肉桂醛对荧光假单胞菌生物膜形成的抑制作用

本文拟研究肉桂醛在非抑菌浓度条件下对荧光假单胞菌（Pseudomonas fluorescens）FML05-2生物膜形成的抑制作用。首先测定了肉桂醛对模式细菌紫色杆菌（Chromobacterium violaceum）CVO26紫色素产生的影响;再对荧光假单胞菌FML05-2形成的生物膜以及与生物膜形成有关的重要因素胞外多糖进行了测定。结果表明:在非抑菌浓度40、20μg/m L条件下,肉桂醛对紫色杆菌CVO26紫色素的产生具有抑制作用,抑制率分别为31.53%、17.90%;对荧光假单胞菌FML05-2生物膜形成的抑制率分别为44.22%、21.77%;对荧光假单胞菌FML05-2胞外多糖产生的抑制率分别为15.72%、5.34%。因此,肉桂醛在非抑菌浓度条件下对荧光假单胞菌FML05-2生物膜的形成具有抑制作用。      

Purification and characterization of the extracellular proteinase of Pseudomonas fluorescens NCDO 2085

Pseudomonas fluorescens NCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 degrees C and pH 7.0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3.5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5.40 +/- 0.05 and a mol. wt of 40 200 +/- 2100. It is heat-stable having D-values at 74 and 140 degrees C of 1.6 and 1.0 min respectively; 40 and 70% of the original activity remained after HTST (74 degrees C/17 s) and ultra high temperature (140 degrees C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from other Pseudomonas spp.     

Thermal stability of an extracellular proteinase from Pseudomonas fluorescens AFT 36

A metalloproteinase, isolated from a shaken milk culture of Pseudomonas fluorescens AFT 36 by chromatography in DEAE and CM-cellulose and Sephadex G-150, was very unstable in 0.1 M-phosphate buffer, pH 6.6, being completely denatured above 70 degrees C in 1 min. It was also unstable in a Ca-containing buffer (synthetic milk salts, SMS) between 50 and 60 degrees C (minimum at 55 degrees C), but stability was very high above 80 degrees C in this buffer. D-values were determined at 10 degrees C intervals in the range 70-150 degrees C in SMS from which a Z value of 31.9 degrees C and an Ea of 8.82 X 10(4) J mol-1 were calculated; the half-life at 150 degrees C was 9 s. Instability at 55 degrees C was due to autolysis as evidenced by gel electrophoresis, gel filtration and increase in 2,4,6-trinitrobenzenesulphonic acid-reactive amino groups. The extent of inactivation experienced at 80 degrees C was inversely related to the rate of heating to 80 degrees C, i.e. length of time spent in the neighbourhood of 55 degrees C. Addition of increasing concentrations of caseinate substrate reduced inactivation of the enzyme at 55 degrees C, presumably due to substrate binding. Attempts to stabilize the enzyme at 55 degrees C by addition of EDTA or by adjusting the reaction pH to 4.2, at which the enzyme has little proteolytic activity, were unsuccessful, although autolysis was prevented. Unlike the proteinase from Ps. fluorescens MC 60, AFT 36 proteinase did not inactivate itself on cooling to 55 degrees C from 80, 100 or 150 degrees C, but did regain autolytic activity on cooling to below 50 degrees C to an extent dependent on the duration of holding at the lower temperature. It is suggested that on heating to approximately 55 degrees C, a conformational change occurs which renders the enzyme susceptible to proteolysis by still active enzyme; at higher temperatures the enzyme, although susceptible to autolysis, is inactive; an active conformation is restored on cooling to below 50 degrees C.     

Autolysis of the proteinase from Pseudomonas fluorescens.

