米曲霉两步活化法对黄芩苷的生物转化及转化产物的纯化与鉴定

利用米曲霉把黄芩苷转化为黄芩素。转化条件为：液体发酵，pH6，30℃，利用综合马铃薯培养基与玉米芯培养基两步发酵。菌种采用两步活化法，将经综合马铃薯培养基培养48h的米曲霉加入至玉米芯培养基培养48h后，再加入底物黄芩苷继续培养3d。玉米芯发酵液加甲醇混匀后过滤，通过高效液相色谱检测，结果表明米曲霉已经将黄芩苷转化为黄芩素，转化率为40．72％。同时用乙醚萃取法从玉米芯发酵液中萃取到黄色固体提取物，经过高效液相色谱与标准品对照鉴定，该提取物为黄芩素。     

米曲霉产中性蛋白酶的适宜条件

研究了米曲霉产蛋白酶的分布,优化了米曲霉产中性蛋白酶的适宜培养条件以及培养基的最优组成。研究发现米曲霉产中性蛋白酶的能力为最强。米曲霉产中性蛋白酶的适宜培养条件为:m(麸皮)∶m(豆粕粉)=4∶1,水的质量分数为60%,培养基中各无机盐质量分数为:KNO30.2%,MgSO40.05%,Na2HPO40.13%,pH值为6.0,接种量为每10 g培养基接种1.0×108个孢子,最佳培养温度为30℃,最佳培养时间为48 h。在此培养条件下,最高酶活力达3 999.2 U.g-1。     

米曲霉中性蛋白酶特性的研究

研究了米曲霉中性蛋白酶的特性。结果表明,米曲霉中性蛋白酶的最佳提取方法为加入pH 7.2的缓冲液研匀,置恒温(30℃)培养箱中提取30 min,每10 min搅动1次,最后用4层纱布过滤。酶反应最适温度为55℃,最适pH值为7.2。NaCl和EDTA对其有一定的抑制作用。酶的热稳定性好,在45℃的温度下处理24 h后,酶活力仍有45.5%。米曲霉中性蛋白酶与培养物一起保存时,稳定性也较好,在4℃保存一个月后,酶活力几乎无变化。     

米曲霉中性蛋白酶特性的研究

研究了米曲霉中性蛋白酶的特性。结果表明，米曲霉中性蛋白酶的最佳提取方法为加入pH7．2的缓冲液研匀，置恒温（30℃）培养箱中提取30min．每10min搅动1次，最后用4层纱布过滤。酶反应最适温度为55℃，最适pH值为7．2。NaCl和EDTA对其有一定的抑制作用。酶的热稳定性好．在45℃的温度下处理24h后，酶活力仍有45．5％。米曲霉中性蛋白酶与培养物一起保存时，稳定性也较好，在4℃保存一个月后，酶活力几乎无变化。     

鱼干曲霉菌分离株鉴定及产毒特性

【目的】探明湛江市售鱼干中曲霉菌(Aspergillus spp.)分离株产AFB₁的特性。【方法】采用马铃薯-葡萄糖琼脂(PDA)对鱼干中的曲霉菌进行分离纯化,根据菌落形态、显微形态观察和ITS序列分析对分离株进行鉴定,采用液相色谱-串联质谱(LC-MS/MS)法分析菌株的AFB₁合成量。【结果】鱼干中曲霉菌的分离率高达47.66%,经形态学鉴定发现以黄曲霉(Aspergillus flavus)A03、烟曲霉(Aspergillus fumigatus)H32、杂色曲霉(Aspergillus versicolor)M23、欧式曲霉(Aspergillus europaeus)M11、棘孢曲霉(Aspergillus aculeatus)B21等为主,其中红笛鲷干(Lutjanus sanguinaus)中黄曲霉菌的含量高达1200 CFU/g。黄曲霉A03在红笛鲷干培养基中常温(27~30℃)培养21 d,产AFB₁能力达到3.8 ng/mL。【结论】鱼干中存在产AFB₁的黄曲霉菌,警示鱼干中存在AFB₁污染的风险。     

