桦褐孔菌液体深层培养研究

研究了不同碳源、氮源、生长因子和无机盐对桦褐孔菌液体深层培养菌丝体产量的影响。采用“二次回归正交组合设计”对培养基进行优化,结果表明,当培养基配方为小麦淀粉水解糖100.83g/L(还原糖含量为24%)、酵母粉18.70g/L、玉米浆11.01g/L、KH2PO44.59g/L、MgSO4·7H2O1.5g/L、pH6.0,在30℃、160r/min的恒温培养振荡器上振荡培养120h,生物量可达19.80g/L。     

Medium optimization of antifungal activity production by Bacillus amyloliquefaciens using statistical experimental design

In order to overproduce biofungicides agents by Bacillus amyloliquefaciens BLB371, a suitable culture medium was optimized using response surface methodology. Plackett-Burman design and central composite design were employed for experimental design and analysis of the results. Peptone, sucrose, and yeast extract were found to significantly influence antifungal activity production and their optimal concentrations were, respectively, 20 g/L, 25 g/L, and 4.5 g/L. The corresponding biofungicide production was 250 AU/mL, corresponding to 56% improvement in antifungal components production over a previously used medium (160 AU/mL). Moreover, our results indicated that a deficiency of the minerals CuSO(4), FeCl(3) · 6H(2)O, Na(2)MoO(4), KI, ZnSO(4) · 7H(2)O, H(3)BO(3), and C(6)H(8)O(7) in the optimized culture medium was not crucial for biofungicides production by Bacillus amyloliquefaciens BLB371, which is interesting from a practical point of view, particularly for low-cost production and use of the biofungicide for the control of agricultural fungal pests.     

簇生沿丝伞液体发酵培养及其发酵物体内抗肿瘤活性研究

采用中心组合旋转设计法确定了簇生沿丝伞液体深层培养菌丝体的最佳方案。采用H22荷瘤小鼠进行体内抗肿瘤试验,对簇生沿丝伞发酵物的抗肿瘤活性进行了探究。结果表明,当培养基配方为麦芽糖59.27g/L,蛋白胨:酵母粉=1:1为8.04g/L,磷酸二氢钾:硫酸镁:硫酸铵=1:1:1为2.27g/L时,生物量达到最高,为15.06g/L。簇生沿丝伞发酵物的抑瘤率与剂量存在明显的量效关系,200mg/kg时,抑瘤率达最高,为54.71%,且白细胞数和IL-2的含量与对照组相比均显著增高。     

翅鳞伞深层发酵胞外多糖优化研究

采用PlackettBurman设计(PlackettBurman Design, PB)对影响翅鳞伞［ Pholiota squarrosa (Pers. Ex Fr.) Quel.］ AS 5245菌株发酵产糖的内在和外在相关因素进行了筛选,所选取的20个相关因素为葡萄糖、果糖、麦芽糖、酵母膏、胰蛋白胨、KH₂PO₄、K₂HPO₄、(NH₄)₂SO₄、NaNO₃、FeSO₄、MgSO₄、MnCl₂、ZnCl₂、FeCl₃、CuSO₄·5H₂O、维生素B1、起始pH、发酵温度、时间和装液量。在此基础上,再采用响应曲面法(Response Surface Methodology,RSM)对影响发酵产糖的内在关键影响因素酵母膏、果糖、MgSO₄、麦芽糖、ZnCl₂和发酵基质起始pH值的最佳水平范围作了进一步的研究与探讨,通过对二次多项回归方程求解得知,在上述自变量分别为6.0g/L、11.5g/L、0.5g/L、9.6g/L、38.6mg/L和5.3时,胞外多糖最大预测值为876.32μg/mL发酵醪,此预测可信度不仅被统计分析所验证,也实践所证实。     

萝卜抗真菌蛋白1(Rs-AFP1)在大肠杆菌中的融合表达及其对大丽轮枝菌抑制活性的研究(英文)

