Abstract:The serine protease with clip domain is a new serine protease family and plays an important role in innate immunity. One EST sequence with high homology with serine protease gene of other species was found from the cDNA library of Sinonovacula constricta and then the complete ORF and 3′-UTR sequence were obtained by PCR. The 5′-UTR sequence was got by 5′-RACE. The cDNA of this gene was 1 228 bp, which consists of a 66 bp 5′-untranslated region (UTR)，a 1 002 bp open reading frame (ORF) and a 160 bp 3′-UTR. The translated protein is composed of 333 amino acids containing a signal peptide. Sequence analysis of the protein revealed that the protein contained a clip domain and a Tryp_SPc domain. The three disulfide bonds were formed by six conserved cysteines in the clip domain and the catalytic triad (HDS) was contained in the Tyrp_SPc domain.This gene was designated as ScSP. The quantitative reverse transcriptase (qRT-PCR) analyses showed that the ScSP can be expressed in six tissues. The expression level of ScSP gene was highest in liver, then in gonad, but lowest in water pipe, mantle and gill. The expression of ScSP gene in liver tissue was up regulated at 4h and 8h following the challenge with Vibrio anguillarum. These results indicated that ScSP is a new serine protease with clip domain and might be involved in innate immunity, which contributes to understanding the structure and function of serine protease in the future.

Abstract:Full-length cDNA FREP1 (fibrinogen-related protein) was cloned from larval Paralichthys olivaceus by the way of RACE PCR,and 1 131 bp length FREP1 cDNA with a 786 bp open reading frame encoded a putative protein containing a fibrinogen C terminal domain signature(WWYSRCGSAGLNG).The whole amount in situ hybridization showed that FREP1 was only expressed in intestine of 2 dph and 5 dph (days post hatching) larvae, but expressed in fin, skin, gill and intestine of 9 dph and 13 dph larvae. qRT PCR reveals that FREP1 was expressed mainly in fin, skin, gill and intestine of adult fish , but was expressed very lowly in liver, heart, kidney, spleen and muscle. If muscle expression was a control sample , compared with the other tissues the expression of FREP1 in fin and skin were highest (P<0.01). But compared with gill and intestine, the expression of FREP1 in heart, kidney, spleen, liver and muscle is much lower(P<0.05). Since it was mainly expressed in skin, gill and intestine, which was associated with innate immunity system , FREP1 may be an important member of innate immune system of P. olivaceus.

Abstract:Green mud crab, Scylla paramamosain, is an important commercial species in aquaculture. Therefore, more and more researches focus on its molecular mechanism of reproduction. ERK pathway is essential for the regulation of gonad development. The 14-3-3 was proved to be bonded to phosphorylated protein and involved in transmission of ERK pathway. The full length cDNA of tyrosine 3 monooxygenase/tryp tophane 5 monooxygenase activator protein (14-3-3ζ) from green mud crab were obtained by SMART RACE in this study. The full length cDNA of 14-3-3ζ gene is 1 092 bp encoding a polypeptide of 248 amino acids. The results of real time quantitative PCR revealed that 14-3-3ζ gene was constitutively expressed in examined tissues, and its expression level in ovary was higher than that in the other tissues (P<0.05), while the expression level of 14-3-3ζ gene in secondary vitellogenesis stage was higher than that in proliferation stage (P<0.05). Consequently, we may infer that 14 3 3ζ plays a key role in ovarian development of the crab.

Abstract:ZP protein is a family of proteins which possess a conserved ZP domain and generally play an important role in the fertilization process. In this study, a new ZP cDNA from Branchiostoma japonicum, which codes 927 amino acid residues, was found. By using SMART software, it was confirmed that the ZP protein contains three domains (ZP Domain, Transmembrane Domain and Low Complexity Region). Furthermore, the sequence diversity of this ZP gene inBranchiostoma japonicumpopulation was analyzed. As a result, the ZP sequences amplified from 9 Branchiostoma japonicum individuals are 2 481-2 784 bp, which code 826-927 amino acids. There are 55 variable cites (5.93%, 55/927) identified by amino acid sequence alignment. 9 and 101 amino acids fragment deletion in two individuals may be due to the diverse alternative splicing of ZP gene. In conclusion, the sequence diversity of ZP proteins may be correlated with its structure and function diversity.

