Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
by
Antonia Yarbeh Tetteh
r
li
(
"... Mathematical facts worthy of being studied are those which reveal unsuspected
relations between other facts, long since known, but wrongly believed to be
unrelated to each other."
(
ABSTRACT
· .~
Chitin, a polymer of N-acetyl-D-glucosamine b now heing llsed in the
food industry as inexpensive polysacchél ride. The research pursued relates to the
establishment of interrelationship between various factor~ affccting chitin
the yield of chitin extraction. Optimization of chitin extraction was carried out
using crab, lobster, and shrimp solid waste~. The study wa~ dlvloed into two
stages: (a) optimization of chitin extractability with respect to partidc ~ize (h)
effeet on cr ab and lobster chitin extractability; a partide size of 2.0mm gave the
highest yield in chitin extraction. The mean yield of chi tin trom crah and loh~ter
at particle size of 2.0mm were 28.8% and 23.2%, respectively. Shrirnp chitin
extractability was not affecterl by particle size with the mean yicld heing 25.2%.
rotatable design was applied to the four variables. Ali variable~ had an effeet on
time of extraction at levels of 2.25N, 1:5 • 1:6 (wjv), 29°C, and 5 1/2 h
i
-------------------------------------------
water holding eapacity of the nltimate chitin and ehitosan products were
measured as a means of determining their applicability in foods. Viscosity,
moleeular size and water holding capacity were respectively, 17.1ep, 1.31 x 106
daltons, and 404 - 415% (wjw) for chitin; 1.54ep, 4.17 x 104 daltons, and 501.6-
Il'
I~
ii
RESUME
La chitine, un polyrr1he de N-ac~thyl-D-glucose amine, e~t actuellement
utilisé'e dans l'industrie alimentaire comme polysacc!mrid t ' peu on~reuse. L~I
/
recherche menee relate les interconcetions entre les different~ factuer~ affectant
l'extractions de la chitine et la comhinai~on de l'optimisation~ de~ factl\cr~ re4ui~
~ 1
pour maximiser l'extraction d" la chitllle. L'extraction~ de la chitine a elc
optimis~e avec des d'echets solides de crahes, h()mard~, ct crcvcttc~ . Cette
ltude est divise'e en deux parties: (a) optimisation du rendemeIlî d'cxtracti()n~ de
la chitine en fonction de la taille des particule!l. (h) optimisation~ dc~ ftat~ de
d!min~ralisatiolJ et de deprotlinisation de la chitine extracte du homard grSce a
l'~tude de R~ponses de Surface. La taille des particule~ a lin effet ~ignificatif Mir
extractabilid' de la chitine de crahe et de homard. une taille de particule de
2.0mm donnant le plus haut rendement d'extraction de la chitine. Le plu~ haml
rendement d'extraction de chitine de crahe et de homard. avec des particlllc~ de
cette taille, respectivement de 2K8% et 23.2%. L'extractahilit/ de la chitinc de
crevette n'est pas affectle par la taille de!l particules, son rendement maximum
1 1
est de 25.2%. La methode des Reponses de Surface permet une etude
simultanle des effets de (a) la concentration en extractant, (h) du rapport
coquille: extractant (c) des templratures d'extraction ct (d) du tcmp~
d'extraction, sur les etats de deprotiinisations et de dé'min~rali!lati()n!l de la
chitine ainsi que sur l'augmentation du rendement de chitine. Un plan central a
, I\'-
composentes rotatives fut applique' aux quatre paramètre~. Tou~ le~ paramètre~
ont un effet sur le rendement de la d:mine'ralisation et la deprot~ini~ati{)ns. Le
maximum de rendement de deprot~inbati{)n est obtenu avec un modèle tel que
les "
parametres concentrations en NaOH, rapport coquille:extractant,
tempe'rature et temps d'extraction ont respectivement le!l valeur!l de 1.75%, 1:6 -
1:7 (plv). 55°C, et 5 h 1/2. Le maximUITl de de'min(rali~ation~ fait tgalcment
iii
( dltermine par un mod~le multifacteur comprenant les parametr~s concentration
pour la chitine sont: viscosite' 17.1cp, poids molfculaire 1.03 x 106 daltons, et
capacite a la retentions d'eau de 404 - 415% (p/p), pour le chitosane les valeurs
respectives de ees difftrents param~tres sont: 1.5cp, 4.17 x 104 daltons et 501.6 -
504.9% (p/p).
.,
.,
.,f
~
iv
----------------------..............
ACKNOWLEDGEMENTS
1 would like to thank the Almighty God. for the guidance. protection and
numerous bll~ssings he gave me throughout the course of thi!o. work. With Him.
this work bf~came a possibility.
1 \\,')uld like 10 thank my supervisors, Dr. B.K. Simpson and Dr. J.P. Smith
for all the help they gave me 10 make this work possihle. Their support wa~ very
mu ch appreciated.
My sincere thanks go to the Head of Department (Ag.). Dr. Intcal Alli
for providing facilities during the course of thb work. Hb constant ~upport and
encouragement V/as appreciated.
1 would like to thank the Canadian International Development Agcncy
(CIDA) for providing financial support.
1 thank the University of Science and Technology. Kumasi. Ghana. the
Head of Biochemistry Department. Dr. J.H. Oldham. and other Maff memher~
for their participati0n in the program. Special mention i~ made of Dr. E.K.
Marfo for the moral support and constant encouragement he offercd me during
the course of tbis work.
To Mr Eby Noroozi and Mr Abdelnaby Khalyfa. 1 say thank you for the
technical help you offered.
1 wou Id like to thank Dr Thaddeus Varney for having the patience to
proof read this work.
1 would Iike to thank Mr Melvin Munsaka of Mathematic~ and Statistic~
v
-- ------------------------------.,
encouragement.
Above ail, my sincere thanks go ta my dear husband, Paa Kow Tetteh,
and my son, Kweku Bedu Tetteh, who were very support ive and helped in ail
ways to make this work possible. Their inestimable encouragement will always
be remembered.
,(
, t
vi
Tahle of contents
Page
Abstract
Resume llJ
Acknowledgements v
List of figure~ . Xl
APPENDIX
CHAPTER 1
INTRODUcnON .
CHAPTER2
LITERATURE REVIEW. 4
2.0 Introduction. 4
'-,f-
vii
~;,.
3.4 Hydroehloric acid demineralization of lobster shells 44
3.5 Sodium hydroxide deproteinization of lobster shells 45
3.6 Decoloration of crude chitin 45
3.7 Central eomp0!:lite rotatable design for optimization studies 46
3.8 Nitrogen determination 46
3.9 A'ih determination 49
3.lO Viscos;ty and molecular weight determination :9
If
viii
List of Tables
Table Page
chitosan solutions. 27
4. Demineralization conditions used by various authors . 28
chitin extraction 47
8. Factors and values of the coded levels used in the Central
composite rotatable design to optlmize demineralization ~tep in
chitin extraction . 48
9. Uncoded level combination for a four vari?ble Central
extraction . 55
extraction . 56
12. Estimated ridge of maximum response for the various factors used in
ix
deproteinization . 64
extraction 69
14. Coded level combinat ion for a four variable Central Composite
16. Estimated ridge of maximum response for the various factors used in
demineralization 83
x
List of figures
, ~
ratio and time held constant at 1:15(wjv) and 6 h
< ,\"
of NaOH and shell:extractant ratl, , with temperature and time he Id
.....
constant at 6S o C and 6 h respectively, required to produce a
'
, .1' xi
( constant at 6S o C and 6 h r,~spectively, required to produce a
degree of deproteinization of 6.74% residual total nitrogen
in chitin 65
( on yield of demineralization 75
15. A three dimensional Response Surface graph showing the effeet
, 1\'"
xii
of tempe rature and time with concentration of Hel and shell:extractant
ratio he Id constant at 2N and l :20(wIv) respectively,
on yield of demineralization 76
16. A three dimensional Response Surface graph showing the effect
of shell:extractant ratio and time with concentration of HCI
and temperature held constant at 2N and 2SoC ref,pectively,
on yield of demineralization 77
17. A two dimensional contour plot showing the levels of concentration
of HCI and shell:extractant ratio with temperature and time held
constant at 2S oC and 4 h respectively, required to produce a degree
xiii
( 25. Viscosity of crab chitosan against concentration 95
26. Molecular weight distribution of chi tin and chitosan . 96
. ,~
, 1\'
• l'
(...
