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

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1 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. 2 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. 5. The structure The main content of the dissertation was divided into three chapters (142 pages) accompanied with the conclusions (3 pages), the references (19 pages) and the appendix (56 pages). CHAPTER: LITERATURE REVIEW 1.1. The components and value of by-products from manufacturing shrimps By-products from shrimp production consists of head, shell and a minor amount of broken meat. Although the ratio between them was dependent on species, ages, seasons and methods of processing the total of them was in range of 40-60% of the whole of raw materials. 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 3 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 4 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 onevariable-at-a-time technique and response surface methodology; Data analysis conducted by using specialized soft wares. 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 5 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. 2.3. Analytical methods Data were collected through standardized and modern methods, including HPLC, X-ray, FT-IR, 1 H NMR, and SEM. 2.4. Statistics analysis Experiments were run in triplicate using three different lots of sample. The statistically differences between means (p<0.05) were tested using analysis of variance (ANOVA) with the Pairwise Multiple Comparison Procedures (Tukey Test). SigmaPlot, Origin Pro 8.0, Design Expert 8.0.7, and MINTAB 16.1 were used to design experiments and analyze data. 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, 6 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 (27o 30 C), 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. 25 50 Total weight Protein TVB-N Loss of weight (%) 20 E DE 40 E D 15 30 10 cd C 5 B A bc b 20 d bcd 10 a Content of TVB-N (mg/100g) F a a 0 0 0 2 4 6 8 Time of delay (h) Figure 3.1: Effects of storing time at room temperature (27-30oC) on the weight and protein losses of heads of white leg shrimp and the changes of TVB-N value. Different letters indicate significant differences (p < 0.05). 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 7 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. 4.3 a a a e e cde bcd e 4.2 4.1 ab a 4.0 70 3.9 60 3.8 50 3.7 40 3.6 30 3.5 0 1 2 3 a a 92 a 90 b 20 Protein content (%) cde cd bc 80 e Yield of antioxidant recovery (%) Yield of nitrogen recovery (%) 22 90 b 88 86 18 bc bcd 84 cde 16 def ef 82 ef ef 14 80 f g 12 78 76 g 10 74 8 4 Degree of deproteinization (%) 100 72 0 1 2 3 4 Time (h) Reaction Time (h) Nitrogen recovery at the ratio of 1:0 Nitrogen recovey at the ratio of 1:1 Nitrogen recovery at the raio of 1:2 Protein content at the ratio of 1:0 Protein content at the ratio of 1:1 Protein content at the ratio of 1:2 Antioxidant recovery at the ratio of 1:0 Antioxidant recovery at the ratio of 1:1 Antioxidant recovery at the ratio of 1:2 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 60oC and native pH DP at the ratio of 1:0 DP at the ratio of 1:1 DP at the ratio of 1:2 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 60oC and native pH Different letters indicate significant differences (p < 0.05). 0.18 B 1.2 A a ab 0.16 a abc a b b cdefbcdef 0.8 bcd bc bcde 0.14 bcdef bcde bcd bcdef f def ef 0.6 OD at 700nm DPPH (M/g materials) 1.0 abc abcd cde 0.12 abc abcde abcde bcde cde 0.10 cde de e 0.08 0.4 0.06 0.2 0.04 0.0 0 1 2 3 4 0 1 2 3 4 Reaction time (h) Reaction time (h) The ratio at 1:0 The ratio of 1:1 The ratio of 1:2 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). 