鲟鱼中荧光假单胞菌生长预测模型构建及货架期预测

为快速预测有氧贮藏鲟鱼的货架期、预防鱼肉变质,通过将鲟鱼有氧冰藏条件下的特定腐败菌（荧光假单胞菌）接种于灭菌鲟鱼片后置于0、4、10、15、20 ℃有氧贮藏,分析其生长动态及货架期终点的感官评分、pH值和挥发性盐基氮值,在以修正的Gompertz方程为一级模型的基础上,分别以平方根方程和Arrhenius方程为二级模型,建立并验证荧光假单胞菌的生长预测模型。结果显示：荧光假单胞菌菌数的最小腐败值为（7.35±0.05）（lg（CFU/g））;同时,分别在8 ℃和波动温度条件下对模型验证的结果表明：基于平方根方程的荧光假单胞菌生长预测模型的残差分布在－0.09～0.10之间,准确度Af为1.14、1.17,偏差度Bf为0.97、1.02,货架期预测相对误差为－7.95%、－3.28%;而基于Arrhenius方程的模型误差较大,其中残差分布在－0.17～0.24之间,Af为1.21、1.31,Bf为0.94、1.08,货架期预测相对误差为24.87%、7.54%。因此,基于平方根方程建立的模型可以更有效地预测有氧包装鲟鱼在0～20 ℃贮藏温度条件下的荧光假单胞菌的生长及相应货架期。     

Modeling high-intensity pulsed electric field inactivation of a lipase from Pseudomonas fluorescens

The inactivation kinetics of a lipase from Pseudomonas fluorescens (EC 3.1.1.3.) were studied in a simulated skim milk ultrafiltrate treated with high-intensity pulsed electric fields. Samples were subjected to electric field intensities ranging from 16.4 to 27.4 kV/cm for up to 314.5 μS, thus achieving a maximum inactivation of 62.1%. The suitability of describing experimental data using mechanistic first-order kinetics and an empirical model based on the Weibull distribution function is discussed. In addition, different mathematical expressions relating the residual activity values to field strength and treatment time are supplied. A first-order fractional conversion model predicted residual activity with good accuracy (A_f = 1.018). A mechanistic insight of the model kinetics was that experimental values were the consequence of different structural organizations of the enzyme, with uneven resistance to the pulsed electric field treatments. The Weibull model was also useful in predicting the energy density necessary to achieve lipase inactivation.     

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.     

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.     

Microbial competition: effect of Pseudomonas fluorescens on the growth of Listeria monocytogenes

Listeria monocytogenes Scott A was cultured alone and in coculture with Pseudomonas fluorescens ATCC 33231 to characterize quantitatively the effects of microbial competition on the growth of this psychrotrophic pathogen. The bacteria were cultured in brain–heart infusion broth (BHI), using a 3×3×3×2 complete factorial design to assess the impact of temperature (4, 12, 19°C), initial pH (5·0, 6·0, 7·0), and sodium chloride content (5, 25, 45 gl⁻¹) on the interaction between the two micro-organisms. Samples were periodically plated on BHI agar and Vogel Johnson agar to obtain total counts and L. monocytogenes counts, respectively. Growth curves were generated by fitting the data to the Gompertz equation, and the derived growth kinetics were compared. WhenP. fluorescens did influence the growth of L. monocytogenes, the primary effect was a suppression of the maximum population density (MPD) reached by the pathogen. Suppression of L. monocytogenes was generally associated with low incubation temperatures (4°C) and sodium chloride levels (5 and 25 gl⁻¹). Slight increases (<1·0 log cfu ml⁻¹) in the MPD attained by L. monocytogenes were observed when grown in the presence of P. fluorescens at higher temperatures (12 and 19°C) and sodium chloride levels (25 and 45 gl⁻¹) when the pH was 5·0. The current study supports earlier work that indicates that reliance on microbial competition as a barrier to control L. monocytogenes in refrigerated foods will require detailed knowledge of how the interaction between the pathogen and the microflora is affected by environmental and food characteristics such as storage temperature, pH, and water activity.     

