KIGALI INSTITUTE OF SCIENCE AND TECHNOLOGY (KIST)

FACULTY OF APPLIED SCIENCES

DEPARTMENT OF APPLIED CHEMISTRY
ENVIRONMENTAL OPTION

EXTRACTION OF CHITIN AND ITS MELANIN COMPLEXES FROM

INSECTS IN RWANDA
Submitted by: INTWALI Parfait Jimmy
GS 20060182
Supervisor: Prof. Elena KOVALEVA

A research proposal submitted to the Department of Applied Chemistry, KIGALI

INSTITUTE OF SCIENCE AND TECHNOLOGY in partial fulfillment for the award of
the Degree of Bachelor Science in ENVIRONMENTAL CHEMISTRY.

1
 Academic year 2009
TOWARDS A BRIGHTER FUTURE

2
DECLARATION
I, INTWALI Parfait Jimmy, hereby declare that the work entitled “EXTRACTION OF
CHITIN AND ITS MELANIN COMPLEXES FROM INSECTS IN RWANDA” presented
in this report is my original final research project. To the best of my knowledge, this work has
never been presented in any other institution or university for the same award.

Signature…………………………….. Date………………………………

Supervisor:
This research proposal has been submitted with our approval as university supervisor.

Professor KOVALEVA Elena

Chemistry department
Kigali Institute of Science and Technology (KIST)
P.O.Box 3900, Kigali

Signature……………………………… Date……………………………

3
 ABSTRACT

The extraction of chitin and its melanin complexes was carried out using two types of
insects such as bees and cockroaches. Chitin – melanin complexes were extracted with the yield
2.3 % in the case of bees and 3.8 % in the case of cockroaches .

Chitin and its melanin complexes were identified by FTIR spectroscopy using reference
sample of chitin-melanin complexes extracted from bees.

Comparative analysis of percentage of chitin and melanin complexes was carried out for
the two types of insects. The ratio of purity in chitin-melanin complexes in cockroaches and bees
is 0.9 and 0.83 respectively; this is due to working conditions. The amount of chitin-melanin
complexes in cockroaches is higher compared to that in bees ; 90% of chitin was obtained from
cockroaches and 80% of chitin was extracted from bees. This was due to the difference in
chemical compositions of the two types of insects and also due to working conditions such as
washing conditions.

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 DEDICATION

I dedicate this thesis to God almighty, for responding to me each time I called upon his
help; and to my mother for her support and advice during all my existence.

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 ACKNOWLEDGEMENTS

My most sincere gratitude goes to my supervisor Professor ELENA KOVALEVA for

supporting me during this project.

Thanks to KIST, Chemistry Department for all the efforts they made and their support
during this period.

To the government of Rwanda for the scholarship.

To my sister Diana NIYONIZEYE and my Mother MUKABIRASA Agnes for their

supportive ideas.

To all my friends, colleagues and all KIST administration.

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 TABLE OF CONTENTS
DECLARATION.............................................................................................................3

ABSTRACT...............................................4

ACKNOWLEDGEMENTS...............................................................................................6

TABLE OF CONTENTS.................................................7

LIST OF FIGURES AND TABLES...........................................8

LIST OF ABBREVIATIONS ...............................................9

INTRODUCTION.........................................................................................................10

LITERATURE REVIEW.................................................................................................11

2.2.Uses of Chitin..................................................................................................13

2.3.1.Use of chitin in agriculture........................................................................15

2.3.2.Use of chitin in industrial processes..........................................................16

2.3.3.Use of chitin in medicine...........................................................................16

2.4. JUSTIFICATION................................................................................................16

2.5. OBJECTIVES OF THE PROJECT......................................................................17

2.5.1. General Objective:...................................................................................17

2.5.2. Specific Objectives:..................................................................................17

3.3. Extraction process.......................................................................................18

4. RESULTS AND DISCUSSION ..................................................................................20

4.1. FTIR results.....................................................................................................20

4.2. FTIR results interpretation...........................................................................23

4.3. Elementary analysis results and their interpretation......................................23

5. CONCLUSION AND RECOMENDATIONS...............................................................26

5.1. Conclusion:.....................................................................................................26

5.2. Recomendations.............................................................................................26

REFERENCES............................................................................................................. 26

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 LIST OF FIGURES AND TABLES

Figure 1: Reference spectrum for chitin-melanin complex obtained from a material of bee

Figure 2: The FTIR spectrum for chitin-melanin complex from bee samples from Rwanda.

