5 hr. Further alkali treatment does not deacetylate

significant and only degrades the molecule chain .

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II. Preliminary studies of chitin sterilized by irradiation

Over the past several years, chitosan has been receiving increased attention for its

applications in the chemical, biomedical, and food industries. Chitin’s key properties are

its ability to act as a cationic flocculent, viscosifier and selective chelator of metal ion.

The major commercial application for chitin currently is used for waste processing, but

there is growing interest as non-toxic cationic polymer in the hair treatment and skin care.

Clear solution form clear film that adhere to skin or hair, primary due to chitin’s cationic

character. Chitin is an excellent moisturizer, attributes

to wound healing, make an attractive biopolymer for cosmetics a personal care

application. Hun-Lee and coworkers reported for the antimicrobal activity of chitin as

natural preservatives in cosmetic product against some microorganisms . Formula

preserved with 0,5 % chitosan had an effective antimicrobial activity against the Gram

(+) and Gram (-) bacteria but not fungi.

In order to develop application of chitin as cosmetic additive it is necessary to choose

the best way of its sterilization. Chitin easily undergoes thermal destruction, chemical

sterilization by ethylene oxide or formaldehyde was proved to reduce its

biocompatibility. On the other hand, the radiation sterilization leads even to improvement

of some biocompatibility factors.

The aim of our work was to determine of irradiation sterilization dose of chitin from

shell waste of prawn (penaeus monodon). Analysis of change in infrared spectra and

thermal stability before and after irradiation also studied. The knowledge of these

processes is essential to optimize production technologies of chitin containing

biopolimer used as additive for cosmetic.

Material and Experiment

Chitin extracted from prawn shell ( Penaeus monodon ), it was got from Muara Karang ,

North Jakarta. They were initially washed by water and then dried at 800 C overnight and

conditioned at room temperature for 24 hr. All other chemicals NaOH, HCl were

analytical grade reagents from E Merck.

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The irradiation of sample for sterilization was carried out in a 60 Co – Gamma

irradiation source. The dose of irradiation were 2, 4, 6, 8, and 10 kGy with a dose rate of

2,0 kGy / hr

Result of Experiment

Results concerning the effect of radiation sterilization by various doses on the total number of bacteria of chitin are presented in Table 3.

Table 3. Total number of bacteria

No Irradiation dose

( kGy )

Bacteria Yeast/ Mold

1 0 1,5 x 104 3 x 103

2 2 1,5 x 103 3 x 10

3 4 1,5 x 10 0

4 6 0 0

5 8 0 0

6 10 0 0

It was found that at a dose of 4,0 kGy the number of yeast and mold was eliminated

and for bacteria was at 6,0 kGy. On the basic of the above result, it shows that the dose

of irradiation sterilization was not so high . So it was irradiation seems to be best

sterilization method for sterilized of chitin.

III. Studies on radiation graft-copolymerization of monomer with selective functional group onto chitin as adsorbent.

The natural chelating marine polymer chitin, Polly(N-acetyl-D-glucosamine) and its

deacetylated derivative chitosan are useful for removing heavy metal ion waste from

discharge water . Chitin , the most abundant naturally is undoubtedly one of the most

promising and attracting resources present in quantity. Among some interesting

properties of chitin , chelating ability arising from its characteristic structure is especially

noteworthy. Many researchers have explored the feasibility of this approach. Kurita.K

et.,all, conducted experiments with a number of heavy metals.

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The adsorption behavior of chitin and its concerns with various degree of

deactylization. This high adsorption capacity was ascribable primarily to its remarkable

hydrophilicity in cooperation with the relatively high amino group content. It is indicate

that the importance of hydrophlicity and suggest that , in order to develop adsorbents of

high capacity , it is make indicate the importance of hydrophilicity essential to make

chitin derivatives highly hydrophilic and yet insoluble in water.

Chitosan is natural polysaccharide and has the same skeleton structure as

cellulose, by a radiation modification such as graft-copolymerization of

hydrophilic monomer expected to improve its hidrophilicity.and performance for

application as an ion exchange adsorbent. In the field of Radiation Process, free

radical formation is the key role of the modification technique. Graft modified of

chitosan with hydrophilic functional monomers was suitable method to develop its

as ion exchange adsorbent

The experiment have been done were list above :

A. The effect of total irradiation dose on graft copolymerization of acrylic acid onto

chitin .