xiv
1
CHAPTERI
Introduction
structure, modification and application and these have shown that, chitinou~
et al. 1984). Current chi tin and chitosan processing conditions and chemical
deacetylation procedures, cause sorne modifications such as depolyrnerization
and deacetylation of native chitin. For the purpose of preparing chitin of less
modified structure, mild treatments are preferable. Sorne investigators have
attempted to remove protein by enzymatic digestion (Hackman, 1960; Takeda
and Abe, 1962; Takeda and Katsuura, 1964), however, complete removal of
pro teins was not attained. Other investigators have also tried to produce
chitosan by fungal fermentation, involving the enzyme chitin deacetylase (Araki
and Ito, 1975; McGahren et al. 1984), but these have shown inconsistent trends
in degree of deacetylation, with low yields (Knorr and Klein, 1986) as weil as
variations in molecular weight (Arcidiacono et al. 1988), making chemical
methods of extraction of chitin and chitosan more popular among producers
experimental designs and multiple regression analysis, was used in this study to
experimental run. RSM has been applied to studies of canola sauce production
(Ma and Ooraikul, 1986), protein extraction from brewer's spent grain (Diptee l't
al. 1989), bacterial growth (Shroder and Busta, 1973), ca~ein extrusion (van de
Voort and Stanley, 1984), and shelf life extension of English type crumpets
(Smith et al. 1988). Thus in summary, this work was carried out to investigate the
, f
4
CHAPTERl
Literature review
2.0 Introduction
The Shellfish Industry
The rapid perishability of fish and shellfish compared with meat has at aIl
times and places made preservation against putrefaction an urgent necessity. At
a very early age in history, man learned the survival value of storing day-to-day
1962). Most people, if they tasted fish at ail, did so only when it was dried, salted
or pickled. The development of nice-houses" and freezers were a step forward in
retaining sorne of the freshness of fish and shellfish but the market for these
products was not satisfactory. Canning was the result of an attempt to preserve
fish satisfactorily without adversely affecting the freshness and palatability by
introduced the concept of preservation by canning, fish and lobsters were among
the preserved foods he presented to the French government. Although canning
proved to solve many of the above mentioned problerns, and allowed large scale
f
,
5
largest catch of crustace ans in the world (Cutting, 1(62). Shrimp canning planb
in the USA generally process from 9,000 to 18,000 kg of raw shrimp per day. The
largest plants are capable of processing up to 55,000 kg a day. Raw ~hrimp
the turn of the century to over 500,000 tons annually in recent year~ (CRESA.
1971). King crab and Dungeness crab are also harvested to a large extent in the
.\-
United States, but the Soviet Union now appears to be the large~t catcher of
King crab. Japan holds the second place in crustacean catching and King (;rah
dominates the canned pack. Polish deep sea fishery ha~ engaged in halve~ting
marine living resources su ch as, the Antartic krill and squid cor.laining chitin
since 1975. Presently there is a production of about 5 tons per day of peeled krill
meat, leaving behind a great deal of shell wastes, from which practical bnlation
of about 150 kg of chi tin is possible (Brzeski, 1(88). Poland abo harvest~
large quantities of squid from a dozen to 100,000 tons per year, and this can be a
source of raw material for production of chitin and chitman. Squid pen contain~
about 40% chi tin, but the feasibility of chi tin and chitosan production from it
depends strictly on the market situation, since the market is more familiar with
crustace an chitin.
Ali the above mentioned countries produce large quantities of wastes per
- day and per year. Since the wastes are not biodegradable, disposai becomes a
worldwide problem.
6
.
\ 2.0.2 Waste disposai problem of the shellfish industry
As a result of processing shellfish, large quantities of shells and other
waste mate rials are left over which do not find any use presently. Approximately,
about 75% of the total weight of shellfish is discarded as waste, and in sorne
cases, as in crustacean rneat industries, the waste mate rial can represent an
amount greater than 80% of the landing (Simpson, 1978), from 20 - 30% of the
dry weight of the waste is chitin, depending upon the processing method.
Disposai of shellfish wastes which contains chi tin as a major constituent
represents a significant problem to proceswrs who are limited in their
alternatives by environmental and economic restrictions (Revah-Moiseev and
Carroad, 1981). Current methods of handling shellfish wastes include ocean
dumping, incineration and landfilling (Kreag and Smith, 1975). Simpson (1978)
stated that, disposai operations in the near future will have to meet increasingly
( more stringent ecological standards. Problems and regulations governing current
shellfish wastes handling rnethods are briefly discussed below
1. Ocean dumping: this i5 regulated by the Environmental Protection
Agency (EPA). ft is r"ohibited in the United States to throw wastes back inlo
the sea. It is also costly in energy and dollars for the hauling and storage ùf the
wastes.
2. Incineration: it requires a government permit and yields only lime as
integuments (insects and crustaceans). Fungi, molds and yeasts also possess
chitinous cell walls (Ruiz-Herrera, 1978). Chi tin and chitosan are also present in
environment, arthropods are the main chi tin producers, with a chitin production
of 51 g.m -2.yr- 1, and this fluctuates with seasonal changes (Yamamoto and Seki,
1979).
daphnia has been reported to have annual chitin production of 3.2g.m-2 .year- 1
(
which would represent a total of 55,360 kg of chitin for the whole water body of
17.3 km 2 (Peterson, 1983). Another rich source of chi tin in the marine
has reported that, chi tin values of copepods on dry weight basis ranges from
12.22% occurring in the cladoceran species. The rnean annual chitin production
the dominant producers be;ng crustace ans, bryozoans and hydrozoans (Jeunial'x
et al. 1988). In this group of animaIs the crustaceans show the highest chitin
production. Extensive work has been carried out on Euphausiids (krill) of the
North Atlantic Ocean and North sea (Lindley 1978; 1982a). With a chitin
content of 7.08% determined for Euphasia superba (Antartic kri1l, Yanase, 1975)
Atlantic ocean and North sea (Jeuniaux et al. 1988). Antartic krill has been u~ed
for chitin and chitosan production on an industrial scale (Anderson ct al. }<)78:
Brzeski, 1982). At present chitin is u5tually isolated from crab. loh~ter and ~hrimp
shells owing to their high content of chi tin and ta their availability in relatively
high amounts from fisheries and canned food industrie~.
Poulicek and Jeuniaux (1988), have reported the use of marine ~ediments
selected fungi is shown in Table 1. Chitosan b also present in mo~t fungi and the
order Mucorales ha~ been found ta synthe~ize chitosan as ccli wall component
(Bartnicki-Garcia, 1968), and the se organism~ have been regarded as alternative
source of chitosan. Examples of strains of Mucoraceae are Phycomycc~,
Rhizopus, Absidia and Mucor, with the members of the genu~ Ahsidia producing
the highest amounts of chitosan (Shimahara ct al. 1988). The~e have heen ~hown
to have a chitosan content of between Il and 14% of the dry weight of the cell
wall.
10
Table 1. Chi tin content of sorne selected fungi (Kong, 1975; Naczk et al. 1981).
Chltm content
Fungi (%)
Aspergz1lus niger 42.0.*
( Penicillium notatum 18.5.
Penicillium clzrysogenum 20.1*
Saccharomyces cerevisciae 2.9
Mucorrouxii 44.5
such as mannans, glucans and galactan~ in the fungal cell walls white in animais,
shell, proteins are the main components and account for 50 - 80% of the matrix
dry weight, while chi tin occurs at low and variable percentages of hetween 0.10 -
40% of the matrix d~' weight (Poulicek, 1982; Jeuniaux, 1963; Goffinet and
Jeuniaux, 1979).
'. intermediate (Peter et al. 1985), Conclusions drawn from ehemical model
reactions suggest that crosslinking of the cuticular proteins results from Michael
type conjugate addition and Sehiffs base reaction with free peptidic amino
groups (Anderson, 1979; Brunet, 1980; Lipke et al. 1983). It has been propmed
hydroxy oxygen (Peter et al. 1985). Chitin may also contribute to the Mability of
, ,F the sclerotized inseet cuticIe (Hackman and Goldberg, 1977; Vincent and
the form of calcium carbonate and to a lesser extent as calcium pho~phate . This
chitinoproteic complex for calcium ions (Poulicek et al. 1985). Squid pens are
non-calcified and contain about 60% protein and 40% chitin (Hunt and Nixon,
1981).
