柱前衍生-高效液相色谱法测定浒苔多糖的组成

研究了浒苔多糖样品经三氟乙酸完全水解而无损失的最适条件,并且在优化流动相洗脱程序等色谱条件的基础上,建立了1-苯基-3-甲基-5-吡唑啉酮(PMP)柱前衍生-高效液相色谱法分析浒苔多糖的单糖组成及含量的方法。该方法采用配备紫外检测器的高效液相色谱仪,在250 nm波长下进行测定,外标法定量。结果表明,在此条件下,8种单糖能够良好分离,各单糖在1-100μg/ml范围内具有良好的线性关系,相关系数r≥0.999,回收率在76.0%-108%之间,相对标准偏差RSD10%。采用该方法对浒苔多糖样品进行组成及含量分析,发现无论样品原料为何种浒苔,浒苔多糖均以鼠李糖为主,同时还含有葡萄糖醛酸、葡萄糖、木糖、半乳糖以及少量的甘露糖、半乳糖醛酸和岩藻糖。     

离子色谱法同时测定水中7种阴离子的含量

为建立快速准确地同时测定水中F-、Cl-、NO2-、SO24-、PO34-、Br-、NO3-等7种阴离子含量的离子色谱分析方法,通过配制不同浓度的多种淋洗液,选择合适的淋洗液、淋洗液流速和离子色谱仪参数,使用ICS-3000离子色谱仪系统、Dionex IonPac AS-23阴离子分离柱和IonPac AG-23保护柱进行了检测试验,分析这种方法的线性、检测限、回收率和精密度。结果表明,用0.45μm微孔滤膜过滤水样,以4.5mmol.L-1Na2CO3+0.8 mmol.L-1NaHCO3为淋洗液,淋洗液的流速为1.0 mL.min-1,混合标准溶液的进样量为50μL,进行电导抑制检测,可以有效分离这些阴离子,标准曲线在一定范围内具有良好线性,线性相关系数(R2)为0.999 3～0.999 7,相对标准偏差(RSD)0.2%～0.6%,各离子的回收率为90%～110%,检测限为0.011～0.052 mg.L-1。     

海参皂苷的提取条件研究

海参皂苷极性较大,多采用醇提法进行提取,为减少海参皂苷制备过程中多糖和蛋白质的干扰,简化后续分离过程,本实验考察了乙醇浓度、温度、料液比对干海参中海参皂苷提取的影响。结果表明:乙醇浓度为0%、80%时海参皂苷得率较高,其中0%乙醇提取时提取液中多糖含量较少,更有利于后续分离;20℃、30℃提取时皂苷得率高,30℃提取时杂质更少;料液比为1∶15时提取得到的海参皂苷含量较高,提取时间控制在8 h内,可有效减少杂质产生且能获得较多皂苷。     

海参水煮液酶解多肽的抗氧化活性

以海参水煮液的冻干粉为原料,选取中性蛋白酶、胃蛋白酶和胰蛋白酶分别酶解制备多肽,测定其对·OH、O2-·和DPPH·自由基的清除能力,并与合成抗氧化剂TBHQ对比.结果表明,3种蛋白酶酶解制得的海参水煮液多肽对·OH、O2-·和DPPH·自由基的清除能力均随样品质量浓度的升高而增强;其中,中性蛋白酶酶解制得的海参水煮液多肽清除·OH、O2-·和DPPH·自由基的IC50值依次为8.565、6.658和2.015mg/ml,其对O2·的清除能力优于TBHQ.经液相色谱法测定,中性蛋白酶、胃蛋白酶和胰蛋白酶酶解制得的海参水煮液多肽分子量分别分布在＜2 500、＜3 500和＜1 000 D的小分子肽中.     

水溶性牡蛎多糖的降血压活性

牡蛎中含有丰富的多糖。从牡蛎中制备的水溶性多糖经HPLC测定,其单糖组成为葡萄糖,以葡聚糖分子筛测定其分子量为(3.93~10.84)×106 Da。以牡蛎多糖(7g/kg)对高血压模型大鼠灌胃均具有较明显的降低收缩压和舒张压作用。     

鸭绿沙塘鳢精子生理学特征

为探究鸭绿沙塘鳢(Odontobutis yaluensis)精子生理学特征, 提高精子活力, 测定了其精液浓度、精子密度、 pH 和精子活力, 以精子激活率(activation rate, AR)、快速运动时间(fast movement time, FT)以及精子寿命(life time, LT)作为评价精子活力的指标, 检测了水温、pH、离子(NaCl、KCl、CaCl₂、MgCl₂)、非离子(葡萄糖、Tris、甘油)、 碱度(NaHCO₃)共 10 种单因子条件下的精子活力, 并在此基础上进行 17 组复合因子精子激活液的筛选。结果表明, 鸭绿沙塘鳢精巢前段精子活力、精子密度、精液浓度均高于中后段, 精巢整体精子密度(1.83±0.03)×10⁹ 个/mL, 精液浓度 23.10%, pH 6.9±0.1。单因子精子激活液中, 鸭绿沙塘鳢在水温 20 ℃、pH 6.0、NaCl 68 mmo/L、 KCl 54 mmol/L、CaCl₂ 27 mmol/L、葡萄糖 28 mmol/L、甘油 65 mmol/L 条件下精子 AR, FT 和 LT 均最高, 在 MgCl₂ 11 mmol/L、Tris 4 mmol/L 激活液中精子激活率最高。复合因子精子激活液中, 精子在 KCl 40 mmol/L、甘油 33 mmol/L、Tris 4 mmol/L 条件下 AR, FT 和 LT 均最高, 精子活力最高。综上所述, 在进行鸭绿沙塘鳢人工授精时, 可直接使用精巢前段(生精部), 同时应用复合因子精子激活液(KCl 40 mmol/L、甘油 33 mmol/L、Tris 4 mmol/L)提高精子活力。     

