Optimization of chitin and chitosan extraction from by product from white leg shrimp (penaeus vannamei) industry in vietnam to improve its quality and efficiency

The dissertation "Optimization of chitin and chitosan extraction from by-product from white leg shrimp Penaeus vannamei industry in Vietnam to improve its quality and efficiency" was con

INTRODUCTION

1 The rationale

Vietnam is one of the leading shrimp production in the world with two main species of black tiger

(Penaeus monodon)and white leg shrimps (Penaeus vannamei) The production of cultured shrimp reached more

than 480,000 tons in 2012, among them 130,000 tons came from white shrimp and it will be more in future Annual amount of by-products from shrimp production was estimated up to 200,000 tones which consists of

head, shell and broken meat Shrimp heads and shells is composed of protein, fat, chitin, protease and pigments,

astaxanthin Therefore, the efforts to convert those wastes into useful products, especially bioactive molecules, are rational and important because environment pollution will be prevented as well as more benefits was achieved

The by-products from shrimp industry was actually used for chitin and animal feed production in Vietnam and commercial chitin is mainly isolated from crustacean shells through chemical treatments Consequently, added value and sensitive by-products such as protein hydrolyzates and pigments were not recovered Moreover, the chemical procedures caused the side effects on chitin quality and serious chemical pollution Therefore, a great interest still exists for the innovation and optimization of the recovery bioactive compounds from shrimp wastes, especially from white shrimp - the new farmed species, that will facilitate Vietnam's chitin industry following up the sustainable development

Integration between chemical and biological methods in recovery of chitin and other bioactive compounds from crustaceous wastes were explored increasingly, however, in oder to put it into practice more information relevant to the kinetics and the extra assistance need to be cleared

At the present, the trend of technology innovation is paying more attention on applying physic factors

on chemical and biological process Of these factors, ultrasound was topics of universal interests Ultrasonic waves was proved that is one of green and efficient energy sources on several fields including textile, food and chemicals industries Studying of applying sonication on chitin and chitosan production is expected eagerly opening a new way for innovation of recovery bioactive compounds

The dissertation "Optimization of chitin and chitosan extraction from by-product from white leg shrimp (Penaeus vannamei) industry in Vietnam to improve its quality and efficiency" was conducted with

the aim of finding out the way to integrate enzymatic and chemical methods with physic method which support the innovation of chitin - chitosan production technology in Vietnam

2 The scope and objectives

In the dissertation, three main steps in the chitin and chitosan production, including deminerilization, proteinization and deacetylation, were optimized by using integrating technology in order to improve product quality, reduce consumption of chemicals, recovery protein and prevent environment pollution

The objectives include: (1) determine the components of white leg shrimp (mass, proximate, amino acid and minerals components); (2) optimize chitin recovery process from shrimp heads and shells; (3) study the kinetics of deproteinization under the catalysis of pepsin; (4) optimize and characterize the heterogeneous deacetylation under the facilitation of sonication; and (5) recommend the efficient processes to recovery chitin and protein simultaneously as well as chitosan through applying integrated technology

3 The objects of the study

The main objects were shrimp heads and shells collected after the manufacturing of white shrimp

(Penaeus vannamei), in the average size of 81-120 bodies/kg

4 The scientific and realistic significances and innovations

- The data of the composition of chemicals, amino acids, minerals and heavy metals of three main parts

of white leg shrimp (heads, shell and meat) as well as the effects of pH and temperature on the activity of the endogenous proteases from heads of white shrimp leg cultivated in Khanh Hoa province were collected

- Gaining the optimization condition for recovering chitin and protein hydrolysates from white leg shrimp shells with pepsin In addition, the information relevent to kinetics of the process and the linkage between protein, minerals and chitin in shrimp shell were cleared

- New data and information about the supporting capacities of ultrasound in chitin enzymatic extraction and heterogeneous deacetylation were collected

