两步培养法测定真菌产孢营养需求

生防真菌产孢条件的测定一般都是通过连续培养方法,即在同一种限定培养基上完成其生长和产孢过程。文中我们提出分离真菌的生长和产孢阶段,测定产孢营养需求的两步培养新方法。6种生防真菌首先在平板上进行营养生长,然后转移至营养成分和浓度确定的新鲜培养基中继续产孢过程来测定菌株产孢阶段实际营养需求。通过与连续培养方法比较,发现只有淡紫拟青霉Paecilomyces lilacinus、金龟子绿僵菌Metarhizium anisopliae二者产孢条件一致,而厚孢轮枝菌Pochonia chlamydosporia、球孢白僵菌Beauveria bassiana、蜡蚧轮枝菌Lecanicillium lecanii、绿色木霉Trichoderma viride菌株产孢的营养条件存在显著的差异。基于这一方法,确定了绿色木霉最佳产孢条件,即起始碳浓度2g C/L,碳氮比10:1,最佳碳氮源组合纤维二糖和酵母浸膏,为真菌生防制剂生产调控提供了依据。     

营养物质对桑黄菌丝生物量及胞外多糖产量的影响

研究了不同碳源、氮源和无机盐对桑黄深层培养菌丝生物量和胞外多糖产量的影响,结果表明：在培养温度为26℃、摇床转速为160r／min、发酵时间为10d的条件下,以桑黄菌丝生物量为指标,最适碳源、氮源和无机盐分别是果糖或葡萄糖、酵母粉或酵母膏、KH2P04或MgSO4·7H2O;菌丝生物量分别达到1．51、1．31、1．69、1．52、1．52、1．36g／100mL;以胞外多糖为指标,最适碳源、氮源和无机盐分别是葡萄糖、酵母膏、ZnSO4·7H2O,胞外多糖产量分别达到0．49、0．45、0．22g／100mL。     

培养基组分及培养条件对蜡梅花粉萌发及花粉管生长的影响

采用液体培养法研究不同培养基组分和培养条件对蜡梅花粉萌发和花粉管生长的影响。结果表明:(1)PEG-4000是蜡梅花粉离体培养所必需的培养基成分,当培养基中无PEG-4000时,花粉不能正常萌发。(2)培养基内低浓度蔗糖对花粉萌发和花粉管的生长无显著影响,但随着蔗糖浓度的升高,则对花粉萌发和花粉管生长表现出强烈的抑制作用,且浓度越高,抑制效应越强。(3)培养基内其它组分分别在一定浓度范围(0～250g/L PEG-4000、0～50mg/L硼酸、0～30mg/L硝酸钙)内对花粉萌发及花粉管生长有促进作用,但超过上述高限值时则起抑制作用。(4)培养基内镁和钾的浓度对花粉萌发及花粉管生长影响不显著。研究表明,蜡梅最适花粉液体培养基组分为250g/L PEG-4000+50mg/L H3BO3+30mg/L Ca(NO3)2.4H2O,且在pH 5.5、温度15℃和600lx的光照培养条件下蜡梅花粉萌发和花粉管生长最佳。     

隐甲藻深层培养产生二十碳五烯酸的研究

二十碳五烯酸 (Ｃ2 0 :5 ,Ｅｉｃｏｓａｐｅｎｔａｅｎｏｉｃａｃｉｄ ,简称ＥＰＡ)是一种重要的ω 3系列人体必需多不饱和脂肪酸 (Ｐｏｌｙｕｎ ｓａｔｕｒａｔｅｄｆａｔｔｙａｃｉｄｓ,ＰＵＦＡ) [1～ 2 ] 。自从Ｄｙｅｒｂｅｒｇｅ等人报道了二十碳五烯酸对防止和治疗血栓、关节硬化、抗炎症、抗癌、促进脑组织发育等方面具有明显效果以来 ,人们对它的营养和医药价值及生产方法进行了广泛的研究[3～ 6] 。目前 ,二十碳五烯酸主要来源于深海鱼油 ,但其产量不稳定 ,纯化工艺复杂且含有难以消除的鱼腥味 ,难以满足市场的需求。由于二十碳五…     

被孢霉菌发酵产生花生四烯酸的研究

研究了温度、培养基初始pH、碳源、氮源对被孢霉(Mortierella sp.)产生花生四烯酸的影响。正交试验结果表明,Mortierella sp.M10最佳培养基组成为(g/L):葡萄糖100,酵母膏10,KNO_3 4.0,KH_2PO_4 2.0,CaCl_2·2H_2O 0.1,MgSO_4·7H_2O 0.5,FeCl_3·6H_2O0.015,ZnSO_4·7H_2O 0.0075,CuSO_4·5H_2O 0.0005。采用最佳培养基及发酵条件,细胞干重和花生四烯酸产量分别为33.51g/L和0.827g/L。同时对摇瓶发酵过程进行了分析。     

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.     