12
carotenoids, since the colours of most carotenoids are red, orange or yellow
, ,~
(Muzzarelli, 1977). According to Muzzarelli (1977), crabs, like many other
animais, convert ingested yellow plant carotenoids into oxygenated and thus
more polar orange or red keto derivatives and in sorne cases conjugate these to
brown seaweed eaten by crabs is a rich source of cawtene, but the funct'fm of
crustace an shells with the usual demineralization reagellts, namely, disodiurn salt
citric acid, dissolves the calcium carbonate but leave aIl pigments firmly bound
to the dernineralized shells, indic :tting that, the carotenoids are not bound to the
scleroproteins, but to chitin itself. Fox (1973) reported that the carotenoids are
matreials would suggest that, non-covalent type bonds are more important in the
complex formation
( convert the waste into valu able rnaterials and to develop pc;>tential applications
for it to increase its marketability. In Japan, chitosan was produced industrially
13
for the fifst time in the world in 1971 by Katakurachikkarin Inc. (Hokkaido) and
Kyowa Rei:o Inc. (Tottori), (Hiram), 1988) from chitin. Since the n, chitin and
chitosan have been produced by a number of companies and, in 1986, there were
States, Protan Laboratories and Kypro Company form the main chitin/chito!lan
industries (Sanford, 1988). In Poland chitin and chitosan are being produced on
a laboratory scale from krill and squid pens (Brzeski, 1988). Commercial chilin
, .1- and chitosan vary in quality with each of these companies and they supply these
products in the form of powders, flakes. fibers, films, beads and sheet!l.
these companies produced 1,270 ton~ of chitin. Out of this, 1,170 ton!l were u!led
production, and 40 tons remained as excess. The yield of chitosan was 700 tom,
and out of this amount 500 tons was used as flocculants, and 100 tons for
cosmetics, foods, and feeds production, with an excess of 100 tons (Hiram),
bonding in its solid state. Chitin is crystalline and insoluble in water and
soluble in strong mineraI acids and in anhydrous formie acid but insoluble in
alkali. By repeatedly freezing and thawing it in al kali solutions it can be wholly
disso!ved (Danilov and Plisko, 1954). The ease of dissolution depends on the
degree of crystallinity. Only beta-chitin dissolves in anhydrous formie acid.
low viscosity chitin solutions and hydrolytic degradation proceeds relatively slow
, \
(c )molecular shape
Currently, the intrinsic viscosity is related to the average molecular weight (Mw)
bya Staudinger equation (Billmeyer, 1971)
Mvol = Mw = average Mw
K = 8.93 x 10.4
'a' = 0.71
15
Austin et al. (1988) reported that squid chitin, which is beta-chitin is found to be
dissolved LiCI has been discovered hy Rutherford and Austin (lQ78a). Austin
, ,1' (1984; 1988); Barton (1983) and Burell (1955) develaped the concept of
approximating that of the polymer have the best chance of compatibility. formic
acid, a popular solvent for chitin was found ta have a 6 of 24.8 - 25:7 for
alpha-chitin and 23.2 - 26.0 for beta-chitin. Very often the solubility JJarameter
solvents, ego 1,2-dichloroethane have been found to be very useful (Austin et al.
gel partides often rernain suspended in the solution. When formic acid (96%,6
beta-chitin is obtained. The LiCI/tert amide systems are ail strong swelling
appears the best solvent for beta-chitin, giving a very viscous solution (Austin,
1988). The solvents power is derived from the addition of LiCI, as the two liquids
alone are only swelling agents for chitin. The Liel apparently reduces or breaks
, 'l'
, l'
16
fibers. Chitin and chitosan are known to bind 2 to 5 times their weight of water
with chitosan having the greatest water binding capacity (Knorr, 1(82).
with protein residues which remain with it even after the most dra~tic alkali
treatment.
5% N,N-dimethylacetamide-LiCI and since then thi!l solve nt hu!>. been used for
viscosity measurements (Shimahara and Takiguchi, 1988; Rutherford and
Austin, 1978b) since it both swells and dissolves chitin without any hydrolytic
an enlarged effective volume due to charge repulsior and stretching out of the
molecule (Muzzarelli, 1977). Upon addition of suffic;c!1t salt to neutralize this
compound through the constant Km. Km depends on the nature of the solute
( P = degree of polymerization
1977). Lee (1974) obtained average molecular weight of beta-chitin from Loligo
pen as 2.5 x 106. Chitosan prepared from this by deacetylating with 45% NaOH
under nitrogen for 4, 6 and 8 hours at 1400 C gave average molecular weights of
7.25 x loS, 4.92 x loS, and 2.35 x loS daltons respectively. Molecular weights of
chi tin and chitosan are different and a severe degradation of the chain takes
place during the production process. This happens during the de calcification step
(
when the shells are submitted to the action of acid solutions at high
concentrations and at both room and elevated temperatures, or during the
deacetylation step which involves harsh treatment with alkali at high
concentrations and temperatures.
( chitinous polymers are obtained via physical methods which are based on high
shearing forces, centrifugation and lyophilization which results in drastic changes
19
in the physical properties of the material. Sorne of the changes in properties are
improved dispersibility, and unusual viscosity stability at prolonged and elevated
growth of Bifidobacteria in the gut (Austin et al. 1981). These bacteria block the
required for digestion of milk lactose. This may be significant for both humans
and animais with lactose intolerance. Above 10% additions chitin de presses iTOn
It has also been found that, when chi tin is added to bread whose gluten
has been partly replaced by other protein sources, there is prevention of
depression of loaf volume (Knorr and Betschart, 1978; 1981). When used a!l
,
~ ... l
, i
food additives
additives stems from the concept that functional ingredients which are absorbed
intact or metabolized may interact with target tissues or organs and constitute a
20
,
l
Egg
,-
breaking 100-200e 6.7 7.1 70 - 90
4
55-75 (Bough,1976)
~ operation
~hiller effluent
Scalder effluent
~n conjunction with 2 • 20 mg.L of Betz 1130, a cationie polymer
Packing wastewater
~rocessing and curing waste
fWith mg/L of WT-3000, a negatively charged polymer
21
potential risk. (Takeda and Abe. 19(2). The use of chitin/chitosan ,L, non-
investigated in animal feeding trials with the food dye. FD&C Red No. 40. This
showed that when the dye is attached to the chitinous polymer. absorption of the
'l-
polycationic carbohydrate polymer has been found to be particularly effective in
aiding the coagulation of protein from food process wastes (80ugh, 1976; Fujiti.
1972). Table 2 indicates that, chitosan can reduce suspended solids by 70-98%.
The traditional coagulants used are multivalent inorganic salts such as AI and Fe
sulfates.
Peralta et al. (1989) found that both acid-soluble and water-soluble chitosan salts
chitosan salt or water soluble chitosan salt and the conventional silica
.f'" soljgelatinjbentonite treatment for the clarification of fruit juices revealed that,
22
2.3.6 Miscellaneous
The unique properties of chi tin and chitosan make them attractive
sources of dietary fiber, functional ingredient, a carrier for food additives with a
potential for cholesterol reduction in humans. The humectant properties of
chitin/chitosan and their derivatives allow them to be used in food processing to
improve moisture uptake.
The chelating properties of chitosans prove advantageous in the removal
of heavy rnetals, dyes, pigments, acids or organic solids such as proteins. This has
resulted in utilization of approximately, 50% of the currently produced chitosans
in clarification, coagulation or flocculation processes in processing wastes and
waste water treatment.
The gelling properties of chitosan ami its water soluble derivatives allow
a wide range of application in food biotechnology, the most attractive being
, r
23
coating of foods and pharmaceuticals, and gel entl apment of biochemicals, plant
embryos and whole celIs, mieroorganisms and algae. Sueh entrapment offer!\
have led to recommending chi tin films as oyen and other food wraps.
crustace an species su ch as crab, shrimp, lobster, prawn and crayfish. The shells
of these animais are presently available in quantitie~ sufficient to support a
20 - 30% on a dry basis. Crab shell forms the main source of industrial
production of chi tin in Japan, because they are relatively rich in chitin and less in
calcium carbonate, and easy 10 obtain in large quantities from the crab meat
.
...- '
world. Its edible tail is commerciallv.' u~ed and the waste mate rial is suitablc for
24
1. Demineralization
2. Deproteinization
3. Decoloration
Sorne classical methods for preparation of chitin include the method of
Hackman (1954), Whistler and Be Miller (1962), Horowitz et al. (1957), Foster
and Hackman (1957), Takeda and Abe (1962), Takeda and Katsuura (1964) and
Broussignae (1968). The key steps in the extraction of chitin from crustacean
2.4.1. Demineralization
Hackman (1954), Whistler and BeMiller (1962), and Broussignac (1968) aIl used
carbonate is eonvert~d into soluble calcium ehloride and carbon dioxide gas is
evolved. With progressive increase in the concentration of the acid, the degree of
the use of acid concentration above 1.25N adversely affects the ~Jscosity of the
L.
25
solid wastes
ckaning
drying
pulverizing
dernineralization
washing
deproteinization
washing
drying
decoloration
washing
drying
chitin
-
Fig. 1. A generalized scheme of chitin recovery.
