一种海藻内生真菌二萜生物碱类化合物的应用

本发明涉及杀虫剂领域，具体地说是一种海藻内生真菌二萜生物碱类化合物及其制备和应用。具体结构式如（I）所示，其制备方法为将米曲霉（Aspergillus oryzae）cf-2接种于培养基中静止发酵，发酵液经乙酸乙酯萃取浓缩，菌丝体用有机溶剂提取，再经乙酸乙酯萃取浓缩，合并浓缩物，得到粗提物；粗提物进行硅胶柱层析，用有机溶剂进行梯度洗脱，收集洗脱液，洗脱液经薄层层析检测；将以洗脱液体积比5-8：1梯度的洗脱组分进行凝胶柱层析、硅胶柱层析和薄层层析分离纯化得目标化合物。本发明获得的海藻内生真菌二萜生物碱类化合物，经杀虫活性实验得出化合物在100微克/毫升时对卤虫的致死率为42.9%。

一种海藻内生真菌二萜生物碱类化合物及其制备和应用

本发明涉及杀虫剂领域，具体地说是一种海藻内生真菌二萜生物碱类化合物及其制备和应用。具体结构式如（I）所示，其制备方法为将米曲霉（Aspergillus oryzae）cf-2接种于培养基中静止发酵，发酵液经乙酸乙酯萃取浓缩，菌丝体用有机溶剂提取，再经乙酸乙酯萃取浓缩，合并浓缩物，得到粗提物；粗提物进行硅胶柱层析，用有机溶剂进行梯度洗脱，收集洗脱液，洗脱液经薄层层析检测；将以洗脱液体积比10-15：1梯度的洗脱组分进行凝胶柱层析、硅胶柱层析和薄层层析分离纯化得目标化合物。本发明获得的海藻内生真菌二萜生物碱类化合物，经杀虫活性实验得出化合物在100微克/毫升时对卤虫的致死率为61.9%。

海藻内生真菌二萜生物碱类化合物及其制备和应用

本发明涉及杀虫剂领域，具体地说是一种海藻内生真菌二萜生物碱类化合物及其制备和应用。具体结构式如（I）所示，其制备方法为将米曲霉（Aspergillus oryzae）cf-2接种于培养基中静止发酵，发酵液经乙酸乙酯萃取浓缩，菌丝体用有机溶剂提取，再经乙酸乙酯萃取浓缩，合并浓缩物，得到粗提物；粗提物进行硅胶柱层析，用有机溶剂进行梯度洗脱，收集洗脱液，洗脱液经薄层层析检测；将以洗脱液体积比5-8：1梯度的洗脱组分进行凝胶柱层析、硅胶柱层析和薄层层析分离纯化得目标化合物。本发明获得的海藻内生真菌二萜生物碱类化合物，经杀虫活性实验得出化合物在100微克/毫升时对卤虫的致死率为42.9%。

一种天然海藻内生真菌二萜生物碱类化合物及其制备和应用

本发明涉及细菌抑制剂领域，具体地说是一种天然的海藻内生真菌二萜生物碱类化合物及其制备和应用。具体结构式如（I）所示，其制备方法为将米曲霉（Aspergillus oryzae）cf-2接种于培养基中静止发酵，发酵液经乙酸乙酯萃取浓缩，菌丝体先用有机溶剂提取，再经乙酸乙酯萃取浓缩，合并浓缩物，得到粗提物；粗提物进行硅胶柱层析，用有机溶剂进行梯度洗脱，收集洗脱液，洗脱液经薄层层析检测；将以洗脱液体积比0-10：100梯度，洗脱下的组分进行凝胶柱层析、硅胶柱层析和薄层层析分离纯化得目标化合物。本发明所得二萜生物碱类化合物具有显著的抑菌活性。