Figure 3: The FTIR spectrum in cockroach sample extract in Rwanda.

Table1: Organisms and there chitin percentage

Table 2: Absorption bands of FTIR spectra of the extracted materials and reference bee extract

Table 3 : Average percentages of C,N,H in bees and cockroaches in Rwanda given by Perkin-
Elmer Elemental Analyzer (C,N,H), USA.

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 LIST OF ABBREVIATIONS
o
C: degree celcius

% : percent

C: Carbon

(C77H98O33N14S) n: melanin

(C8H13O5N)n : chitin

EA: Elemental analyzer

FTIR: Fourier Transform Infrared spectroscopy

g: grams

H: Hydrogen

H2O: Water

HCl : hydrochloric acid

ml: milliliter

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N:Nitrogen

NaOH: Sodium Hydroxyde

NMR: nuclear magnetic resonance

pH: hydrogen potential

USA: United States of America

INTRODUCTION

Rwanda is a country whose population is based mainly on agriculture. However it

currently faces crucial problems in the field of agriculture. Erosion problems, soil deterioration,
deforestation led to a decrease in crops and productivity. Chitin and chitosan are ones of the most
important key materials in the 21st century. They have multifunctional properties with
applications in the pharmaceutical science, environmental problem-solving area (such as water
cleaning) to fibers, films and hydrogels, in cosmetics industry, food additives and as agricultural
materials. Commercial chitosan is derived from the shells of shrimp and other sea crustaceans,
including Pandalus borealis (Shahidi, F.& Synowiecki, J.J. ,1991). In Rwanda, chitosan and its
complexes can be found in lowest plants, mushrooms (Vetter J., 2007), insects such as bees
(Teslenko A.Yu., Voevodina I.N., 1995) and industrial wastes, especially from brewing of beer
and manufacture of wine and ethanol can be used for extracting chitosan containing products.

Chitin is one of the most abundant polysaccharides found in nature. It is often considered
a cellulose derivative, although it does not occur in organisms producing cellulose. The

10
difference between cellulose and chitin is that the 2-hydroxy group of the cellulose has been
replaced with an acetamide group,( Shahidi, F.& Synowiecki, J.J. ,1991)

Figure 1: β-N-acetyl-D-glucosamine unit of chitin

Insects are an available and promising source of chitin and chitosan. Melanins are high-
molecular-weight heterocyclic polymers with irregular structure whose chemical structure
remains to be determined completely. With respect to chemical structure, properties, and species
affiliation, melanins are classified as allo-, pheo-, and eumelanins (Britton G, 1983).

LITERATURE REVIEW
2.1. sources of chitin

2.1.1.Chitin in insects

Insect cuticles form an exoskeleton that exhibits only a limited capacity to keep pace with
body growth because it is a more or less rigid structure due to the presence of chitin and
sclerotized proteins. To allow growth and development, insects are therefore periodically forced
to replace their old cuticle with a new and looser one during molting (ecdysis). The nascent, non-
sclerotized integument underneath the old cuticle is strongly furrowed and can only expand when
molting is complete. Ecdysis is initiated by apolysis, the process that separates epidermal cells
from the old cuticle by molting fluid secretion and ecdysial membrane formation. The molting
fluid contains proteases and chitinases, enzymes that digest the main constituents of the old
endocuticle (Reynolds and Samuels, 1996).

Chitin is among insoluble and indigestible non-starch polysaccharides naturally occurring

polymers. It is translucent, pliable, resilient and quite though. In its pure form, it is leathery, but
when encrusted in Calcium Carbonate it becomes much harder. (Campbell, 1996). Recent

11
studies point out that chitin is the most abundant amino polysaccharide estimated to be produced
annually almost as much as cellulose. It has become of great interest not only as an underutilized
resource, but also as a new functional material of high potential in various fields (Majeti N.V.
Ravi Kumar, 2000).

