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 60 o C 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 one- variable-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, H 1 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 (27- 30 o 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. Time of delay (h) 0 2 4 6 8 Loss of weight (%) 0 5 10 15 20 25 Content of TVB-N (mg/100g) 0 10 20 30 40 50 Total weight Protein TVB-N a a b bcd cd d bc B C D DE E F A E a Figure 3.1: Effects of storing time at room temperature (27-30 o C) 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. Reaction Time (h) 0 1 2 3 4 Yield of nitrogen recovery (%) 30 40 50 60 70 80 90 100 Yield of antioxidant recovery (%) 3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 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 Antioxidant recovery at the ratio of 1:2 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 Time (h) 0 1 2 3 4 Protein content (%) 8 10 12 14 16 18 20 22 Degree of deproteinization (%) 72 74 76 78 80 82 84 86 88 90 92 Protein content at the ratio of 1:0 Protein content at the ratio of 1:1 Protein content at the ratio of 1:2 DP at the ratio of 1:0 DP at the ratio of 1:1 DP at the ratio of 1:2 a a a b bc b 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). Reaction time (h) 0 1 2 3 4 DPPH (M/g materials) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 a b b bcd bcde bcde bcdef cdef def ef bcdefbcdef f bc bcd A Reaction time (h) 0 1 2 3 4 OD at 700nm 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 a cde cde a ab abc abc abcd abcde abcde 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 60 o C, 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 70 o C 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 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. Reaction time (h) 0 2 4 6 8 10 12 14 16 18 20 22 24 Content of minerals (%) 0 5 10 15 20 25 30 Degree of demineralization (%) 40 50 60 70 80 90 100 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. 9 Pepsin concentration (U/g protein) 0 5 10 15 20 25 30 35 Degree of Deproteinization/Demineralization (%) 0 20 40 60 80 100 120 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. 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 (X 1 , in range of 30-40 o C), E/S ratio (X 2 , in the range of 5-25U/g protein) and incubation time (X 3 , in range of 6 - 18h). The fitted model, expressed in coded variables, is represented by the equation: = 65.33 + 21X 1 +9.875X 2 + 11.375X 3 + 5.75X 1 X 2 + 3,75X 1 X 3 – 9.417X 1 2 – 8.167X 2 2 – 11.667X 3 2 (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-R Squared , 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 40 o C, 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 N o X 1 , o C X 2 , U/g.protein X 3 , h Y, (%) * N o X 1 , o C X 2 , U/g.protein X 3 , h Y, (%) * 1 -1 -1 0 41,89 9 0 -1 -1 41,08 2 1 -1 0 65,30 10 0 1 -1 58,57 3 -1 1 0 45,64 11 0 -1 1 57,11 4 1 1 0 86,95 12 0 1 1 76,09 5 -1 0 -1 34,99 13 0 0 0 73,15 6 1 0 -1 61,91 14 0 0 0 73,17 7 -1 0 1 46,77 15 0 0 0 73,76 8 1 0 1 85,55 * Mean ± SD (n=3) Table 3.9: Observed and predicted values of the confirmation experiments Trials Condition DP (%) * Observed Predicted 1 X 1 =40 o C ; X 2 = 10U/g.pro; X 3 =15h 82,41 ± 0,97 81,25 2 X 1 =40 o C ; X 2 = 12,5U/g.pro; X 3 =14h 86,58 ± 0,51 85,39 3 X 1 =40 o C ; X 2 = 15U/g.pro; X 3 =15h 89,87 ± 0,19 89,52 4 X 1 =40 o C ; X 2 = 15U/g.pro; X 3 =16h 90,22 ± 0,14 89,74 5 X 1 =40 o C ; X 2 = 20U/g.pro; X 3 =16h 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). Time of treatment (min) 0 20 40 60 80 100 Enzyme activity (U/mg) 36 38 40 42 44 46 With sonication Without sonication Figure 3.12: Effects of sonication time on pepsin activity (37kHz, 35W) Time of treatment (min) 0 5 10 15 20 25 30 Enzyme activity U/mg) 36 38 40 42 44 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 25min- 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 [...]... 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. .. figures in Table 3.28 the rough benefits of applying the innovated technology for 1000 kg of shrimp by- products were estimated and summarized in Table 3.29 The proposed innovation in chitin extraction brought the profit in both aspects of economy and environment It helped to solve the shortcomings in environment issues and create foundation for developing sustainably chitin and chitosan industry in Vietnam. .. that allowed to control efficiently the chitin and chitosan production Chitin and chitosan produced according to the proposed procedures had high quality that met the specifications from the leading companies In addition, the yield of chitin recovery was incereased by 2,8 and 32,87% respectively to shell and heads; Solubility of chitosan was incerased by 1,5% and và yield of purified chitosan also raised... 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... 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. .. applied to recover chitin, protein and chitosan according to the proposed procedures were presented in Table 3.28 These figures shown that the quality of chitin produced by the proposed procedures was enhanced in compared with that of chitin produced by the reference procedure, including: (1) higher purity (the residues of protein and 22 minerals of the former chitin were below 1% whereas the protein content... (%) 3,52±1,54 a 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... 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... vannamei) cultivated in Khanh Hoa province were collecte: - In case of shrimps in size of 60÷160 bodies/kg: the mass ratio of head and shell were average of 38,70±6,46 (%) in comparison with the mass of whole shrimps, whereas, the ratio of each part was 27,50±3,93 and 11,21±2,63 (%) respectively; - In case of shrimps in size of 81÷120 bodies/kg, the content of protein, minerals and chitin of head and. .. conducted in shells of white leg shrimp by pepsin obeyed Pseudo-first-order and protein existed in shrimp shell in a structure of layers Degree of deproteinization (DP) and its rate (r) in the period of first hours had the equation as following: - 2 Ultrasound (37kHz, RMS 35W) had capacity to facilitate the activity of pepsin (increase 8% after sonication of 25 min) as well as intensify the rate and the . 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. 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. 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