利用聚合酶链式反应 (PCR)获得了萝卜 (RaphanussativusL .)抗真菌蛋白 1(Rs_AFP1)基因编码区核苷酸序列。将整个阅读框架片段和去除了N_端信号肽序列的片段分别装入原核表达载体pET_32b( )中 ,在大肠杆菌中表达 ,发现带有信号肽的Rs_AFP1不能在大肠杆菌中表达 ,而当这一序列去除后 ,表达出约 2 7kD的Rs_AFP1的融合蛋白。用凝血酶处理融合蛋白以去除N_端His.tag的部分序列 ,然后用处理后的融合蛋白进行了抑制真菌生长的实验。结果表明 ,在加入 0 .3g/L的Rs_AFP1的融合蛋白的培养液中 ,大丽轮枝菌 (VerticilliumdahliaeKleb .)的生长受到抑制 ,分别比加入对照细菌蛋白和PBS下降 5 7.5 %和 6 9.8% ;孢子的萌发也受到抑制。显然 ,细菌表达的融合蛋白对大丽轮枝菌的生长有抑制作用。     

桑木层孔菌液体培养条件的研究

对桑木层孔菌(Phellinus mori)液体发酵条件进行了研究,以生物量和胞外多糖为指标,通过L16(45)和L9(34)正交表进行了两次正交试验,筛选出桑木层孔菌最适液体培养条件为:麦芽糖30 g/L,酵母浸粉和蛋白胨15 g/L(质量比2 1),KH2PO4和CaCl25.5 g/L(质量比1 1),初始pH6.0;通过单因素试验筛选出最适装液量为120 mL/250 mL,最适接种量为10%。在此条件下液体发酵培养7 d后,桑木层孔菌生物量达到23.375 g/L,胞外多糖产量达到3.993 g/L。     

圆红冬孢酵母菌发酵产油脂培养基及发酵条件的优化研究

采用均匀设计和单因子试验法,系统考察了圆红冬孢酵母菌(Rhodosporidiumtoruloides)在不同碳氮比条件下产油发酵情况以及添加无机盐对产油发酵的影响,通过均匀设计软件对二次多项回归方程求解及单因素分析得知在培养基组成分别为葡萄糖70g/L,硫酸铵0.1g/L,酵母粉0.75g/L,磷酸二氢钾0.4g/L,七水硫酸镁1.5g/L,初始pH6.0,在灭菌(121℃15min)后添加ZnSO41.91×10-6mmol/L、CaCl21.50mmol/L、MnCl21.22×10-4mmol/L、CuSO41.00×10-4mmol/L。发酵摇瓶装液量为250mL三角瓶装培养基50mL,接种量为10%(种龄28h)。在上述条件下,30℃振荡(200r/min)培养120h,所得菌体油脂含量高达76.1%,脂肪得率系数可达22.7。     

Fermentative hydrogen production and bacterial community structure in high-rate anaerobic bioreactors containing silicone-immobilized and self-flocculated sludge

A novel continuously stirred anaerobic bioreactor (CSABR) seeded with silicone-immobilized sludge was developed for high-rate fermentative H2 production using sucrose as the limiting substrate. The CSABR system was operated at a hydraulic retention time (HRT) of 0.5-6 h and an influent sucrose concentration of 10-40 g COD/L. With a high feeding sucrose concentration (i.e., 30-40 g COD/L) and a short HRT (0.5 h), the CSABR reactor produced H2 more efficiently with the highest volumetric rate (VH2) of 15 L/h/L (i.e., 14.7 mol/d/L) and an optimal yield of ca. 3.5 mol H2/mol sucrose. The maximum VH2 value obtained from this work is much higher than any other VH2 values ever documented. Formation of self-flocculated granular sludge occurred during operation at a short HRT. The granule formation is thought to play a pivotal role in the dramatic enhancement of H2 production rate, because it led to more efficient biomass retention. A high biomass concentration of up to 35.4 g VSS/L was achieved even though the reactor was operated at an extremely low HRT (i.e., 0.5 h). In addition to gaining high biomass concentrations, formation of granular sludge also triggered a transition in bacterial community structure, resulting in a nearly twofold increase in the specific H2 production rate. According to denatured-gradient-gel-electrophoresis analysis, operations at a progressively decreasing HRT resulted in a decrease in bacterial population diversity. The culture with the best H2 production performance (at HRT = 0.5 h and sucrose concentration = 30 g COD/L) was eventually dominated by a presumably excellent H2-producing bacterial species identified as Clostridium pasteurianum.     

