基于纤维素酶和果胶酶活力的康氏木霉固体发酵培养基配方研究

为找到廉价的优化培养基配方生产高活力的复合纤维素酶,该实验采用配方试验和双温度培养法对康氏木霉F244菌株进行固体发酵,测定滤纸酶、棉花酶、羧甲基纤维素酶、β-葡萄糖苷酶、果胶酶活力并通过SPSS软件建立回归方程,其全相关系数分别达到0.852,0.941,0.964,0.703,0.899,而后利用无约束规划求解找到复合酶生产配方.结果表明在固体培养基中Tween80对纤维素酶和果胶酶的生产均存在抑制作用,理想固体产酶培养基是稻草粉、麸皮、(NH4)2SO4及水分的混合物;复合纤维素酶生产的适宜配方为稻草粉16%,麸皮24%、(NH4)2SO4 3%、含水率57%,该配方对应的滤纸酶、棉花酶、羧甲基纤维素酶、β-葡萄糖苷酶及果胶酶的期望酶活分别达到72,129,342,112和108g.     

一株高活性纤维素降解细菌的筛选鉴定及酶学特性

利用小麦/玉米秸秆还田土壤样品,通过富集培养和刚果红平板染色法筛选分离出纤维素降解细菌XWS-12;对分离的菌株进行16S rRNA基因序列系统发育分析,初步鉴定为伯克氏菌属(Burkholderia),定名为Burkholderia sp.XWS-12。以玉米秸秆和麸皮为碳源,研究了氮源、发酵时间、初始发酵温度、培养基初始pH等条件对该伯克氏菌产纤维素酶的影响。结果显示,该菌株产纤维素酶最适氮源为硝酸钠,培养时间为60 h,培养温度为37℃,培养基初始pH为4,该菌株的CMC酶活力最高,可达25 U/ml。其粗酶液的最适反应温度为50℃,最适反应pH为5,在pH 4~8的范围内酶活力较稳定。粗酶液的热稳定较差,当温度超过50℃时,该酶活力显著下降;当温度为50℃时,保温1 h,该酶活力损失53%。     

纤维素降解菌CMC-4的分离鉴定、诱变和酶学特性研究

从秸秆还田土壤中初筛获得一株高效纤维素降解菌CMC-4,根据菌株形态、理化性质及16S rDNA序列分析,初步鉴定为地衣芽孢杆菌(Bacillus licheniformis)。以此为出发菌株,经亚硝酸钠诱变获得一株稳定高产纤维素酶突变株CMC-4-3。对CMC-4和CMC-4-3的产酶条件和酶学性质进行比较分析,结果表明:CMC-4-3纤维素酶活力较CMC-4提高67.5%;其最适产酶条件是:37℃、pH 6.0、葡萄糖为碳源、蛋白胨为氮源、接种量2.0%、装液量60 ml/250 ml。该菌株所产纤维素酶的最适反应pH为6.0,且在4.0~7.0酶活力较稳定;最适反应温度为50℃,在20~80℃范围内均保持稳定;金属离子Fe~(2+)、Mg~(2+)、Co~(2+)、K~+对酶有激活作用,其余离子均有不同程度抑制作用,而Cu~(2+)和Ca~(2+)抑制作用最强,酶活力减弱近50%,是该酶的强效抑制因子。诱变前后菌株产酶条件和酶学性质等部分表型发生了变化,而突变菌株显示出了更宽泛的环境适应范围。据此,获得一株高效产纤维素酶、耐受范围广的具纤维素降解能力的地衣芽孢杆菌,而CMC-4-3和CMC-4的表型可作为深入探讨基因型变化的线索。     

