琼胶降解菌AT-22的筛选和产酶性质

从青岛近海的红藻(Gelidium amansii)中分离筛选到1株高活力的海洋琼胶降解菌AT-22，对其生长、产酶特性和酶活力影响因素进行初步研究。结果表明，AT-22所产琼胶酶为诱导酶，0．2％葡萄糖的添加对菌株产酶有抑制作用。该酶作用的最适pH值为6．0-7．0，最适温度为40℃，最适底物质量分数为1％～1．2％，Ca^2 的添加对酶促反应有较强的促进作用，而Fe^3 、Mn^2 、Cu^2 和Hg^2 等有不同程度的抑制作用。     

褐藻胶裂解酶产生菌的发酵优化研究

以筛选的羊栖菜腐败菌系不动杆菌属X8菌株为研究对象,对其产褐藻胶裂解酶的发酵培养条件进行优化研究.结果表明,该菌株最适液体培养基组成为(m/V):0.6%褐藻酸钠,0.7%蛋白胨,0.2% KH_2PO_4,0.2 % NaCl,初始pH 7.0.该菌株于25 ℃,150 r/min 振荡培养16 h后,褐藻胶裂解酶酶活可达157.32 U/ml,较优化前提高了1.59倍.Mg~(2+)、Ca~(2+)和Fe~(2+)对菌株产酶有一定的抑制作用.     

海洋细菌DT-7产琼胶酶的培养基组分优化

以海洋细菌Brevundimonas sp. DT-7为实验菌株，通过单因子试验，确定了培养基主要成分的优化范围，再利用SAS8.2统计软件的筛选试验设计筛选出三个显著因子X1(琼脂粉)，X4(CaCl2)，X8(蛋白胨)，然后采用响应面分析法进行实验设计，最后通过响应面回归过程进行数据分析，建立了二次响应面回归模型，得出优化后培养基组分(w/v)：琼脂粉 0.536%，氯化钠 2.5%，磷酸高铁 0.1%，氯化钙 0.052%，磷酸氢二钾 0.1%，七水硫酸亚铁 0.03%，酵母膏 1%，蛋白胨 0.484%；经培养基组分优化后，在250ml三角瓶装液量为50ml、初始pH 7.5、摇床转速为120r•min-1、培养温度为23±1℃、培养28h的条件下，菌株产酶活力从优化前的372.0 U•ml-1提高到503.3 U•ml-1，酶活力为前者的1.35倍，从而为进一步研究琼胶酶产生菌产业化提供了基础参数。     

一株海洋过氧化氢酶高产菌的鉴定及产酶条件优化

对来自青岛近海海域底泥的一株产过氧化氢酶菌株YS0810进行形态学观察、16S rDNA序列同源性分析及生理生化特性的鉴定,在250 ml摇瓶中进行发酵产酶条件优化。初步确定该菌属于不动杆菌属Acinetobacter。发酵培养的最佳碳、氮源分别为蔗糖20 g/L和蛋白胨15 g/L,无机盐MgSO4·7H2O、NaCl、KH2PO4最佳浓度分别为0.9、5.0和1.0 g/L;菌株在培养基起始pH=7.0、4%接种量、50 ml装液量和25℃的条件下发酵24 h获得较高的酶产量。在最佳培养条件下酶产量为2469 U/ml,是优化前的5倍。     