26
, ,~
treatment to which the raw material has been submitted to, prior to the
deacetyl(ltion step. An overview of the various demineralization conditions
used by various authors are given in Table 4. To control the deacetylation and
depolymerization, Lusena and Rose (1953) have suggested the use of HCI at pH
not lower than 3. Moreover, instead of the use of HCI, formie acid (Horowitz et
al. 1957) ûr EDTA (Foster and Hackman,1957; Takeda and Abe, 1962; and
Takeda and Katsuura, 1964) have also been used for demineralization.
2.4.2. Deproteinization
Deproteinization of the demineralized shells may be accomplished by
dilute aqueous NaOH solution. Hackman (1954), Whistler and Be Miller (1962),
Horowitz et al. (1957), aIl used dHute aqueous solution of NaOH in removal of
prote in, with constant stirring and for prolonged hours in an inert atmosphere.
, " The disadvantages of this method are that, it requires large amounts of alkali,
and causes removal of acetyl groups though it removes proteins and peptides as
desired (Muzzarelli, 1977), and therefore in ail cases, a partially deacetylated
produc\ is obtained. The effect of alkali treatment on the macromolecular length
and viscosity of the uItimate chitosan is less pronouneed than that of the Hel
treatment. Muzzarelli (1977) also reported that, between 20 - 80 mesh particle
size, alkali treatment had no effeet on the extent of deaeetylation and viscosity of
27
, Hf
, _Ii'
28
(
Table 4. Demineralization conditions used by various authors.
Variables * Authors
Xl X2 X3
• I~
,~
1.63 1:15 25 2 Kamasastri and Prabhu, (1961)
2.00 1:25 25 5 Sannan et al. (1976)
2.00 1:25 25 2 Sannan et al. (1976)
1.14 1:4 20 1.5 Brzeski (1982)
1.00 25 12 Mima et al. (1982)
(
2.00 1:10 25 5 Hackman (1954)
1.25 25 1 Madhavan and Ramachandran, (1974)
4.5 20 Whistler and Be Miller, (1962)
, \
(
r ,f
29
crustacean shells by use of proteolytic enzymes. Takeda and Ahe (1962), Takeda
with papain, pepsin, or trypsin. These enzymes are specifie for proteins and
, II-
therefore do not deacetylate the chitin, however, complete removal of protein i~
2.4.3. Decoloration
Carotenoid pigments as weil as other lipids occur in the crustacean
firmly associated with the tough and pliable chitinous pie ces, indicating that, the
T ,F carotenoids are bound to chitin itself. These may be removed by organic solvents
such as ethanol or acetone. Other ways of removing these pigments are by the
use of cold formie acid on the carapace, and mixtures of ammonium sulfate and
I~
sulfuric acid on chitosan. Whistler and BeMilIer (1962) used a combination of
95% ethanol, acetone and ether to decolorize chitin hut these cOlild not remove
ail the pigments. Since the se solvents cannot remove ail pigment~ 10 give a
Variables * Authors
Xl X2 X3 '4 Xs
4 80 3 Mima et al (1982)
4 1:3 100 12 17 Hackman, (1954) i
1,
1
,
"
10 25 72 20 Whistler and Be Miller, (1962)
10 1:50 100 2.5
!
J
Horowitz et al. (1957)
3 2:3 100 0.5 Madhavan and Ramachandran, (1974) i
*Xl = Cone. of NaOH (%)i X2 = Ratio of shell:extractant
(w/v); X3 =
Temp. (OC); X4 = Time (hr); X5 = Yield (%).
(
.. ~
i
... ~
31
hydrogen peroxide (Brine and Austin, 1981), and ethyl acetate (Brzeski, 1982).
, f Bu: No et al. (1989) reported that, the treatment which gives the most acceptable
absolute acetone for 45 minutes, followed by treatment with 70% acetone, and
then bleaching with 0.315% sodium hypochlorite in the cold (Blumberg et al,
1951).
•
2.4.4 Preparation of chitosan
Chitosan is prepared by deacetylation of chilin. Chitin possesses 2,3-trans
Von~Furth and Russo (1906) indicated that, three out of four acetyl groups can
progresses only gradually after this, reaching 80% in five hoUTs. Deacetylation
can rarely extend beyond 80% (Muzzarelli, 1977), unless the alkali fusion
hydrochloric acid and nitric acid have revealed that, acidic treatment leads to
extensive degradation of the polymer chain even at O°e. Any acidic treatment
12
c
, l'
carried out on chitin or on chitinous raw material leads to a partial or extended
depolymerization. Even though this polymerization occurs during alkali
treatment of the material the extent of degradation induced by NaOH treatment
chi tin, with repeated washing of the intermediate products with water. The total
time for preparing this chitosan was reduced to less than five hours, at a reaction
tempe rature of 1100 C. white the conventional continuous procedure required a
prolonged time at that same temperature. The shortened reaction time in this
method avoids degradation of the molecular chains of the obtained chitosan
chromatography.
Another method of preparing chitosan is by microbial fermentation.
deacetylation process occurs in tandem with chitin synthesis. The fungal order
be readily cultured on cheap nutrients and the celI wall mate rial can be
recovered by simple chemical procedures. McGahren et al. (1984) carried Ql1t
cells in a medium containing a carbon source, such 3!\ glucose or molasses, a high
fermentation are as follows: the percentage yield is very low and varies with the
age of the culture, the size of the culture vessel used (oxygen availability). and
the type and strength of acid. White et al. (1979) determined that 1 N
hydrochloric acid was the acid of choice. But Arcidiacono et al. (1988) observed
that hydrochloric acid degrades the cell wall mate rial during extraction and th us
lead to low yields. They evaluated acetic acid and found out that 2% W:lS most
10% of total dry weight of the biomass, and 30% to 35% of the cell wall was
reported by Arcidiacono et al. (1988). White et al. (1979) also produced chitosan
< 'f-
by fermentation of Mucor rouxii. The chitosan products obtained from
Klein, 1986). lbis method also produces wide variations in the average
advantages in that the chitosan produced have very low degree of acetylation
(5% to 10%; White et al. 1979), while that of shellfish chitin ranges from near
-
, .11'
and controls over biosynthesis, problems with yield, degree of acetylation and
• IF
34
• f
( With the classical experimentation procedure only one variable can be tested at
a time and this requires a large number of experiments which could be expensive
35
and time consuming. The change in one obtained optimum level will modify the
next optimum level, and so the optimum concentration level must be re-
determined at that factor level and so on. The optimum product might not be
1. Two or three factors that are most important to the product under
study are identified. If the factors are not known, preHminary expeTiments must
2. The range of factor levels which will determine the number of sampi es
to be tested are defined. If the range is too broad, the optimum will not be
clearly defined by RSM. In most food products, the factor levels are restricted by
physical (e.g., tempe rature ) and cost limitations and government regulations.
Because of these limitations, the optimum may lie outside the JeveJ~ tested and
therefore cannot be determined. Once the factor levels are set, preliminary Tuns
36
;
with samples representing the mid-points of these levels should be performed to
'"
establish that the levels are appropriate.
3. The specifie test samples are selected and tested using the appropriate
experimental design. These designs sele(..! a subset of sampI es to be tested from
the set of aIl possible samples which could be tested. While covering the range of
factor levels specified by the experiment, the design emphasizes those tests
close st to the mid-points of these ranges, and thereby decreasing the total
dependent only on the number of variables and the selected response equation.
-,f'
The center point for each explanatory variable level is given a code of zero while
the highest and lowest levels of interest for each independent variahle are coded
plus or minus one respectively for three level designs. The highest and lowest
levels are given maximum and minimum codes respeetively (Thompson, 1(82).
A coded level combination for a four-variable central composite design to
optimize the yield of chitin extraction in these studies is given in Tahle 6.
Table 6. Coded level eombination for a four variable Central Com~osite Design to
optimize the yield of demineralization and deproteinization in cbitm extraction.