8 In a word, the combination of autolysising at proper condition (Temperature of 60oC, 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. 30 Content of minerals (%) 90 20 80 Content of Minerals Degree of demineralization 15 10 70 60 5 50 0 Degree of demineralization (%) 100 25 40 0 2 4 6 8 10 12 14 16 18 20 22 24 Reaction time (h) 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 (27-30oC) 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. Degree of Deproteinization/Demineralization (%) 9 120 100 80 60 DP of Pepsin DM of Pepsin Total DP Total DM 40 20 0 0 5 10 15 20 25 30 35 Pepsin concentration (U/g protein) 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. A B Figure 3.8: SEM (20kV) images of shrimp shell before (A) and after (B) treated with HCl 0,25M for 2h 3.3.3. Optimization of pepsin process After analysising experimental data (Table 3.8) through the function of respone surface methodology (RSM) in software Design Expert 8.0.7 an quadratic model was expoited. Equation (3-2) expressed the relationship between degree of deproteinization with independent variables including temperature (X1, in range 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 – 9.417X1 – 8.167X2 – 11.667X3 (Equation 3-2). 2 2 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 10 Table 3.8: The Box-Behnken design of the experiments and response of deproteinization X1, o C -1 1 -1 1 -1 1 -1 1 No 1 2 3 4 5 6 7 8 X2, U/g.protein X3, h Y, (%)* No X1, oC X2, U/g.protein X3, h Y, (%)* -1 -1 1 1 0 0 0 0 0 0 0 0 -1 -1 1 1 41,89 65,30 45,64 86,95 34,99 61,91 46,77 85,55 9 10 11 12 13 14 15 0 0 0 0 0 0 0 -1 1 -1 1 0 0 0 -1 -1 1 1 0 0 0 41,08 58,57 57,11 76,09 73,15 73,17 73,76 * Mean ± SD (n=3) Table 3.9: Observed and predicted values of the confirmation experiments Trials Condition DP (%)* 1 2 3 4 5 X1=40oC ; X2= 10U/g.pro; X3=15h X1=40oC ; X2= 12,5U/g.pro; X3=14h X1=40oC ; X2= 15U/g.pro; X3=15h X1=40oC ; X2= 15U/g.pro; X3=16h X1=40oC ; X2= 20U/g.pro; X3=16h Observed Predicted 82,41 ± 0,97 81,25 86,58 ± 0,51 85,39 89,87 ± 0,19 89,52 90,22 ± 0,14 89,74 93,29 ± 0,16 92,48 * Mean ± SD (n=3) 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). 46 46 With sonication Without sonication ef 44 44 Enzyme activity U/mg) Enzyme activity (U/mg) With sonication Without sonication 42 40 def abcde cde bcde abc 42 ab a abc abc abcd abcd abc 40 38 38 36 36 0 20 40 60 80 100 Time of treatment (min) Figure 3.12: Effects of sonication time on pepsin activity (37kHz, 35W) 0 5 10 15 20 25 30 Time of treatment (min) Figure 3.17: Effects of sonicating pepsin on deproteinization from shrimp shell (20U/g protein, 40oC, 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 25minsonicated 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 11 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 Nacetyl 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 Nitrogen recovery a (%) Ratio of antioxidant recoveryb (%) 64,2±2,7 3,52±1,54 a Chỉ tiêu Antioxidant capacity For solution having In comparison to concentration of 1mg/ml BHA (1mg/ml) (%) DPPH TNLK DPPH TNLK (mM) (OD700nm ) 0,13±0,01 0,1640±0,015 46,79±4,19 46,02±1,67 In comparison to total of nitrogen in raw materials; b In comparison to the weight of raw materials. 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. 0.0 k1 Ln (P/Po) -0.5 -1.0 k2 -1.5 -2.0 k3 -2.5 -3.0 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (h) Figure 3.22: Logarithmic variation of the protein content in chitin as a function of the deproteinization time performed at the optimal condition (Pepsin concentration of 0.42g/L, pH2, 40oC). The initial protein 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. 