萝卜抗真菌蛋白1（Rs-AFP1)在大肠杆菌中的融合表达 及其对大丽轮枝菌抑制活性的研究

The Raphanus sativus L. antifungal protein 1 (Rs-AFP1) gene was isolated by polymerase chain reaction (PCR). The complete open reading frame and the fragment encoding the putative mature protein were inserted into the prokaryotic expression vector pET-32b(+), respectively. Subsequent expression showed that the Rs-AFP1 was produced in E. coli as a 27 kD fusion protein only when the N-terminal signal peptide was removed. After treatment with thrombin to remove part of the N-terminal His.tag sequence, the bacterially expressed Rs-AFP1 was used for fungal growth inhibition assay which was conducted on Verticillium dahliae Kleb., a soil-born fungus causing the cotton wilt disease. Results showed that, in the liquid medium, the Rs-AFP1 fusion protein at a concentration of 0.3 g/L clearly inhibited the growth of V. dahliae and the germination of spores. Thus the bacterially expressed fusion protein had the antifungal activity against V. dahliae.     

链霉菌A01-chit33CT产纳他霉素和几丁质酶协同表达发酵条件优化

生物农药由于具有良好的生态效应和安全性,因此比化学农药更受到人们的青睐,生物农药的发展契合低碳、循环、清洁绿色经济发展理念。因此,寻求利于食品安全和环境保护,同时高效控制植物病害的新型生物农药成为时下及未来研究的热点。链霉菌以产生纳他霉素等抗生素起到生防作用。链霉菌株A01-chit33CT既可以产生纳他霉素又可以高表达几丁质酶活,生防效果大大增加。为确定链霉菌A01-chit33CT产纳他霉素和几丁质酶协同表达的发酵条件,初步探索了碳氮源和发酵条件对菌株产生纳他霉素和几丁质酶的影响。结果表明,葡萄糖促进纳他霉素的产生而抑制几丁质酶的表达,因此分两阶段添加葡萄糖和几丁质粉来达到二者协同表达。研究确定最佳发酵培养基为:葡萄糖40 g/L,几丁质粉10 g/L(发酵4 d添加),黄豆粉30 g/L,大豆蛋白胨10 g/L,CaCO35 g/L,MgSO4.7H2O 0.5 g/L,K2HPO40.5 g/L。最优发酵条件为:初始pH 6.0,温度28℃,转速180 r/min。在此条件下,链霉菌A01-chit33CT产纳他霉素达1.52 g/L,同时几丁质酶活达990 U/ml,二者比优化前的水平分别提高了1.95倍和2.27倍。     

4-丁内酰胺水解酶法制备γ-氨基丁酸

以吡咯烷酮为唯一碳源,从采集的土样中分离、筛选得到具有4-丁内酰胺水解酶活性的菌株.采用响应面分析法对该菌株的产酶培养基组分进行了优化研究并对酶促转化反应条件也进行了研究.结果表明:编号为HHSW-16的菌株水解活性最高.优化后的培养基组成为:葡萄糖11.50 g·L-1,牛肉膏6.35 g·L-1,酵母粉5.58 g...     