2.1.2.Chitosan in insects

A principal derivative of Chitin produced by alkaline deacetylation of Chitin is Chitosan.

Chitosan is highly positively charged, which makes it an effective binder to negatively charged
molecules, such as metals, biochemicals, macromolecules, and cells. This binding can be used in
the removal of heavy metals, and pollutants in drinking water.

Chitosan occurs in nature in the cell walls of some fungi, insects etc. It is prepared by the N-
deacetylation of “chitin with 40-50% aqueous alkali .Chitosan possesses a large set of useful
properties and applications as compared to cellulose and its natural precursor chitin.(G.
Josefsson, 1980).It is obtained by deacetylating chitin with a hot alkali solution (Muzzarwelli
R.A.A. et al, 1980) . Application of chitinous products in foods and pharmaceuticals as well as
processing aids has received considerable attention in recent years as exotic synthetic compounds
that are losing their appeal (Elsevier Science Ltd, 1999).

Figure2: Structure of chitosan

2.1.3.Melanine in insects

Insect cuticle contains melanins that are covalently bound to protein and chitin; due to their
unique physicochemical properties, they protect insects from ultraviolet light, ionizing radiation,
the toxic effects of heavy metals, as well as endogenous and foreign chemical compounds
(Tyshchenko V.P., 1986).Melanin is found in plants, animals, and protists and act as a defence
mechanism against predators. Its molecular formula is C77H98O33N14S.

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2.2.Uses of Chitin

2.2.1.Wound Healing

Chitin has been found to have an acceleratory effect on the wound healing
process.Standard silk and catgut sutures coated with regenerated chitin or chitin show wound-
healing activities only slightly lower than the all-chitin fibers.

Surgical gauze coated with regenerated chitin demonstrates a substantially greater

amount of activity than an uncoated control group, (Muzzarwelli R.A., Tanfani, F., G.,1980).

2.2.2.Burn Treatment

Chitin is a very attractive candidate for burn treatment. This is true since chitin can form
tough, water-absorbent, biocompatible films. These films can be formed directly on the burn by
application of an aqueous solution of chitin acetate. The solution, although acidic, provides a
cool and pleasant soothing effect when applied to the open wounds of burn patients. Another
advantage of this type of chitin treatment is that it allows excellent oxygen permeability. This is
important to prevent oxygen-deprivation of injured tissues.

Additionally, chitin films have the ability to absorb water and are naturally degraded by
body enzymes. This fact means that the chitin needs not be removed. In most injuries (and
especially burns), removing the wound dressing can cause damage to the injury site.

2.2.3.Cell Binding Activity of Chitin

Deacyletylated chitin, or chitin, has been shown to aggressively bind to a variety of

mammalian and microbial cells. This property of chitin may lead to a variety of biomedical
applications. These possible applications will use chitin as a hemostatic, bacteriostatic, and
spermicidal agent.

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2.2.4.Chitin as a Hemostatic Agent

Chitin can be referred to as a polycation. Since the early 1950s, polycations have been
known to bind to red blood cells. Many studies since have shown that polycations are effective
cellular agglutinating agents. In the early 1960s, chitin was investigated for its agglutinating and
binding abilities. It was found that chitin, even at very low concentrations, had the ability to
agglutinate red blood cells. This led to chitin's consideration as a hemostatic agent. The
agglutination of red blood cells by polycations is dependent both on polymer structure and
molecular weight. Out of six common polycations, only chitin was able to effectively initiate gel
formation of heparinized blood.

Chitin with a molecular weight of 35,000 was only able to produce a loose coagulum in
heparinized blood, while chitin with molecular weights of 600,000 or above produced firm
coagulum.

2.2.5.Mechanism of Cell Binding

The mechanism of binding has also been researched. It is well known that the repulsive
force between red blood cells is due to the high net negative charge on the cell membranes. This
high negative surface charge is predominantly due to the presence of neurminic acid residues on
the cell membrane. Researchers removed this acid by means of the enzyme neuraminidase. This
process removed the high net negative charge of the red blood cell.