- The simple procedures to recovery chitin and protein efficiently through simultaneous combination of autolysis with the endogenous protease and physical force were put forward

- The benefits of the integration technology between physics (mechanical force and ultrasound), enzyme (endogenous and commercial proteases) and chemicals (NaOH, HCl) in chitin and chitosan production were demonstrated The processes of chitin and chitosan production were controlled by the mathematic equations

The proximate components of different shrimp species is not the same, however, protein always is the majority (33-49.8% dry basis), follwing by minerals and chitin (respectively 21,6-38% and 13,5-20% , db) Therefore, shrimp by-products were the value source to recovery of both chitin and protein

There are a significant amount of endogenous enzymes in shrimp head, especially proteases These proteases include both endoprotease and exoprotease and their activities were equal to commercial proteases

however they were easy to be lost due to denaturation and drift out In case of white leg shrimp (Penaeus vannamei), the favourate condition of proteases was temperature of approximately 60oC and pH of 7,5-8, which was the same pH of fresh shrimp heads Therefore, utilization of endogenous proteases in shrimp heads for recovery of chitin and protein hydrolysate will be more economic than using commercial enzymes

1.2 Pepsin and its application on recovery of protein and chitin

Pepsin was an endopeptidase and belong to aspartate protease Pepsin is most efficient in cleaving peptide bonds between hydrophobic and preferably aromatic amino acids such

as phenylalanine, tryptophan, and tyrosine Pepsin is a monomeric, two domain, mainly β-protein, with a high

percentage of acidic residues (43 out of 327) leading to a very low pI The catalytic site is formed by two

aspartate residues, Asp32 and Asp215, one of which has to be protonated, and the other deprotonated, for the protein to be active This occurs in the 1–5 pH interval, dependent on substrates In the 5–7 pH interval the conformation of pepsin is poorly characterised Above pH 7, pepsin is in a denatured conformation that retains some secondary structure This denaturation is not fully reversible The bioactive capacity of hydrolysates from pepsin were more than that from other proteases such as Alcalase, α-chymotrypsin, or trypsin Based on the mentioned characteristics pepsin has a potential of application in chitin production to combine deprotenization with deproteinization which will support for time - saving and recovery bioactive compounds

Commercial pepsin is extracted from the glandular layer of hog stomachs through conventional method therefore the price is rather high in comparison with other commercial proteases which were recovered from mass of microorganisms With the latest success in seeking new sources of pepsin (from fish viscera or

microorganisms such as Botrytis cinerea or Aspergillus niger) and innovation in purifying enzymes based on

Aqueous two-phase system it is expected that the price of pepsin will become reasonable in near future

1.3 Ultrasound and its potential application

Ultrasound is an oscillating sound pressure wave with a frequency greater than the upper limit of the human hearing range (>20kHz) In fields of food and biotechnology, ultrasound with low frequency - high power (20-100kHz) were applied widely, especially for extraction and adjusting physical and chemical characteristics of materials as well as the activity of enzymes Generally, the mechanism of ultrasonic is based

on the high energy waves that create cavitations in the liquid solution Dependent on the feature of the system sonicated (characteristics of liquid, presence of air and solid debris) as well as sonication condition (manipulation of wave duty cycle, time of exposure and acoustic power of ultrasonic system) the mechanism can

be changed

Replying on multifunctional mechanism, sonication is able to create a change in spatial structure of objectives (materials or enzymes) or/and increase the contact between them This effect leads to facilitate reaction rate and time-saving significantly

Ultrasonication offers great potential in the processing of liquids - solid system, by improving the mixing and chemical reactions in various applications and industries Application of ultrasound is enable to cut down the severity of reaction condition (temperature, time, chemicals), improve quality along with cost-saving