, ·1'
•
VarIable •
Run# *. Xl X2 X3 '4
1 -} -1 -} -1
. ,~ 2 1 -1 -1 1
3 -1 1 -1 1
4 1 } -1 -}
5 -1 -1 1 1
6 1 -1 1 -1
7 -1 1 1 -1
« 8
9
1
1
-2
2
1
0
0
1
0
0
1
0
0
11 0 -2 0 0
12 0 2 0 0
13 0 0 -2 0
14 0 0 2 0
15 0 0 0 -2
, 1\0 16 0 0 0 2
17 0 0 0 0
18 0 0 0 0
19 0 0 0 0
20 0 0 0 0
, ,li'
39
( Y:
, If
•,
, '1
41
0.4813%. They concluded that RSM was an efficient experimental design when
several variable~ are to be evaluated simultaneously. Diptee et al. (1989)
evaluated the application of RSM in protein extraction studies from brewer's
spent grain with central composite rotatable design to optimize temperature,
time, BSG:extractant ratio and concentration of disodium hydrogen phosphate
in the extractant solution, with particle size held constant at 1.5mn,. A yield of
approximately 60% protein could be obtained from the dried brewer\ spent
grains. Smith et al. (1988) also used response surface methodology in shelf Iife
extension studies of a bakery product. The four variables were evaluated at twu
levels around the optima. They concluded that RSM is an elegant tonl to
determine and simultaneously solve multivariate equation~ which specify the
optimum shelf life for a specified set of factors through mathematical model5t.
LDSO of 16gjkg body weight of mice. It is estimated that, only 0.05 to 0.1 %
chitosan will be present in animal diets when used as a protein coagulating aÎd
in insignificant differences in growth rate, blood and liver composition from the
control group (Landes and Bough, 1976). Since chitosan i!l a natural chelating
, ,t
.. required for homoeostasis, but Muzzarelli (1977) has reported that when chitin
and chitosan powders are brought into contact with alkali metals (eg, Na +, K + )
and alkali earth metals (e.g., Mg2 +, Ca2 +), these metals are not collected to
any extent and do not prevent collection of transition metal ions when
simultaneously present.
Evaluation tests with chitin and chitosan powder in order to find possible
irritating or allergizing effects have been carried out and the results were
2.7. Limitations
Though chi tin and chitosan have very promising applications, sorne
, II:-
limitations for their use have been encountered (Knorr, 1984). The main
limitation is that the properties of chi tin and chitosan vary with source as well as
method of preparation. So far aIl the methods of extraction of chi tin by various
authors and industries are different from each other. This has given rise to
inconsistent yields and variation in properties. ft is therefore suggested that,
maxirnization of yield of chitin extraction from crustacean wastes with constant
chitinous polyrners will open a wider door to increased applications in foods and
feeds.
\1'
,
i
43
• CHAPTER3
Industriallobster, crab and shrimp solid \Vastes were used throughout this
study and were obtained from WestmorIand Fisheries, Cap Pele, New
Brunswick. The waste products were transported in frozen form and upon
receipt were transferred to a freezer (u20oC) and stored at this temperature umil
required for use as source material for chitin recovery. The solid waMe~ were
thawed at room temperature, cleaned by scraping under running tap water, and
pre-dried in a hot air oven overnight at 6S°C. The shells were then dried
thoroughly in a vacuum oven (Precision Scientific Inc., Model No. 19) at 65()C
and 30mmHg for 4 hours. The dried shells were then pulverized with a waring
blender and sieved manually through a sieve of 2.0, 1.7, 1,41, and 1.0mm mesh
sizes to give uniform shell sizes. The pulverized ~ample~ \Vere then stored in
Isolation of chi tin from the above named crustacean shells involved the
. ,\-
, f
, f
44
extractant solution (ii) time of extraction (iii) ratio of shell:extractant, and (iv)
concentration of extractant. The four levels of particle shell sizes used were:
2.0mm,1.7mm, 1.41mm, 1.0mm. Samples (20g) of lobster, crab and shrimp were
subjected to demineralization by mixing the shells with the demineralizing agent
, IF
(2N HCI) in a ratio of 1:15 (w/v) at room temperature (23°C) for 6h with
constant stirring. This was followed by washing under running tap water, rinsing
with deionized water and filtration through 120 mesh sieve. The extract was
then deproteinized with 3.5% NaOH at 6S oC in a sheIl:extractant ratio of 1:15
(
(w/v) for 2 h with constant stirring. The extract was then washed, rinsed with
deionized water and dried at 65 0 C for 4 h in a vacuum oven. This was followed
by decoloration and drying and the residual weight measured.
Samples (20g) of dried and pulverized lobster shell waste (2.00mm) were
, II'
mixed with various concentrations of HCI (0.5, 1.0, 1.5, 2.0, "and 2.5N) in 500ml
beakers using shell:extractant ratios of 1:5, 1:10, 1:15, 1:20, and 1:25 (w/v). The
mixtures were heated at temperatures of 20, 25, 30, 35 and 400 C for 2, 4, 6, 8,
filtration (through 160pm mesh sieve), washed, rinsed with deionized water and
dried al 6S oC for 4 h in a vacuum oyen. The residues were analyzed for ash and
45
. total nitrogen content as described in 3.9 and 3.8 respectively. The residues were
(1:5, 1:10, 1:15, 1:20, anf1 1:25 w/v). The mixtures were heated at temperature!\
of 45, 55, 65, 75, and 85 0 C for 2, 4, 6, 8) and 10 h respectively, and then filtered
through 160um mesh sieve. The residues were recovered by filtration and
washed with water, rinsed with deionized water and dried at 65°C for 4 h in a
vacuum oyen. The residual weight was rneasured to estimate lh~ los~ in weight
upon deproteinization and the cru de protein content was abo rncasured as
described in 3.8
erude chitin was decolorized using the method of Karnasastri and Prabhu
(1961) and Blumberg et al, (1951). Crude chilin samples were refllJxed with
ahsolute acetone (1S0rnl) for 45 min and dried at room temperature fûr 2 h and
then treated with 70% acetone. Samples were then washed, rinsed with
deionized water and then mixed with 0.315% sodium hypochlorite solution
(containing 7% available chlorine) for 5 min. The residues were then removed
.r by filtration (through a 160Jlrn mesh sieve) and then washed, rinsed with
deionized water and dried at 65°C for 4 h in a vacuum oyen. The re~idual
, l'
.
\. 3.7 Central composite rotatable design for optimization of demineralization
and deproteinization steps in chitin extraction
A four-factor 5-level central composite rotatable design (CCRD) by Box
et al, (1978) was employed for optimizing each factor in the CCRD. Factors and
levels of each factor in the design (Tables 7 and 8) were selected on the basis of
work done by previous authors (Horowitz et al. 1957) and also those required to
, 11f
prevent depolymerization and deacetylation of the native chitin chain. In the
CCRD, particle size was kept constant at 2.00mm and the shell:extractant ratio
varied from 1:5 to 1:25 (wjv). For demineralization, temperature varied from
200 C to 40°C, concentration ranged from 0.5N to 2.5N, and extraction time from
2 to 10 h. For deproteinization, temperatures varied from 4SoC to 8S oC,
concentration NaOH from 1% to 5%, and extraction time from 2 to 10 h. The
coded levels of -2, -1. 0, +1, and +2 used in the four factor CCRD (Table 6)
were obtained from Box et al, (1978) and values of coded levels of variables used
in the experimental design are shown in Tables 7 and 8 for demineralization and
deproteinization respectively. The total number of experimental runs
determined from this design was 20. Duplicate measurements were taken for
each experimental fun. On completion of the extraction processes, ash and erude
protein contents were determined as outlined in 3.9 and 3.8, respectively.
f
47
'-';'''\..
, t
Table 7. Values for coded levels of factors used in the central composite rotatable
design for optimization demineralization.
Coded levels *
-<,'''-
Factor -2 -1 0 +1 +2
"'"
Cone. of HCl (N) 0.5 1.0 1.5 2.0 2.5
< v
Ratio of shen: 1:5 1:10 1:15 1:20 1:25
extractant (w Iv)
Temp(OC) 20 25 30 35 40
Time (hr) 2 4 6 8 10
, ,..
48
, I~
Table 8. Values for coded levels of factors used in the central composite rotatable
Coded levels *
Factor -2 -1 0 +1 +2
Difference
f
• f
49
crucible was weighed and ignited for 30 min at 600°C. ft was then cooled and
transferred into a dessicator with the aid of tongs for 15 - 20 min and reweighed
the crucible and the sam pie was pre-ashed in a fume hood. When the !lumple
ceased giving off smoke, it was placed in a preheated 6000 C muffle furnace for 6
h. When ashing was complete the crucible was transferred directly into a
were compared to that of commercial chi tin and chitosan obtained from Sigma
(Sigma Chemical Company Ltd, St Louis, MO, USA). The viscosity of various
Laboratory, Mass., USA) at room temperature using a dise spindle (No. 3). Each
sample solution was transferred to the sample cham ber of the ~mall !lample
adapter (SC-4) and the torque required to rotate the spindle at a constant ~peed
• III'
3.11 Determination of water holding capacity
Water holding capacity of laboratory prepared chitin and chitosan were
compared to those of commercial chitin and chitosan (Sigma Chemical
company). The water absorption characteristics of chitin and chitosan were
determined by the method of Sosulski (1962). Excess water (20 - 30ml) was
added to the sample (1.5g) in weighed centrifuge tubes (radius, 1.2cm). The
suspension wa~ thoroughly mixed by shaking vigorously for 4 times with 10 min
rest period between each mixing. The suspension was then centrifuged at 730g
(JA-20, 220 C) for 25 mins after which the supernatant was decanted and the
tube air-dried for 10 min until no residual liquid could be seen. Water
51
accomplished by the use of Duncan's New Multiple Range test (Steel ami
Torrie, 1980). Regression coefficients and correlation coefficients werc a1so
computed using the Statistical Analysis System (SAS, 1982), on a McGiII
University mainframe. The 3 dimensionai response surface graph!. and 2
dimensional contour plots were done using an Splus package on an Sparc Sun
Station Unix machine.