The effect of total irradiation dose on graft copolymerization of acrylic acid onto

chitin by pre-irradiation technique at nitrogen atmosphere has been carried out. The aim

of this research was to find out the optimum of total dose of grafting with water as

solvent at various times of reaction and temperature. Instead of that the effect of total

dose on the molecular weight and the free radicals formation were evaluated. The

influence of monomer concentration and solvent composition were studied at optimum

total dose. The result showed that the percentage of grafting increased with increasing of

the total dose and the optimum condition was achieved at the total dose of 12 kGy . The

solvent composition has significant effect on the degree of grafting . The best

composition was obtained in the acetic acid /methanol weight ratio 1 : 1. The percentage

of grafting increased with increasing the monomer concentration. Visual observation

showed that the formation of homopolymer was affected by the reaction temperature and

concentration of monomer.

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B. The effect of solvent composition on grafting of acrylic acid onto chitin by

irradiation technique.

The effect of solvent composition on grafting of acrylic acid onto chitin by irradiation

technique have been carried out. The aim of the research is to find out the optimum

solvent composition to the increase swelling properties of chitin so that acrylic acid

could carry in the active site of chitin. In this experiment the trapped radical and

peroxide graft irradiation co-polymerization method were employed . The influence of

solvent compositions e.g. water – methanol, water – acetic acid, and methanol –acetic

acid and the grafting method that affected to the degree of grafting have been studied.

The result showed that the percentage of grafted chitin by pre- irradiation peroxide

method is higher than trapped radical’s method . The methanol-acetic acid in the

composition of 7 / 3 was the good solvent for grafting of chitin. The percentage of

grafting was found 47,2 %.

C. Study on radiation grafting of acrylamida onto chitin

Studies on radiation grafting of chitin were examined. Chitin is amino

polysaccharides and thus anticipated to have high potential as specialty polymeric

materials compared with cellulose. They are, however, utilized scarcely because of

problems associated with poor solubility, hidrophilicity , and low reactivity. Graft

copolymerization induced by radiation is one of the methods for polymer modification in

order to improve its properties such as hydrophilicity and reactivity. In this present

study , modified chitin was prepared by grafting of acrylamide using gamma ray. The

grafted functional group onto chitin are expected to increase its hydrophilicity and

reactivity.

Isolation of chitin involved basic operations such as deproteinization, demineralization,

and decolorization by acetone . The irradiation of samples was carried out in 60Co

gamma irradiation source . In the present experiment, the pre- radiation –peroxidized

method was employed. In this method chitin was irradiated in presence of atmospheric

oxygen at room temperature. A monomer acrylamide solution than introduced into

irradiated chitin and the graft polymerization was carried out in a nitrogen atmosphere at

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a certain time of reaction. The grafted chitin obtained was washed and dried in vacuum

and the percentage of grafting was measured by gravimetric method.

The percentage of grafting has been determined as a variation of total dose, monomer

concentration and temperature as a function of time of reaction. The results showed the

optimal total dose for the grafting process was 12 kGy. The percentage of grafting

increases as increasing total dose, acrylamide concentration and temperature. The

optimal conditions were total dose of 12 kGy. acrylamide concentration of 30 %,

temperature of 70 0C and reaction period of 3 hours. The yield of grafting was found

94,3 %. The presence of acrylamide on chitin was demonstrated by IR spectrum and the

thermal stability by DSC.

THE RESEARCH STILL BE DOING

I. Removal of heavy metals from solution by using modified chitosan (Chito-g-Aac )

Selective removal of heavy metal such as Cu, Hg, Cd and Pb from solution at

laboratory scale was carried out. The most promising method of removing of heavy

metal from solution is the use of a cation –exchange materials having a chelating group

such as Chitos-g-Aac or Chitos-g-Aam.. A previous study in our laboratory has

indicated that Cu ion can be successfully remove from the solution using chitosan

modified with value of 8 times compare only non modified chitosan. The goal of the

study was to investigate the possibility of removing toxic heavy metal such as Hg, Cd,

and Pb

II. Study of composition on formulation of chitosan as film forming for nail polish

The effect of solvent composition on formulation of chitosan as film forming has been

carried out. The aim of this research was to find out the best composition of solvent using

chitosan as film forming for nail polish. The solvent used were ethyl alcohol, butyl acetic

acid, and acetic acid. The physical and mechanical properties of the resulting film were

examined, e.g. hardness, adhesion and glossy of the film, viscosity and flow properties of

the formulations.

