铜绿假单胞菌基因型与生物被膜形成能力的分析

通过改良的组织平板培养法及ERIC-PCR方法对48株PA生物被膜的形成进行定量分析并绘制其指纹图谱。结果发现48株PA菌株生物被膜形成能力不同,且在17%相似水平上分为A、B、C、D、E五个基因型,其中生物被膜形成能力较弱的菌株大多属于前三个基因型,而D和E基因型则包含了大部分生物被膜形成能力较强的菌株,这表明不同生物被膜形成能力的PA在5个基因型中的分布并非随机的,菌株生物被膜形成能力和基因型之间具有一定的相关性。     

氯化钙对食品致腐荧光假单胞菌生物被膜形成的影响

研究氯化钙（CaCl2）对食品腐败株荧光假单胞菌（Pseudomonas fluorescens）生物被膜形成特征的影响。采用结晶紫法、菌体计数、苯酚-硫酸法、共聚焦扫描显微镜和实时荧光定量聚合酶链式反应（polymerase chain reaction,PCR）检测Ca2＋对荧光假单胞菌的生物被膜形成、多糖分泌、被膜结构及相关基因表达的影响。结果表明,0.1 mmol/L Ca2＋刺激荧光假单胞菌生物被膜,随着浓度增加被膜形成增强,其中1 mmol/L促进最明显,而高于10 mmol/L作用减弱,并且Ca2＋不影响浮游细菌生长;同时,0.1 mmol/L和1 mmol/L Ca2＋对胞外多糖、薄膜和泳动性均呈现促进效果,而高浓度下呈现抑制;共聚焦扫描显微镜观察荧光假单胞菌对照组、1 mmol/L和20 mmol/L Ca2＋处理组的成熟生物被膜厚度分别为20.0、40.0 μm和25.0 μm,其中添加1 mmol/L Ca2＋显著增加PF07被膜厚度、菌体和胞外聚合物分泌量,使被膜结构更致密;实时荧光定量PCR检测显示,1 mmol/L Ca2＋刺激菌体黏附素lapA、藻多糖alg、鞭毛flgA基因表达量增加3～4 倍,并且Ca2＋均显著刺激AHLs合成酶luxI基因的表达,提示Ca2＋影响生物被膜与群体感应密切相关。可见,食品介质中CaCl2通过影响菌体黏附行为、胞外分泌物、基因表达导致荧光假单胞菌生物被膜形成特征和结构的改变,该研究为复杂的食品介质中腐败菌生物被膜形成和黏附提供依据。     

不同环境条件对隆德假单胞菌生物被膜形成能力的影响

为研究环境条件对食品腐败菌隆德假单胞菌（Pseudomonas lundensis,PL）生物被膜形成能力的影响,采用微孔板结晶紫法测定不同的营养条件、接种浓度、pH、NaCl和Mg2+浓度条件下其生物被膜量,用激光共聚焦扫描显微镜观测其在不锈钢材料上的黏附和结构特征。结果表明,PL生物被膜的形成量在营养胁迫下降低,稀释TSB培养基50倍造成的营养胁迫使其生物被膜量从1.75±0.35降低至0.24±0.17。PL接种量从1.3×107 CFU/mL降至2.5×104 CFU/mL时,其生物被膜的形成量无显著差异（P>0.05）,在pH为8.0时PL形成生物被膜量最多,1.25%以上的葡萄糖、4%以上的NaCl和0.5%的Mg2+能够显著（P<0.05）抑制PL生物被膜的形成。激光共聚焦显微镜观测结果表明该菌在一定浓度下具有在不锈钢表面形成典型生物被膜的能力。PL生物被膜形成能力受营养条件、葡萄糖浓度、pH、NaCl浓度、Mg2+等环境因子的影响。     

植物提取物对浅黄假单胞菌生物被膜的作用

本文研究具有抑菌效果的药食同源植物提取物对冷却肉中关键腐败菌——假单胞菌生物被膜的抑制、清除效果.从市售冷却肉中分离一株具有较强生物被膜形成能力的假单胞菌,经鉴定为浅黄假单胞菌8-4.采用结晶紫染色法观察蒲公英、金银花、丁香、肉桂、薄荷、桑叶等6种提取物对浅黄假单胞菌生物被膜的抑制和清除作用.结果表明:金银花提取物和蒲...     