The researchers then looked at the effect of chitin on the modified blood cells. It was
determined that chitin did not cause any gelling of the blood cells. Therefore it was concluded
that the gel formation of red blood cells is due to the interaction of the positively charged chitin
polymer with receptors containing neuraminic acid residues on the cell surface.

2.2.6.Other Biological Applications

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 Chitin provides a diverse spectrum of uses in the biological arena. In addition to the
wound healing and burn treatment provided by chitin, it has been shown to reduce serum
cholesterol levels. To a certain degree, it has also been shown to stimulate the immune system.
chitin, when coated on seeds, results in increased crop yields. Apparently, this is due to chitin
inducing a protective response in the germinating plant. chitin has been proven effective for
many different applications. This is due in large part to its favorable biological and chemical
properties. By understanding these properties, biomedical engineers will be able to fashion better
tools to help the medical profession. Below are the useful biological and chemical properties of
chitin.

2.3.The Future of Chitin and chitin

Chitin and its derivatives show promise for the future. Products produced using chitin
have been shown to increase wound healing in animals and humans. Chitin has also
demonstrated a physiological compatibility with living tissues. Chitin's ability to form sulfate
esters which are non-thrombogenic appears to make it a promising candidate for prosthetic
structural devices of any shape or size. Therefore, chitin could serve as replacements for bone,
cartilage, arteries, veins, and musculo-fascial replacements. The uses of chitin and chitin are only
limited by the creativity of the biomedical engine.

2.3.1.Use of chitin in agriculture

Most recent studies point out that chitin is a good inducer for defense mechanisms in
plants(Linden, J., Stoner, R., Knutson, K. Gardner-Hughes, C., 2000) It was recently tested as a
fertilizer that can help plants develop healthy immune responses, and have a much better yield
and life expectancy. The EPA regulates chitin for agricultural use. Chitosan is derived from
chitin, which is used as a biocontrol elicitor in agriculture and horticulture.

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2.3.2.Use of chitin in industrial processes

Chitin is used industrially in many processes. It is used in water purification, and as an

additive to thicken and stabilize foods and pharmaceuticals. It also acts as a binder in dyes,
fabrics, and adhesives. Industrial separation membranes and ion-exchange resins can be made
from chitin. Processes to size and strengthen paper employ chitin.

2.3.3.Use of chitin in medicine

Chitin's properties as a flexible and strong material make it favorable as surgical thread. Its
biodegradibility means it wears away with time as the wound heals. Moreover, chitin has some
unusual properties that accelerate healing of wounds in humans. Occupations associated with
high environmental chitin levels, such as shellfish processors, are prone to high incidences of
asthma. Recent studies have suggested that chitin may play a role in a possible pathway in
human allergic disease. Specifically, mice treated with chitin develop an allergic response,
characterized by a build-up of interleukin-4 expressing innate immune cells. In these treated
mice, additional treatment with a chitinase enzyme abolishes the response (Sashiwa, H. A. J.,
2004).

Table1: Organisms and there chitin percentage

Organisms containing chitin Percentage of chitin composition (%)

Fungi 5-20
Worms 20-38
Squid/Octopus 3-20
Scorpions 30
Spiders 38
Cockroaches 35
Water Beetle 37
Silk Worm 44
Hermit Crab 69
Edible Crab 70

Source: "Chitin", www.psrc.usm.edu/macrog/sea/chtin.htm

2.4. JUSTIFICATION

16
 This project was done to prove how raw materials in our country Rwanda such as insects
can be used for extraction of chitin and its melanin complexes.

2.5. OBJECTIVES OF THE PROJECT

2.5.1. General Objective:

The extraction of chitin and its melanin complexes from insects of Rwanda and
characterization of the extracted products using elemental analysis and FTIR spectrophotometry.

2.5.2. Specific Objectives:

1. Sampling of two types of insects of Rwanda.

2. Extraction of chitin and its melanin complexes from these types of insects.

3. Characterization of chitin and its melanin complexes using elementary analysis and FT-IR
spectroscopy.

4. Comparative analysis of percentage of chitin and melanin complexes in the extracts of

cockroaches and bees of Rwanda.