加压CO2对乳中荧光假单胞菌杀菌效果的研究

为了研究加压CO2对乳中荧光假单胞菌的杀菌效果,首先采用单因素试验研究处理压力、处理时间和处理温度分别对杀菌效果的影响,然后通过正交试验确定最佳杀菌条件。结果表明:当压力为7 MPa,时间为70 min,温度为40℃时,加压CO2对荧光假单胞菌杀菌率为99.80%。     

黑曲霉糖化酶基因在荧光假单胞菌中的表达

黑曲霉糖化酶基因cDNA按正确的读码框架克隆到广宿主表达载体pBBR1MCS-2,构建出含黑曲霉糖化酶基因的表达载体pBBR1MCS-2GA,用三亲和接合转化法将重组质粒导入2-酮基-D-葡萄糖酸高产菌株荧光假单胞菌AR4。得到重组菌ARW4,经SDS-PAGE凝胶电泳和Westernblot分析显示,黑曲霉糖化酶基因在ARW4中得到表达,表达产物主要以不溶性包涵体形式存在。     

不同品牌CN琼脂上铜绿假单胞菌色素表达的研究

目的 对铜绿假单胞菌在不同品牌CN琼脂上的色素表达及其原因进行研究。方法 比较ATCC 10145、ATCC 15442、CMCC 10104 3种铜绿假单胞菌标准菌株在5种不同品牌CN琼脂上的色素表达, 并测定CN琼脂的磷含量。结果 5种CN琼脂的磷含量在123~1560 mg/kg之间, 最高的磷含量与最低磷含量相差10余倍。只产青脓素的铜绿假单胞菌ATCC 15442在各CN琼脂上均为黄色菌落, 在紫外线下产荧光。只产绿脓菌素的铜绿假单胞菌ATCC 10145, 在含磷量低的CN琼脂上呈现为蓝色菌落, 表现为产绿脓菌素; 而随着磷含量的增加, CN琼脂上绿脓菌素的表达逐渐减弱。既产绿脓菌素又产青脓素的铜绿假单胞菌CMCC 10104在含磷量低的CN琼脂上呈现为蓝色菌落, 表现为产绿脓菌素; 而随着磷含量的增加, CN琼脂上绿脓菌素的表达逐渐减弱, 青脓素的表达逐渐增强, 菌落颜色逐渐变黄并在紫外线下产荧光。结论 铜绿假单胞菌在5种不同品牌的CN琼脂上的表达色素的情况与CN琼脂的磷含量有关, CN琼脂含磷量越低, 越有利于绿脓菌素的表达, 青脓素的表达受到抑制; CN琼脂含磷量越高, 越有利于青脓素的表达, 绿脓菌素的表达受到抑制。     

乙醛对荧光假单胞菌的抑菌活性及机理

该研究通过分析乙醛对荧光假单胞菌(Pseudomonas fluorescens)菌体细胞膜完整性、膜电位、总蛋白含量、Na+K+-ATP酶变化以及细菌生物被膜的影响,探究乙醛对P. fluorescens的抑菌活性及其抑菌机理。结果表明:乙醛对P. fluorescens的最小抑菌浓度MIC值为0.5μL/m L,最小杀菌浓度MBC值为1μL/m L;电导率和二乙酸荧光素染色实验发现乙醛处理可引起P. fluorescens细胞膜出现破裂;添加1×MIC、2×MIC、4×MIC乙醛3 h后降低菌体膜电位,平均荧光强度从72.10 AU分别下降至35.57、15.31和7.46 AU,影响细菌的代谢活力;不同浓度乙醛处理后胞内蛋白质的含量3 h内分别下降至0.40、0.35和0.34 mg/mL,3~12 h期间呈现平缓、略有下降,表示细胞膜的完整性遭到破坏;乙醛降低了Na⁺K⁺-ATP酶活性,导致胞内ATP代谢异常,不能正常为细胞活动供给能量,促使荧光假单胞菌的凋亡;0.25μL/m L的乙醛对生物被膜形成抑制率为30.11%,显著降低P. fluorescens生物被膜形成。由此可见,乙醛对P. fluorescens可能的抑菌机理是改变菌体细胞膜完整性,为拓展水产品的防腐保鲜提供新思路。     

Purification and characterization of the lipase from Pseudomonas fluorescens HU380