一种天然海藻内生真菌次生代谢产物二萜生物碱类化合物的应用

本发明涉及杀虫剂领域，具体地说是一种海藻内生真菌次生代谢产物二萜生物碱类化合物的应用。所述海藻内生真菌次生代谢产物二萜生物碱类化合物具有杀虫作用，海藻内生真菌次生代谢产物二萜生物碱类化合物如式（I）所示；本发明通过分离于海洋红藻异管藻的真菌米曲霉（Aspergillus oryzae）cf-2发酵经提取、分离获得的二萜生物碱类天然化合物，经杀虫活性实验得出此二萜生物碱类化合物在100微克/毫升时对卤虫的致死率为74.2%。

绞股蓝离体培养中影响皂甙产量的几个因素研究

绞股蓝(Gynostemma pentaphyllum)系葫芦科绞股蓝属植物，药用价值广泛，但其野生资源日趋减少，绞股蓝主要药效成分为绞股 蓝皂甙。利用组织和细胞培养生产绞股蓝皂甙是合理开发利用和保护绞股蓝资源的可能途径之一。本文对绞股蓝组织培养中培养基 的蔗糖和激素的组成以及各种胁迫条件：渗透压、重金属离子、真菌诱导物等对皂苷产量的影响进行了初步研究。其中，渗透压、 重金属离子、真菌诱导物对绞股蓝愈伤组织皂甙产量的影响尚未见报道。1. 蔗糖对绞股蓝愈伤组织之生长影响显著，2,4-D对绞股 蓝愈伤组织皂甙含量、产量影响显著。增加蔗糖用量，减少2，4-D的用量可提高皂甙产量。2. Mn++ 用量的提高抑制绞股蓝愈伤组 织的生长，但可促进皂甙含量、产量的提高。Mn++用量提高至MS培养基的20-30倍时可使皂甙产量增加近一倍，而提高Cu++浓度的 作用不明显。3. 甘露醇用量增加抑制绞股蓝愈伤组织的生长，但可使皂甙含量、产量提高。0.680mol·l-1甘露醇可使皂甙产量提 高83%，而Nacl较大抑制愈伤组织的生长并使皂甙产量降低。4. 米曲霉粗提物对绞股蓝愈伤组织生长先略微促进，然后抑制，而根 霉粗提物则使愈伤组织生长受抑制；两者对皂甙含量、产量的作用相似：在较低浓度范围内升高，然后下降。米曲霉粗提物可提高 产量一倍，根霉粗提物可提高42%。这些结果为高产细胞系的筛选和生长、生产培养条件的优化积累了资料。在综述部分，对植物 细胞培养中组织和器官分化、细胞结构变化、生化水平的变化与次生物合成和积累的关系作了讨论。Gynostemma pentaphyllum blongs to Gynostemma, Cucurbitaccae. It has a wide medical use, but its wild resource is threatened by people's excessive use. Its effective medical components are gypenosides. For reasonable use and protect its resource, it is a possible way to product gypenosides by plant tissue and cell culture. This paper has a primary study on the components of sucrose and hormones and a variety of stress conditions: osmostic pressure, heavy metal ion, fungal elicitors in the medium for the calli culture. The effects of osmostic pressure, heavy metal ion and fungal elicitors on the calli of Gynostemma pentaphyllum have not been reported. 1. Sucrose had a significant effect on the growth of the calli, 2,4 D had notable effects on the gypenosides content and production of the calli. Increased the concentration of sucrose and decreased the concentration of 2,4 D improved the production of gypenosides. 2. Increased the concentration of Mn++ inhibited the growth of the calli, but improved the content and production of gypenosides. The optimum concentration was 20-30 times as MS medium which improved the production 100%. Increased the concentration of Cu++ had not a notable effect. 3. Increased the concentration of mannitol inhibited the growth of the calli, but improved the content and production of gypenosides. The optimum concentration was 0.680mol·l-1 which improved the production 83%. Nacl apparently inhibited the growth of the calli and decreased the production of gypenosides. 4. The crude preparation of Aspergillus oryzae inhibited the growth of the calli that in low concentration. The crude praparation of Rhizopus formosensis inhibited the growth of the calli throughout. Their effects on the content and production of gypenosides are alike, but the former is higher than the latter. On the optimum concentration, each crude preparation improved the production 100% (Aspergillus oryzae), 42%(Rhizopus formosensis). These results has accumulated some informantion on the select of high yield cell strains and choose the best culture conditons for the growth and gypenosides product of the calli. In the review, it is discussed that the differentiation on tissue-organal, cellular and biochemical levels related to the synthesis and accumulation of secondary metabolites in plant culuture.