桃金娘果实花色苷提取物对两种假单胞菌生物被膜的抑制

为研究桃金娘果实花色苷提取物对隆德假单胞菌(Pseudomonas lundensis)和荧光假单胞菌(Pseudomonas fluorescens)生物被膜的抑制作用,采用肉汤稀释法测定了桃金娘果实花色苷提取物对两种假单胞菌的最小抑菌浓度和最小杀菌浓度,在亚抑菌浓度下采用结晶紫微孔板染色法测定了桃金娘果实花色苷提取物对两种假单胞菌的抑制作用和粘附情况。结果表明,桃金娘果实花色苷提取物对两种假单胞菌均有一定的抑制作用,对隆德假单胞菌和荧光假单胞菌的最小抑菌浓度分别为1500和1000 mg/mL,在500 mg/mL的亚抑菌浓度下,桃金娘果实花色苷提取物对隆德假单胞菌和荧光假单胞菌生物被膜具有显著抑制效果,能够显著降低两种假单胞菌在接触面上的粘附量(p<0.05)。桃金娘果实花色苷提取物具有开发为隆德假单胞菌和荧光假单胞菌生物被膜抑制剂的潜在价值。     

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。     

金银花和蒲公英提取物对肉源性假单胞菌生物被膜的清除作用

为明确金银花和蒲公英提取物对假单胞菌生物被膜的清除效果,采用单因素实验确定假单胞菌生物被膜的最佳培养温度、培养时间和固定方法;在此条件下,采用结晶紫法结合扫描电镜研究金银花和蒲公英提取物在不同质量浓度、不同添加时间下对肉源性假单胞菌生物被膜的清除作用。结果表明,30 ℃培养24 h生物被膜测定值最高。提取物对生物被膜的清除效果随质量浓度的增加而逐渐增强(P<0.05),在生物被膜形成0 h加入的清除效果显著强于在24 h加入的清除效果(P<0.05)。当提取物浓度高于最小抑菌浓度(MIC)时,主要通过抑制菌体生长而清除生物被膜;当低于最小抑菌浓度时,主要通过破坏生物被膜立体结构而清除生物被膜。提取物破坏假单胞菌生物被膜结构,使生物被膜形成孔洞,从而清除生物被膜。本研究证明金银花和蒲公英提取物能够清除假单胞菌生物被膜,对肉的保鲜具有潜在应用价值。     

Nisin与和厚朴酚联用对铜绿假单胞菌生物被膜的协同抑制作用

目的:铜绿假单胞菌生物被膜(BF)难以清除,是引起食品持续性污染的重要原因。Nisin作为一种天然食品防腐剂可抑制多种食源性致病菌,然而其对革兰氏阴性菌效果差。通过Nisin与和厚朴酚联用来增强对铜绿假单胞菌ATCC 9027生物被膜的抑制和清除能力,并探究其作用机制。方法:首先,筛选可抑制铜绿假单胞菌BF的中药成分及最小抑制浓度;然后评估Nisin与和厚朴酚联用对铜绿假单胞菌生物被膜的协同抑制作用及其效果随时间的变化,以及对BF结构、组成成分、相关基因表达的影响。结果:和厚朴酚对铜绿假单胞菌ATCC 9027生物被膜的抑制效果最强,选择其作为与Nisin联用的中药成分。Nisin与和厚朴酚的MBIC50分别为1 mg/mL和12.5 μg/mL。多个组合均有协同抑制作用,其中1.5625 μg/mL和厚朴酚与0.125 mg/mL Nisin对BF的协同抑制效果最佳,在18 h达到最高,并且二者联用显著影响BF结构,降低胞外多糖和eDNA的含量,同时BF相关基因lasR、pelA、algC、pqsA、lasI的表达量降低。结论:Nisin与和厚朴酚联用对铜绿假单胞菌ATCC9027生物被膜具有协同抑制作用,其可降低BF相关基因转录水平以减少EPS和eDNA的分泌,从而抑制和清除BF。本研究旨在为减少由铜绿假单胞菌生物被膜引起的食品安全问题提供解决策略。     