麒麟菜多糖的研究——Ⅰ.琼枝多糖的性质及其红外光谱

琼枝多糖是琼枝(Eucheuma gclatinae)分离得到的一种硫酸酯化半乳聚糖。构成这种多糖的单糖主要是 D-半乳糖和3,6-脱水-D-半乳糖。分子量为 1.99×10~5,比旋光度为正值。大多数碱金属阳离子和碱土金属阳离子能使此多糖的胶凝能力提高。用碱或石灰处理时,琼枝多糖便部分脱去硫酸酿基团,3,6-脱水-D-半乳糖的含量增加。红外光谱分析证明,琼枝多糖在1240cm~(-1) 、930cm~(-1)和845cm~(-1)有吸收峰。用氯化钾分级时,琼枝多糖可分为溶于氯化钾溶液和不溶于氯化钾溶液两个级分;前者的红外光谱除了同后者一样在 1240cm~(-1),930cm~(-1)和845cm~(-1)有吸收峰外,在 820cm~(-1)处还有吸收峰。根据琼枝多糖的组成,性质和红外光谱,可以推断,溶于氯化钾溶液的级分是类 mu-卡拉胶;不溶于氯化钾溶液的级分是类 kappa—卡拉胶。     

海带岩藻聚糖硫酸酯电渗析分离工艺

采用电渗析法对盐沉后的海带酶解液中岩藻聚糖硫酸酯进行分离,探索工业化分离生产岩藻聚糖硫酸酯的方法,以解决目前乙醇沉淀法存在的安全性和高成本等问题。以脱盐率、糖保留率为检测指标,采用响应面法优化了电渗析工艺参数。结果显示,在操作电压80 V、料液流量3.2 L/min、固形物浓度10 mg/ml、p H=5的条件下,电渗析连续循环3次,海带酶液解干基中多糖含量由40.17%提高至47.11%,硫酸根含量由3.36%提高至4.97%。电渗析后海带酶解液干基中的金属离子含量为Ca 0.13 mg/g、Mg 16.04μg/g、Cu 0.29μg/g、Se 11.7μg/g、Cr 8.06μg/g,Pb未检出,较电渗析前均有降低。电渗析对脱除褐藻胶的海带酶解液进行脱盐纯化处理,可有效地分离纯化出岩藻聚糖硫酸酯。     

不同盐度激活液、K+浓度及保存时间对日本鳗鲡精子活力的影响

在6种不同盐度(34、32、30、28、26和22)激活液、3种不同K+浓度(25 mmol/L、30 mmol/L和35 mmol/L)稀释液和不同保存时间(0 h、24 h、48 h、72 h和96 h)条件下,对日本鳗鲡(Anguilla japonica)精子的活力进行观察和测定.结果表明,激活液盐度为30时精...     

拟穴青蟹卵巢蛋白质双向电泳的样品制备方法

为探索适用于拟穴青蟹卵巢蛋白质双向电泳的样品制备方法,比较了TCA/丙酮沉淀法和3种裂解液抽提法对总蛋白的提取效果,4种方法分别得到133、369、306和417个蛋白点。采用裂解液Ⅲ(7 mol/L尿素、2 mol/L硫脲、4% CHAPS、40 mmol/L Tris-base、65 mmol/L DTT、2% IPG Buffer、Protease in hibitor cocktail、1 mmol/L EDTA)提取蛋白所得图谱效果最好,背景清晰,蛋白点最多。     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.     

Effects of cortisol on hepatic carbohydrate metabolism and responsiveness to hormones in the sea raven,Hemitripterus americanus

The sea raven, Hemitripterus americanus, is a sit-and-wait, low metabolic rate, marine teleost. The objective of this study was to determine i) whether cortisol implantation (50 mg. kg^-1) for 7 days altered hepatocyte metabolism, and hepatocyte responsiveness to epinephrine, glucagon and insulin, and ii) whether 8 weeks of food-deprivation modified the above response. Cortisol implantation significantly increased hepatocyte total glucose production and oxidation from alanine compared to the sham group. There was no cortisol effect on glycogen breakdown, suggesting that the activation of other pathways, including gluconeogenesis, are required to account for the increased glucose production. Epinephrine-mediated (10^-5M) glycogen breakdown and insulin-mediated (10^-8M) total glucose production were enhanced in hepatocytes of cortisol implanted sea ravens, but there were no change in any glucagon (10^-7M) effects. The enhanced glycogen breakdown in the absence of similar increases in total glucose production with epinephrine indicates mobilization of carbohydrate reserves for endogenous use by the liver. Food-deprivation for 8 weeks significantly decreased condition factor, plasma cortisol concentration and liver glycogen content in the sea raven, but had no effect on plasma glucose concentration. Hepatocyte total glucose production and flux rates from alanine increased significantly with food-deprivation. Moreover, food-deprivation increased responsiveness of total hepatocyte glucose production to the actions of glucagon and insulin, but not to epinephrine; none of these effects were modified by cortisol implantation. Our results indicate that cortisol in the sea raven exerts both a direct and an indirect or permissive effect on hepatocyte metabolism by modifying hepatocyte responsiveness to epinephrine and insulin stimulation. Cortisol implantation did not modify the effects of glucagon or food-deprivation in this species.