纤维素降解真菌A25-2的分离、鉴定及其产纤维素酶的酶学特性

本研究以Avicel-刚果红选择培养基为初筛培养基,从云南哀牢山国家级自然保护区和广西猫儿山国家级自然保护区的土壤样品中分离筛选得到4200株真菌,从中筛选出透明圈与菌落直径比较大、透明程度较为清晰的12个菌株。通过液体培养发酵,测定其上清液中的羧甲基纤维素酶活力、滤纸酶活力和Avicel酶活力,最终筛选出一株产该三种酶且其活力均最高的真菌菌株A25-2。通过对菌株A25-2形态学观察和其内转录间隔区（internal transcribed spacer,ITS）序列同源性比对分析,将菌株A25-2鉴定为哈茨木霉（Hypocrea lixii）。酶活测定结果表明菌株A25-2产纤维素酶的酶活力较高,在最适作用pH4.5和最适作用温度55℃下,其羧甲基纤维素酶活力为2.26IU/mL,滤纸酶活力为0.58IU/mL,Avicel酶活力为0.39IU/mL。薄层层析实验表明A25-2具有完整的纤维素酶系统。因此,真菌A25-2可作为饲料加工等生产和纤维素酶相关研究的备选菌株。     

N+注入选育漆酶高产菌株及其产酶优化研究

以糙皮侧耳Pleurotus ostreatus WY01为出发菌株,通过N+注入诱变处理担孢子、RBBR-PDA平板变色法初筛、ATBS法测定漆酶活性复筛,获得1株漆酶高产诱变菌株ADW-08。用高碳低氮无机盐制备的油菜秸固体培养基（SM）培养,其峰值酶活力比出发菌株高出2.80倍,达7.78 U/g,且产酶稳定。对ADW-08固体培养产酶条件的研究表明,以葡萄糖为碳源明显优于蔗糖、麦芽糖、麸皮和可溶性淀粉;酒石酸铵较有利于ADW-08漆酶的分泌;适宜初始pH为5.0或6.0;ABTS和藜芦醇对产酶均有明显的诱导作用,而吐温-80对产酶有一定的抑制作用;正交试验结果表明,葡萄糖、酒石酸铵和pH最佳参数分别为15.0g/L、0.2g/L和5.2,酶活峰值为8.33 U/g。     

稻草秸秆纤维素分解菌的分离筛选

本研究基于获得高效木质纤维素分解菌的目的,以刚果红纤维素琼脂和滤纸条培养基为初筛培养基,从分离获得的124株真菌中筛选出透明圈与菌落直径比值较大、滤纸条分解能力较强的11个菌株。经液体发酵,测定其酶活力,复筛得到羧甲基纤维素酶活和滤纸酶活均较高的4个菌株;并进行了不同碳源和不同pH对筛选菌株产酶能力的影响试验,发现不同菌株对不同纤维素物质的分解能力不一样,同一菌株对不同纤维素碳源的利用能力也不相同。     

混合酶对经不同预处理的甜高粱秆渣的水解

采用正交试验设计,探索不同的液固比、pH值、温度和混合酶配比对甜高粱秆渣的水解情况,并在此基础上研究了混合酶对经漆酶和高压蒸汽预处理的甜高粱秆渣的水解情况。结果表明,混合酶水解的最佳条件为液固比11∶1、pH值3.6、30℃、果胶酶∶纤维素酶∶半纤维素酶=1∶1∶2,该条件下的水解效率要好于纤维素酶单独的水解效率,经漆酶和高压蒸汽预处理的甜高粱秆渣的酶解效率得到显著提高,浸润于不同溶液中的甜高粱秆渣经高压蒸汽预处理并酶解,其葡萄糖收率有所不同,且在溶液中加入KMnO4后再进行高压蒸汽处理,能进一步提高葡萄糖收率。     

纤维素酶高产菌株桧状青霉9-3的诱变选育及产酶条件研究

本文以桧状青霉9-3为出发菌株,采用硫酸二乙酯（DES）诱变,通过筛选得到一株酶活力高且遗传稳定性良好的菌株H16,其滤纸酶活力、β-葡萄糖苷酶酶活力与蛋白产量均较出发菌株相比均提高了4倍左右。并通过对发酵培养基以及发酵条件的优化,确定了最佳的产酶条件为：微晶纤维素浓度为2%,玉米浆干粉浓度为1.5%,发酵温度为30℃,初始pH为5.5,装液量为30 mL/250 mL,发酵周期为5 d。在优化的条件下,该菌株的纤维素酶和蛋白产量均进一步提高25%左右。     