3. METHODS AND MATERIALS

3.1 Preparation of chemicals for extraction

Solutions were prepared in to facilitate the procedures of extraction. Each solution of 8%

w/v NaOH and 6, 7 % w/v HCl is prepared in 1.5 l of water.

Preparation of 6, 7%w/v HCl solution in 100 ml in water needs first of all to dilute five
times 33% HCl solution from laboratory to reach the percentage w/v solution needed; then after
to prepare 6,7% w/v HCl solution in 1,5 l using the analytical expression (formula) below:

C1V1= C2V2 , (3.1)

where V1: the initial volume of water
C1: the initial concentration HCl

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 V2: Final volume of water
C2: Final concentration of the solution
In our case, the volume of HCl (V1) necessary is calculated as follows:
C2×V2

V1 = 
C1
6.7 % w/v*1500 ml

V of HCl = = 300 ml

33 % w/v
Preparation of a 1.5 liter solution of 8% NaOH involves firstly mixing 8 grams of NaOH
in 92 ml of water. Then, using the formula above, 1.5 liter solution of NaOH concentrated at
8% w/v is calculated as follow:

100 ml of water > 8grams NaOH

1500 ml of solution > 8 grams of NaOH × 1500 ml ⁄ 100 ml

> 120 grams of NaOH

3.2. Pre-treatment of insects for extraction

Insects were collected and killed using chloroform, after that the samples were weighed
with the digital balance. Chloroform (CHCl3) is a non flammable clear and volatile liquid at
ordinary temperature and pressure. It reacts by blocking the respiratory system and does not have
any effect on the chemical composition of the sample.

3.3. Extraction process

A raw biomass of insects (150g of each sample) was treated twice with 8% solution of
NaOH at 90ºС for 1 h (deproteinization process) After the two procedures wash the product to
ratio of: product- water: 1-20. After this procedure treat product with HCl 6.7% (300ml acid into
1200 ml water) and heat for three hours at temperature of 25 0C, (demineralization process). After

18
then filtrate and measure pH, at this level the pH is 1 and we have to wash with distilled water up
to pH 7. The final product after we remained with 3.5g in bees ; 5.7 in cockroaches after
extraction process (Teslenko A.Yu., Voevodina I.N.,etc. (1995) Method of preparing glucane-
chitosan complex. Patent RU 2043995 C1, 12p.) .

3.4. Characterization of extracted products using Fourier Transform Infrared

Spectroscopy (FTIR)

Identification of different functional groups were made during this procedure through
spectral bands. FTIR spectrum of chitin melanin complexes extracted from bees was used as a
reference spectrum.

3.5. Elementary analysis

The percentage of C,N and H in the extracted samples was determined and through those
percentages the chemical formula of the compound can be deduced.

3.6.Equipment and Materials

3.6.1. Equipment:

1. Perkin-Elmer Elemental Analyzer (C,N,H), USA

2. Perkin-Elmer FTIR spectrophotometer, USA

3. Heater, aspirator, funnel;

4. Analytical Balances, Beakers.

3.6.2. Reagents:

Sodium hydroxide (NaOH) ;

Hydrochloric acid (HCl).

Potassium bromide (KBr) for FTIR.

Chloroform

3.6.3. Materials:

Two types of insects (bees and cockroaches).

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4. RESULTS AND DISCUSSION

4.1. FTIR results

After extraction procedures, final products were solid and insoluble. The raw material was
150g each before extraction. After this process 3.5 g of extract of bees and 5.7g of extract of
cockroaches were obtained. This means that the yields of extracted materials were 2.3% and 3.8
% in the case of bees and cockroaches, respectively.

FTIR analysis was done by comparing the obtained spectra to the reference spectra of
chitin and its melanin complexes without impurities.

The FTIR results are presented in the form of spectral bands and each functional group
has its own signature, to carry out the FTIR tests Perkin-Elmer FTIR spectrophotometer, USA
was used. By comparing our product with the reference sample of chitin and its melanin
complexes, the obtained products were found out to have approximately the same spectral bands
as the reference spectra, small differences found in our products spectra were due to working
conditions and methods used in extraction and this can be defined as impurities. The FTIR
results in our two samples showed that cockroaches contain higher amounts of chitin and its
melanin complexes than that in bees; this may be due to the difference in chemical composition,
type of food they eat and living conditions of the two types of insects. This may also be due to

20
working conditions because the two samples were prepared in different intervals and solutions
prepared separately. Below are the spectra and figures of the obtained FTIR results.