一株拮抗芒果炭疽菌海洋细菌的鉴定和发酵培养基优化

从湛江近海海水和海泥分离的229株细菌中,选出对芒果炭疽菌(Colletotrichum gloeosporioides)具有较强拮抗作用的海洋细菌BSW03,抑菌圈直径为14.0 mm。根据菌体形态、生理生化特性和16s rDNA序列分析,BSW03鉴定为枯草芽孢杆菌(Bacillus subtilis)。从9种培养基中筛选出BDPB作为BSW03的发酵培养基,通过单因子碳源、氮源试验和L9(34)正交试验对发酵培养基成分进行优化。结果表明:发酵培养基最佳配方为乳糖20 g/L,牛肉膏15 g/L,K2HPO3.3H2O 0.75 g/L,MgSO4 0.75 g/L,pH自然。转速170 r/min、温度28℃的恒温摇床培养3 d后的无菌发酵液抑菌效果最佳,抑菌圈直径为27.3 mm。     

海洋土曲霉C23-3与不同类型海洋微生物共培养对其次生代谢产物的影响

【目的】研究海洋土曲霉C23-3与不同类型海洋微生物共培养对其次生代谢产物的影响,挖掘土曲霉菌株C23-3的次生代谢潜力。【方法】将副溶血弧菌、枯草芽孢杆菌和胶红酵母的活菌或灭活菌体分别以不同时间与土曲霉C23-3共培养;采用DPPH自由基清除法和Ellman法分别评价各提取物的抗氧化和乙酰胆碱酯酶抑制活性,采用滤纸片法比较各提取物的抗菌活性,采用HPLC指纹图谱分析各提取物化学成分的差异。【结果与结论】发现不同类型的微生物与土曲霉共培养均可激活一些原本沉默的活性次级代谢产物;菌株及共培养物的化学拮抗能力、HPLC指纹图谱中的特征代谢产物变化揭示在活菌培养体系中土曲霉占竞争优势;而且灭活菌体中可能存在某些信号物质,在12 d时对土曲霉群体自感应信号分子丁内酯-I的产生具有比活菌更强的刺激作用。研究结果为深入挖掘海洋真菌代谢潜力及抗老年痴呆等活性先导化合物的发现提供基础。     

乙酰微小杆菌生物合成5-甲基尿苷的研究

用乙酰微小杆菌(Exiguobacterium acetylicum)完整细胞作为酶源,催化鸟苷和胸腺嘧啶合成抗艾滋病药物关键中间体5-甲基尿苷,并对影响菌体生长和5-甲基尿苷转化率的因素进行了考察。采用紫外诱变方法获得高表达核苷磷酸化酶菌种,使5-甲基尿苷的转化率提高到了75.8%;对培养基进行了碳源和氮源的优化,结果表明,3 g/L葡萄糖、20 g/L玉米浆、4 g/L NH4Cl有利于菌体生长;通过响应曲面(Response Surface Methodology,RSM)法获得了较为合适的转化条件:0.03 g/L MnSO4加入到pH值为8.0的磷酸缓冲液,两种底物浓度均为67 mmol/L,反应温度为56℃。     

湛江近海域沉积物含氯多环芳烃对斑马鱼胚胎的毒性研究

为探究湛江近海域海洋沉积物中含氯多环芳烃的生物学毒性效应,选取湛江近海域2个地点(TS和JSW)采集样品,以含氯多环芳烃为目标,制备沉积物提取物,将斑马鱼胚胎胚胎暴露于不同浓度的沉积物提取物中,观测供试斑马鱼胚胎的死亡和畸形情况。结果表明:TS采样点沉积物提取物的LC50为15.98 mg/mL(48 h)和14.41mg/mL(96 h),JSW采样点沉积物提取物的LC50为19.89 mg/mL(48 h)和16.36 mg/mL(96 h),且致死效应呈现浓度依赖关系;TS和JSW 2个采样点沉积物中含氯多环芳烃能导致斑马鱼胚胎卵黄囊水肿、心包水肿、脊椎弯曲等致畸效应,其致畸效应与暴露浓度基本呈浓度依赖关系。湛江近海域沉积含氯多环芳烃物提取物对斑马鱼胚胎具有明显的致死作用和致畸毒性。     