52
CHAPTER FOUR
, l'
RESULTS AND DISCUSSION
size
The extraetability of chitin from crustacean solid wastt.s was investigated
by using a completely randornized design. In this experiment, lobster, crab and
shrimp solid wastes were used. Fig. 4 shows the effect of partic1e size and type of
crustacean solid waste on the yield of chi tin extraction. The results show that,
the extractibility of chi tin with respect to particle size varies with the type of
crustaeea. Among the three crustaceans used, analysis of variance revealed that
partic1e size had a significant effeet on chi tin extractibility in lobskr and erab
chitin yield (Appendix A and B). There were significant differences between the
means of the ehitin yields. Analysis with the Duncan's new multiple range test
revealed signifieant differenees between the me ans of chitin yields of lobster
between particle sizes 2.0mm and 1.0mm, and also between 1. 7mm and 1.0mm
with yield of ehitin at particJe size 2.0mm being the highest. With the yield of
crab chitin with respect to particle size, analysis of variance (Appendix B) also
showed significant differences between the Mean yields of chitin extraciion from
the various particle sizes al 0.01 level. Duncan's new multiple range lest located
differences between the means of the yields at particle sizes of 2.0mm and
1.7mm, 2.0mm and 1.0mm 1.41mm and 1.7mm, and 1.41mm and l.Omm. with
Mean yield at particle size of 2.0mm being the highest. On the other hand, a
different pattern was observed with extractability of chitin from shrimp waste at
the various particle sizes. Analysis of variance (Appendix C) showed that there
were no significant differenees between the me ans of the yields of chi tin at aIl
the various particle sizes. This observation has also been observed with crawfish
L
"_ ....... ;.~-- .... " .. ~,-<-. ..... ~ _J"",~_"",_~""_--;~~_""""~,,,,,,,,,,,,-~,,,,,,,,",,,,,,,,,,,,,--,,,~,,,,,_,,,,,,,,_-""'l'~""~~-tt-"t" ""'''''~'4l! AYJiN
,
~
...
\
'"
1
'ÎI
sample
28
-------- lobster
-+- crab
---*- shrimp
26
24
22
~~
20 --~--~--~----'------'
L-I
( solid waste (No et al. 1989). Since it is in the interest of the chitin industry to
starting materials for chitin production. Since this was not observed with shrimp
and crawfish shells the choice of a particle size at which to produce chitin may be
left to the discret:()n of the industry depending on whether the interest is to
• • f'
4.2 Optimization of deproteinization
for fitting the second order response surface is shown in Table 6. Uncoded and
( coded level combination used in each experimental run are shown in Tables 9
and 10 respectively. The estimates based on coded and uncoded data are
identical. Table 10 also shows the effectiveness of deproteinization expressed as
percentage residual total nitrogen of chitin with the corresponding coded values
extraction (X3) and time of extraction (~). The second order polynomial model
Examination of the fitted model (Table Il) with the t-test (25 d.f.) indicated that
temperature and time of extraction (X h X2, X3, and ~), aU the quadratic
55
Variable
••
y ~ ,,,....
Run • # Xl X2 X3 X4
1 2 0.10 55 4
2 4 0.10 55 8
3 2 0.05 55 8
4 4 0.05 55 4
5 2 0.10 75 8
6 4 0.10 75 4
7 2 0.05 75 4
8 4 0.05 75 8
9 l 0.06 65 6
10 5 0.06 65 6
11 3 0.20 65 6
12 3 0.04 65 6
13 3 0.06 45 6
14 3 0.06 85 6
15 3 0.06 65 2
16 3 0.06 65 10
17 3 0.06 65 6
18 3 0.06 65 6
19 3 0.06 65 6
, .lI'- 20 3 0.06 65 6
Table JO. Coded level combination for the four variable Central Composite
Rotatable Design for optimization of deproteinization
Variable --
Resldual Total Nltrogen
Run • Xl X2 X3 J4 of chitin
• "1'
Il Predicted Observed
1 -0.5 -0.250 -0.5 0.25 5.73 5.69
2 0.5 -0.250 -0.5 1.25 6.03 5.99
3 -0.5 -0.875 -0.5 0.25 5.89 5.86
4 0.5 -0.875 -0.5 0.25 5.77 5.74
5 -0.5 -0.250 0.5 1.25 5.76 5.72
6 0.5 -0.250 0.5 0.25 5.47 5.43
7 -0.5 -0.875 0.5 0.25 5.36 5.33
8 0.5 -0.875 0.5 1.25 5.26 5.23
9 -1.0 -0.750 0.0 0.75 3.23 3.26
( 10 1.0
0.0
-0.750 0.0 0.75 6.70 6.73
, 11 1.000 0.0 0.75 3.83 3.84
12 0.0 -1.000 0.0 0.75 5.47 5.32
13 0.0 -0.750 -1.0 0.75 5.44 5.48
14 0.0 -0.750 1.0 0.75 6.34 6.37
15 0.0 -0.750 0.0 -0.25 6.29 6.32
16 0.0 -0.750 0.0 1.75 6.10 5.84
17 0.0 -0.750 0.0 0.75 5.55 5.53
18 0.0 -0.750 0.0 0.75 5.57 5.53
19 0.0 -0.750 0.0 0.75 5.39 5.53
20 0.0 -0.750 0.0 0.75 5.60 5.53
, II'
Coding of the factor levels were done by a default system using the formula in
equation 6:
coded value = (original value - M)jS
[6]
where M i!l the average of the highest and lowest values for the variable in the
design and S is half their difference
-Each run was reolicated twice for a total of 40 runs
.. X 1 = Concentration of NaOH (%); X2 = shell:extractant ratio (w/v); X3 =
t
Temperature (oC); X4 = Time (h)
;(
','
57
Table 11. Analysis of least squares estimates of the second order model parameters
for demineralization
7.85 ***
< .i'
. ~ 1.3(0.66)
X1
2 -0.52(0.03) -3.32 *.
X2
2 -0.89(25.91) -5.40 ***
X3
2 0.39(0.00) 2.51 *
.*
~2 0.55(0.00) 3.51
XI X3 1.03(0.01) 1.50ns
X2 X3 -4.46(0.63) -4.40 **
.*.
X3X4 -6.06(0,00) -8.82
R2 0.92
,- li-
, IF
.\
"
.li
, .Ir-
L
61
· ,;.
, .1
62
. ,~
, ,),0
. \
the estimated surface does not have a unique optimum. a ridge analysi~ was also
performed to determine the region in wUch the optimum Iie~. and to indicate
shows the estimated ridge of maximum response for the variolls factor~. Ridge
analysis indicates that maximum yields will result from rehttively low
'--If-
concentrations of approximately 1.75%, a relatively low shell:extractant ratio of
hetween 1:6 and 1:7(w Iv), a relatively moderate tempe rature of ahout 5SoC. and
reaction limes of about 5 1/2 hours. It b noted from the analY!lis of variance for
the model that aH the factors are highly significant. If further experimentatioin i~
undertaken, it :night he best to fix ail factor~ at their critical values and to
for fitting the second order response surface is shown in Table 6. U ncoded and
, .
coded level combination used in each experimental run are shown in Tables 13
, .~
, ,f
64
Table 12. Estimated ridge of maximum response for the various factors used in
deproteinization
COOëd Estlmated Oncoded factor values
radius response Xl X2 X3 J4
, 1.
0.0 5.47(0.13)21 3.00 0.12 65.00 6.00
0.1 6.12(0.15) 2.83 0.12 64.03 6.11
0.2 6.82(0.19) 2.68 0.12 63.02 6.27
0.3 7.63(0.27) 2.55 0.12 62.02 6.44
i
l,.