Abstract:Genetic structure of five hard clam stocks were detected using nine pairs of SSR primers and six ISSR primers. The shell color of one population is red and their offspring are red or yellow, while another population is yellow and their offspring are yellow also. The PIC，observed and expected heterozygosity of five populations detected from SSR ranged from 0.643 0 to 0.704 9 , 0.745 5 to 0.794 6 and 0.717 5 to 0.752 0,respectively. The percentages of polymorphic loci detected from ISSR were from 92.11% to 97.37%. The highest Shannon Index from PR population was 0.503 3 and the lowest from RF1Y was 0.412 6. Both two markers showed high polymorphism. Compared with their parents’, the genetic structure of the three offspring populations didn’t change. The results of cluster from the two kinds of markers were the same: the red shell color parents and their offspring gathered together at first, while the yellow parents and offspring gather together, then those two sub branches clustered into one group. Overall, the genetic diversity of the parents and their offspring was very high, and compared to their parents’, the genetic structure of the offspring didn’t change. That is to say, those offspring could be used for artificial breeding as parents seccessively.

Abstract:Melanocortin 1 receptor (MC1R), one of the key genes in animal melanin synthesis pathway, plays an important role in determining animal body color and coat color. Oujiang color common carp (Cyprinus carpio var. color), characterized as five kinds of body color patterns with black patches (e.g. “whole red”, “whole red with big black patch”, ”whole red with scattered black spots” ,“whole white” and “whole white with big black patch”), has been considered as a good material or model in studying animal melanin synthesis mechanism. In this paper, MC1Rgene was cloned and sequenced, and its expression profile in the four body color patterns ( “whole red”, “whole red with big black patch”, “whole white” and “whole white with big black patch”) and tissues were also analyzed in Oujiang color common carp. The result showed that the full length cDNA of MC1R was 1 914 bp, containing a 5′un translated region (UTR) of 637 bp, a 3′-UTR of 311 bp and an open reading frame (ORF) of 966 bp. This gene has only one exon encoding 321 amino acids and predicting 7 transmembrane domains. There were 98% nucleotide similarity with Carassius auratus and 93% similarity with Danio rerio. However, Only one nucleotide nonsense mutation (C/G) in the coding region was found among different color patterns, indicating the same amino acid sequence among them. The result of qRT PCR indicated that the expression of MC1R in eyes was significantly higher (P<0.05) than that in skin, muscles, gills, kidney, bladder, heart and liver. It was surprising that there was no significant expression difference among the different color patterns (P＞0.05). This finding showed that MC1Rgene is not a determinant factor in controlling color patterns of Oujiang color common carp,and it is necessary to conduct further studies in other color related genes or factors.

Abstract:Six species of genus Anguilla, including Anguilla rostrata, A. anguilla, A. bicolor pacifica, A.marmorata, A. australis and A.japonica are cultured in China. Misidentification on the fry will impact farmers’ income because of different Anguilla species with different prices in China. In this study, we tried to use two molecular markers (1179bp length mitochondrial COⅠ gene and 633bp length COⅡ gene) to discriminate the six species based on 54 fry individuals. The percentage of variable sites in COⅠ and COⅡwas 17.98% and 16.45% respectively. And the percentage of parsimony informative sites was 12.16% and 10.11% in COⅠ and COⅡ. The negative bias for G was observed in both COⅠ and COⅡ genes in checked samples, and their G frequences were 18.45% and 16.67% respectively. The pairwise distances based on either COⅠor COⅡ among six eel species were almost more than 2% except for only one between A. rostrata and A. anguilla based on COⅡ, and the former was a little higher than the latter. But the genetic distances within each species were all less than 1% based on two genes. Although there existed a small difference between two cluster results of six eel species,which was based on COⅠ gene or COⅡ gene, all individuals of each species formed a strong monophyletic group on NJ and Mrbayes trees. Taken together, both COⅠand COⅡgenes are good molecular markers to be used in species identification in genus Anguilla, and our results may be helpful for eel aquaculture industry in China.

Abstract:The present study was focused on the cloning of vitellogenin cDNA sequence of Eriocheir sinensis by the methods of RT RCR and RACE. The structure of the sequence was also analyzed. It was found that the vitellogenin cDNA of Eriocheir sinensis is of 7 955 bp, which contained 7 689 bp of an open reading frame (ORF), 62 bp of the 5′UTR and 204 bp of the 3′UTR, respectively. The further analysis showed that this gene maybe encode a kind of protein of 2 562 amino acid residues， compared with other crabs whose Vg sequences are known, their physicochemical properties and spatial structure are similar, and all contain 2 kinds of structural domains: the N terminal domain of vitellogenin with the function of lipid traffic, and the von Willebrand factor D type domain. The identities of the amino acid sequence of vitellogenin were about 33%-77% compared with other known crustacean species.This confirmed the functional consistency in crabs and individual differences,and makes our research about growth more in depth.It can be applied in more rational production guidance and can create greater economic values.