高产中性纤维素酶的巨大芽胞杆菌基因工程菌发酵工艺条件优化

为了加快纤维素酶工业化应用的步伐,解决纤维素酶发酵工业产量低、生产成本高等问题,本研究采用正交设计法对高产中性纤维素酶的巨大芽胞杆菌(Bacillus megaterium)基因工程菌的培养基和发酵条件进行优化.结果表明,最佳发酵培养基成分为:麦麸2.5%、玉米浆1.5%、磷酸二氢钾0.75%、硫酸镁0.04%、氯化钠0.25%;最佳发酵条件为:温度37℃,pH 7.0,罐压0.03～0.05 Mpa,转速600 r/min,装液量3 L,接种量10%,培养4 h后以0.25 g/L/h的流速流加木糖10 h,发酵16 h后,以恒溶氧方式流加补料8 h,共补加料320mL,发酵过程控制溶氧浓度≥30%.该工程菌在此最佳发酵条件下培养,纤维素酶活力可达3846.48 U/mL,是优化前摇瓶发酵的4.3倍.本研究可为工业化生产纤维素酶提供依据.     

产黄纤维单胞菌CR-14菌株诱变选育及发酵条件优化

为提高产黄纤维单胞菌CR-14纤维素酶活力,对菌株进行亚硝酸、紫外线及复合诱变处理,筛选产纤维素酶活较高的突变株,并对其发酵条件进行优化。结果表明,经亚硝酸和紫外线复合诱变得到一株产纤维素酶活较高的菌株Y-UA-18,其酶活力为10.57 U,为原菌株产酶活力的1.67倍。通过正交试验得到菌株Y-UA-18产酶最佳培养基为:秸秆粉1.0%、复合氮源0.6%、KH2PO40.1%、Mg SO40.1%、Na Cl 0.08%;最佳培养条件为:初始p H值7.0、培养温度30℃、发酵时间3 d。通气量对菌株Y-UA-18产酶影响不明显,但在厌氧条件下发酵液酶活显著降低,0.1%TW-80对菌株Y-UA-18的产酶没有影响。     

Structure-function analysis of the vanillin molecule and its antifungal properties

The aim of the present study was to evaluate which structural elements of the vanillin molecule are responsible for its observed antifungal activity. MICs of vanillin, its six direct structural analogues, and several other related compounds were determined in yeast extract peptone dextrose broth against a total of 18 different food spoilage molds and yeasts. Using total mean MICs after 4 days of incubation at 25 degrees C, the antifungal activity order was 3-anisaldehyde (1.97 mM) > benzaldehyde (3.30 mM) > vanillin (5.71 mM) > anisole (6.59 mM) > 4-hydroxybenzaldehyde (9.09 mM) > phenol (10.59 mM) > guaiacol (11.66 mM). No correlation was observed between the relative antifungal activity of the test compounds and log P(o/w). Furthermore, phenol (10.6 mM) was found to exhibit a greater activity than cyclohexanol (25.3 mM), whereas cyclohexanecarboxaldehyde (2.13 mM) was more active than benzaldehyde (3.30 mM). Finally, the antifungal order of isomers of hydroxybenzaldehyde and anisaldehyde was found to be 2- > 3- > 4- and 3- > 2- > 4-, respectively. In conclusion, the aldehyde moeity of vanillin plays a key role in its antifungal activity, but side-group position on the benzene ring also influences this activity. Understanding how the structure of natural compounds relates to their antimicrobial function is fundamentally important and may help facilitate their application as novel food preservatives.     