12 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 Rate constants Pepsin treatment NaOH treatment (1M, 15mL/g demineralized shrimp shells)** ([E] =0.42 g/L, E/S = 1.68/1000 g/g demineralized shrimp shells)* 40oC k1 (10-2min -1) 50oC 70oC 0.72 ± 0.092 (r = 0.98) 2.10 ± 0.04 2.68 ± 0.05 -3 -1 3.05 ± 0.57 (r = 0.98) 3.12 ± 0.16 1.52 ± 0.08 -4 -1 6.5 ± 0.01 (r = 0.83) 0.48 ± 0.1 1.53 ± 0.27 k2 (10 min ) k3 (10 min ) * Mean ± SD (n=3); ** According to Percot, 2003. 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 k2 = 40,983 (min-1) and kd (=k3*Km) = 1,535 (min-1). (Equation 3-9) (Equation 3-10) 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 90 With sonication With hot water Control a DD (%) a 80 b 70 60 50 Samples 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 13 software (Table 3.13) indicated that in compared with the control Crystalline Index (χcr) of chitin which were pretreated with ultrasound and hot water reduced by 1,38 and 2,54%, respectively. It was supposed that pretreatment of chitin enable to decrease crystal area therefore the efficience of deacetylation were improved. Thus, proposed pretreatment was that soaking chitin in hot water of 60oC for 60 min. A B Figure 3.28: SEM (10kV) images of chitin treated for 60 min at 60oC with hotwater (A) and ultrasound(B). Table 3.13: Crystalline Index of chitin after treatment Sample Control Hot water Ultrasound CrI020 (%) 90,09 88,68 90,13 χcr (%) 74,12 71,58 72,74 CrI110 (%) 95,85 95,05 95,97 3.5.2. Screening the supporting posibility for deacetylation of sonication Figure 3.30 shown that when chitin was deactylaed with NaOH solution having concentration in range of 35-65%, w/w, ultrasound had impact on both solubility and degree of deacetylation but they were in different extent. The influence of sonication on degree of deacetylation was significant when NaOH concentration was NaOH ≤45%, DD of the sonicated samples were considerably higher than those of the controls (p<0,05), the value of DD were in direct proportion to NaOH concentrations. However, when NaOH concentration were NaOH ≥50% the effects of sonication and NaOH concentration were not significant, DD of the sonicated samplea and controls were not significant different (p>0,05). In the same deacetylation condition of NaOH concentration and reaction time,the solubility of the soncated samples were always higher than those of the controls. However, the influence decreased when the NaOH concentration were increased and when the concentration reached to 65% there was no remarkable differnce (p>0,05). In comparison with DD, sonication had more impact on solubility, the impact was continued until NaOH concentration was up to 60%. 90 A efg e efg ef fg efg g 100 efg f hi g ij h 50 55 60 ij B c c 70 80 Solubility (%) Degree of deacetylation (%) d 80 60 50 a a e c c 60 40 40 b a b 30 20 20 35 40 45 50 55 60 65 NaOH concentration (%, w/w) 35 40 45 65 NaOH concentration (%, w/w) Without sonication With sonication Figure 3.30: The relationship between DD and solubility with NaOH concetration and deacetylating means as function of time during 6h at 80oC. Different letters indicate significant differences (p < 0.05). 14 The properties of chitosan were produced in the present of sonication and in condition without sonication (80oC, 4h, NaOH=60%, w/w) were characterised by XDR and FT-IR spectra. XRD spectra in Figure 3.31 indicated that there was a small change in case of the sonicated sample at the position of 020, from 2θ=9,54o to 2θ=10,08o which was accompanied with a slight reduction of Crystalline Index, after 4 hours of deacetylation degree of crystallinity (χcr) of the sonicated and control samples were 71,38% and 72,81%, Lin (counts) respectively. A 160 140 120 100 80 60 40 20 0 -20 10.08273 20.1758 38.59359 Lin (counts) 0 10 160 140 120 100 80 60 40 20 0 -20 20 