1.4 Shortcomings in chitin-chitosan production in Vietnam

Heads and shells after manufacturing black tiger and white leg shrimps were the materials for chitin production In shrimp processing enterprises, due to the sensitive characteristics to deterioration shrimp heads were always separated from the whole after receiving (exclusive HOSO product) Whereas, shrimp shells were separated later which was dependent on types of products At the end, they were mixed together and kept for long time (interval of 4 to 8 hours) at ambient temperature in waste house The by-products were often deteriorated seriously before transferring to the place where fishmeal and chitin were produced This way of treatment caused the recovery of useful compounds to be lost the efficiency and to pollute environment concurrently

The industry of chitin and chitosan production in Vietnam has not been developed and still employed backward technology The majority of chitin processing factories were in Mekong delta and the South The annual average output of a factory is approximately 2,000 tons Chemical extraction were used prevalently while

the procedures which were combined between chemical and biological methods have been applied in a quite limitative level HCl and NaOH were the main reagent used to liquidate minerals and protein, respectively The minerals was removed in the condition of HCl 4-6% at ambient temperature for one day The solution of NaOH 4-5% was used to exclude protein at room temperature or at higher one Infact, energy was only used in case of having a demand of high quality chitin The protein liquids were collected, conveyed to containing towers, concentrated into thick liquid which were not in good quality and used for animal feeds, after that

The primary product was chitin but its quality was still poor and not stable; the residuals of protein and minerals remained high, over 1%, in addition it was easy to be changed into bad color and cost price was so high therefore its ability of application and marketing was low Fewer and fewer factories which produce chitin could

be alive A large number of them must be closed due to violating the regulation of environment The predominant reasons of polluting came from off-odor, protein drain and chemical wastes The urgent requirements involve in finding out solutions which enable to solve thoroughly the pollution and improve the quality

In brief, by-products from shrimp processing industry was only utilized to recovery chitin Up to now no much attention was pay on recovery protein with its biological value The products has not been competitive and limitative in application Besides, the studies conducted have only focused on establishment parameters of chitin extracting procedures, the process kinetics as well as the interaction between process factors have not been investigated

CHAPTER II: MATERIALS AND METHODS 2.1 Materials

White leg shrimp (Penaeus vannamei), cultivated in Khanh Hoa province, were used in two forms: (1)

whole shrimp to determine the mass components (size of 60-160 bodies/kg), the proximate component, the composition of amino acid amine and minerals (size of 81-120 bodies/kg); and (2) shrimp by-products (size of 81-120 bodies/kg, head and shell separately) to recovery chitin and protein Materials were used in fresh condition after collecting from NhaTrang Seafoods Company (F17), Nha Trang, Khanh Hoa

2.2 Methodology

The figures and data were collected through experimental methods which were combined between variable-at-a-time technique and response surface methodology; Data analysis conducted by using specialized soft wares

one-The research objects were characterized on the component of mass, the proximate component, and the composition of acid amine and minerals as well as the changes during storing time when they were kept in the conditions imitating the real parameters at the shrimp factory

Finding out the procedures recovering chitin were conducted on heads and shell separately The aim was

to be estimate the capacity of integrating enzymatic and chemical methods with physical methods Demineralization were carried out with HCl in the way how to reduce the side effects of the acidity on the polysaccharide of chitin Removing protein were implemented by biological methods: using endogenous proteases for heads and commercial pepsin for shells The outcomes were the optimization procedures for applying autolysis and pepsin process to exclude protein in solid parts and recovery bioactive protein hydrolysates Chitin were converted into chitosan through heterogeneous deacetylation in the presence of

ultrasound Sonication were used to facilitate deacetylation process at two point: previous and during the process In addition, the kinetic information relevant to protein hydrolyzing with pepsin and deacetylation were collected

Based on the data collected from my own experiments and from liturature review, procedures for chitin and chitosan production were proposed The products were characterized through deterniming the criteria involving to purity, molecular weight, degree of acetyl/deacetyl, spectrum of IR, X-ray and NMR as well as some important physicochemical fuctions; hydrolysates were analyzed its antioxidant capacity through DPPH and total reducing power tests