化学诱导对2株海洋真菌次级代谢产物及其生物活性的影响

【目的】以2株海洋真菌为研究对象,探究不同种类及浓度的化学表观遗传修饰剂对其次级代谢产物化学多样性和生物活性的影响。【方法】基于化学表观遗传修饰策略,采用两种不同的化学表观遗传修饰剂5-氮杂胞嘧啶核苷(5-azaC)和丁酸钠,分别对Penicillium sp.019和Aspergillus terreus ZN4-5-4两株海洋来源真菌进行表观遗传修饰。通过观察真菌在培养基中的生长状态变化,并根据薄层层析(TLC)指纹图谱、高效液相色谱(HPLC)图谱、乙酰胆碱酯酶(AChE)抑制、1,1-二苯基-2-三硝基苯肼(1,1-Diphenyl-2-picryl-hydrazyl,DPPH)自由基清除和卤虫致死活性筛选模型等技术手段,从中筛选出代谢产物丰富、AChE抑制活性与抗氧化活性产物丰富且毒性小的发酵培养条件。【结果】添加1 mmol/mL的丁酸钠诱导剂可使菌株019和ZN4-5-4的次生代谢产物的种类增加,且同时能提高该菌株次级代谢产物的AChE的抑制活性和DPPH自由基清除活性。因此,确定菌株019和ZN4-5-4的最优发酵条件均是添加浓度为1 mmol/mL丁酸钠的PDB培养基。【结论】化学表观遗传修饰策略指导下,添加诱导剂5-azaC或者丁酸钠对2株海洋真菌次生代谢产物的产生有影响。     

Ultrastructure of Single Cells, Callus-like and Monosore-like Cells in Porphyra yezoensis Ueda on Semi solid Culture Medium

It had been demonstrated that individual cells or protoplasts isolated from Porphyra thallus by enzyme could develop into normal leafy thalli in the same way as monospores, and that isolated cells develop in different way in liquid and on semi solid media. The authors observed the ultrastructure of isolated vegetative cells cultured on semi solid media and compared them with those of monospores and isolated cells cultured in liquid media. The results showed that subcellular structures were quite different among cells in different conditions. In their development, isolated cells on semi solid media did not show the characteristic subcellular feature of monospore formation, such as production of fibrous vesicles. Callus like cells formed on semi solid media underwent a distinctive modification in cellular organization. They developed characteristic cell inclusions and a special 2 layer cell covering. Golgi bodies, ER, starch grains, mitochondria. Vacuoles were not commonly found in them.     

An Investigation on Tolerance and Accumulation of a Facultative Marine Fungus Aspergillus flavus to Pentavalent Arsenic

Tolerance of a facultative marine fungus Aspergillus flavus towards As (V) was tested.Luxuriant growth of the test isolate was observed in culture media with As (V) concentrations of 25 mg L-1 and 50 mg L-1,indicating its tolerance to the metal.Accumulation rate of arsenic was always higher when exposed to As (V) at 50 mg L-1 than at 25 mg L-1.The study reveals Aspergillus flavus as a promising candidate for environmental bioremediation.Arsenic contents (mg g-1) in the fungus when exposed to 50 mg L-1 As (V) were measured as 11.1773,4.0983,and 8.0000 mg g-1 on day 3,6 and 9,respectively.The highest content was observed initially,i.e.on day 3,followed by a decline and a rise again.These results provide baseline information for further explorations regarding the exploitation of the fungus for arsenic removal.     

鱼汁、海泥抽出液和人尿对小环藻生长的影响

在室内条件下,通过单因子实验和正交实验的方法,研究了鱼汁、海泥抽出液和人尿对小环藻(Cyclotella sp.)种群生长的影响。结果表明,350 mL的培养体积下,当鱼汁添加量为2~3 mL时,小环藻生长效果最好,相对生长率(K)较对照组升高了33.3%,平均倍增时间(G)降低了34.9%,但随着浓度升高,藻细胞开始出现死亡。海泥抽出液添加量为20 mL时,K值最大,较对照组增加35.6%,G值下降了25.15%。人尿添加量为10 mL时,K值最大,较对照组增加17.9%,G值下降了15.77%。统计分析表明,鱼汁、海泥抽出液和人尿对小环藻实验种群的生长均有极显著影响。海泥抽出液对小环藻影响大,其次是鱼汁和人尿。正交实验表明,在鱼汁、海泥抽出液和人尿的添加量分别为2.5、15.0和1.0 mL的组合实验组,小环藻的生长效果最好。     