30 40 50 2THETA 9.54498 B 20.21717 0 10 20 30 40 50 2THETA 55 50 3800 3500 3200 2900 2600 2300 2000 1800 1600 Wavenumber cm-1 1400 1200 1000 900 800 669.51 630.70 612.42 583.04 543.54 531.02 798.13 773.65 896.01 1030.95 1082.15 1154.35 1262.14 1321.87 1421.75 1380.14 1657.23 2922.26 3448.70 40 45 Transmittance [%] 60 65 Figure 3.31: XRD spectra of deacetylated products in conditions of [NaOH]=60% at 80oC for 4h with (A) and without sonication (B) 700 600 500 Figure 3.32: FT-IR spectra of deacetylated products in condition of [NaOH]=60% at 80oC for 4h with (red line) and without sonication (black lie) FT-IR spetra of chitosan samples treated in condition with and without sonication in Figure 3.32 shown that these of two chitosans had similar peaks as those which were characterised in the spectum of chitosan by Rinaudo (2006). It means that ultrasound at the frequency of 37kHz (35W) did not have any signifficant impact on chemical bonds in chitosan moleculars. However, in the FT-IR of chitosan deacetylated with sonocation the absorption bands at position of 1560 and 1312 cm-1 (ascribed to amide II and amide III) were lower and the peak at 1415cm-1 was sharper, all of those proved that sonication facilitated deacetylation and there was an increasing DD in the sample sonicated. 3.5.3. Kinetics of deacetylation process with sonication The changes of DD values of chitosan deacetylated with and without sonication (37kHz, RMS 35W) in the condition of 80oC, 4h, NaOH=35-60%, w/w, were in the same pattern: DD rapid increased as a function of time until it reached the maximum and leveled off; the happening moment was indirect proportion to NaOH concentration: the higher NaOH concentration the faster, except NaOH concentration of 35%, w/w. In the same 15 condition of NaOH concentration and time, the samples treated in the presence of sonication always leveled off sooner and reached the higher maximum value of DD in compared with those of the control samples (Figure 100 100 40 80 80 30 60 60 20 10 DD (%) 50 DD (%) DD (%) 3.33). 40 20 A 0 40 20 D 0 0 60 120 180 240 300 360 C 0 0 60 Reaction time (min) 120 180 240 300 360 0 Reaction time (min) 80 60 120 180 240 300 360 Reaction time (min) 100 80 DD (%) DD (%) 60 40 60 40 Without sonication With sonication 20 20 B 0 E 0 0 60 120 180 240 300 360 0 60 Reaction time (min) 120 180 240 300 360 Reaction time (min) Figure 3.33: The DD of the resulting chitosans during heterogenous alkaline deacetylation reactions in condition with and without of sonication at 80°C and different NaOH concentrations (A) 35%, (B) 40%, (C) 45%, (D) 50% và (E) 60% (w/w). * Mean ± SD (n=3) The rate values of deacetylation reaction in different conditions (Table 3.16) shown that deacetylation process reduced the rate as function of time and it nearly happened in the first hour, especially in the first fifteen minutes. In the first period (0-15 min), the rate of deacetylation always was in bigger values when NaOH concentration was increased as well as when sonication was applied whereas in the latter periods (2, 3 and 4) the rates were in reverse tendency it went down when deacetylation were carried out in the condition of higher NaOH concentration and sonication, except the case of NaOH 35%. After 4 hour of deacetylation, the rates of the reactions were too small, reaching nearly zero even if sonication was used. Table 3.16: The rate of deacetylation reaction (%/min) (x102)a Deacetylation condition NaOH con centration (%) (w/w) Periodb Without soncation 35 With sonication 40 45 50 60 35 40 45 50 60 1 18,58 44,53 173,58 271,19 311,32 78,58 196,35 252,32 355,94 416,37 2 54,41 63,81 51,87 49,23 59,66 55,58 50,62 42,19 37,62 37,51 3 2,25 11,30 10,76 4,96 3,14 1,61 8,43 8,70 2,56 1,41 0,24 