罗伦隐球酵母对草梅采后灰霉病害的生物防治

测定了罗伦隐球酵母对草莓采后由灰葡萄孢霉引起的灰霉腐烂的抑制效果。在马铃薯葡萄糖琼脂固体培养基（PDA）体外抑菌实验中，把生长有罗伦隐球酵母的营养酵母葡萄糖固体培养基(NYDA)切块放置在涂布有灰霉孢子的马铃薯葡萄糖琼脂固体培养基上一起培养，罗伦隐球酵母并不能抑制霉菌的生长；而在马铃薯葡萄糖液体培养基(PDB)体外抑菌实验中，罗伦隐球酵母活细胞有效地抑制了灰霉孢子的萌发。酵母细胞菌悬液对灰霉的抑菌效果好于酵母培养液，而酵母热杀死液和滤液没有抑菌作用。酵母菌悬液的细胞浓度对其抑菌作用有显著的影响：无论草莓在20℃条件下贮藏3 d或是在2℃条件下贮藏20 d，应用在草莓上的酵母细胞浓度越高，则草莓的霉变率越低。罗伦隐球酵母菌悬液对草莓整果储藏自然条件下霉变的抑制试验也有类似的结果。因此，罗伦隐球酵母可以代替化学杀菌剂作为草莓采后的霉菌抑制剂。     

Evaluation of the potential of fungal and plant laccases for active-packaging applications

Laccases from Trametes versicolor (TvL), Myceliophthora thermophila (MtL), and Rhus vernicifera (RvL) were investigated with regard to their potential utilization as oxygen scavengers in active packages containing food susceptible to oxidation reactions. The substrate selectivity of the laccases was investigated with a set of 17 reducing substrates, mainly phenolic compounds. The temperature dependence of reactions performed at low temperatures (4-31 °C) was studied. Furthermore, the laccases were subjected to immobilization in a latex/clay matrix and drying procedures performed at temperatures up to 105 °C. The results show that it is possible to immobilize the laccases with retained activity after dispersion coating, drying at 75-105 °C, and subsequent storage of the enzyme-containing films at 4 °C. TvL and, to some extent, MtL were promiscuous with regard to their reducing substrate, in the sense that the difference in activity with the 17 substrates tested was relatively small. RvL, on the other hand, showed high selectivity, primarily toward substrates resembling its natural substrate urushiol. When tested at 7 °C, all three laccases retained >20% of the activity they had at 25 °C, which suggests that it would be possible to utilize the laccases also in refrigerated food packages. Coating and drying resulted in a remaining enzymatic activity ranging from 18 to 53%, depending on the drying conditions used. The results indicate that laccases are useful for active-packaging applications and that the selectivity for reducing substrates is an important characteristic of laccases from different sources.     

Optimisation of the Solid-Contact Test with Arthrobacter globiformis (7 pp)

Background, Aim and Scope   A number of biotests are available for the characterisation of solid matters such as soil or sediment. Among these, bacterial biotests using single test species often analyse the toxicity of water-soluble contaminants in aquatic extracts, but there is also a need for a fast and inexpensive bacterial solid-contact test. In this study, a solid contact test with added bacteria (Arthrobacter globiformis) was optimised (through miniaturization) for the development of a test kit with conserved bacteria. As in other tests, the results can be influenced by natural soil factors, often masking anthropogenic impacts. For this reason, a further goal of this study was the investigation of the influence of natural soil characteristics on the result of the solid contact test. The project is part of the joint research project 'Optimization of ecotoxicological test methods for routine use' (abbreviated as ERNTE-Forschungsvorhaben: 0330305). Materials and Methods: This method is based on an existing German standard (DIN 38412 L 48) using Arthrobacter globiformi for testing whole soils and sediments. The test principle is the measurement of the dehydrogenase activity of the test organism A. globiformis after an incubation time of two hours with the solid material. To attain the miniaturization in microplates, dye measurement was changed from spectrophotometrical determination of the substrate resazurine to the fluorimetric measurement of the product resorufin. A second step towards optimisation was the use of freeze-dried bacteria. Seven selected uncontaminated soils were tested in order to determine the influence of natural soil characteristics on the results of the solid contact test with A. globiformis. Freshly spiked and polluted field soils were analysed in order to obtain information about the sensitivity of the test. Results: It is possible to perform the contact test in microplates. The fluorimetric dye measurement can be carried out in the presence of the solid material, so the work-intensive step of centrifugation and filtration is no longer necessary. The measurement in the optimised contact test is based on the kinetics of the enzyme reaction. The investigation showed that conserved bacteria have the same activity and sensitivity as cultivated bacteria. Discussion: The study of the uncontaminated soils demonstrated the influence of various soil characteristics on the results of the solid contact test. This information is the basis for the selection of the control and reference soils and is crucial for setting the threshold value in toxicity testing. The investigation of freshly spiked and contaminated soils showed a different sensitivity dependent on the kind of the contamination. Conclusions: The solid contact test was successfully optimised using microplates, whereas now less than six hours are necessary for the analysis. The optimised test is rapid and sensitive, requiring small samples and no stock culture of the bacteria A. globiformis if using freeze-dried bacteria. In this study, the effect of natural soil factors such as pH-value was shown. This information is used to define the threshold value for toxicity. Therefore, the optimised contact test can be used for an efficient assessment of soil or soil substrates. Further studies will clear up if this optimisation is also valid for aquatic sediments and waste. Recommendations and Perspectives: Due to its short analysis time, the test is suitable for screening different kinds of solid matter and can be used for on-site analysis. - The optimised contact test with freeze-dried bacteria as part of a battery of tests is appropriate for the assessment of contaminated soils, sediments and waste.     