0.4 8.55(0.37) 1..43 0.12 61.02 6.62
0.5 9.58(0.48) 2.31 0.13 60.04 6.81
0.6 10.75(0.61) 2.19 0.13 59.06 7.00
0.7 12.03(0.76) 2.08 0.14 58.08 7.18
0.8 13.45(0.92) 1.97 0.15 57.11 7.37
0.9 14.99(1.09) 1.87 0.15 56.15 7.56
1.0 16.66(1.29) 1.76 0.15 55.18 7.75
, ~
astandard error
Fig. 9. A two dimensional contour plot showing the levels of concentration of NaOH
and shell:extractant ratio with temperature and time held constant at 6SoC and 6 h
respectively, required to produce a deproteinization of 6.74% residual total nitrogen
in chitin.
< lF-
1 1
OB OL 09 OS
Fig. 11. A two dimensional contour plot showing the levels of tempe rature and time
with concentration of NaOH and shell:extractant ratio he Id constant at 5% and ',
1:15(w/v) respectively, required to produce a deproteinization of 6.74% residual ,)
total mtrogen in chilin.
~)
~----------------------------------------------------------------~7
o
CO
o
r-
o
ID
c)
li)
O~ B 9
Fig. 12. A two dimensional contour plot showing the levels of concentration of
NaOH and time with shell:extractant ratio and tempe rature he Id constant at
1:15(w/v) and 6S oC respectively, required to produce a deproteinization of 6.74%
residual total nitrogen in chi tin.
1.....
i
68
-ln
\
T IF
O~ 9 9
69
Table 13. Uncoded level combination for a four variable Central Composite
Rotatable Design to optimize demineralization in chitin extraction.
•
Vanable ••
Run • Il Xl X2 X3 X4
l 1.0 0.10 25 4
1 .'1 2 2.0 0.10 25 8
3 1.0 0.05 25 8
4 2.0 0.05 25 4
5 1.0 0.10 35 8
6 2.0 0.10 35 4
7 1.0 0.05 35 4
8 2.0 0.05 35 8
9 0.5 0.06 30 6
( 10 2.5 0.06 30 6
11 1.5 0.20 30 6
12 1.5 0.04 30 6
13 1.5 0.06 20 6
14 1.5 0.06 40 6
15 1.5 0.06 30 2
16 1.5 0.06 30 10
17 1.5 0.06 30 6
18 1.5 0.06 30 6
19 1.5 0.06 30 6
20 1.5 0.06 30 6
, If
, ~
Table 14. Coded level combination for a four variable Central Composite Rotatahle
Design to optimize the yield of ciemineralization in chi tin extraction
Resldual % ash of
Run * # Xl X2 X3 ~ chitin
Predicted Observed
.-
2
-0.50
0.00
-0.3750
-0.3750
0.25
0.25
-0.50
0.00
0.72
0.29
2.54
2.12
3 -0.50 -0.6875 0.25 0.00 0.24 4.41
4 0.00 -0.6875 0.25 -0.50 0.07 4.24
~ 5 -0.50 -0.3750 0.75 0.00 0.34 2.17
6 0.00 -0.3750 0.75 -0.50 1.93 3.76
tt 7 -0.50 -0.6875 0.75 -0.50 0.97 5.14
t 8 0.00 -0.6875 0.75 0.00 0.27 4.44
9 -0.75 -0.6250 0.50 -0.25 330.16 27.17
10 0.25 -0.6250 0.50 -0.25 2.46 -0.52
11 -0.25 0.2500 0.50 -0.15 37.12 36.41
12 -0.25 -0.7500 0.50 -0.25 2.73 4,(>4
13 -0.25 -0.6250 0.00 -0.25 1.14 -1.84
14 -0.25 -0.6250 1.00 -0.25 2.46 -0.53
15 -0.25 -0.6250 0.50 -0.75 2.36 -0.60
16 -0.25 -0.6250 0.50 0.25 3.00 0.01
17 -0.25 -0.6250 0.50 -0.25 2.02 0.60
18 -0.25 -0.6250 0.50 -0.25 2.62 0.60
19 -0.25 -0.6250 0.50 -0.25 2.19 0.60
20 -0.25 -0.6250 0.50 -0.25 2.15 0.60
, ,1"
71
ratio, temperature of extraction, and time of extraction (XI, X2, X3, and ~),
two quadratic terms, (concentration of HCI)2, (shell:extractant ratio)2, and
(XI 2, and Xi,) and two interaction terms (XIX2 and X3~) were significant.
The lack ofsignificance of the interaction terms XIX3, Xl~, X2X3 and X2~
could be attributed to the antagonistic effects produced by these variables on
that, the quadratic model dues not fit the data very weil, so firm statements
about the underlying process cannot be based only on the above analysis.
Moreover, Table 14 shows that the observed values for residual % total nitrogen
are far from the predicted values indicating an inadequacy in the model. A third
order regression analysis may be carried out to improve the lack-of-fit but the
resultant regression equation becomes cumbersome and difficult to visualize.
,
, t'
72
Table 15. Analysis of least squares estirnates of second order polynomial model
parameters
Xl -99.69(32.35) -3.08 **
X2 -1715(605.42) -2.83 **
X3 -5.26(2.50) -2.10 *
X2
2 2672.99(334.42) 7.99 ***
XI X3 -0.23(0.88) -0.27n~
X2 X3 -0.73(16.34) _O.04 oS
2 -0.01(0.02) -0.87°S
X3
R2 0.92
, ~ ~f·
Only the significant terms in the second order polynomial were used to
the data revealed that the stationary point is a sadd le point. Examples of
vs temperature with shell:extractant ratio and time held constant at 1:20(w Iv)
and tempe rature with a relatively low shell:extractant ratio resulted in improved
variables are shown in Figs. 17 - 20. Table 16 shows the estimated ridge of
, ,\>-
maximum response for the various factors. Ridge analysis indicates that
1:6 (w Iv), a relatively moderate tempe rature of about 29 0 C, and reaction times
of about 5 1/2 hours. ft is noted from the analysis of variance for the model that
, Ll:-
Fig. 13. A three dimensional response surface graph showing the effeet of
concentration of Hel and shell:extractant ratio with temperature and time held
constant at 25°C and 4 h respectively, on the yield of demineralization in chitin
'- '. extraction.
75
, II'
Fig. 14. A three dimensional response surface graph showing the effect of
concentration of Hel and tempe rature with shell:extractant ratio and time held
constant at 1:20 (w Iv) and 4 h respectively, on the yield of demineralization in chitin
extraction.
-
76
• Ilf
• II'
Fig. 15. A three dimensional response surface graph showing the effect of
tempe rature and time with concentration of Hel and shell:extractant ratio held
constant at 2N and 1:20(w/v) respectively, on the yield of demineralization in chitin
extraction.
77
, ,\-
)
>
t,"
l
(
,
~
l't
~
~
< I\-
t,
~
~
'I""i)l-
r'~
~
~,
~,
f
f
1
Fig. 16. A three dimensional response surface graph showing the effect of
shell:extractant ratio and time with con~~ntration of HCI and temperature held
constant at 2N and 2S oC respectively, on the yield of demineralization in chitin
1 extraction.
1
p
78
indicated by the elevating behavior of the graph~ over the response surface. At
critical levels of 1.75% NaOH, 1:6 - 1:7(v';v), 55 0 C anù 5 1/2 h,
deproteinization !'eemed to be highest. Though an extraction time of 5 1/2 h
seem to be long compared 10 the relatively short period of extraction used by
other researchers, a comb:natlon of the levels of factors indicated above with a
time period of 5 1/2 h will produce the highest degree of deproteinization. On
the otheï hana in demineralization, the behavior of contour plots especially
those of Figs. 18 and 20 reveal that degree of demineralization does not follow
any particular pattern with changes in combination of shell:extractant ratio with
time. The critical factors in determining the degree of demineralization appear
to be concentration and temperature. Since the model for demineralization was
not adequate, ridge analysis was carried out on the data, and it revealed the
direction in which the optimum levels of factors lie and the direction in which
further experimentation should be performed. A combination of concentration
of HCI, shell:extractant ratio, temperature and time at levels of 2.25N, 1:6 -
1:7(w/v), 29 0 C, and 5 1/2 h which is obtained from the ridge analysis seem to be
one which will cause destruction of the molecular structure of the chi tin chain
because of the high level of concentration of HCI (Madhavan and
Ramachandran, 1974). A lower degree of demineralization which may be
obtained at less destructive conditions may be preferable .
• I\'
Fig. 17. A two dimensional contour plot showing the levels of concentration of Hel
shell:extractam ratio with temperature and time held constant at 25°C and 4 h
respectively, required to produce a degree of demineralization of 0.07% residual ash
content.