工厂化循环水养殖条件下云纹石斑鱼消化道产酶菌的分离鉴定

采用16S r DNA-PCR菌群分离鉴定的方法,对循环水养殖条件下云纹石斑鱼(Epinehelus moara)幼鱼的胃、幽门盲囊、前肠、中肠和后肠的菌群结构进行了鉴定,用产酶菌筛选培养法对产消化酶的菌株进行了分离鉴定,并测试了各菌株消化酶的活力。研究发现,云纹石斑鱼幼鱼消化道内可培养的主要菌群为假单胞菌属(Pseudomonas)、微小杆菌属(Exiguobacterium)、不动杆菌属(Acinetobacter)、寡养单胞菌属(Stenotrophomonas)和葡萄球菌属(Staphylococcus),其中产消化酶的菌株占可培养菌的55.6%。在产酶菌中,同一株菌产3种酶的有5株;产2种酶的有9株;中肠和后肠的菌株数最为丰富,胃次之,幽门盲囊和前肠菌群种类较少;产脂肪酶的菌株都集中在中肠。产消化酶的菌株主要以产蛋白酶和淀粉酶为主,且产酶量丰富,产蛋白酶活力最高达(87.732±1.134)U/m L;淀粉酶活力为(77.176±0.599)~(73.458±0.574)U/m L;产纤维素酶的菌仅一株,且酶活力较低。分析得知,消化道的菌群结构直接影响了外源性消化酶的种类与活性。本研究为工厂化循环水养殖条件下产酶有益菌的筛选提供了理论依据。     

一株产几丁质酶菌株的筛选鉴定与产酶条件优化

几丁质酶在降解几丁质和生物防治上具有重要作用。采用平板筛选法从对虾养殖池塘底泥中筛选几丁质酶产生菌株,以培养时间、培养温度和胶体几丁质含量为自变量,响应面法优化该菌株的产酶条件,建立二次回归方程。试验结果表明,通过菌株的菌落形态、亚显微形态特征和16S rDNA序列同源性分析,确定该菌株为蜂房芽孢杆菌。3个因素对菌株产酶的影响依次为培养时间培养温度胶体几丁质含量。菌株的最佳产酶的条件为:培养基中胶体几丁质含量1.62%,培养温度40℃,培养时间72h,在此条件下,菌株产生的几丁质酶活力为13.07U/mL。该研究为筛选菌株的进一步利用提供科学依据。     

虾壳的微生物转化和菌种的筛选

从水产品加工场长期堆放虾壳的土壤中,分离出一株能分解虾壳有较高活力的菌株。对该菌株(ST90-5)进行了形态、培养特征、生理生化特性等鉴定,此菌为放线菌(Streptomyces sp.)。这一菌株是用甲壳质为唯一碳源的筛选培养基中筛选出来,培养基成份为Na_2HPO_4、KH_2PO_4、NH_4NO_(?)、MgSO_4·7H_2O、FeSO_4·7H_2O、CaCl_2、甲壳质、微量金属盐。该菌株产酶最适条件为pH7、温度30℃、120r/min旋转摇床培养4—7天。经过发酵的培养液测定β-N-乙酰氨基葡萄糖苷酶和β-N-乙酰氨基半乳糖苷酶的活性。     

野生银鲳消化道内潜在产酶益生菌产酶条件的初步研究

采用酶学分析法研究了单因子(温度、p H、发酵时间)变化对野生银鲳(Pampus argenteus)消化道内同时分泌蛋白酶、淀粉酶、纤维素酶和脂肪酶中两种以上的5株菌株产酶活力的影响,即通过测定酶活力的大小找到每株菌最适产酶条件范围。结果显示,在温度31~37℃、p H 7~8、发酵时间3 d的条件下,5株产酶菌的蛋白酶活力最大;在不同温度和p H条件下,5菌株分泌的淀粉酶活力各不相同,但都随发酵时间的延长而增大,3~5 d达最大值;4株分泌纤维素酶的菌株纤维素酶活力受环境影响总体变化不大,在温度31~37℃、酸性条件(p H 5)和发酵时间4 d的条件下为活力最大;2株分泌脂肪酶的菌株在28~31℃、中性(p H 7)、发酵时间1 d条件下其脂肪酶活力最大,最大可接近180 U·m L-1,但活力都随发酵时间而显著下降。结果表明,不同产酶菌所分泌的消化酶活力所需的最适条件有很大不同,了解和掌握银鲳肠道中菌群的产酶条件,对开发潜在产酶益生菌有着积极的作用。     