Application of artificial neural network coupling particle swarm optimization algorithm to biocatalytic production of GABA

The biotransformation of L-sodium glutamate (L-MSG) to gamma-aminobutyric acid (GABA) catalyzed by the cells of Lactobacillus brevis with higher glutamate decarboxylase activity was investigated. The results showed that pH, temperature, and FeSO(4) x 7H(2)O concentration had significantly positive effect on GABA yield. The individual and interactive effects of pH, temperature, and FeSO(4) x 7H(2)O concentration were further optimized in terms of GABA yield. In the present work, an artificial neural network (ANN) and response surface methodology (RSM) models were developed, which incorporated pH, temperature, and FeSO(4) x 7H(2)O concentration as input variables, and GABA yield as output variable. The optimized ANN topology included four neurons in the hidden layer and the best network architecture was 3-4-1. The trained ANN gave total root-mean square error (sigma) equal to 1.84 for GABA yield while the RSM gave sigma equal to 2.63. The results demonstrated a slightly higher prediction accuracy of ANN compared to RSM. The modeled maximum GABA yield was identified by applying particle swarm optimization algorithm to the ANN model developed. The modeled maximum GABA yield reached 91 mM under the following optimal conditions: 25 mL Na(2)HPO(4)-citric acid buffer (100 mM, pH 4.23), 120 mM L-MSG, 0.83 g/L FeSO(4) x 7H(2)O, 10 microM PLP, the resting cells obtained from a 60-h culture broth, 2.68 g dry cell weight (DCW)/L, and without agitation at 40 degrees C for 5 h. The previous high value of GABA yield that was observed was 81.8 mM. The optimized conditions allowed GABA yield to be increased from 81.8 to 90.57 mM after verification experiments test.     

响应面法优化类芽胞杆菌HB172198产褐藻胶裂解酶发酵培养基

为了提高类芽胞杆菌新种HB172198产褐藻胶裂解酶活力,本研究采用响应面法对该菌株液体发酵培养基进行了优化实验。在单因素实验和Plackett-Burman试验筛选出海藻酸钠、胰蛋白胨、NaCl、MgSO4·7H2O等4个显著影响产酶因素的基础上,通过Box-Behnken设计及响应面法进行回归分析,得出产褐藻胶裂解酶最佳发酵培养基,其成分为:海藻酸钠7.50 g/L、胰蛋白胨13.57 g/L、NaCl 29.75 g/L、MgSO4·7H2O 0.08 g/L。优化条件下该菌株最大酶活性达14.60 U/mL,是优化前的1.87倍。本研究为菌株HB172198产褐藻胶裂解酶的大规模生产和工业应用提供了重要的理论依据。     

海洋芽胞杆菌B-9987产新型抗菌环脂肽Marinhysin A的培养基优化

Marinhysin A(MA)是由海洋芽胞杆菌B-9987产生的具有抗真菌活性的新结构环脂肽类化合物,该化合物不仅抑菌谱广而且盆栽防效良好。采用响应面分析法(Response Surface Methnology)对海洋芽胞杆菌B-9987产脂肽MA的培养基进行了优化。Plackett-Burman (PB)实验表明,蔗糖和酵母粉是显著影响MA产量的因素。中心组合实验(Central Composite Design)分析表明,当培养基中蔗糖和酵母粉的含量分别为42.0g/L和42.3g/L时,MA的浓度最大可达68.19mg/L,与实验值75.74mg/L相近。优化后,MA产量(250ml摇瓶发酵)较优化前的54.12mg/L提高了28.5%。用优化后培养基进行5L罐发酵,MA浓度可达182mg/L,较优化前的130mg/L 提高了28.4%。     

Shelf-life enhancement of bio-inoculant formulation by optimizing the trace metals ions in the culture medium for production of DAPG using fluorescent pseudomonad R62