The gene encoding the proteinase from Pseudomonas fluorescens was cloned and sequenced in an effort to identify the cleavage sites involved in its autolysis at 50 degrees C. A single open reading frame consisting of 1449 nucleotides, encoding a protein of 482 amino acids, was found. Analysis of the N-terminal amino acid sequence of the purified proteinase indicated the presence of a prosequence consisting of 13 amino acid residues. The molecular weight of the mature protein was calculated as 48,900 based on the deduced amino acid sequence, which was consistent with that of the purified proteinase as determined by sodium dodecylsulfate-PAGE. Greater than 90% loss of proteolytic activity was observed upon heating at 50 degrees C for 2 min compared with the unheated enzyme. Incubation of the proteinase at 50 degrees C led to disappearance of the intact enzyme, as shown by sodium dodecyl sulfate-PAGE, whereas it was stable in the presence of the protease inhibitor o-phenanthroline. Autolytic fragments were fractionated by reverse-phase HPLC and subjected to N-terminal amino acid sequence analysis in an effort to determine the cleavage sites. The cleavage profile was not definitive; however, amino acid residues with small side chain groups, such as glycine or alanine, were frequently found adjacent to the cleavage sites.     

纳米硒化荧光假单胞菌KBD-1菌株对烟草病毒病的抑制作用

为挖掘具有纳米硒化能力的生防菌株,开发烟草病毒病的绿色防治材料,从烟草病毒病重病田块健株根际土壤中筛选获得到一株荧光假单胞菌Pseudomonas fluorescens KBD-1,对其进行了纳米硒化,采用Real-time PCR和Western blot测定了两者对烟草常见病毒基因表达的影响,并采用接种法测定了其对烟株相应病毒病的防治作用。结果表明,纳米硒化后的KBD-1菌体外部存在高密度电子颗粒,在X射线11.22 keV处出现硒的特征吸收峰。纳米硒化后的P.fluorescens KBD-1菌液浓度在5×10⁵ cfu/mL（单质硒浓度0.25 mg/L）时,对烟草花叶病毒（TMV）、马铃薯Y病毒（PVY）、黄瓜花叶病毒（CMV）、番茄斑萎病毒（TSWV）4种病毒病的防治效果均在88.0%以上,对CMV防治效果最高达91.4%,该浓度处理对烟株促生效果显著。荧光假单胞菌KBD-1可成功将亚硒酸钠还原生成纳米硒,并能增强原始菌株的抗烟草病毒活性和促生效果。     

Inhibition of Pseudomonas fluorescens by Hydroxycinnamic Acids and Their Alkyl Esters

Alkyl esters of six hydroxycinnamic acids and cinnamic acid were evaluated in a liquid medium for their effectiveness in inhibiting the growth of Pseudomonas fluorescens, a bacterium commonly implicated in food spoilage. Compounds were tested at concentrations of 0–250 ppm, using turbidity as a measurement of growth. At 125 ppm, esters of p-coumaric and caffeic acids were more inhibitory than either methyl or propyl paraben. Methyl esters of methoxylated cinnamic acids were much less inhibitory than the methyl esters of the corresponding positional hydroxylated cinnamic acids. The results indicate that esters of certian of these naturally occurring hydroxycinnamic acids may be useful as food additives to extend shelf life by inhibiting microbial growth.     

A rapid colorimetric assay for the extracellular lipase of Pseudomonas fluorescens B52 using beta-naphthyl caprylate

Conditions necessary for the determination of crude extracellular lipase activity from Pseudomonas fluorescens B52 using beta-naphthyl caprylate (beta-NC), an 8-carbon ester, as the substrate were examined. Maximum enzyme synthesis occurred at 20 degrees C in pyruvate mineral salts medium containing 1 mM-CaCl2. Bile salts were necessary for enzyme activity; 6 mM-Na taurocholate or Na deoxycholate gave maximum activity but the latter compound was inhibitory at higher concentrations. Activity was optimal in N-Tris[hydroxymethyl]methyl-2-aminoethane sulphonic acid buffer pH 8.0 at 40 degrees C. A comparison of beta-NC with beta-naphthyl butyrate (a 4-carbon ester) and beta-naphthyl myristate (a 14-carbon ester) showed that beta-NC was the best substrate; Km values of 0.0415, 0.141 and 0.200 mM and Vmax values of 67.2, 20.1 and 5.28 mumol ml-1 h-1 were obtained for those substrates respectively. The enzyme was inhibited 50% in its activity against beta-NC by 0.009 mM-EDTA, 0.0007% (w/v) mixed alkyltrimethylammonium bromide and 0.00275% (w/v) Triton X-100. The biochemical properties determined using beta-NC as substrate are consistent with those reported for the lipases of other strains of Ps. fluorescens using natural substrates.     