~----------~--------------------------------------~9
o
o
C\I
, ,~
li)
cl
20 25 30 35 40
"CI
N .....
o e
o
.....
~:-----------------------------------------
'--",
Fig. 19. A two dimensional Contour plot showing the levels of temperature and lime
with concentration of Hel and shell:extractant ratio held constant at 2N and
1:20(wIv) respectively, required ta produce a degree of demineralization of 0.07%
residual ash content.
~ ______________________________________________________ ~81
o
'It
...
·.
o
(1')
\
O~ a 9
(
L,
, ..
Fig. 20. A two dimensional contour plot showing the levels of ~hell:extractant ratio
and time with concentration of Hel and temperature held constant at 2N and 25°('
respectively, required to produce a degree of demineralization of 0.07% re~idlJal a~h
content.
82
·P
o
(\J
o
li)
T"'"
• • 10
o
..-
o
, 10
LI)
o
ci
: O~ B 9
83
Table 16 Estimated ridge of maximum response for the various factors u~\'Ù ill
demineralization .
. \-
radius response
astandard error
, ...
84
85
most researchers (Hackman, 1954; Whistler and BeMiller, 1962~ Shimahara nnd
Takiguchi, 1988). NaOH removes proteins and peptides a~ desired but it al~o
removes :\cetyl groups and may weIl lead to fragmentation of the chitin chain
(Foster and Hackman, 1957). Optimal conditions for deproteinization has heen
value of 6.9% for pure chitin. Treatment No. 10 with conditions: 5% NnOH,
10). AlI the other combinations gave a residual % total nitragen content lower
than 6.71 % (Table 10). No et al. (1989) reported that. at a fixed temperature of
content showed that total nitrogen levels of the decalcified ~hells gradually
residual % total nitrogen content of Run No.s 9 and Il were very low, about half
the theoretical value of that of pure chitin (Table 10). This observation could be
attributed ta the fact that there was extreme hydrolytic deamination on the
presents a problem since chitin is not a chemical entity but a product defined by
. (
86
, l' nitrogen, molecular weight and viscosity, and water holding capacity of the
isolated chitins. The results were then compared to those of commercial crab
chitin and chitosan .
.. \
content lower than the theoretical value of 6.9% for pure chitin, which is an
(
indication of hydrolytic deamination or contamination in the product
(Rutherford and Austin, 1978b). Since ail the values obtained were lower, it is
established that, ail the various combinations accomplished total removal of
1,
proteins, though No et al. (1989) have stated that it is highly improbable that ·1
, l' t'l
chi tin samples can be prepared without sorne residual pwtein remaining since 1
prote in is bound by covalent bonds to chitin (Austin et al. 1981; Brine and
Austin, 1981; Hackman, 1960).
Water binding capacity of lobster chitin and chitosan obtained from the
optimization studies was compared to that of commercially available crab chi tin
( and chitosan (Sigma). Water binding capacity of lobster chitin and chitosan
ranged between 400 ~ 510% (w/w) with chitosan having the greater water
, II'
------,
87
binding capacity (Fig 21). Generally, it was found that, the water binding
capacity of lobster chitin and chitosan was relatively lower than that of crab
chitin and chitosa'1. Knorr (1983) stated that water uptake of chitin and chitosan
was between 400 - 550% (w /w) with chitosan having the greatest value. The
products as weIl as differences in the amount of salt forming group~ boum] to the
products and proportion of covalently bound proteins (Austin et al. 1l.>81). Since
~-IY
protein residues remain with chitin even after drastic alkali treatment,
water holding capacity of the products (Knorr, 1982). The high water binding
capacity of lobster chitin and chitosan may be exploited in food application for
use as humectants (Sanford, 1988). Knorr, (1982) observed that, the ~pecific Joaf
incorporating chi tin, because of its high water and fat binding capacitie!l.
4.6.3.1 chitin
the constant, Km. Km depends on the nature of the solve nt, the type of bond and
(Rutherford and Austin, 1978b), ~ince it both swells and dÎ!l~olve!l chitin without
500
400
300
200
100
89
obtained for viscosities and molecular weight of lohster and crah chi tin and
chitosan in the stated solvents. The viscosity of lobster chi tin was comp~uahle tn
those of commercial crab chi tin, with very !iule differences in hoth values. Thi~
observation suggests that, the degree of polymerization of lob~ler chitin wa~ abo
comparable to the crab chitin, indicating that, the method of extraction did not
hydrolyze the macromolecule any further than that of commercial crab chitin.
The conditions of extraction of lobster chitin wa~ therefore good. The intrinsic
zero concentration. The intrinsic viscosity of crab chitin was fl)und to be 22.22cp
and that for lobster chitin was 17.77cp, which sugge~ts a slightly lower degree of
Staudinger equation are 1.79 x106 and 1.31 x 106 dalton~ respectively. The!ote
results are close to those reported by Knorr (1984). Since the viscosity is directly
lobster chitin was 27% lower than that of'commercial crab chitin indicating that,
at Ieast, about 27% hydrolytic depolymerization of the lobster chitin had taken
place.
4.6.3.2 Chitosan
The soivent system, O.2M acetic acid + O.IM sodium acetate was
800
400
200
O~i--------~--------~--------~--------~------~
o ~ 1 ~ 2 ~
concan tration of ch itin (g/100ml)
\0
o
'\
~ ... ~ " '.Ji
'-
~
800
400
200 .... · ............... .-....... ... .. ... .. .. . ........ ... .. ....... . . .... ,..
O~I----------~--------~--------~----------~--------~
o 0.5 1 1.5 2 2.5
concentration of chitin (g/100ml)
....ID
, 'f
92
, "'f
LOBSTER CRAB
( chitin chitosan chilin chitosan
, of
(,.
93
volume due to charge repulsion and stretching out of the moiecule~. When
sufficient salt is added to neutralize the charge effeet the viseosity behavior i!oo
normal. Table 17 and Figs. 24 - 25 show the results ohtained for vi!ooco~Jtie~ of
commercial crab chitosan and lobster chitosan. The result!\ show that vi~coslty of
the lobster ehitosan was significantly lower than that of the crah chitosan. Thi!oo
lower than that of crab chitosan and therefore the method of preparation eamed
The intrinsic viscosities of both chitosan!l were ohtamed from Fig~. 24 and
chitosan was much lower than the crab chitosan suggesting a significant
deproteinization procedure used, since the molecular weight of the lohster ehitin
was close to that of crab chitin. An interesting fef.ture of this high vi~co~ity of
lobster chi tin is that, it can be envisaged to have a range of applicationll: a!oo
inexpensive thickening and suspending age 11 in foods. Battista (1975) and Austin
et al. (1981) reported that, such high vis~osity chitin suspension and gels have
unusual stability at prolonged and elevated temperature~ and this property i!oo
•
Fig. 24. Viscosity of lobster chitosan
against concentration
viscosity of chitosan (centipoises)
200,~--------------------------------------------------~
150
100
50
1 /i
o IL-
o
---1
- - concentration
\0
~
, "1
>;j
~
'" 'l'
200
./
150
100
50
- - concentration
10
\Jl
.~~, -'\,~t ...
, '"""" '"
140-···················· ........ ,
120 ············c··········· ..
100
80 .... ···························· ................................... .
40-···· ........................ .
OLI-------
97
CHAPTER FIVE
CONCLUSION
optimum levels of factors for chi tin extraction, and hence to estahli!\h a single
Methodology. This stati~tical tool was found to he capable of !\uhstituting for one
Results obtained from regression analysi!-J revealed that, the model for
deproteinization was v~ry good and hence result~ ohtained could he relied on
and used to a great extent in chitin research and production. On the other hand,
product obtained from the optimization studies which were well within the range
of those of crab chitin suggest that, lobster chitin could be u!-Jed in food
applications. It also confirme'! that, the choice of factor~ and leveb of factors in
the experimental design, which were based on literature value~ and practical
experience, were adequate and in providing optimum levels of factors for chitin
extraction.
magnitude of aH the faccors under study will lead ta a higher power of extraction,
as it is also indicated by the elevating behavior of the graphs over the respon!-Je
surface. At critical levels of 1.75% NaOH, 1:6 - 1:7(w/v), 55°C and 5 1/2 h,
, ,\,
99
APPENDICES
.......
100
• II'
D. Duncan's new multiple range test to locate ditTerences in treatment means for
lobster chitin (level of significance = 0.05)
Treatment * 1 2 3 4
Mean yield 20.99 22.23 22.52 23.19
a ** ab b b
E. Duncan's new multiple range test to locate difTerences in treatment means for
lobster chitin (level of significance = 0.01)
Treatment * 1 2 3 4
,c
102
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