Abstract:The effects of salinity on the survivial of newly hatched larvae of Pseudosciaena crocea and the hybrid F₁ of Pseudosciaena crocea (♀) × Miichthys miiuy (（♂）) were investigated. The newly hatched larvae were transferred from sea water (salinity 27) to other seven salinities (12, 17, 22, 27, 32, 37, 42). The results showed that the survival rate, mean survival time (MST) and survival activial index (SAI) of the newly hatched larvae of large yellow croaker and hybrid F₁are decreased following the increase of salinity. In the salinity range of 12 to 32, the SAI values of hybrid F₁ was up to 62.42 to 86.56 which was significantly higher than the large yellow croaker (46.18 to 53.89). At the salinity 12, the MSTof large yellow croaker and hybrid F₁ reached the maximum value，（10.69±0.16） d and （13.47±0.35） d , and at the salinity 42, the MST reached a minimum value， (4.91±0.27） d and （6.30±0.27） d. The continued downward trend of survival rate, median death time (DT₅₀), mean survival time (MST) and survival activial index (SAI) of the newly hatched larvae of large yellow croaker and hybrid F1 were compared at the same salinity. The results show that the newly hatched larvae of hybrid F₁ had stronger ability to survive during salinity changes, and showed strong heterosis.

Abstract:In the present study, we investigated the individual day food consumption (I_DF), specific growth rate (SGR), food conversion (FC), and gonadosomatic index (GSI) between males and females S.argus1 year old, as well as activities of trypsin, amylase and lipase in stomach, hepatopancreas and intestinae in S. argus below 1 year old, 1-2 years old, and above 2 years old. Result showed that individual day food consumption, specific growth rate and food conversion in female S.argus was significantly higher than males (P<0.05).GSI increased from March to May, and a significantly higher GSI could be found in females at the end of experiment. Pepsin in stomach, amylase and trypsin (except hindgut) in hepatopancreas and intestinae exhibited higher activities in females than males in below 1 year old and 1-2 years old fish , but no difference was found in lipase, pepsin, amylase and trypsin between males and females S.argus above 2 years old. Activities of pepsin, trypsin and amylase were highest in fish below 1 year old, then in 1-2 years old, and the lowest in that above the 2 years old. However, a reverse trend was found in lipase, and a significant difference was found in the above enzymes (except some tissues) between 1 year old and 2 years old fish. Results suggested that faster growth rate in females S.argusbelow 2 years old than males was related to the higher IDF, FC and activities of protease and amylase, but that in fish above 2 years old had nothing to do with digestive enzyme.

Abstract:We have cultivated the female broodstock of half smooth tongue sole intensively by using nereid from 11th April 2012 to 3rd October 2012. It turned out that the females grew rapidly and the gonad development had been brought forward, the duration of spawning had been extended, egg laying amount, fertilization rate, hatching rate, etc. had risen notably,and mortality rate had a significant reduction which was the most important. After 4 months culturing, the average body weight of Group A rose from 1.25 kg to 1.85 kg, the net gain and average daily gain were 0.6 kg and 4.92 g/d respectively, which were significantly higher than those of Group B,which were 0.34 kg and 2.79 g/d respectively. Before spawning, 118 individuals had matured, the mature rate was 59.60%. However, only in 67 individuals in Group B the gonad had matured, the mature rate was 33.84%. In Group A, 11 individuals were dead, the mortality rate was 9.32%, by contrast, there were 28 individuals dead and the mortality rate was 41.79%, which was much higher than 32.47% over Group A. Gonad of parental fish of Group A began to develop on 26th May, which was 19 days earlier than Group B, and it began to spawn on 10th September, which was 6 days earlier. The spawning duration of Group A was 24 days, 13 days were extended compared with Group B. Mean fecundity and mean floating eggs were 411.3 mL and 263.2 mL separately, which was 60.6 mL and 71.2 mL higher than Group B respectively, also the average of floating rate of Group A (80.97%) was notably higher than Group B (67.65%). The fertilization rates of the 2 groups were 60.37% and 47.29% separately. Group A was 13.08% higher than Group B. The hatching rates of the 2 groups were 53.28% and 22.40%, Group A was 2.38 times as much as Group B. Both of the 2 groups’ aberration rates were low, 2.36% and 4.73% separately, however, Group A was less than half of Group B.