微生物酶法拆分消旋芳香族氨基酸的研究

通过单因子和多因子摇瓶正交试验，确定了米曲霉液态发酵产氨基酰化酶的最佳发酵条件。优化发酵培养基组成（ρ/g L-1）： 葡萄糖40,蔗糖10,可溶性淀粉20,蛋白胨2.5,马铃薯液1 000mL, pH自然。培养基装量50mL/250mL三角瓶，接种量4%。培养温度30℃，转速100 rmin-1，发酵时间42h。每50mL培养物的总酶活由优化前的2627U提高到7338U，是优化前的2.79倍。 研究了米曲霉氨基酰化酶的部分酶学性质，该酶催化反应的最适pH为7.0，最适温度为40℃，低浓度的Co2+（5×10-4mol/L）对酶活激活作用显著，催化反应过程中，底物浓度大于0.2 mol/L时，存在高浓度底物抑制酶活力现象。 初步探索了包埋法固定化米曲霉氨基酰化酶的载体，在实验的五种载体中，以海藻酸钠为载体包埋固定化米曲霉氨基酰化酶酶活保留率高，且操作简单，成本低廉。对包埋法固定化米曲霉氨基酰化酶酶学性质进行了研究，较游离米曲霉氨基酰化酶，最适温度未发生改变，最适pH向碱性范围偏移至8.0，对酸碱和热的稳定性增强，最适底物浓度增大到0.4 mol/L。 根据氨基酰化酶能立体专一水解L-氨基酰化物的特点，利用米曲霉氨基酰化酶对消旋苯丙氨酸进行了拆分。在米曲霉氨基酰化酶选择性的作用于底物N-乙酰-L-苯丙氨酸，得到L-苯丙氨酸后，通过732阳离子树脂和结晶法分别将L-苯丙氨酸和N-乙酰-D-苯丙氨酸分离，N-乙酰-D-苯丙氨酸通过酸水解脱去乙酰基得到D-苯丙氨酸，拆分得到光学纯度为98%的L-苯丙氨酸（收率84.8%）和光学纯度为92.3%的D-苯丙氨酸（收率89.5%）。 separate factors tests and orthogonal experiments,the optimum fermentation conditions of aminoacylase –producing Aspergillus oryzae were determined, as follows（ρ/g L-1）,glucose 40,sucrose 10,soluble starch 20,peptone 2.5，potato juice 1000ml, inoculation volume 4%and fermentation temperature 30℃,rotation speed 100rmin-1.The highest total enzyme activity ,7338μ,was obtained after fermentation for 42 h, increased by 279% compared with the original value of 2627μbefore optimization. We dicussed partial characteristics of aminoacylase. The optimal pH and temperature of aminoacylase were 7.0 and 40℃ respectively. Low- concentration Co2+ (5×10-4mol/L）activated the aminoacylase remarkably while high-concentration substrate lowered the aminoacylase . Five vectors has been used for immobolizing the enzyme and calcium alginate showed to be the best one for it had the slightest influence on the enzyme activity, easy to operate ,and low in price, comparing with other fours. The enzymatic charateristic study showed that its optimum temperature didn’t change, but the optimum pH and substrat concentration were higher after immobilization. The stability of immobolized enzyme to acid, alkaline and heat rised as well. The aminoacylse from Aspergillus oryzae was used to resolute racemic phenylalanine to obtain D-phenylalanine. After catalyzing process, we took two methods to separate D-phenylalanine .In end,L-phenylalanine was obtained with 98% optical purity in 84.8% yield, D-phenylalanine was obtained with 92.3% optical purity in 89.5% yield.