The quality of chitin and chitosan produced were evaluated and compared with the chitin and chitosan standards which have been promulgated by two companies, AxioGen (India) and Ensymm (Germany) The differences of the amount of consumption chemicals between that of the proposed procedures and that of the reference procedure were used to estimated the efficiency, specially focussing on environment aspect

2.5 Chemicals and equipments

Pepsin was 107185 0100 from Merck (Germany) Chemicals and reagents were purchaed from Merck or LoBa company (India)

Ultrasound was creared by ultrasound bath (Model S15-S900H, Elma Co., Germay) and has the frequency of 37kHz and RMS of 35W

CHAPTER III: RESULTS AND DISCUSSION 3.1 Characteristics of the white leg shrimp by - product

The mass average ratio of head and shell of shrimp in range of 81-120 bodies/kg was 27.5±3.93 and 11.21± 2.63 (%), respectively, thus the estimative amount of by-products was 38.70±6.46 percentage of the total number of raw materials processed

The main constituents of shrimp head and shell were ash, protein, and chitin Although there is no significant difference in ash content between the head and shell of shrimp (size of 81-120 bodies/kg): 25.6 % to

32 % dry weight, respectively, the chitin and protein contents of head and shell are largely different The chitin content of shell and head of white shrimp were 27.37 and 11.40%, respectively The chitin content in the shell was three times higher that than in the head but the heads have up to 50% higher in protein content than the shell

The amount of amino acid in heads and shells was approximate 50 and 30 percentage of that in shrimp meat, respectively In general, there were slight differences in amino acid composition among three parts of shrimp and most of essential amino acids were present Glycine/Arginine, Glutamic/Glutamine,

Aspartic/Asparagine, and Alanine predominated of the amino acid profile However, the amount of Tyr, Phe, Leu and Val of head and shell part were higher than that of meat one The contents of K and Cu in the shell and head were nearly the same whereas the contents of Na, Ca and Fe were significantly different A small amount

of heavy metal amount (As, Cd, and Pb) were detected in head and under the restricted levels to food In the shell, only Pb was found and the level was equal to that in the head The contents of Se and Hg were under the limit of detection

Therefore, both protein and chitin should be recovered from by-product from the production of white leg shrimps through reasonable procedures to keep their biological functions and to improve their quality as well as

the process efficiency

3.2 Recovey of chitin and bioactive hydrolysates from white leg shrimp heads

3.2.1 Effects of storage time

The quality of shrimp heads declined seriously when the time of keeping them at room temperature

(27-30oC), was increased The TVB-N value of shrimp head increased continously and nearly exceeded the level of restriction to food after 4 hours (28.7 mg/100g in compared with the limited level of 30 mg/100) In consequences, the loss of protein and total weight were rather significant (5.08±1.26% and 15.59±0.44% after 4 hours, respectively) Therefore, shrimp heads should be handled as soon as posible, not more than 4 hours after removing out of the body so that the quality and pollution were controlled

50 Total weight

Protein TVB-N

a a

b

bcd cd d bc

B C

D DE

E F

3.2.2 Studying procedure to recovery proteinand chitin from heads of white leg shrimp

Data corresponding to the zero-hour samples in Figure 3.2 and Figure 3.3 shown that the combination of using physical force for 2 mimutes to stir strongly shrimp heads and filtering the mixture through net having the pore size of 1mm was the efficient manner which helped to divide shrimp heads into two parts: the solid was carapaces and the liquid wad protein The liquid part contained more than 70 percentage of the total protein amount of heads wheeras the mass of the solid part was about 7,45± 1,89 percentage of the whole weight of heads and its protein content was only 20% (db)