Abstract:The choices of mussel（Anodonta woodiana）shell width when Chinese bitterling (Rhodeus sinensis) spawn in different shell width groups (50-100 mm) and in mixed shell width groups were studied. The result showed that Chinese bitterling’s spawning rate in groups of shell width less than 50 mm and more than 90 mm were lower than the groups of shell width 50-90 mm significantly. The spawning rate in groups of 60-80 mm were higher than other groups significantly, and the group of 70-80 was the highest. In the same way, the highest spawning rate of shell width was 60-70 mm in study of mixed shell width groups, which was higher than others significantly. The spawning rate declined when the shell width greater than 70 mm or less than 60 mm. The results in both of experiments indicated that Chinese bitterling(weight 1 g) might choose the right mussel, the best shell width of which was 70 mm.

Abstract:Sexual reproduction characters of Sargassum cinereum were observed by tissue culture in vitro. Effects of light intensity 20, 40, 60, 80, 120, 160 μmol/(m²·s) on sexual reproduction and early development of S. cinereum germlings were studied. The results showed that four to six eggs were released from each female conceptacle. Freshly released unfertilized eggs had single nucleus, which then changed into two to eight nuclei by nuclear division. The first two cell divisions after fertilization were transverse, and the following two cell divisions were all longitudinal. Through subsequent countless cell divisions, a pyriform germling was formed. Light intensity insignificantly influenced the size of eggs, the size and the cell division velocity of zygotes. However, variations in rhizoid number, rhizoid length and thalli growth were highly significant under different irradiances. The first branches on germlings were observed after being cultured for 8 days. Suitable growth of i>S. cinereum occurred under irradiances from 40 μmol/(m²·s) to 120 μmol/(m²·s). Optimal growth of these germlings occurred under irradiances from 40 μmol/(m²·s) to 60 μmol/(m²·s) in early development stage and from 80 μmol/(m²·s) to 120 μmol/(m²·s) after one leaflet was formed, respectively. These results may provide theoretical basis for artificial seedling culture of i>S. cinereum.

Abstract:This experiment researched the effects of Astragalus Polysaccharide (APS) on the growth and non specific immune parameters of Procambarus clarkii, with different additive concentrations (0%, 0.2%, 0.4% and 0.8%). Results showed that, 0, 2, 4, 6, 8, 12, 16, 20 d, weight gain ratio (R_WG) and specific growth ratio (R_SG) of the crayfish fitted the modified Gauss model well (R²2=0.999 9). At the concentration of 0.40%-0.75%, R_WG and R_SG reached the maximum values of 37.13% ± 7.75% and 1.57% ± 0.28% respectively. Curve of crayfish molt count (NM) was consistent with the log normal model (R²=0.997 6), with significantly different between experimental and control groups (P＜0.05), and weight gain was also significantly different (P＜0.01). Non specific immune parameters of superoxide dismutase (SOD) and acid phosphatase (ACP) changed with APS isn’t significantly (P<0.001).vely promote the growth and non specific immunity of crayfish. In addition, the suitable additive concentration was 0.40%-0.80%.

Abstract:The phytoplankton of 12 salt lakes in Naqu region, Tibet had been investigated during April to May in 2009. A total of 58 species of phytoplankton were identified, belonging to 39 genera of 6 phyla. Bacillariophyta (34 species) was dominant in species richness by 58.62 percentage, followed by Cyanophyta (11 species) and Chlorophyta (8 species). Navicula sp., Nitzschia sp., Lyngbya limnetica, Oocystis parva were the main dominant species. The average density and biomass of phytoplankton were 9.70×104 cells/L and 0.162 8 mg/L, respectively. This paper also discusses the phytoplankton community characteristics of Tibet salt lakes, and the relationship between floristic composition characteristics of phytoplankton and environmental factors, especially the salinity. The results show that the correlationship between phytoplankton species, density, biomass and salinity were not significant(P＞0.05). In salt lakes, the salinity is an important limiting factor in determining the structure of phytoplankton community, and it is controlled by numerous ecological factors.