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The results shows that the best formulation for chitosan as film forming were as follows;

concentration of chitosan 2 %, the composition of the solvent was ethyl alcohol / acetic

acid = 1 / 3 with the value of the hardness of the film B, adhesion 78,5 %, glossy 68,2 %

and the viscosity of the formulation was 1113 cps with a good flow properties.

In this research we continue study of effect an organic pigment and some additive like

hardener etc for make a good film for nail polish .

THE RESEACH WILL BE DOING

1. Science and technology for rural areas development

This research will begin next year it was our division proposal to our government for

developing of rural people in north sea side Java Island , which that area rich with shell

shrimp or crab waste. In that proposal we will introduce and teach them how to make

chitin from that waste after that they can make by themselves and sells the product to

private company as distributor of chitin.

Chitin from distributor was send to our institute for irradiation and we process to

make oligo-chitosan with high degree of deacetylization and we send to distributor again

for distribute oligo-chitosan to industry in Indonesia. For this project we begin with the

capacity of 1000 kg chitin/ month / area, and we plans for four area there are 2 in

Cirebon and 2 in Semarang.

2. Research and development of oligo-chitosan for bioplastic materials.

One objective of research is to make biodegradable films from renewable sources

such as oligo-chitosan. These films can then be applied to replace some petroleum-

based film or to new specialized niche areas made available by increasing

environmental concern.

We choice oligo-chitosan as basic material for bioplastic because from our

experience research on chitosan as film forming we found that oligo-chitosan can

form a good film. Casting on glass plate at elevated temperature can form Chitosan

films. For getting a good film some additive must be added on the formulation such

as crosslingking agents and plastisizer.

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CONCLUSION

It is clear that the research give highlights the probability of utilizing. The waste

product of the sea food industry (prawn, crabs shell) to products which are useful in a

number of different area.

Irradiation treatment was applicable on upgrading of the chitosan. Such as

sterilization, degradation to make oligo-chitosan and modification of chitin by

irradiation grafting.

REFERENCES

1. Goosen,M.F.A. , Aplication of Chitin and Chitosan, Technomic Publishing Company,

Inc, Lancaster, Pennsylvania, USA. 1997, 297 –305.

2. Purwatiningsih, “ Isolasi kitin dan komposisi senyawaan kimia dari limbah udang

windu”, Tesis .Jurs. Kimia – ITB ,1992, 5 – 30.

3. Kurita, K; Koyama,Y ; and Taniguchi, A. Journal of Applied Polymer Science.

1986 , 31, 1169 – 1176.

4. Hong, K.N.O ; Mayers, S.P; Lee, K.S. Journal of Agricultural and Food Chemistry,

1989 , 37 ,(3) , 575 – 579.

5. Gatot Trimulyadi Rekso; Anik sunarni; Kadariah; Isni Marliyanti; and Rahayuningsih

Chosdu, Preliminary Studies of Chitin Sterilized by Irradiation, Proceedings book of

4th Scientific Conference of The Asian Societies of Cosmetic Scientists, Bali,

Indonesia, 7 - 9 April ,1999.

6. Gatot Trimulyadi Rekso; dan N.M. Surdia, Pengaruh Dosis Total Iradiasi

Kopolimerisasi Cangkok Asam Akrilat Pada Khitin, Prosiding Temu Ilmiah Jaringan

Kerjasama Kimia Indonesia Seminar Nasional VIII Kimia dalam Industri dan

Lingkungan, Hotel Santika Yogyakarta, 16-17 November,1999.

7. Gatot Trimulyadi Rekso; dan N.M. Surdia; Pengaruh Pelarut Pada Kopolimerisasi

Cangkok Asam Akrilat Pada Khitin Dengan Teknik Iradiasi, Seminar Kimia Bersama

ITB-UKM IV, Yogyakarta, 9-10 Februari ,1999.

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8. Dewi Wulandari, dan Gatot T.M, Pengaruh Iradiasi Kobalt-60 Terhadap Khitosan

Yang diperoleh dari Isolasi Kulit Kepala dan Badan udang putuh, Tesis Universitas

Pancasila Fakultas Farmasi, Jakarta, 1999.

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