Statistical experimental design was used to optimize the concentration of trace elements for production of antifungal compound, 2,4-diacetylphloroglucinol (DAPG), from fluorescent pseudomonad R62 in shake-flask cultivation. The selection of the trace metal ions, influencing DAPG production, was done using Plackett-Burman design (PBD). Only Zn(2+), Mn(2+) and MoO(4)(2-) were the most significant components (p<0.05). A quadratic model was used to fit the response. Application of response surface methodology (RSM) revealed that the optimum values of the salts of the trace elements Zn(2+) (ZnSO(4)·7H(2)O), Mn(2+) (MnCl(2)·4H(2)O), and MoO(4)(2-) (Na(2)MoO(4)·2H(2)O) were 83, 42 and 135μM, respectively, to achieve 125 mg/L of DAPG, which was nearly 13-fold more compared to its production in basal synthetic medium in shake flask. The studies in 14L bioreactor resulted in 135 mg/L of DAPG at the end of 36 h of cultivation. The culture broth containing 125 mg/L of DAPG was found to be sufficient for keeping the bio-inoculant viable in non-sterile talcum powder-based formulations (which contained 25μg DAPG/g carrier) when stored at 28°C for 6 months. The structure of the purified DAPG was confirmed using (1)H NMR and mass spectrometry.     

Evaluation of in vitro alpha-glucosidase inhibitory,antimicrobial, and cytotoxic activities of secondary metabolites from the endophytic fungus,Nigrospora sphaerica,isolated from Helianthus annuus

This study aimed to evaluate alpha-glucosidase inhibition and antimicrobial activity as well as cytotoxic activity of extracts from the endophytic fungus, Nigrospora sp., isolated from leaves of Helianthus annuus, which is widely cultivated for food and used as a medicinal plant. The fungus (TSU-CS003) was identified based on internal transcribed spacer ribosomal DNA sequences and fungal biomass, and fermentation broth was subjected to extraction by solvents (hexane and ethyl acetate). All extracts were tested for their antimicrobial activity, alpha-glucosidase inhibition, and cytotoxicity activity. In addition, the active extract was analyzed by using gas chromatography mass spectrometry (GC-MS) TSU-CS003 was identified as Nigrospora sphaerica. The fermentation broth extract (BE) showed strong antimicrobial activity against Staphylococcus aureus and methicillin-resistant S. aureus (Gram-positive bacteria) with minimum inhibitory concentration (MIC) values in the range of 16–32 μg/mL and a few yeasts with MIC values ranging from 64 to 128 μg/mL, especially Talaromyces marneffei with an MIC value of 4 μg/mL. The effects of BE were observed by SEM. The results showed that this extract affected the cell morphology of T. marneffei. The half-maximal inhibitory concentration (IC50) of BE from alpha-glucosidase inhibition was recorded as 17.25 μg/mL and also showed significant cytotoxicity against A549 human cancer cell lines with an IC50 value of 22.41 μg/mL. Furthermore, BE was analyzed by using GC-MS and divided into three main compounds, including 5-pentyldihydrofuran-2(3H)-one, (Z)-methyl 4-(isobutyryloxy)but-3-enoate, and 2-phenylacetic acid. This was the first report of the endophytic fungus N. sphaerica from H. annuus. It is a potential source of active metabolites, which gave the strong antifungal activity, antioxidant activity, and cytotoxicity to A549 cancer cell lines.     

响应曲面法优化灵芝廉价型深层发酵培养基的研究

为了获得生产用廉价型灵芝发酵培养基,采用中心组合旋转设计法和响应曲面法对低成本培养基组分进行了优化。优化的四个组分为玉米粉(x1)、麸皮粉(x2)、豆饼粉(x3)和蔗糖(x4)。结果表明,灵芝菌体发酵和多糖发酵的培养基预测模型分别为:Y1=15.1–0.31x1–0.34x2+0.36x3–0.44x4–1.26x12–1.98x22–0.85x32–1.15x42–0.59x2x3和Y2=2.0–0.08x1–0.08x2+0.04x3–0.09x4–1.13x12–0.33x22–0.08x32–0.16x42–0.16x2x3–0.10x1x4。从中获得菌体发酵的最优配方为:玉米粉19.7g/L,麸皮粉11.3g/L,豆饼粉6.3g/L,蔗糖19.5g/L;多糖发酵的最优配方为:玉米粉19.6g/L,麸皮粉11.0g/L,豆饼粉6.7g/L,蔗糖19.1g/L。150L发酵罐中试放大结果表明,灵芝菌体的产量为16.92g/L,多糖产量为1.86g/L。所得培养基为灵芝产品的高效低成本生产提供了基础。     