8,08 3,90 3,24 1,87 0,16 3,77 6,55 3,53 1,88 4 a Mean ± SD (n=3) ; b Period 1: 1 (0-15 min); 2 (15-60 min); 3 (60-240 min); 4 (240 -360 min) Deacetylation processes in condition with and without sonication followed Pseudo-first order kinetics and were expressed by the equation dX/dt = k* X, where X was DD value of the sample at the moment t of deacetylation and k was the rate constant; however, the value of k always changed in NaOH concentration, time and the manner of deacetylation (with or without of sonication) and it had the same trend as that described above for the rate of deacetylation (Table 3.17) 16 Table 3.17: The rate constant of deacetylation reaction (min-1) (x103)a Deacetylation condition NaOH con centration (%) (w/w) Periodb Without soncation 35 With soncation 40 45 50 60 35 40 45 50 60 1 43,81 78,11 151,36 178,58 187,19 106,36 158,77 174,11 195,62 205,55 2 36,78 30,63 13,11 9,12 9,60 22,01 11,82 8,48 5,83 5,10 3 0,70 2,36 1,75 0,71 0,40 0,39 1,35 1,29 0,34 0,17 0,07 1,27 0,53 0,42 0,22 0,04 0,52 0,82 0,44 0,22 4 a Mean ± SD (n=3) ; b Period 1: 1 (0-15 min); 2 (15-60 min); 3 (60-240 min); 4 (240 -360 min) Therefore, ultrasound (37kHz, 35W) had the capacity for supporting deacetylation, it increased the rate of the process and the uniform of products, facilitated the solubility but not changed the nature of deacetylation as well as chemical bonds in chitosan molecular in comparison with conventional deacetylation. 3.5.4. Effects of NaOH concentration, temperature and reaction time on DD and solubility of chitosan produced by heterogenous deacetylation with the facility of sonication The influcence of temperature (70-80oC), reaction time (2-6h), and NaOH concentration (40-60%) on deacetylation process in the presence of sonication was investigated with two-level factors model and the results shown in Table 3.19 and Table 3.20 revealed that all of NaOH concentration, time and temperature as well as interaction of temperature and time, interaction of concentration and time and interaction of temperature and concentration played dominant role on DD and solubility of chitosan within the experiment range (p<0.05); Among of them, NaOH concentration was the most important term with its level of 26,73% and 52,65% on DD and solubility, respectively. The second important term on solubility was temperature but in case of DD that was the term of time. The effect levels of temperature on DD and solubility were 11,10% and 3,73%; whereas those of time on DD and solubility were 5,85% and 10,13%, respectively. Table 3.19: Analysis of variance from chitosan’s Table 3.20: Analysis of variance from chitosan’s data for response degree of deacetylation ( data for response solubility ( (p=0,05) Parameters Effect (%) Coefficient Prob>F (p=0,05) Parameters X0 X1 X2 X3 X1X3 X2X3 Effect (%) 3,751 10,138 26,731 -1,967 -9,017 Coefficient 70,080 1,875 5,069 13,365 -0,984 -4,509 Prob>F 0,001 0,001 0,001 0,001 0,004 0,001 X0 X1 X2 X3 X1X3 X2X3 11,100 5,850 52,650 -6,300 -3,650 68,225 5,550 2,925 26,325 -3,150 -1,825 0,001 0,001 0,007 0,001 0,005 0,031 X1: Temperature (oC); X2:Time (h); X3: Concentration (%); The best-fit regression equations for the relationship of these terms with DD ( ) (%) and solubility ( ) (%) obtained from the statistical analysis were Equation (3-11) and Equation (3-12), respectively. 