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.     

A Study on the Application of C17 Medium for Anther Culture

Media with varied levels of minor elements and KNO3, NH4NO3, KH2PO4, MgSO4·7H2O, CaCl2· 2H2O and Fe-Salts were screened to obtain high yield of callus from wheat anther by using orthogonal tests. Of all the seven minor elements treated, the one without NaMoO4·2H2O and with 11.2 mg/l MnS04·4H2O was found to be the most effective. Among the five kinds of organic materials tested, biotin appear- ed to have the highest influence on the induction frequency of anther callus and its opimum dosage was 1.5 mg/l. Based on these data, C17 medium was developed and its induction frequency of callus with good quality from wheat anther reached 12.19% in the Institute in 1980. The maximum diffrentiation freguency of anther callus obtained was 50% on C17 medium. C17, medium compositions are as follows (Mg/l): 1400 KNO3, 150 CaCl2-2H2O, 150 MgSO4·7H2O, 300 NH4NO3, 400 KH2PO4, 27.85 FeSO4·7H2O, 37,25 Na2-EDTA, 11.2 MnS04·4H2O, 8.6 ZnSO4·7H2O, 6.2 H3BO3, 0.83 KI, 0.025 CuSO4·5H2O, 0.025 CaCl2·: 6H2O, 8 glycine, 0.5 nicotinic acid, 0.5 thiamine hydrochloride and 1.5 biotin. The re- differentiation medium is supplemented with 2 2,4-D+0.5 KT+7000 agar+90000 sucrose, pH5.8. The dedifferentiation supplemented with 0.5 IBA+2 KT+7000 agar+ 30000 sucrose, pH 5.8.     

类球红细菌SOD发酵条件优化

本文对类球红细菌3757产SOD进行了发酵条件优化,结果得到了较优的培养基组成(g/L):苹果酸3,胰蛋白胨4,磷酸氢二钾0.9,磷酸二氢钾O.6,硫酸镁0.2,无水氯化钙0.075,硫酸亚铁0.012,EDATA 0.02,微量元素溶液10 mL,生长因子溶液10 mL,pH 7.5。其中,微量元素溶液配方(g/L):硼酸2.8,硫酸锰1.6,钼酸钠0.76,硫酸锌0.24,硫酸铜0.04;生长因子溶液配方(g/L):维生素B_1 1,烟酰胺(VPP)1,生物素0.016,对氨基苯甲酸1。较优培养条件为:接种量5%,转速150 r/min,种龄24 h,发酵温度32℃,发酵时间24 h。优化后酶活力较优化前提高了88.0%。     

绿僵菌无纺布菌条的制作(I)液体种子培养条件

研究了金龟子绿僵菌菌株M a83液体培养最佳营养及环境条件,为无纺布培养提供良好的液体种子。以生物量为指标,通过液体摇瓶试验确定了绿僵菌M a83菌株液体种子培养参数,对不同的氮源、碳源及其浓度和初始pH进行单因素分析。结果表明,绿僵菌M a83菌株液体种子培养最佳营养条件为4%黄豆粉,2%白砂糖,0.5%MgSO4.7H2O,0.05g/LKC l,0.1g/L FeSO4.7H2O,1.0g/L KH2PO4,pH6.3~6.5。70L发酵罐培养,干生物量第三天可达35g/L,显著优于筛选前培养基(SDA,28g/L,a=0.05)。     

假丝酵母YQ5产S-腺苷甲硫氨酸的发酵培养基优化

利用单因子实验和正交实验对假丝酵母(Candida sp.)突变菌株YQ5摇瓶发酵产生S-腺苷甲硫氨酸的培养基成分进行了优化。单因子实验结果表明, 发酵最适pH值为6.0, 最佳碳源为 8%蔗糖, 最佳氮源为1.5%胰蛋白胨, 酵母粉最适浓度为2%, MgSO4·7H2O、CaCl2、FeSO4·7H2O、CoCl2、CuSO4·5H2O、H3BO3等作为无机离子对胞内S-腺苷甲硫氨酸的积累均有促进作用。利 用正交实验获得了最终的发酵培养基配方。在最适条件下发酵48 h, S-腺苷甲硫氨酸含量可达1740.0 mg/L。     

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...     