堆肥微生物的培养与磷酸酶活性的研究(I)——曲霉的产磷酸酶活性的研究

对培养过程中丝状真菌的产磷酸酶活性进行了比较,筛选了一株产磷酸酶活性较高的曲霉菌株HQ,对其产酶特性及对不同难溶性磷底物的磷释放量进行了研究。液体培养结果表明,该菌株在以植酸钠为底物时,中性磷酸酶活性为0.54mg m l-1,磷释放量46.65μg m l-1。固体培养结果表明,以畜禽废弃物为培养基的磷酸酶活性最高,其中性磷酸酶活性为37.4mg g-1。该菌株有潜力作为堆肥微生物的候选菌株。     

Simultaneous determination of dextrose, sucrose, maltose, and lactose in sausage products by liquid chromatography

A liquid chromatographic (LC) method for the simultaneous determination of dextrose, sucrose, maltose, and lactose in sausage products has been developed. Dextrose, sucrose, maltose, and lactose are extracted from comminuted meat products with 52% ethanol. After filtration, the extracts are purified by passing them through a C18 Sep-Pak cartridge and 2 ion exchange resin Econo-columns in series. After concentration and filtration, extracts are analyzed by LC using a normal phase amino column and a differential refractometer detector. Homogeneously ground samples of cooked and fresh sausages are fortified with dextrose, sucrose, maltose, and lactose at 4 different concentrations. Average recovery for dextrose, sucrose, maltose, and lactose at all 4 levels of fortification was greater than 80% with a coefficient of variation less than 10%.     

川芎提取液对脐橙的防腐保鲜效果

为了开发植物源脐橙贮藏保鲜技术,该文研究了川芎提取液在离体和活体条件下对采后脐橙的主要致腐菌意大利青霉和指状青霉的抑制作用,以及对贮藏期脐橙品质和生理生化的影响。结果显示:在离体条件下,川芎提取液对2种供试菌种有显著的抑制作用(P0.05),最低抑菌质量浓度均为25.00 mg/m L;在活体条件下,川芎提取液能有效抑制2种病原菌在脐橙上的生长,且质量分数为4%效果较好,与体积分数为50%乙醇处理的对照相比,差异显著(P0.05);在脐橙采后贮藏中,川芎提取液对脐橙的防腐保鲜效果与其质量分数相关,其中质量分数为4%的效果较好,与蒸馏水处理的对照相比,能够显著(P0.05)降低采后脐橙的腐烂率、失重率和丙二醛含量,延缓总酸的下降,以及诱导过氧化物酶活性上升,有利于维持可溶性固形物和总糖质量分数,而对维生素C和果皮色泽的变化无显著影响(P0.05)。结果表明,川芎提取液用于脐橙贮藏保鲜切实可行,研究结果为脐橙天然保鲜剂的开发及脐橙在贮藏、运输和销售期间防腐保鲜提供参考。     

Phosphatase activity does not limit the microbial use of low molecular weight organic-P substrates in soil