Abstract:Yangcheng Lake is the third largest freshwater lake of Taihu basin, which is an important water source of the Suzhou area and the only preparing source of drinking water for the city of Kunshan. Yangcheng Lake plays an important role in domestic water, industrial water, and irrigation and fisheries resources. Since 1980s, with the further use of the water resources in Yangcheng Lake, the nutrients were gradually enriched in the lake, which accelerated the eutrophication process of the lake. Algal blooms outbursted in Taihu Lake and other lakes in 2007, and all sectors of society attach great importance to the safety problems of water environment in recent years. Yangcheng Lake which is famous for culturing high quality crab (Eriocheir sinensi) had a large area about 5 400 hm² for the purse seine aquaculture. In order to protect the ecological environment and the source of drinking water ,the government decided to reduce the area to about 2 133 hm² in 2008. We conducted a research about crustacean zooplankton’s community structure, temporal changes and their relationship with environmental factors in Yangcheng Lake, and analyzed the differences inside and outside of the pen about the crustacean zooplankton with a purpose to reveal whether the crustacean zooplankton of Yangcheng Lake has a response to the pen. We also accumulate data for the aquatic ecosystem variation of Yangcheng Lake and provide a theoretical basis for the sustainable development of the ecological fishery. The main physical and chemical characters (TP, TN, SD and Chl.a), species composition, density, biomass and the diversity index inside the pen were not significantly different from those outside the pen（P>0.05）.

Abstract:Four kinds of sediment heavy metal samples (Zn, Cu and Pb and Cd ) were collected from lakes, rivers, and aquaculture ponds in Kunshan for analysing distribution pattern and evaluating pollution status of these sediment heavy metals . The results show that, the Cu value at sampling sites of lakes varied between 6.54-333.99 mg/kg, and the mean value was136.60 mg/kg. The Cu value at sampling sites of rivers varied between 11.00-475.88 mg/kg, and the mean value was 126.78 mg/kg. The Cu value at sampling sites of aquaculture ponds varied between 31.73-155.78 mg/kg and the mean value was 84.73 mg/kg. The ecological risks of these four heavy metal elements in Kunshan waters was Cu > Cd > Zn > Pb. The potential ecological risk index of Pb, Zn, Cu was under 40. It showed the three kinds of heavy metals were at low ecological risk. And the potential ecological risk indexes of Cd all exceed 320. What’s more, the potential ecological risk indexes of Cd in all sites were more than 600,and present serious ecological risks. The general potential ecological risk level of heavy metals was in the order: aquaculture ponds > lakes > rivers.

Abstract:Based on the seawater and sediment monitoring results of Shengsi Lvhua Island’s mariculture zone in spring, summer, autumn of 2011, the results showed that, mariculture zone’s water temperature, salinity, pH, chemical oxygen demand (COD), dissolved oxygen (DO) , dissolved inorganic nitrogen (DIN) and chlorophyll a(Chl.a) changed with the seasons, eutrophication of the mariculture zone was serious, and the eutrophication level was spring＞summer＞autumn；the sea water’s organic pollution level (A) in summer was the lowest, but attained lightly polluted level in spring and autumn. The seawater environment was mainly affected by the Changjiang River, breeding pollution and so on, and the higher level of dissolved inorganic nitrogen was the main reason for eutrophication and organic pollution, and was also a reason of the area’s high incidence of the red tides. The content of mercury (Hg), arsenic (As), copper (Cu), lead (Pb), cadmium (Cd), chromium (Cr) in sediments (6 kinds of heavy metal) accorded with the first class of the marine sediment quality of National Standards. According to the Hakanson’s evaluation method, the potential ecological risk was low, but the content of chromium (Cr) reached medium pollution level, so monitoring should be strengthened.

Abstract:The morphological characteristics of Vorticella campanula attached on Eriocheir sinensis were redescribed by the observations of living bodies and protargol and silver nitrate impregnated bodies. Some new morphological supplemented characteristics were: One contractile vacuole was 7.5-8.7 μm in diameter. Freedom movements of some yellow, green, brown and black granules were within the protoplasm. Food vacuoles were 9-15 μm in diameter. Stalks were 5.1-7.6 μm in width. Myonemes were 2.7-3.2 μm in width, on which some black granules in the diameter of 0.5-0.7 μm were densely distributed. The clear striaes were on the outside of pellicle and the granulars were in the inside of pellicle. The outer two kineties were longer than the inner kinety of P3. The three kineties of P2 were equal in length and end of junction of P1 and P2. The outer two kineties were longer than the inner kinety of P1. The argyrophilic grains were obvious and densely and unevenly distributed. Phylogenetic analysis indicates that Vorticella campanula was more original than other Vorticella campanula and was divided from Vorticella at the earliest stage.