70,081 + 1,875 X1 +5,069 X2 +13,365 X3 - 0,984 X1X3 - 4,509 X2X3 = 68,225 + 5,55 X1 +2,925 X2 +26,325 X3 - 3,154 X1X3 - 1,825 X2X3 (Equation 3-11) (Equation 3-12) Respond surface for the changes in DD and solubility as a function of NaOH concentration, time and temperature indicated that in order to produce chitosan having DD > 70% and solubility > 85% the deacetylation must be conducted in condition of NaOH≥ 50% and reaction time ≥ 4h at 80oC. 3.5.5. Optimal conditions for deacetylation with sonication 17 In order to investigate the optimal condition of deacetylation, DD and solubility of samples obtained from various conditions (Table 3.21) were statistically analyzed using respone surface methodology with MINITAB 16.1 software. The best fit regression Equation (3-13) and Equation (3-14) for the optimum DD ( ) (%) and solubility ( ) (%), respectively, within the experiment range were obtained. = 85,8035+ 3,0240 X1 +2,069 X2 - 1,2260X12 - 1,2865X22 -0,5759X1X2 2 (Equation 3-13) 2 = 96,5179+ 3,0600 X1 +1,3300 X2 +0,6400X1 - 0,5100X2 -1,2175X1X2 Table 3.21: Results of the experimental matrix with Central Composite design Solubility (%) 91,56 No -1 DD (%) 78,31 (Equation 3-14) 1 X1, % -1 2 +1 -1 85,41 100,00 9 +1 0 87,13 100,00 3 -1 +1 83,32 96,43 10 0 -1 81,60 94,12 100,00 11 0 +1 86,30 97,23 96,14 12 0 -1 85,95 95,78 0 0 84,94 95,78 0 0 85,76 96,76 No 4 5 X2, h +1 0 +1 0 88,12 86,34 0 DD (%) 80,89 Solubility (%) 93,65 X1, % X2, h 8 0 6 0 0 85,98 97,13 13 7 0 0 86,12 97,45 14 X1: Concentration (%); X2:Time (h) Results of statistical analysis summaried in Table 3.23 allowed to confirm the accuracy of Equation (313) and Equation (3-14). These two Equation could explain 97,85% and 95,76% for the experimental figures corresponding to the respones of DD and solubility, respectively. Table 3.23: Results of statistical analysis for Equation (3-13) and (3-14), (p=0,05) Terms PRESS R-Sq (%) R-Sq(pred) (%) R-Sq(adj) (%) Lack of fit Respone Degree of deacetylation Degree of Solubility , %) , %) 5,62 5,43 98,84 97,72 94,18 93,03 97,85 95,76 0,48 0,91 Table 3.25: Effects of deacetylation condition on the viscosity average molecular weight of chitosan (Mv, kDa) Deacetylation condition Temperature (oC) NaOH Concentration (%) Time (h) 0 2 4 6 8 Viscosity average molecular weight of chitosan (Mv), (kDa) With sonication Without sonication 80 70 80 50 60 50 50 60 1652, 00 592,61 468,53 421,82 377,37 * 1652,00 539,12 404,21 361,84 323,55 1652,00 473,26 - 1652,00 439,90 - 1652,00 378,53 - Mean of duplicate Due to deacetylation and depolymerization reactions always happened simultaneously and affected on the properites of chitosan, the average molecular weight were studied in the resulting chitosan. Results in Table 3.25 shown that viscosity average molecular weight of chitosan (Mv) was dependent on the condition of deacetylation (temperature, time, concentration and the presence of sonication). 18 In the same other condition of deacetylation (for 6h, NaOH concentration of 60%, (w/w)) viscosity o o average molecular weight of chitosan produced at 70 C were significant higher than that of chitosan collected at 80 C (473,26 so với 421,82kDa); chitosan deacetylated in conventional condition, without sonication, had the average molecular higher than that in case of sonication but the differences were not remarkable (439,90 and 378,53 kDa in compared with 421,82 and 361,84 kDa regarding to NaOH concentration of 50% and 60%, respectively at the temperature of 80oC for 6h). Average molecular weight of chitosan reduced when reaction time incerased in some extent and the rate of reduction was different and dependent on duration of deacetylation. The rate of depolymerization was rather high during the first two hour, the average molecular weight reduces about 64-67% only after two hours of deacetylation (from 1652kDa to 592,61 and 539,12 kDa regarding to NaOH concentration of 50% and 60%, respectively). After that, the rate of depolymer had a slower trend, reduction of approximate 20 and 25% were observed after 6h and 8h, respectively, when deacetylation were conducted at NaOH concentration of both 50% and 60%. The results of regression analysis of logarithmic variation of of avarage molecular weight as a function of the deacetylation time performed during the period of 2 to 8 hours revealed that the trentency of deacetylation reaction in case of sonication was the same as that in case of conventional deacetylation and they followed Pseudo-first-order reaction (Figure 3.38). 