Effect of carbon dioxide on growth and extracellular enzyme production by Pseudomonas fluorescens B52

The effect of carbon dioxide (30 mM.l-1) on growth and extracellular protease and lipase production by Pseudomonas fluorescens B52 growing in a simulated milk medium at 7 degrees C in fermenter was investigated. The addition of carbon dioxide was shown to have a differential effect with extracellular enzyme production, particularly lipase, being inhibited to a greater extent than bacterial growth and final cell numbers.     

Purification and characterization of an extracellular protease produced by Pseudomonas fluorescens M3/6.

Pseudomonas fluorescens strain M3/6 was inoculated into reconstituted NDM and incubated at 7 degrees C for 46 d. A significant amount of extracellular protease was produced, mainly during the latter part of the culture's life cycle. The protease was purified using ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The isolated protease had activity on azocasein, alpha-, beta-, and kappa-caseins and a plasmin substrate but did not have plasminogen activator activity. The protease had a molecular weight of 45 kDa, an isoelectric point of pH 8.25, a broad temperature and pH range for activity, and was less heat stable in the isolated form than in the cell-free extract.     

Colonization of Beef Muscle Surfaces by Pseudomonas fluorescens and Pseudomonas fragi

Attached and unattached cell densities were determined for Pseudomonas fluorescens and Pseudomonas fragi growing on the surface of beef muscle stored at 4 and 25°C, in presence of NaCl, KCl, and CaCl₂. A mechanical rinsing procedure was developed for this purpose. Both species colonized the surface at both temperatures and were enhanced at low (4°C) temperature. Attached cells represented up to 90% of the total until a density of 10⁵-10⁶ CFU cm^?2 was reached. At that point, a proportion of attached cells to unattached cells declined but colonization of the surface continued. In presence of CaCl₂, ratios of attached to unattached cells did not decline, suggesting a significant role for the calcium ion in colonization. Ability to colonize meat surfaces may be a significant competitive advantage for meat spoilage bacteria such as Pseudomonas fragi and Pseudomonas fluorescens.     

Growth of an extracellular proteinase-deficient strain of Pseudomonas fluorescens on milk and milk proteins

An extracellular proteinase-and lipase-deficient mutant of a psychrotroph, Pseudomonas fluorescens strain 32A, has been isolated and the absence of the proteinase enzyme confirmed by growth on differential media, enzyme assay and polyacrylamide gel electrophoresis. Competition between the parent and the mutant was observed when equal numbers of the 2 strains were inoculated together into raw skim-milk at 6 degrees C. Bitterness was detected at 6 degrees C in pasteurized skim-milk inoculated with the parent cells concurrent with the detection of proteolytic activity. In the case of the mutant, slight bitterness which did not increase with increasing cell numbers was detected in the absence of proteolysis. Mutant cells failed to grow on Na caseinate as the sole source of carbon. It was concluded that the extracellular proteinase, while not essential for growth in milk, does provide a selective advantage to the producer organism. This enzyme is, however, essential for growth on milk proteins and contributes to the development of bitterness in pasteurized milk.     

低温等离子体对气调包装中荧光假单胞菌的抑杀机理

为探讨低温等离子体(CAP)对气调包装中荧光假单胞菌的杀菌条件以及CAP在气调包装中的杀菌机理,以杀菌率为指标,采用三因素三水平正交试验法优化CAP处理条件;采用细胞活性、细胞壁通透性、细胞膜完整性、抗氧化酶活性及微观结构等指标研究CAP对荧光假单胞菌的抑菌机理。结果显示：CAP协同气调包装的最佳杀菌条件为处理电压50 kV、处理时间180 s、气调气体体积比V(N2)∶V(CO₂)=3∶2,在此条件下杀菌率为99.49%。CAP对荧光假单胞菌的抑菌机理表明,低温等离子体处理使细胞活性降低;破坏了细胞壁和细胞膜的完整性,核酸和蛋白质泄露量增加,Na+/K+-ATP酶(ATP)下降了13.10 U/mg prot,碘化丙啶(PI)升高,碱性磷酸酶(AKP)活力下降为12.09金氏单位/g prot;细胞内抗氧化酶超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性下降;改变了细胞的形态,使细胞黏连和凹陷。结论：低温等离子体协同气调包装可有效抑杀荧光假单胞菌,致使细胞失活或死亡,且处理时间越长效果越好。     