Abstract:In order to solve the problem of a large amount of pond sediment deposition, which caused water deterioration and fish disease outbreak, a mechanized regulating system for pond sediment during pond culture was designed. Solar energy was used to provide power to the systems. The sediment was lifted to upper water and reused again. The routing of the mechanized regulating system was regular and controllable at water surface. Based on the predetermination of capacity of water flow, working radius, light intensity, still water speed and other parameters, the design of mechanized regulating system was accomplished. The overall structure and working principle of four core parts were described, which was composed of lifting device for sediment, power plant, traveling mechanism and control system . The mechanized regulating system’s working coverage, water flow, empty noise, speed of traveling mechanism, traveling speed of lifting device for sediment, distance of remote control by wireless were tested，and every indicator can meet design requirements. The results showed that the mechanized regulating system for pond sediment was safe and reliable, which can release the nutrients of sediment and reuse the sediment.

Abstract:Chub mackerel (Scomber japonicus) is an important economic species in the coastal waters of China, and its sustainable utilization and scientific management has been attractive and attentions by peoples. In this paper, based on Gordon Schaefer bio economic model, we use the catch data of purse seine fishery and economic data from China, Japan and Korea from 1998 to 2008 to calculate the Maximum Sustainable Yield (MSY), Maximum Economic Yield (MEY), Bio economic balance point (BE) and their corresponding fishing efforts. Combined with the above results, the bio economic social synthesize model based on different weight is established by considering the impact of biology, economy and society, to compare the economic benefit and resources status of different management objectives at short term (1-5 years), middle term(10 years) and long term(20 years). The results showed that the resources of chub mackerel is overfishing now. If the MEY is considered as the management objective for maximum long term economic benefit, the resources status keeps well but with low social employment rate. If the BE is considered as management objective for maximum short term economic benefit, it will provide a plenty of employment, but long term economic benefit is the lowest and the resources status will be worst. It is found the comprehensive benefit of fishing program 9 in all aspects is the best. The results showed that the most suitable fishing efforts is 19 889 fishing nets by considering the impact of all factors, and the suitable CPUE should keep between the corresponding fishing effort of MEY and MSY, and slightly close to MSY.

Abstract:To establish a HPLC method for the determination of free aromatic amino acids in aquatic product. Optimal conditions such as pH, flow rate and concentration of mobile phase were confirmed. Accuracy, precision and limit of quantitation were surveyed and evaluated. The method is based on an extraction of amino acids from aquatic product with a solution of 5% perchloric acid followed by neutralization by the addition of KOH just prior to HPLC analysis. HPLC system consisted of a Waters Atlantis T3 C18(4.6×250 mm, 5 μm) column. The mobile phase consisted of 85% 0.05 mol/L sodium phosphate buffer and 15% methanol, adjusted to pH 6.0 with phosphoric acid. The flow rate was 0.9 mL/min, the column temperature was 27 ℃, and the detection wavelength was 256.7 nm. The three amino acids were separated in 12 min. The standard curves of Tyr, Phe, Trp were linear within a range of 10-500, 50-1 000 and 5-250 μg/mL, and the correlation coefficient deviations range from 0.999 86 to 0.995. The limits of detection were 2, 10 and 1 μg /mL. The average recovery of spiked sample deviations ranges from 88.0% to 105.2%, In conclusion, this method proved to be simple, rapid, reliable and with high accuracy and precision. It has practical value for detecting the three amino acids in aquatic product.

Abstract:This study outlines a method to prepare chitooligosaccharides with a specific degree of polymerization (5-6). It was carried out by hydrochloric acid hydrolysis in a chitosan degraded system and a following procedure by gel filtration chromatography to fractionate chitosan oligomers. The degradation of chitosan over a range of temperatures (40 ℃, 60 ℃ and 80 ℃) and hydrochloric acid concentrations (6 mol/L, 9 mol/L and 11 mol/L) was investigated. The results showed that chitosan was degraded effectively and various chitooligosaccharide mixtures with different oligomer constitutions were obtained. Among them, the optimum conditions were 9 mol/L HCl at 60 ℃ and the yield of chitopentaose and chitohexose reached 16.2%. A procedure using Sephadex G 15 onto size exclusion column was further developed to narrow the range of oligomers and obtain expected fraction containing mainly chitosan pentamer and hexamer detected by thin layer chromatography. Furthermore, it was identified by high performance liquid chromatography. Alongside peaks for chitopentaose and chitohexaose, a new peak designating an unknown compound occurred. It was identified as chitooligosaccharide by 13C and 1H nuclear magnetic resonance. Enzymatic depolymeriza tion by chitosanase produced mainly trimer and tetramer, further confirming the presence of a heptamer.