Alternative medium for production of Pleurotus ostreatus biomass and potential antitumor polysaccharides

Pleurotus species are recognized for producing beta-glucans with important medicinal properties as a constituent of the cellular wall of the fruiting body or of the mycelium. The aims of this work were to select a culture medium that maximized the production of biomass and polysaccharides produced by Pleurotus ostreatus DSM 1833 and to evaluate the selected medium in two values of initial oxygen transfer rate -K(L)a (10.2 and 19.3 h(-1)). A 2* *4 factorial design was constructed to evaluate the supplementation of wheat extract with corn steep liquor--CSL (10 or 20 g L(-1)), yeast extract--YE (2 or 5gL(-1)), ammonium sulfate--AS (0 or 5 g L(-1)) and glucose (20 or 40 g L(-1)). In terms of maximum productivity in biomass and global productivity in polysaccharides, the best values were obtained when 5 g L(-1) of YE and 40 g L(-1) of glucose were used. In terms of maximum concentration of biomass, the best results were obtained when 20 g L(-1) of CSL and 40 g L(-1) of glucose were used. The best results in terms of production of biomass and polysaccharides were achieved when lower initial K(L)a (10.2 h(-1)) was used.     

凋落物化学组成对土壤微生物学性状及土壤酶活性的影响

通过模拟试验的方法研究了单一施加杉木(Cunn inghan ia lancceola ta(L am b)Hook.)叶凋落物,杉木(C.lancceola ta)和桤木(A lnus crem astogyne Burk ill)混合凋落物,杉木(C.lancceola ta)和枫香(L iqu id am ba f orm osana H ance)混合凋落物,杉木(C.lancceola ta)、桤木(A.crem astogyne)、枫香(L.f orm osana)混合凋落物对土壤化学性状和土壤微生物量碳、代谢熵(qCO2)、土壤酶活性的影响。研究结果表明,土壤微生物学性状比土壤化学性状对不同凋落物处理的效应反应更敏感;与单一杉木叶凋落物比较,混合凋落物处理的土壤微生物量碳明显增加,土壤脲酶、蔗糖酶、脱氢酶活性升高;土壤代谢熵(qCO2)和土壤多酚氧化酶活性有下降趋势;另外,研究结果也表明,不同树种的叶凋落物混合对土壤质量的影响存在差异,有桤木叶的混合凋落物对土壤质量的改善效果似乎更明显。     

Acid-catalyzed steam pretreatment of lodgepole pine and subsequent enzymatic hydrolysis and fermentation to ethanol

Utilization of ethanol produced from biomass has the potential to offset the use of gasoline and reduce CO(2) emissions. This could reduce the effects of global warming, one of which is the current outbreak of epidemic proportions of the mountain pine beetle (MPB) in British Columbia (BC), Canada. The result of this is increasing volumes of dead lodgepole pine with increasingly limited commercial uses. Bioconversion of lodgepole pine to ethanol using SO(2)-catalyzed steam explosion was investigated. The optimum pretreatment condition for this feedstock was determined to be 200 degrees C, 5 min, and 4% SO(2) (w/w). Simultaneous saccharification and fermentation (SSF) of this material provided an overall ethanol yield of 77% of the theoretical yield from raw material based on starting glucan, mannan, and galactan, which corresponds to 244 g ethanol/kg raw material within 30 h. Three conditions representing low (L), medium (M), and high (H) severity were also applied to healthy lodgepole pine. Although the M severity conditions of 200 degrees C, 5 min, and 4% SO(2) were sufficiently robust to pretreat healthy wood, the substrate produced from beetle-killed (BK) wood provided consistently higher ethanol yields after SSF than the other substrates tested. BK lodgepole pine appears to be an excellent candidate for efficient and productive bioconversion to ethanol.