However, simultaneous combination of autolysis and physical force enabled not only to improve the efficiency of nitrogen recovery in the liquid part but also to reduce the protein content of the solid one to significantly lower level than that in case of using physical force individually Increasing treatment time, the

efficiency of nitrogen recovery, the ratio of antioxidant products, and the degree of deproteinization from shrimp heads became better and better at any level of supplied water In spite of that, at the ratio of water to shrimp heads was 1:1 (v/w) the efficiency of protein recovery, including both nitrogen recovery and antioxidant products, was in the better tendency, its value was always the highest one corresponding with all of the water ratios used as well as the protein residue on the carapaces was lowest Protein hydrolysate collected after two hour treatment at this ratio had the best capacity of scavenging DPPH radical (Figure 3.5) When the autolysising time was more than two hours the efficiency of protein recovery and degree of deproteinization at the ratio of 1:1 did not increase significantly on the contrary the antioxidant capacity was in the decreasing trend

Nitrogen recovery at the ratio of 1:0

Nitrogen recovey at the ratio of 1:1

Nitrogen recovery at the raio of 1:2

Antioxidant recovery at the ratio of 1:0 Antioxidant recovery at the ratio of 1:1

a

a

a

a bc ab

cde cd bcd

e e cde e e

Figure 3.2: Effects of treatment time and water ratios

used on the efficiency of recovery of nitrogen and

antioxidant products when autolysising shrimp head

at temperature of 60 o C and native pH

Protein content at the ratio of 1:0 Protein content at the ratio of 1:1 Protein content at the ratio of 1:2

bcd

ef cde def

g

ef ef

g

f

Figure 3.3: Effects of treatment time and water ratios used on protein residues and degree of deproteinization when autolysising shrimp head

at temperature of 60 o C and native pH

Different letters indicate significant differences (p < 0.05)

bcdef

f bc

bcd A

de bcde abc

e cde

The ratio at 1:0 The ratio of 1:1 The ratio of 1:2 B

Figure 3.5: Effects of treatment time and water ratios used on the capacity of scavenging DPPH radials (A) and total reducing power (B) of the hydrolysate Different letters indicate significant differences (p < 0.05).

The protein and minerals content of the carapaces which were collected after autolysising at the optimal condition (Temperature of 60oC, the ratio of water to shrimp heads was 1:1, native pH, 2 hours) and separated by physical force were 13,78± 0,75%, and 34,23±0,2% % (db), respectively These carapaces were handled more deeply to recover chitin According to the literature, the carapaces were proposed to handle under the condition combining deminerilization of HCl 0,25M at room temperature during 12h with deproteinization of NaOH 1% at

70oC for 8h The content of protein and minerals in chitin extracted by the proposed procedure were under 1% (0,59 ± 0,17% and 0,45±0,12%, respectively)

In a word, the combination of autolysising at proper condition (Temperature of 60 o C, the ratio of water

to shrimp heads was 1:1, native pH, 2 hours) and using physical force to stir and filter allowed to recover protein hydrolysate having antioxidant activity along with chitin efficiently from fresh shrimp heads: the recovery efficiency of nitrogen and antioxidant products were approximately 86,19±1,67% and 4,09±0,12%, respectively, moreover, nearly 90 percentage of total shrimp heads could be prevented to treat with chemicals However, it is need to carry out more deep studies on bioactive capacity of shrimp protein hydrolysate collected

to seek solutions that enable to commercialize them

3.3 Recovery of chitin and bioactive hydrolysates from wwhite leg shrimp shells

3.3.1 Treatment with HCl

The curve displaying relationship between the contents of mineral and protein with time during the process of HCl 0,25M (Figure 3.6) shown that deminerilazation mostly happened in the period of first two hours, there were 96 percentage of minerals eliminated, after that only a small amount of them were excluded and the rate of deminerilazation left off at the tenth hour; the remaining contents of protein and minerals were 32,26 and 2,61 percentages (db), respectively When 96 percentage of minerals were removed out of the shells pH of the mixture also reached the stable value (around pH value of 1,77±0,06) Therefore, shells were demineralized in the condition of 0.25M HCl (1:4, v/w), at room temperature, for 2h