A lipase, which markedly splits polyunsaturated fatty acid ester (PUFA) bonds, from newly isolated Pseudomonas fluorescens HU380 was purified. The purification procedure included Phenyl-Toyopearl fractionation, DEAE-Sepharose chromatography, and Superdex-200HR chromatography. The enzyme was purified 24.3-fold with a yield of 14% and a specific activity of 9854 U/mg. Its molecular weight was estimated on SDS-PAGE to be 64,000. The optimum pH and temperature were 8.5 and 45 degrees C, respectively. The lipase was stable over the pH range of 6.0-7.0 at 30 degrees C for 24 h, and up to 40 degrees C at pH 7.0 for 60 min, when 0.1% Triton X-100 was present. The lipase preferably acted on short to middle-chain fatty acid simple methyl-esters and triglycerides, and cleaved mainly 1,3-ester bonds and to a lesser extent the 2-position ester bond of triolein. The lipase was inhibited by Co2+, Ni2+, Fe3+, Fe2+, and EDTA, and activated by Ca2+. Its N-terminal amino acid sequence was determined to be GVYDYKNFGTADSKALFSDAMAITLY, which exhibited considerable similarity with those of the lipases from other P. fluorescens strains, but no significant homology with other lipases. This lipase was able to decompose fats and oils that contained eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) without significantly affecting the contents of these fatty acids. The results suggest that the lipase may be useful when applied to the processing of industrial fats and oils containing EPA and DHA, such as fish oil splitting.     

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.     

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。      

壳聚糖复合保鲜剂对荧光假单胞菌的抑菌活性

该实验以质量分数1.5%壳聚糖、0.14%ε-聚赖氨酸、0.15%D-异抗坏血酸钠组成复合保鲜剂,以荧光假单胞菌为实验菌株,探究壳聚糖复合保鲜剂对水产品中的优势菌的抑菌性能和机理。实验方法：通过测定最小抑菌浓度(MIC)、抑菌圈、细菌生长曲线考察保鲜剂对荧光假单胞菌的抑菌活性,测定OD₂₆₀值、ATP酶和AKP酶活性的变化,细胞超微结构(SEM)和SDS凝胶电泳法研究保鲜剂的抑菌机制。结果表明：壳聚糖复合保鲜剂对荧光假单胞菌的最低抑菌浓度(MIC)为2.24 mg/m L。壳聚糖复合保鲜剂有着显著的抑菌活性,壳聚糖复合保鲜剂导致菌体的细胞壁膜通透性增大、完整性被破坏,菌体内ATP和AKP酶活性被抑制,显著低于(P<0.05)对照组;SDS凝胶电泳表明壳聚糖复合保鲜剂使菌蛋白条带颜色变浅且造成部分蛋白条带消失;细菌超微结构(SEM)显示壳聚糖复合保鲜剂使菌体发生变形破裂,内容物大量流出,导致菌体死亡。结论：该研究证明了壳聚糖复合保鲜剂良好的抑菌性能,研究了保鲜剂对荧光假单胞菌的抑菌机理,为水产品可食性涂膜保鲜的研发提供理论支持。     

固定化青霉素酰化酶对荧光假单胞菌群体感应的抑制作用

水产品腐败菌的群体感应现象是加速水产品腐败的原因之一。近年来,由于化学保鲜剂的使用导致许多腐败菌产生抗性,并且其安全性也令消费者担忧,因此研究安全、高效的生物保鲜剂是水产保鲜中的重要课题。本研究用固定化青霉素酰化酶来抑制水产品优势腐败菌——荧光假单胞菌的群体感应现象。利用CV026验证固定化青霉素酰化酶对荧光假单胞菌AHLs的淬灭作用;利用光学显微镜和扫描电镜研究酶对生物被膜的影响;利用牛奶琼脂平板验证胞外蛋白酶的活性;通过分子对接预测酶与不同AHLs的相互作用。结果表明,固定化青霉素酰化酶能够降低荧光假单胞菌AHLs的含量,从而减少紫色杆菌CV026紫色菌素的生成,减缓荧光假单胞菌生物被膜和胞外聚合物的形成。同时也干预了胞外蛋白酶的产生,当酶质量浓度为15 mg/mL时无法观察到明显的蛋白抑制圈。分子对接结果显示,该酶与短链和长链的信号分子都能成功对接,然而,更偏向于长链AHLs。3D对接结果显示,C14-HSL被活性中心周围的氨基酸残基全部包裹,相互以氢键连接,氢键的数量较多,结合作用更强。由此说明固定化青霉素酰化酶具有良好的群体感应淬灭活性,可作为新型保鲜剂应用于水产品保鲜。     