Isolation and some properties of extracellular heat-stable lipases from Pseudomonas fluorescens strain AFT 36

Aeration increased the growth and lipase production in milk by Pseudomonas fluorescens strain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented approximately 71% of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8.0 and 35 degrees C; it had a Km on tributyrin of 3.65 mM and was inhibited by concentrations of substrate greater than approximately 17 mM. The enzyme was very stable over the pH range 6-9; it was relatively heat-labile in phosphate buffer in the temperature range 60-80 degrees C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100-150 degrees C: the D values at 150 degrees C were approximately 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the corresponding Z values in the temperature range 100-150 degrees C were approximately 40 and approximately 42 degrees C and the Ea for inactivation were 7.65 X 10(4) J mol-1 and 6.97 X 10(4) J mol-1 respectively.     

PF7-5对烟草野火病的抑制及田间防治效果

为验证荧光假单胞杆菌PF7-5(CGMCC No.2069)对烟草野火病菌(Pseudomonas syringae pv.tabaci)的生防效果,设置了PF7-5对烟草野火病菌的室内抑菌及田间防治试验.结果表明,稀释5倍的PF7-5代谢产物室内对烟草野火病菌的抑制效果达96%,PF7-5活菌田间抑菌效果达91%.证实PF7-5对烟草野火病具有显著的拮抗作用和防治效果.     

Determination of the extracellular lipases of Pseudomonas fluorescens spp. in skim milk with the beta-naphthyl caprylate assay

A method based on the hydrolysis of beta-naphthyl caprylate (beta-NC) has been developed for quantitating extracellular lipase from Pseudomonas fluorescens. The assay was extremely sensitive to skim milk (SM); as little as 0.02 ml raw SM in a 2.0 ml reaction mixture resulted in an apparent loss of 50% of the lipase activity. Activity improved 3-fold when trypsin (50 micrograms/ml) was included in the reaction mixture. When super-simplex optimization was used to determine the optimum levels of beta-NC, Na taurocholate (NaTC), SM/lipase mixture and trypsin for maximum activity, NaTC was found to be unnecessary for activity. Subsequent addition of 15 mM-NaTC resulted in 80% loss of activity. On the other hand, NaTC was required for native lipase activity in the presence of SM. Native lipase was completely inhibited by heating at 70 degrees C for 2 min, while B52 lipase retained 75% of its activity under the same conditions. The assay was able to detect lipase produced by Ps. fluorescens B52 in SM at 5 degrees C when the cell density exceeded 10(8) colony forming units/ml. The presence of butterfat (3.5%) in the SM assay inhibited B52 lipase by 97%. The beta-NC assay gave results comparable to the tributyrin agar diffusion assay using cell-free extracts of ten strains of common dairy psychrotrophs. The results suggest that the beta-NC assay may be useful for determining lipase activity in raw SM.     

Inactivation of Pseudomonas fluorescens by High Voltage Electric Pulses

A 30 kV pulsed power treatment system was designed and developed to process fluid materials. Our objective was to investigate the effects of high voltage electric pulses on microbial inactivation in an aqueous solution under different operating conditions and fluid properties. Electric field strength at 10 kV/cm for 10 pulses (2 set pulse period and 2 Fsec pulse width) with a spike of reverse polarity resulted in significant microbial control. P. fluorescens in various aqueous solutions were reduced in population by more than six log cycles. However, the critical electric field strength was affected by the nature of the pulse waveform across the treatment chamber which, in turn, was a function of electrode distance and fluid properties. Sudden charge reversal immediately at the end of a pulse provided maximum microbial decay.