2.9 y = 2,8198-0,0317*t (R=0,9573) y = 2,7800-0,0352*t (R=0,9363) NaOH 50% NaOH 60% Lg(Mv) 2.8 2.7 2.6 2.5 2.4 2 4 6 8 Time (h) Figure 3.38: Effects of NaoH concentration and time on viscosity average molecular weight of chitosan deacetylated in the presence of sonication Equation (3-15) and (3-16) indicated the rule of the change in average molecular weight as a function of time when deacetylation was carried out in the condition of 80 oC, NaOH 50% and 60%, respectively. These equations allowed to determine the exact deacetylation condition (time and NaOH concentration) to collect chitosan having required molecular weight. = 2,8501 - 0,0404*t (Equation 3-15) = 2,7767 - 0,0388*t (Equation 3-16) Therefore, four equations marked from Equation (3-13) to (3-16)) enablde to be controlled deactylation o at 80 C in the presence of sonication (37kHz) to achieve chitosan having desirable DD, Mv and solubility. 3.6. Proposing the procedures applied new technology to recover chitin, chitosan and protein and estimating their benefits 3.6.1. The proposed procedures to recover chitin, chitosan and protein 19 Based on the results achieved in my own study it declare that the proposed technology which were integrated by physical, enzymatic and chemical methods allowed to innovate the production of chitin and chitosan from by-products of the manufacturing white leg shrimp. The procedues were put forward in Figure 3.39, Figure 3.40 and Figure 3.41. Fresh shrimp heads after removing out of shrimp body were conveyed as soon as possible to reaction vessels having propellers. They were mixed with an equal amount of clean water, and heated up to 60oC and then kept for 2 hours to conduct autolysisat at native pH. When finishing the autolysis, the mixture was stirred strongly at the rate of approximate 1000 r/min and filtered through sieve plates having the pore size of 1mm to retain the head carapace (solid phase). The liquid phase was centrifugate at 10,000xg at 4oC for 10 min. The suspernatant was a solution containing the protein hydrolysate with antioxidant activity. It depended on the purpose of use the supernatant was dried and purified futher. The solid phase (chitinous residue) was used for chitin extraction and chitosan production. They were treated with HCl 0,25M (at the ratio of 1:4, w/v) for 12h at room temperature and then washed to neutral pH and drained. Next, they were mixed with NaOH 1% (at the ratio of 1:2, w/v) to deproteinization for 8h at temperature of 70oC. Chitin were collected, washed to neutral pH and dried at the moisture of lower 10% before storage in dry place. Fresh shrimpheads Fresh shell Pressing Autolysis (2h, 60oC, native pH, ratio of H2O to materials =1:1(v/w) Demineralization (HCl 0,25M for 2h, at room temperature, the ratio of HCl to materials =4:1 Stiring for 2 min, rate of 1000 r/min Filtering (Pore size of 1mm) Carapaces Washing and pressing Sonicating pepsin solution for 25min Deproteinization by pepsin at pH =2, for14h, 40oC Protein hydrolysate Hydrolysates Drying Drying The powder The powder Treated with HCl 0,25M for 12 h at room temperature, the ratio of HCl to materials=4:1(v/w) Washing to neutral pH Treated with NaOH 1%, 8h, 70oC, the ratio of NaOH to materials = 2:1(v/w) Preparation of pepsin solution (20U/g protein) Separating Solid Deproteinization by NaOH 1%, for 8h at 70oC, the ratio of NaOH to materials = 2:1 Washing to neutral pH Drying Washing to neutral pH Drying Chitin Figure 3.39: Proposed procedue for recovering chitin and protein from heads of white leg shrimp Chitin Figure 3.40: Proposed procedue for recovering chitin and protein from shells of white leg shrimp Shrimp shell after pressing to remove dipping water were demineralization with HCl 0,25M (at the ratio of 1:4, w/v) at room temperature for 2h. Then, the mixture was decanted and the solid phase was collected and washed roughly before treatment with pepsin. The demineralized shells was soaked in the solution of pH2 (at the ratio of 1:3, w/v), all were heated up to 40oC and then mixed with pepsin at the concentration of 20U/g protein. The deproteinization process with pepsin was carried out at 40oC for 14 or 16h which depended on pepsin previously submitted to ultrasound for 25 min or not. When the time was over, the mixture was handled to seperate into the liquid and solid parts. The former part was treated in the same manner which was mentioned above for that collected from shrimp heads. The latter part was mixed with NaOH 1% (at the ratio of 1:2, w/v) to 20 deproteinization for 8h at temperature of 70oC. Chitin were collected, washed to neutral pH and dried at the moisture of lower 10% before storage in dry place. According to the purpose of using chitiosan the required Mv, DD and solubity were determined and from that the parameters of deacetylation at 80oC (reaction time and NaOH concentration) were calculated based on Equation (3-13), Equation (3-14) and Equation (3-15) or Equation (3-16). Chitin (flake form) was previously submitted to hot water treatment for 60 min at 60 oC and decanted before deacetylation reaction was conducted in the specified condition of NaOH concentration and time with the ratio of chitin to NaOH solution 1:15, w/v. Sonication was applied during the deacetylation and the temperature was constant at 80oC. When the deacetylation process was finished chitosan was soaked and washed to neutral pH before drying to the moisture of less than 10%. The product was kept in dry and hermetic packaging Clarify the purpose of use Chitin Determine the required values of DD Mv and solubility of chitosan Soaking in hot water of 60oC for 60 min Based on Equation (3-13), (3-14) , (3-15) or (3-16) Pressing Sonication (37kHz, 35RMS) Calculating the reasonable condition for deacetylation (Time, NaOH concentration) Deacetyl at 80oC Wasing to neutral pH Drying Chitosan 3.6.2. Figure 3.41: Proposed procedue for determining parameters of deacetylation Characteristics of chitin and chitosan produced according to the proposed procedures Chitin and chitosan produced from shrimp heads and shells according to the proposed procedures in Figure 3.39, Figure 3.40 and Figure 3.41 were characterized and their properties were presented in Table 3.26 and Table 3.27. The results in Table 3.26 shown that the quality of chitin produced from by-product of white leg shrimps met the specifications of commercial chitin that were declared by AxioGen Co (India), all of the test iterms were in range of required values. Table 3.26: Quality of chitin produced according to the proposed procetures Specifications of commercial chitin b Test Items Colour Ash (%, db) Protein (%, db) Degree of acetyl (%) Moisture (%) Viscosity average molecular weight of chitosan (Mv, kDa) Hevy metals (ppm) Insoluble materials (%) a Off yellow <1 >90 <10 ≤ 15 <1 Test resuts From shells From heads Pinky white Pinky white 0,57±0,07 0,68±0,05 0,66±0,1 0,79±0,04 97,01±0,85 94,32±0,29 8,70±0,79 8,07±0,56 1652 1232 0,29 <1a Disolved by NaOH 40% in addition of ure, for 72h at 6-10oC; b: According to AxioGen Co, Ấn Độ.
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