野生与养殖银鲳消化道菌群结构中产酶菌的对比分析

实验对野生和养殖银鲳胃、幽门盲囊、前肠、中肠、后肠菌群结构进行了定性对比分析,并对产蛋白酶、淀粉酶、脂肪酶、纤维素酶的菌株进行了鉴定。结果发现,野生和养殖种群的消化道菌群结构存在较大差异,且同一种群消化道各部分之间菌群结构也存在较大差异。尽管养殖和野生银鲳均在幽门盲囊中具有最多的可培养细菌菌株,但野生银鲳消化道内主要菌群为嗜冷菌属(Psychrobacter)和Pseudochrobactrum,养殖银鲳消化道主要菌群为不动杆菌属(Acinetobacter)和假单孢菌属(Pseudomonas),两种群共有细菌仅一株,即Psychrobacter piscatorii strain VSD503,但其分别存在于野生与养殖种群银鲳消化道的不同部位。在产酶菌株筛选中发现,野生银鲳消化道内分离到16株产酶菌,其中44%可培养菌能产蛋白酶,56%能产淀粉酶,11%能产脂肪酶,56%能产纤维素酶,部分菌株可产2株以上的消化酶,其中产3种以上酶的菌株有5株,且产酶量丰富。相对于野生银鲳,养殖银鲳消化道内分离到22株产酶菌,主要以产蛋白酶和淀粉酶为主,70%可培养菌可产蛋白酶,21%可产淀粉酶,仅Bacillus thuringiensis strain VITGS可产纤维素酶,无一株菌产脂肪酶,其中只有Bacillus thuringiensis strain VITGS产3种酶但产酶量相对较少。研究可为银鲳人工养殖中潜在益生菌的筛选提供理论依据。     

一株产低温碱性蛋白酶嗜冷海洋细菌YS-9412-130的分离和培养条件研究

自海水贝类中取样，经分离培养基和酪蛋白培养基复合培养，用检测蛋白酶产生水解圈和Folin-酚碱性蛋白酶活性测定相结合的方法从1000多份样品中初步筛到了产碱性蛋白酶活力高、性能优良的菌株YS－9412－130。对其初步研究结果表明，该菌只能利用有机氮源生长；在接近自然海水盐度、温度20℃、pH在7.0-9.0条件下，菌体生长和产酶较好。     

鲍琼胶酶高产细菌的筛选和鉴定

从江蓠中分离筛选出两株琼胶酶高产菌,均为革兰氏阴性菌,呈细杆状。通过API条带分析,它们除了在对柠檬酸盐的利用上存在差别外,其它的生理生化特征基本相同,鉴定为多食鞘氨醇杆菌(Sphingobacteriummultivorum),其可信度分别是98.7%和99.5%。     

皱纹盘鲍脯氨酰内肽酶的分离纯化及性质

脯氨酰内肽酶(Prolyl Endopeptidase,PEP)广泛分布在动植物和微生物体内,参与细胞生长和代谢调控。PEP在哺乳动物生殖器官的生长发育过程中起着重要作用,但贝类PEP与性腺发育是否有关尚未有相关报道。本研究以皱纹盘鲍为对象,采用荧光定量PCR技术(qRT-PCR)在性腺检测到有最高的基因表达量和较高的酶活性。通过硫酸铵分级沉淀和连续柱层析,从皱纹盘鲍性腺中分离纯化得到分子量约为82 ku,等电点约为5.5的PEP,其最适温度和最适pH分别为25 °C和6.0,在15~25 °C和pH 5~8能够保持较高的酶活性。利用LC/MS/MS分析纯化蛋白,得到182个氨基酸残基,与皱纹盘鲍脯氨酰内肽酶一致。圆二色谱分析显示,温度对PEP二级结构有较大的影响,拟合热变性温度为51.4±0.2 °C。为了进一步探究PEP在鲍鱼性腺发育过程中的作用,对不同发育时期(生长初期、前期、中期和后期)的雌雄鲍鱼,采用免疫印迹(Western Blot)和qRT-PCR分析性腺中PEP的蛋白和基因表达水平。结果表明,PEP在雄性和雌性性腺的各个时期均能被检测到,在雄性性腺的成熟中期和雌性性腺的成熟后期表达量最高。PEP在鲍鱼性腺不同发育时期时表达量的差异表明其可能参与到性腺发育的过程。     