Content of Minerals Degree of demineralization

Figure 3.6: The curve displaying relationship between the contents of mineral and protein with time

during the process of HCl 0,25M at room temperature (27-30 o C) 3.3.2 Estimating the posibility of pepsin

Results in Figure 3.7 shown that the catalysis activity of pepsin facilitated remarkably demineralization and deproteinization, the increasing level were dependent on the concentration of pepsin used At the pepsin concentration of 5U/g protein, extra 40% of protein and 20% of minerals were emilinated in compared to the controll sample and when the pepsin concentration was 25U/g protein degree of deproteinization and demineralization reached the maximum with the value of 85,93±0,25% and 90,34±0,9%, respectively If the action of HCl were included, the total degrees were 91,16±0,65%; and 99,79± 0,02%, respectively Although the difference of the total degree of demineraiazation in two cases of with and without pepsin were not considerable the disproportion had an significant meaning due to the minerals were removed strictly which made the minaeral residue were under 1% and the product met the quality criteria of high-value chitin

Pepsin concentration (U/g protein)

DP of Pepsin

DM of Pepsin Total DP Total DM

Figure 3.7: Effects of pepsin concentration on degree of deproteinization and demineralization

The SEM images of shrimp shell in Figure 3.8 shown that the shell morphology were changed after treated with HCl, alot of pores appeared in the shell which might support for pepsin penetrating into deeper layers

of 30-40oC), E/S ratio (X2, in the range of 5-25U/g protein) and incubation time (X3, in range of 6 - 18h) The fitted model, expressed in coded variables, is represented by the equation: = 65.33 + 21X1 +9.875X2 + 11.375X3 + 5.75X1X2 + 3,75X1X3 – 9.417X12 – 8.167X22 – 11.667X32 (Equation 3-2)

Because the second degree coefficients in Equation (3-2) were all negative, the surface response is elliptic parabolic with a maxium point The regression sum squares (R-square) and the adjusted coefficient (R square-adjusted) were at the level more than 99.9% and the value of lack of fit was 0.47 as well as the results in Table 3.9 clearly indicated that the predicted model well fitted the experimental data The Pred-RSquared, of 0.958 meant that the data estimated by Equation (3-2) had the accuracy of 95.8% in compared to experimental data Results in Table 3.9 were in agreement This once again confirmed the reliability of Equation (3-2) and it was able to be used for controlling the process of handling shrimp shell by pepsin in reality

The optimal conditions were temperature at 40oC, reaction time of 16h, E/S ratio of 20U/g protein at pH=2 After treatment, approximately 92% of protein in shrimp shells were removed and the residues of protein and minerals were 8,2±1,6% and 0,56±0,04%, respectively

Table 3.8: The Box-Behnken design of the experiments and response of deproteinization

3.3.4 The posibility of using sonication to facilitate pepsin activity in chitin extraction

Figure 3.12 shown that sonicating time (at 37kHz, RMS=35W) had a significant impact on the activity

of pepsin, in the interval of first 25 minutes, the catalysis of pepsin were directly proportion to time, the acivity was increased by 8% after 20-25 minutes of treatment, however, extending time caused opposite effect, pepsin activity trend to go down (p<0,05), after 40 minutes of unbroken sonication pepsin activity was no more different with that in case of no sonication and if time prolonged more catalysis of pepsin were lower than that of the control (p<0,05)

Time of treatment (min)

Figure 3.12: Effects of sonication time on pepsin

46

With sonication Without sonication

ab a abc abcde cde bcde

ef

def

abc abc abc abcd abcd

Figure 3.17: Effects of sonicating pepsin on deproteinization from shrimp shell (20U/g protein,

40 o C, pH=2)

Different letters indicate significant differences (p < 0.05)

Figure 3.17 shown that degree of deproteinization that were achieved after 14h of treatment with sonicated pepsin and that gained after 16h of treatment with non-sonication pepsin was no significant and prolonging processing time with sonicated pepsin more than 25 min did not bring any better results (Figure