98 **AVP-495; NPVO; 15.10.09

97
Transmittance, %
96

95

94
Пропускание

93

1376,6
92

1014,2
91
2925,4

1651,7

1557,2

1070,0
90
3096,0

1621,4
89
3438,4

88

87

4000 3500 3000 2500 2000 1500 1000 500

см-1
cm-1

Melanin part Chitin part Melanin part

Figure 1: The reference spectrum for chitin-melanin complex obtained from a material of bee
(Vetter J., (2007)).

**YUG_R-4`; NPVO; 19.10.09

96
94 Transmittance, %
92
90
88
86
84
Пропускание

82
80
78
1307,5

76
1026,2
1455,7

74
72
1376,5

70
1071,7

68
2853,6

66
1557,6

64
62
1634,0
1652,1
3264,9

60
58
56

4000 3500 3000 2500 2000 1500 1000 500

см-1

cm-1

21
 Melanin part Impurities Chitin Melanin
part part
Figure 2: The FTIR spectrum for chitin-melanin complex from bee samples from Rwanda.

** YUG_R-1; NPVO; 19.10.09

95

90

85

80
Пропускание

75

1378,0
70

1027,8
65

1557,5
3440,5

1652,6

1073,4
60
3262,2

2850,4

1622,6
55

50

45
4000 3500 3000 2500 2000 1500 1000 500
см-1

cm-1
Melanin part Impuritie Chitin Melanin

Figure 3: The FTIR spectrum in cockroach sample extract in Rwanda.

Table 2: illustration of obtained FTIR spectra with the reference spectra

Comparison of obtained spectrum compared to the reference spectrum in

cm-1

peak Peak Peak Peak Peak Peak Peak Peak Peak Peak 10
1 2 3 4 5 6 7 8 9

Reference 3438 3279 3086 2905 1651 1621 1557 1376 1070 1014

Bees 3440 3262 - 2950 1652 1622 1557 1378 1073 1027

Cockroaches 3264 - 3099 2981 1652 1634 1557 1376 1071 1026

This table shows the comparison of the obtained results with the reference sample spectra
and helps to identify and compare the peaks observed in our FTIR spectra.

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4.2. FTIR results interpretation

Identification of isolated chitin-melanin complexes was carried out using the method of
FTIR spectroscopy.

The main absorption bands are subdivided in three main ranges for the reference spectra.

It was found that approximately the same type of spectra for chitin-melanin isolated from two
types of insects to the reference spectra that contains no impurities. This indicates that the
obtained compounds contain chitin and melanin parts.

The presence of chitin parts causes the absorption band at 1651, 1557 and 1376 cm -1,
which correspond to vibrations of amide groups «amide I», «amide II» and «amide III»,
respectively.

For chitin part the values of the same absorption bands are 1652, 1557 and 1378 cm-1 for
bees and 1652, 1557 and 1376 cm-1 for cockroaches.

The presence of absorption bands in the 3400-2800 and 1160-1020 cm-1 in the spectra of
the studied insects indicates the existence of melanin part. These absorption bands appear due
to N-H, O-H, C-H and C-O, C-C vibrations which manifest in the spectra both chitin and
melanin. FTIR results showed that the extracted samples were washed out properly and
contained minimal content of impurities.

4.3. Elementary analysis results and their interpretation

The elementary analysis gives information about total percentage of carbon, hydrogen
and nitrogen atoms present in the compound.

This is given in forms of percentages and by those results we can deduce the molecular
formula of our compound. For elemental analysis Perkin-Elmer Elemental Analyzer (C, N, H),
USA was used and we obtained the following results.