Plant roots and soil microorganisms contain significant quantities of low molecular weight (MW) phosphorylated nucleosides and sugars. Consequently, upon death these can represent a significant input of organic-P to the soil. Some of these organic-P substrates must first be dephosphorylated by phosphatases before being assimilated by the soil microbial community while others can be taken up directly from soil solution. To determine whether sorption or phosphatase activity was limiting the bioavailability of low MW organic-P in soil we compared the microbial uptake and C mineralization of a range of ¹⁴C-labeled organic-P substrates [glucose-6-phosphate, adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP)] to that of the parent compounds (adenosine and glucose). In a fertile grassland soil we showed that at low organic-P substrate concentrations (<0.5 mM) phosphatase activity did not limit microbial uptake or mineralization in comparison to their non-phosphorylated counterparts. However, at high substrate concentrations (1-10 mM) the mineralization of the organic-P compounds was significantly lower than that of the non-phosphorylated compounds suggesting that phosphatase activity or microbial transporter capacity limited bioavailability. Sorption to the solid phase followed the series glucose<adenosine<G-6-P<AMP<ADP=ATP. However, sorption of the organic-P compounds to the solid phase did not appear to greatly affect bioavailability. The high adenosine mineralization capacity of the microbial biomass suggests that nucleosides may represent a significant source of C and N to the soil microbial biomass. We conclude that at low organic-P substrate concentrations typical of those in soil, neither phosphatase activity nor sorption greatly limits their bioavailability.     

Strain of Fusarium oxysporum isolated from almond hulls produces styrene and 7-methyl-1,3,5-cyclooctatriene as the principal volatile components

An isolated strain of Fusarium oxysporum from the hulls of Prunus dulcis (sweet almond) was found to produce relatively large quantities of the hydrocarbons styrene and two isomers of 7-methyl-1,3,5- cyclooctatriene (MCOT). Production of styrene and MCOT was reproduced on a small scale using potato dextrose agar as a growth medium and scaled up using 1 L of inoculated potato dextrose broth. The compounds were trapped as volatile organic compounds (VOCs) onto solid-phase microextraction (SPME) for small scale and Tenax for large scale and then isolated using standard high-performance liquid chromatography (HPLC) methods. Styrene was authenticated by a comparison to the retention times, fragmentation patterns, and calculated retention indices of a commercially available sample. The identity of MCOT was verified by a short chemical synthesis and a comparison of spectroscopic data to the isolated sample. A biosynthetic scheme of styrene is proposed on the basis of a (13)C-labeling study. This is the first report of MCOT isolated as a natural product.     

Natural Gradient Drift Tests for Assessing the Feasibility of In Situ Aerobic Cometabolism of Trichloroethylene and Evaluating the Microbial Community Change

The objective of this study is to develop a method for using the single-well natural gradient drift test (SWNGDT) in the field to assess in situ aerobic cometabolism of trichloroethylene (TCE) and to analyze microbial community changes. The SWNGDT was performed in a monitoring well installed in a TCE-contaminated aquifer in Wonju, South Korea. The natural gradient drift biostimulation test (NGDBT) and surrogate test (NGDST) were performed by injecting dissolved solutes (bromide (a tracer), toluene (a growth substrate), ethylene (a nontoxic surrogate substrate to probe for TCE transformation activity), dissolved oxygen (DO, an electron acceptor), and nitrate (nutrient)) into the aquifer. Push?Cpull blocking tests (PPBT) were also performed to examine whether the monooxygenase of toluene oxidizers is involved in the degradation of toluene and the transformation of ethylene. Through the NGDBT, NGDST, and PPBT, we confirmed that the addition of toluene and oxygen in these field tests stimulated indigenous toluene utilizers to cometabolize aerobically TCE, with the following results: (1) the observed simultaneous utilization of toluene and DO; (2) the transformation of ethylene to ethylene oxide and propylene to propylene oxide; and (3) the transformation of TCE. Furthermore, the results of restriction fragment length polymorphism suggested that the microbial community shifts and the microbes capable of transforming TCE are stimulated by injecting the growth substrate, toluene.