泸州老窖古酿酒作坊空气微生物分布特征研究

本文主要研究了泸州老窖古酿酒作坊内外环境空气真菌和空气细菌的群落结构和分布特征。结果如下： 作坊内外环境空气微生物浓度差别显著，并随季节变换而变化，春、夏季微生物浓度较高，秋、冬季较低，空气真菌在夏季达到最高，细菌在春季最高。 古作坊内外环境检测到的真菌均为16 属，但优势菌属不同，作坊外的优势菌属为青霉属(Penicillium)、曲霉属(Aspergillus)、无孢菌(non-sporing)、枝孢霉属(Cladosporium)和链格孢属(Alternaria)；而作坊内优势菌属为曲霉属、青霉属、酵母菌(Yeast)、无孢菌，作坊内还含有较高浓度的根霉属（Rhizopus）、毛霉属（Mucor）、短梗霉属（Aureobasidiu），枝孢霉属和链格孢属等，曲霉属、酵母菌、根霉属、毛霉属为古酿酒作坊重要的酿酒真菌，青霉属、链格孢属为酿酒不利菌群。对古作坊内曲霉属进行了初步鉴定，主要是小冠曲霉（A.cristatellus）、米曲霉（A.oryzae）、黑曲霉（A.niger）和白曲霉（A.cadidus）。 空气细菌10 属21 种，作坊内外环境的优势菌属均为芽孢杆菌属（Bacillus）、微球菌属(Micrococcus)、葡萄球菌属(Staphylococcus)、假单胞菌属(Pseudomonad)，其中芽孢杆菌属在作坊内占有绝对的优势，浓度比在40℅以上，是古酿酒作坊重要的酿酒细菌，另外还检测到较高浓度的乳酸杆菌（lactobucillus），这类菌容易使酒味发涩发苦，为酿酒不利菌。 作坊内外环境空气微生物表现出明显的交流现象。作坊内，青霉属、枝孢霉属、链格孢属、葡萄球菌属等杂菌占有一定比例；而在作坊外,芽孢杆菌属、曲霉属、根霉属(Rhizopus)、酵母菌等处于相对较高水平，绿化环境较好的营沟头作坊内的短梗霉属，枝孢霉属和链格孢属等杂菌含量低于什字头和新街子作坊。 The community structure and distribution characteristic of airborne microbes was investigated in ancient brewage workshops of luzhoulaojiao. The results are as follows: The concentration of airborne microbes was different in interior and exterior environment of ancient workshops, and also varied by seasons. microbial concentration was higher in spring and summer, and lower in fall and winner. The highest levels of airborne bacteria was in spring, but the fungal’s in summer. The identified genus of fungi were 16 in interior and exterior environment of the ancient workshops. But the dominant genus were different , The advantage genus in the interior were Aspergillus, Yeasts, Penicillum and Nonsporing and in the exterior were Penicillum, Nonsporing, Cladosporium, Aspergillus and Aureobasidiu. Rhizopus ,mucor, Aureobasidiu, Cladosporium, Alternaria and all also were at a higher level. Among these, Aspergillus, Yeasts, Rhizopus ,mucor are important vintage flora . Penicillum, Alternaria do harm to vintage. Aspergillus of ancient workshops was identified , the preponderant aspergillus species were A.cristatellus, A.oryzae, A.niger and A.cadidus in ancient brewage workshops. 10 genus 21 species bacteria were identified, the advantage genuses among the interior and exterior of the three workshops were bacillus, microccus, Staphylococcus Pseudomonas. Bacillus, which account for beyond 40℅ of the total bacteria concentration in all sampling pots, was the most dominant genus. Lactobacillus was identified at a high level in ancient workshops, it makes spirit taste bitter and astringent. So it is not a kind of good bacterium for vintage. The fungus in the interior and exterior atmosphere characterized intercommunion phenomenon. Obviously, the concentration of profitless fungus such as Penicillum, Cladosporium, Alternaria appeared in the interior, and the fungus such as Bacillus, Aspergillus, Rhizopus and Yeasts in the exterior were at a relatively high level. the harmfull fungus in yinggoutou workshops such as Aureobasidiu, Cladosporium, Alternaria and all were lower than shenzitou and xinjiezi workshops.