Table 3: average percentages of C, N, H in bees and cockroaches in Rwanda as given by Perkin-

Elmer Elemental Analyzer (C, N, H), USA

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 COMPOUND AVERAGE % OF COMPOUND

BEES COCKROACHES

C 52.65 47.23

H 8.42 7.32

N 5.55 7.20

A high quantity of nitrogen in cockroaches is apparently due to the presence of a large

amount of proteins in the raw material of cockroaches than in bees, this may bring us to presume
that cockroaches have higher amounts of melanin compounds than bees.

Calculation of the ratio of melanin and chitin part in the obtained products

(C77H98O33N14S)x (C8H13O5N)y (H2O)z (4.1)

% C(total)= (% C in melanin part) × x+ (% C in chitin part) × y (4.2)

% H= (% H in melanin part) ×x + (% H in chitin part) × y + (% H in water) ×z (4.3)

% N= (% N in melanin part) × x+ (% N in chitin part) × y (4.4)

The ratio of chitin part is equal to the molar mass of chitin part times index y over the total mass
of chitin – melanin complex. The ratio of melanin part in chitin- melanin complex is equal to
molar mass of melanin part times index x over the total mass of chitin- melanin complex.

After calculations the following values of x,y,z were obtained :

These values for bees and cockroaches were substituted to the general formula of chitin-
melanin complexes (4.1). The following formulas were derived :

- for bees - (C77H98O33N14S) 0.03 (C8H13O5N) 0. 8 (H2O) 0.004 and

- for cockroaches-(C77H98O33N14S) 0.09 (C8H13O5N) 0. 9 (H2O) 0.002

Also percentages of chitin in the extracted materials were calculated using the following
proportion :

(X+Y+Z) - 100%

Y- n% (4.5)

24
In cockroaches:

N=(0.9x100)/0.992=90% of chitin in cokroaches

N=Y/100

N=(Y x 100)/0.992= (0.09x100)/0.992=9% of melanin in cockroaches

N=(Z x 100)/0.992= (0.002 x 100) /0.992=0.2% of water in cockroaches

By adding the all ratios we get: 90+9+0.2=99.2

In bees:

N=(0.8x100)/0.834=80% of chitin in bees

N=Y/100

N=(Y x 100)/0.834= (0.03x100)/0.834=9% of melanin in bees

N=(Z x 100)/0.834= (0.004 x 100) /0.834=0.4% of water in cockroaches

By adding the all ratios we get: 80+3+0.4=83.4

Table 4 : Amount of chitin and melanin parts in chitin-melanin complexes

Chitin-melanin component Cockroaches bees

Chitin part 90% 80%
Melanin part 9% 3%
Water 0.2% 0.4%

After the calculations we obtained a percentage of 0.9 in chitin part, 0.09

melanin parts in cockroaches and 0.8 chitin part, 0.03 melanin part. The remaining
percentages are due to impurities. The purity of chitin and melanin complexes in
Cockroaches is higher than that in bees, this may be due to chemical compositions
of these insects and to working conditions. Cockroaches have higher amounts of
melanin complexes and this is proved by the higher amounts of N in EA results.

25
5. CONCLUSION AND RECOMENDATIONS

5.1. Conclusion:

Chitin and its melanin complexes were successfully extracted from bees and cockroaches
using standard procedures and identified by FTIR using the reference sample. Chitin – melanin
complexes were extracted with the yield 2.3 % in the case of bees and 3.8 % in the case of
cockroaches .

EA results gave the ratio of 0.9 of chitin and 0.09 of melanin complexes in cockroaches;
0.8 of chitin and 0.03 of melanin complexes in bees. It was found that the amount of chitin-
melanin complexes in cockroaches is higher compared to that in bees.

The ratio of purity in chitin-melanin complexes in cockroaches and bees is 0.9 and 0.83
respectively also 90% of chitin was obtained in cockroaches and 80% of chitin obtained in bees.
This was due to the difference in chemical compositions of the two types of insects and also due
to working conditions such as washing procedures.

5.2. Recomendations

To wash the sample correctly in order to reduce impurities which were observed in FTIR

All experiments should be done here in our country and more likely here in KIST to gain more
knowledge and reduce the cost of the project,

Studies should be made on how to use Chitin in waste water treatment process here in Rwanda
and discover its sorbent properties to heavy metals.

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