Colonization and probiotic effects of lactic acid bacteria in the gut of the abalone Haliotis gigantea

Strains of lactic acid bacteria (LAB) were isolated from several different sources and evaluated in vitro for potential probiotic effects in abalones. Two isolates (Lactobacillus sp. strain a3 and Enterococcus sp. strain s6) were highly resistant to bile salt and/or gastric juice and inhibited the growth of three abalone pathogens (Listonella anguillarum, Vibrio harveyi, and V. carchariae). Each of the LAB isolates was used to supplement diet of the abalone Haliotis gigantea for a period of 3 weeks. One group of animals received Lactobacillus sp. strain a3 added to commercial dry feed, one group received Enterococcus sp. strain s6 added to the feed, and a control group received only standard commercial feed. Culturable LAB counts of gut homogenates indicate the a3 colonized in the gut of abalones. Digestive enzyme activities and the concentrations of a number of volatile short-chain fatty acids (VSCFA) were elevated in the gut of abalones receiving feed supplemented with the two LAB strains. These results indicate that dietary supplementation can enable LAB colonization or persistence in the gut of abalone species and can potentially enhance probiotic effects.     

Physiological responses of pink abalone Haliotis corrugata (Gray, 1828) exposed to different combinations of temperature and salinity

Physiological responses of pink abalone Haliotis corrugata were determined under different temperature and salinity conditions. Oxygen consumption rate was not affected by temperature and salinity. Ammonium excretion of pink abalone was inversely related to salinity. The O:N ratio indicated that abalone maintained in lower salinities had an interval of 4.9–7.7, which is indicative of a protein‐dominated metabolism, whereas the O:N in 35‰ was 28.8–35.5 for both temperatures, suggesting that carbohydrates were used as energy substrate. Haemolymph osmolality of abalone exposed to 20 and 24 °C was slightly hyperiso‐osmoconformic in salinity ranges of 20–35‰. The results of this study suggested that for optimized culture, pink abalone should be cultivated at 24 °C at a salinity of 35‰.     

Status of the digestive gland and feed index in juvenile green abalone Haliotis fulgens fed rehydrated macroalgae

One of the bottlenecks in cultivating juvenile green abalone Haliotis fulgens is the lack of well‐adapted natural or formulated food for optimal growth. The goal of this study was to analyse the digestive gland structure of juvenile green abalone fed rehydrated natural feed, Ulva sp. (Chlorophyta), Eisenia arborea, Macrocystis pyrifera, Egregia menziesii (Phaeophyta) and Porphyra perforata (Rhodophyta), using histochemical techniques. Structure of the digestive gland was described, and proteoglycan granules were detected in the digestive cells. The abundance of granules was variable, depending on the feed provided to the abalone, and this was reflected in their growth. Granular content in digestive cells fed Ulva sp. was scarce, leading to low growth rate and high feed conversion ratio (FCR). Digestive cells of juveniles fed E. menziesii led to the best nutritional condition, including many proteoglycans cellular granules, best weight growth rate and a low FCR. Histochemical analysis of the digestive gland, differentiated by a modified Goldner trichrome method that included Alcian blue, was a useful tool for determining the nutritional status of farmed abalone, therefore recommended for assessing adjustments to the natural feed or formulation to meet the nutritional needs of abalone.     

Alanine racemase activity in the microalga <Emphasis Type="Italic">thalassiosira</Emphasis> sp.