鱼源荧光假单胞菌生物被膜形成和致腐表型的研究

荧光假单胞菌是养殖鱼类低温贮藏中的优势腐败菌。本研究比较分析5种荧光假单胞菌的生物被膜形成和腐败表型。采用结晶紫法、苯酚硫酸法、珠涡流法和荧光显微镜观察荧光假单胞菌的生物被膜形成和粘附能力,并测定细菌泳动性、蛋白酶活性、嗜铁素等致腐表型。结果表明,5株荧光假单胞菌在28℃LB肉汤中生长良好,经24 h培养后气-液界面上出现较厚的膜,在微孔板中生物被膜形成较快,其中鱼源PF01、PF06、PF07和PF10分离株在12 h含量最高,而标准菌株PFuk4在18 h最高。随着培养时间延长,细菌胞外多糖的含量逐步积累,在18～24 h达到最高,并较快粘附到不锈钢片表面,其中PF07的粘附量最高。5株荧光假单胞菌还具有较强的泳动性和蛋白酶活性,且均产嗜铁素。在PF07和PFuk4中还检测出短链高丝氨酸内酯(AHLs)活性,可能与其较高的被膜、粘附能力、泳动性及蛋白酶活性有关。本研究结果为从AHLs角度探究荧光假单胞菌的致腐机理打下良好的基础。     

Characterization of lipase of Pseudomonas fluorescens 27 based on fatty acid profiles

The objectives of this research were to isolate lipase from Pseudomonas fluorescens 27, to compare the purity of the partially purified lipase preparation with crude extract, and to determine if bands of lipase activity revealed by disc gel electrophoresis liberated different free fatty acids from milk fat. Lipases were isolated from a shaken skim milk culture of P. fluorescens 27 by using ion-exchange chromatography on DEAE cellulose (Whatman DE 32) and gel filtration on Sephadex G-150. The principal lipase-rich fractions from gel filtration represented 6.2% of total lipolytic activity. Disc gel electrophoresis of partially purified enzyme revealed two protein bands. These protein bands were cut from disc electrophoresis gels and used as an enzyme source for reaction with butter oil. Free fatty acids were isolated from the assay mixture, separated, and quantified by gas chromatography. Data from gas chromatographic analysis indicated that P. fluorescens 27 produces at least two different lipases.     

荧光假单胞菌AR4产2-酮基-D-葡萄糖酸发酵条件的研究

以淀粉水解糖为碳源,研究了不同氮源、碳氮比、金属离子、接种量以及环境因子(培养基起始pH值、通气量、温度)对抗噬菌体菌株荧光假单胞菌(Pseudomonas fluorescens)AR4产2-酮基-D-葡萄糖酸发酵的影响。结果表明,氮源、碳氮比、培养基的起始pH值、通气量及温度对该菌的产酸均有显著影响。在适宜培养条件下,该菌的摇瓶产酸及发酵转化率分别可达13.5%和90%左右。     

荧光假单胞菌噬菌体对大西洋鲑贮藏过程中品质变化情况研究

目的 探究大西洋鲑在4℃贮藏过程中, 外源添加荧光假单胞菌及其噬菌体影响大西洋鲑品质变化相关的常见指标的变化规律, 为噬菌体在实际食品作为保鲜剂的应用提供科学依据。方法 采用色差值、菌落总数(Total viable count, TVC)、挥发性盐基氮(Total volatile basic nitrogen, TVB-N)、质构、硫代巴比妥酸(Thiobarbituric acid, TBA)、pH、剪切力、持水性等致腐能力指标对冷藏温度(4℃)条件下大西洋鲑的新鲜度变化进行综合评价。结果 4℃贮藏期间, 初期微生物生长迅速且数量变化明显, 噬菌体vB_PF_Y1-MI的加入使得鱼肉中TVC下降了大约0.07 lg ( CFU·g-1 ); 大西洋鲑TVB-N含量整体变化趋势明显呈指数型增长, pH值均呈现先下降后上升的变化趋势; 添加荧光假单胞菌组的大西洋鲑TBA值和失水率增加程度较为明显, 经噬菌体处理后增加程度有所降低。结论 噬菌体vB_PF_Y1-MI的加入, 能够降低大西洋鲑鱼肉脂质氧化速率, 可以延缓其腐败变质, 从而延长大西洋鲑保质期。     

Survival of a Pseudomonas fluorescens and Enterococcus faecalis aerosol on inert surfaces

The aim of the work presented here is to study airborne bacteria survival on three inert supports (glass, polyvinyl chloride, and stainless steel). Two Pasteur Institute bacteria were selected: Enterococcus faecalis 10.30.15 and Pseudomonas fluorescens 56.90. We have observed that bacterial aerosol lethality increased proportionally with the relative humidity of the environment, the gram-negative rod appearing more fragile than the cocci. A significant difference in survival rate is measured dependent on the supports tested, the greatest lethality being observed on the PVCs.