拉曼被孢霉F_5发酵生产γ—亚麻酸的研究

对拉曼被孢霉突变株F5发酵生产γ—亚麻酸的最适碳源、氮源、发酵时间及温度、无机盐离子添加、最适碳源浓度及补加碳源时间等发酵条件进行了研究探讨。最适发酵培养基组成为 (g/L) :葡萄糖 1 0 0 ,酵母浸出粉 4 ,蛋白胨 1 ,K2 HPO4 1 ,CaCl2 1× 1 0 - 2 ,MgSO4 5× 1 0 - 2 ,FeSO4 1× 1 0 - 2 ,ZnSO4 7.5× 1 0 - 3,CuSO4 0 .5× 1 0 - 3,MnSO4 2× 1 0 - 3,pH 6.0。培养温度为 2 5℃ ,1 4 0r/min振荡培养 1 0天 ,培养 8天后 (即收获前 2天 )补加 5 %葡萄糖。发酵结果为 :DC 2 4 .5 9g/L ,TL 1 0 .84g/L ,TL/DC 4 4.0 9% ,GLA/TL 1 0 .67% ,GLA产量为 1 1 5 6.63mg/L。GLA产量较初始结果提高 1 5 6.1 5 %。该菌株已达到工业化生产菌株要求     

人己糖胺酶D的原核表达、纯化及酶学特性研究

糖基化修饰是一种重要的蛋白质翻译后修饰,参与生物体中的信号传导、细胞识别等多种细胞活动,糖基缀合物的正常水解是生物体代谢的必需途径.人己糖胺酶D( Hexosaminidase D)是新发现的一种存在于人细胞质中的切除GalNAc糖基化修饰的外切酶,但该酶的酶学特性尚不清楚.利用PCR的方法,将Hex D的cDNA序列构建到质粒pET3C中,重组质粒转化大肠杆菌BL21( DE3) plysS后,通过优化异丙基-β-D-硫代吡喃半乳糖苷(IPTG)浓度(0.1mmol/L)和诱导时间(10 h)获得了高可溶性表达的重组蛋白酶.采用Ni-NTA亲和层析对重组蛋白进行了纯化,SDS-PAGE检测分子量的大小(58 kDa)和纯度(95％以上).以4-甲基伞形酮-2-乙酰氨基-2-脱氧半乳糖(4-MU-O-GalNAc)为荧光底物,测定该酶的最适反应pH值为5.5,最适反应温度为37℃,且该酶的热稳定性较好,在50℃下放置半小时仍有较高活性,1mmol/L的金属离子(CuSO4、FeSO4·7H2O、MgCl2· 6H2O、CaCl2、NiSO4·6H2O、AlCl3·6H2O、ZnSO4·7H2O、MnCl2)及EDTA对该酶活性影响不大,10mmol/L AlCl3、CuSO、FeSO4·7H2O对该酶有不同程度的抑制.在最适条件下(pH 5.5,37℃)下,该酶的Km为0.16mmoL/L,最大反应速率为3.06 μmol/( min·mg).     

Enzymes of Candida albicans cell-wall lytic system produced by Streptomyces thermodiastaticus

The production of the enzymes of Candida albicans cell-wall lytic system by S. thermodiastaticus was found to be affected by some growth conditions and nutritional factors. The highest lytic activity was obtained after 18 h of incubation at pH 5.5 and an incubation temperature of 50 degrees C. The carbon source influenced the production of the enzymes of the yeast cell wall lytic system. Maximum lytic activity was obtained when Candida albicans cell-wall (1 g/100 ml) was used as the sole carbon source. NaNO3 at 0.1 g/100 ml level was the best nitrogen source for the biosynthesis of the enzymes of the yeast lytic system. From all phosphor sources, microelements, and growth factors tested, KH2PO4 (1 g/l), ZnSO4 (1 mg/l) and Tween 80 (0.1%), respectively were found to favour highest enzymes production of the lytic system. The Candida albicans cell-wall lytic system produced by S. thermodiastaticus mainly contained chitinolytic and proteolytic activities.     

Statistical optimization of medium components for the production of biosurfactant by Bacillus licheniformis K51

The nutritional medium requirement for biosurfactant production by Bacillus licheniformis K51 was optimized. The important medium components, identified by the initial screening method of Plackett-Burman, were H3PO4, CaCl2, H3BO3, and Na-EDTA. Box-Behnken response surface methodology was applied to further optimize biosurfactant production. The optimal concentrations for higher production of biosurfactants were (g/l): glucose, 1.1; NaNO3, 4.4; MgSO4 x 7H2O, 0.8; KCl, 0.4; CaCl2, 0.27; H3PO4, 1.0 ml/l; and trace elements (mg/l): H3BO3, 0.25; CuSO4, 0.6; MnSO4, 2.2; Na2MoO4, 0.5; ZnSO4, 6.0; FeSO4, 8.0; CoCl2, 1.0; and Na-EDTA, 30.0. Using this statistical optimization method, the relative biosurfactant yield as critical micelle dilution (CMD) was increased from 10x to 105x, which is ten times higher than the non-optimized rich medium.     