25min-3.17, the small) Therefore, sonicating pepsin for 25 min before deproteinization from shrimp shell enabled to reduce processing time to 2 hours

3.3.5 Improving the proposed procedure to recover chitin and protein from white leg shrimp shells

The second step to deproteinize was ininitated by NaOH 1% (with the ratio of materials to solution = 2:1, v/w), for 8 hours at 70oC Chitin produced was refined upon a high level of purity (both ash and protein contents were under <1%, respectively, 0,56±0,04% and 0,79±0,02%); structure of polysacchride chains was almost not attacted (DA= 97,01±0,85% and viscosity average molecular weight of chitin Mv= 1652Da); the

product was suitable for processing further chitin derivaties which had bioactivity and very useful, such as

N-acetyl glucosamine Protein hydrolysate was valuable to produce bioactive substrates having antioxidant capacity with the yield of 3,52±1,54% (Table 3.10)

Table 3.10: Data relevant to protein recovery from shrimp shell by pepsin

Chỉ tiêu Nitrogen

concentration of 1mg/ml

In comparison to BHA (1mg/ml) (%)

DPPH (mM)

TNLK (OD 700nm )

a

3.4 Kinetics of deproteinization by pepsin

The logarithmic variation of the percentage of protein remaining in chitin which was plotted as a function of the deproteinization time at 40oC by pepsin in Figure 3.22 revealed that the deproteinization from shrimp shells appears to obey first-order reaction kinetics The deproteinization mechanism could be described

by a first-order equation dP/dt = -kP in each of three domains, where P represents the protein contain remaining

in the shrimp shell, t the treatment time, and k the reaction rate constant

concentration is 15g/L equally 250g of demineralized shrimp shells L -1

The results in Table 3.11 shown that the rate constants kept the same value within every phase and decreased abruptly when change to next phase with k1= 0.72x10-2, k2=3.05x10-3, k3=6.5x10-4 and the rate constant of the second phase was only a half of that one of the first phase, whereas at the third phase the rate constant reduced nearly ten times The abrupt change of rate constant shown that the binding between protein and chitin could be structured with different layers

The behavior of the kinetic model in our study shown the same trend as the models which were established for reaction with NaOH However, in our work the curve was broken into three fragments with the difference in rate constants The study of Percot (2003) for NaOH deproteinization shown that in the first stage the values of rate constants were higher than those of pepsin; In the second stage, the latter was a half of the former at 50oC or both were nearly the same at 70oC but in the third the rate constant of pepsin was sinificantly higher (Table 3.11)

Table 3.11: Comparison of rate constants between deproteinization by pepsin and NaOH treatment

Deproteinization by pepsin mostly took place in the two first hours of the process; The results relation to kinetics and regression analysis allowed to draw that degree of deproteinization (DP) and its rate (r) in this

period obeyed Equation (3-9) and Equation (3-10), respectively and the value of the rate constants were k 2 = 40,983 (min-1) and k d (=k 3*Km) = 1,535 (min-1)

3.5 Enhancement of heterogenous deacetylation

3.5.1 Effects of chitin pretreatment

Figure 3.27 shown that pretreatment of chitin before handling with NaOH considerably facilitated deacetylation Degree of deacetylation of the samples which were pretreated by soaking with hot water or by sonication were 20% higher than that of the control when all of them were deacetylated by conventional procedure (NaOH 60% (w/w), 3h) There were not significantly differenct between the efficiency of two manners of pretreatment (p>0,05)

100

With sonication With hot water Control

a a

b

Figure 3.27: Effects of chitin pretreatment on deacetylation

Different letters indicate significant differences (p < 0.05)

SEM images in Figure 3.28 shown that the surface of chitin sonicated rougher and had more wrinkles than that of chitin treated with hot water as well as the results were drawn from XRD spetra by Origin Pro 8.0