3D numerical simulation in acoustic-elastic coupled media with staggered-grid finite-difference method

Acoustic-elastic coupled media is often encountered in most marine explorations, and accurate simulation of acoustic-elastic coupled media is of great significance. At present, the study of acoustic-elastic coupled media still assumes that the solid of the acoustic-elastic coupled media is isotropic, but this assumption is not in accordance with the actual situation. In this paper, we derive the solid media of acoustic-elastic coupled media from isotropic media to anisotropic media, and propose an acoustic-elastic coupled medium based ontransverse isotropic media with vertical symmetric axes(VTI) to improve the accuracy of forward modeling. Based on the relationship between the Thomsen parameter and the coefficient matrix of the anisotropic elastic wave equation, we transform the Thomson parameter into a velocity model with anisotropic properties. We use a staggered grid finite difference method to simulate the propagation of a wavefield in a three-dimensional acoustic-elastic coupled media. We obtain the snapshots of the wave field when the solid of the acoustic-elastic coupled media is an isotropic medium and a VTI media. When the solid of the acoustic-elastic coupled media is considered VTI media, we can observe the qP wave and qS wave that cannot be observed in the isotropic medium from the wave field snapshot. We can also find that the seismic records obtained by the method we use are more realistic. The algorithm proposed in this paper is of great significance for high-precision ocean numerical simulation.     

DYNAMIC CHANGES OF INORGANIC NITROGEN AND ASTAXANTHIN ACCUMULATION IN HAEMATOCOCCUS PLUVIALIS

ＩＮＴＲＯＤＵＣＴＩＯＮＯｖｅｒｔｈｅｐａｓｔｄｅｃａｄｅ ,Ｈａｅｍａｔｏｃｏｃｃｕｓｐｌｕｖｉａｌｉｓｈａｓｂｅｅｎａｓｕｂｊｅｃｔｏｆｎｕｍｅｒｏｕｓｉｎｖｅｓｔｉｇａｔｉｏｎｓｂｅ ｃａｕｓｅｉｔｓｃｏｎｓｐｉｃｕｏｕｓｒｅｄｃｏｌｏｒｐｏｉｎｔｓｔｏｉｔｓｐｏｓｓｉｂｉｌｉｔｙａｓａｈｏｐｅｆｕｌｒｅｇｅｎｅｒａｔｉｖｅｂｉｏｒｅｓｏｕｒｃｅｏｆａｓｔａｘａｎ ｔｈｉｎ .Ｅｘｔｅｎｓｉｖｅｓｔｕｄｙｏｆｃｕｌｔｕｒｅｆａｃｔｏｒｓ (ｓｕｃｈａｓｔｅｍｐｅｒａｔｕｒｅ,ｉｌｌｕｍｉｎａｔｉｏ…     

鱼汁、海泥抽出液和人尿对小环藻生长的影响

在室内条件下，通过单因子实验和正交实验的方法，研究了鱼汁、海泥抽出液和人尿对小环藻（Cyclotella sp．）种群生长的影响。结果表明，350mL的培养体积下，当鱼汁添加量为20mL时，小环藻生长效果最好，相对生长率（K）较对照组升高了33．3％，平均倍增时间（G）降低了34．9％，但随着浓度升高，藻细胞开始出现死亡。海泥抽出液添加量为20mL时，K值最大，较对照组增加35．6％，G值下降了25．15％。人尿添加量为10mL时，K值最大，较对照组增加17．9％，G值下降了15．77％。统计分析表明，鱼汁、海泥抽出液和人尿对小环藻实验种群的生长均有极显著影响。海泥抽出液对小环藻影响大，其次是鱼汁和人尿。正交实验表明，在鱼汁、海泥抽出液和人尿的添加量分别为2．5、15．0和1．0mL的组合实验组，小环藻的生长效果最好。