低能离子诱变选育华根霉高产L-乳酸菌株的研究

对L-乳酸产生菌华根霉(Rhizopus chinensis)适宜产酸的条件进行了初步研究和优化,并利用10 keV氮离子对出发菌L-乳酸产率进行诱变。为比较出发菌株和突变菌株在分子水平的差异,利用108条RAPD随机引物对出发菌株和突变菌株进行了比较分析。结果表明:在葡萄糖90‰、(NH4)2SO42‰、KH2PO40.6‰、Mg-SO4.7H2O 0.25‰、ZnSO4.7H2O 0.05‰、CaCO350.0‰pH值自然,接种量在5%左右,34℃,200 r/min的培养条件下,可获得较高的L-乳酸产量(28.8 g/L)。氮离子注入诱发L-乳酸产量突变的适宜剂量为1.30×1015ions/cm2。筛选获得的突变株RC-H13 L-乳酸含量为33.7 g/L,比出发菌株提高17%,且遗传稳定性较好。在RAPD分析中108条引物有3条在两池间表现多态性,差异条带的频率为0.67%。     

阿拉伯胶酶产生菌株的筛选及其发酵培养基的优化

从土壤中筛选产阿拉伯胶酶的微生物菌株,并通过紫外诱变选育后得到高产突变菌株ZHB05F,依据菌落和孢子形态特征初步鉴定为镰刀茵(Fusarium sp.).通过单因素试验,优化了产酶培养基的主要组分的浓度和pH值,得到最佳的产酶发酵培养基组成为:阿拉伯胶30 g/L,(NH4)2SO4 8 g/L,K2HPO4 1g/...     

IGF and GH mRNA levels are suppressed upon exposure to micromolar concentrations of cobalt and zinc in rainbow trout white muscle

The objective of this study was to assess the effects of cobalt and zinc exposure of rainbow trout (Oncorhynchus mykiss) on insulin like growth factors (IGF) and growth hormone (GH). Mature rainbow trouts were exposed to 0.42, 2.1, 4.2, 21 and 42μmol/L Co(2+) (added as CoCl(2)·6H(2)O) and 0.34, 1.7, 3.4, 17 and 34μmol/L Zn(2+) (added as ZnSO(4)i·7H(2)O). After 6, 12, 24 and 48h of treatment, expressions of white muscle IGF-I, IGF-II and GH mRNAs were measured by means of quantitative Real Time PCR. During the exposure experiments, no mortalities occurred. The most effective metal concentrations, which caused significant alterations, were determined to be 42μmol/L Co(2+) (10mg CoCl(2)·6H(2)O/L) and 3.4μmol/L Zn(+2) (1mg ZnSO(4)·7H(2)O/L). The following results were obtained for these concentrations. Expression of IGF-I did not change at 6h in zinc treatment while the decrease (p<0.05) was observed at 12h and 24h, and this decrease became stronger at 48h. Cobalt exposure caused a decrease in IGF-I mRNA level at 6h, 12h, 24h and 48h (p<0.05). Both zinc and cobalt exposure resulted in significant decreases in GH expression at 6h. Exposure of trout to Zn resulted in a decrease in expression of IGF-II starting from 6h whereas the significant decrease started at 6h in cobalt exposure and this decrease elevated at 24h. The results indicate that micromolar cobalt and zinc exposure causes significant attenuation in the expressions of these three genes' time dependently. Our findings show that IGF-I is the most resistant and GH is the most sensitive component against cobalt and zinc exposure. We conclude that IGF/GH axis might be strongly affected by the short term exposure to low micromolar concentrations of zinc and cobalt due to alterations of these genes.     

A novel two-stage cultivation method to optimize carbon concentration and carbon-to-nitrogen ratio for sporulation of biocontrol fungi

A two-stage solid cultivation method was used to determine the precise requirements of carbon concentration (1–16 g/L) and C: N ratio (0.625: 1 to 80: 1) for the sporulation of six biocontrol fungi. The C concentration and C: N ratio producing the highest conidia yield were 1 g/L and 5: 1 for Paecilomyces lilacinus IPC-P; 2 g/L and 10: 1 for P. lilacinus M-14; 16 g/L and 80: 1 for Metarhizium anisopliae SQZ-1-21; 4 g/L and 5: 1 for M. anisopliae RS-4-1 and Lecanicillium lecanii CA-1-G; and 2 g/L and 10: 1 for Trichoderma viride TV-1. Sporulation was more affected by C: N ratio than by C and N concentration per se. More spores per colony were produced by the two-stage method than by a conventional, single-stage cultivation method. These results should be useful for improving the mass production of these biocontrol agents.