ABSTRACT:   In this paper, the authors report the detection of alanine racemase activity in the marine diatom Thalassiosira sp. Since the Thalassiosira sp. was cultured under germ-free conditions, it appeared that D-alanine was not derived from bacteria but was produced through catalysis by algal alanine racemase. The rate of conversion of L-alanine to D-alanine was approximately the same as that for the reverse reaction, and the enzyme catalyzed the equilibration of the D- and L-forms. The crude enzyme preparation obtained from the cells at the stationary phase of the growth cycle had an optimal pH of approximately 9.5. The Lineweaver–Burk analysis showed that the K _m for D- and L-alanine was 16.5 mM and 29.4 mM, respectively. It appears that the enzyme is highly specific for D- or L-alanine because it does not catalyze the racemization of other amino acids. In addition, after gel filtration, the enzyme did not require exogenous pyridoxal 5'-phosphate (PLP) for its activity, however, the effects of several chemicals suggest that the enzyme may be PLP-dependent. The enzyme is more similar to that found in invertebrates when compared with that found in bacteria. This is the first report on the occurrence of alanine racemase activity in the microalga Thalassiosira sp.     

Characterization of Pseudomonas aeruginosa associated with diseased postlarval abalone in Shenzhen, China

Outbreaks of mass mortality of postlarval abalone, Haliotis diversicolor supertexta, have occurred in south China since 2002 and have forced many abalone farms to close. About 30 representative bacterial strains were isolated from a sample of five diseased postlarval abalone, taken 25 days post-fertilization during an outbreak of postlarval disease in Shenzhen, China, in October 2006. Bacterial challenge tests showed that the predominant strain, designated as strain 22, was highly virulent to postlarvae with an LD₅₀ value of 7.8 × 10⁴ colony forming units (CFU) ml⁻¹, while four of the other isolates were weakly virulent with LD₅₀ values ranging from 1 × 10⁶ to 1 × 10⁷ CFU ml⁻¹, and the remaining 25 isolates were classified as avirulent with LD₅₀ values greater than 1 × 10⁸ CFU ml⁻¹. By means of API 20NE and 16S rDNA and ITS sequencing analyses, strain 22 was identified as Pseudomonas aeruginosa. Antibiotic susceptibility tests showed that strain 22 exhibited around 75% of susceptibility to 16 various antibiotics tested. The results of this study show P. aeruginosa as one of the bacteria involved in the mortality of abalone postlarvae in Shenzhen, China.     

皱纹盘鲍消化酶的研究

本文对人工养殖的、不同生长时期的正常鲍和病鲍的消化酶活力进行了研究。酶学分析表明，在皱纹盘鲍消化器官中存在着胃蛋白酶（酸性蛋白酶），最适ＰＨ２．８～３．５；类胰蛋白酶（碱性蛋白酶），最适ＰＨ７．８～１０；淀粉酶和纤维素酶。蛋白酶及纤维素酶随着鲍鱼的生长发育活性逐渐增强，淀粉酶逐渐减弱。肝中的酶活性高于其它器官，正常个体酶活性远高于患病个体。我们还做了酯酶的聚丙烯酰胺电泳研究。     

Use of macroalgae supplemented with probiotics in the Haliotis rufescens (Swainson, 1822) culture in Northern Chile

In this study, we evaluate the use of macroalgae as vectors of probiotics bacteria into the digestive tract of abalone to improve their survival and growth. It is shown that when abalone Haliotis rufescens of different sizes were fed with a natural diet composed of fronds of the macroalga Macrocystis integrifolia supplemented with a mixture of Vibrio sp. C21‐UMA, Agarivorans albus F1‐UMA and Vibrio sp. F15‐UMA bacteria, there was a significant increase (P<0.05) in the average monthly growth rate and survival (%)in a period of 210 days, compared with the control without a probiotic supplement. The permanence of the probiotics in the digestive tract of the animals was monitored, and it was found that the number of culturable C21‐UMA and F1‐UMA bacteria decreased significantly (P<0.05) in recently weaned and adult abalone, and they almost disappeared completely on day 19 of the bioassay. However, the culturable Vibrio sp. F15‐UMA disappeared completely from the digestive tract on day 22 of the bioassay, and these were the bacteria that remained at the highest concentration compared with the other two bacterial strains over the experimental period. It is therefore shown that it is feasible to use a probiotic mixture to improve the profitability of the H. rufescens culture.