冷却猪肉及托盘表面细菌生物被膜分析和肉源荧光假单胞菌的鉴定与被膜研究

为研究冷却猪肉及其接触面中细菌生物被膜能力并探讨肉类特定腐败菌的生物被膜特征,分析了腐败的冷却肉和销售托盘表面的细菌生物被膜形成能力;利用形态学观察、16S r DNA分析和VITEK2微生物鉴定系统鉴定了冷却肉中一株生物被膜能力较强的荧光假单胞菌(Pseudomonas fluorescens);测定了该菌在培养基和猪肉浸提液中的生物被膜能力;用激光共聚焦和扫描电子显微镜观测了其生物被膜的结构特征。结果发现:冷却肉中及其销售托盘表面的细菌能够形成生物被膜的比例较高,37%和45%的细菌具有较强生物被膜形成能力。肉源性荧光假单胞菌能够在培养基和肉液中形成大量生物被膜,具有较强的生物被膜能力。显微成像结果表明荧光假单胞菌在培养6 h后有明显黏附,18 h后多糖分泌增多、菌体堆积并形成具有生物功能的立体生物被膜。这些特点可能有利于荧光假单胞菌在冷却肉表面黏附并成为优势腐败菌。     

DMDC杀菌对鲜切菜心品质的影响

本文以鲜切菜心为原料,研究了二甲基二碳酸盐(DMDC)作为杀菌剂对其细菌、霉菌和酵母菌总数的影响,并通过测定鲜切菜心在贮藏期间外观品质、营养成分及其褐变相关主要酶的变化,对DMDC用于鲜切菜心的杀菌效果进行评价。结果表明:DMDC可以有效减少鲜切菜心中细菌(革兰氏阳性菌)、霉菌和酵母菌总数,对其贮藏前期的硬度、色泽、叶绿素含量的变化有一定影响,但是对VC和可溶性糖无明显影响;并且可有效减少可溶性蛋白的损失,抑制多酚氧化酶(PPO)、过氧化物酶(POD)的活性。说明DMDC杀菌可以有效杀灭菜心表面微生物,延缓菜心贮藏期褐变相关酶活性的增加和衰老,有利于维持菜心的营养品质。      

Effect of High Pressure Homogenization and Dimethyl Dicarbonate (DMDC) on Microbial and Physicochemical Qualities of Mulberry Juice

In this study, the effect of high pressure homogenization (HPH) and dimethyl dicarbonate (DMDC) on microbial and nutrient qualities of mulberry juice was evaluated. Results showed that repeated HPH passes at 200 MPa or adding DMDC at 250 mg/L significantly inactivated the indigenous microorganisms in mulberry juice (P < 0.05), whereas some surviving microorganisms recovered to grow during storage of 4 °C. The combined treatment with 3 passes of HPH and 250 mg/L of DMDC (HPH‐DMDC) decreased the population of surviving indigenous microorganisms to the level attained by heat treatment at 95 °C for 1 min (HT) with no significant increase (P > 0.05) in the population of microorganisms during subsequent storage at 4 °C. Moreover, no significant changes (P > 0.05) in the physical attributes, including pH, TSS (^oBrix), L*, a*, and b* values were observed in the samples treated by the HPH‐DMDC or by HT. Compared with HT, HPH‐DMDC treatment resulted in a higher degree of retention in total phenolics, and α‐glucosidase inhibitory activity, although the treatment led to higher losses in cyanidin 3‐glucoside, cyanidin 3‐rutinoside, and antioxidant capacity. Overall, HPH‐DMDC treatment can be a useful alternative to conventional thermal pasteurization of mulberry juice, considering its ability to inactive, and inhibit indigenous microorganisms.     

Quorum Sensing Regulation and Inhibition of Exoenzyme Production and Biofilm Formation in the Food Spoilage Bacteria Pseudomonas psychrophila PSPF19

Gram-negative bacteria use the mechanism of acylated homoserine lactone (AHL) mediated quorum-sensing to regulate gene expression in response to population density. The present work aimed to detect signal molecule production and its influence on the production of food spoilage phenotypes such as exoenzyme (lipases and proteases) synthesis and biofilm formation in Pseudomonas psychrophila PSPF19, isolated from freshwater fish stored in refrigerated conditions. The C4-HSL signaling molecule produced by the isolate induced exoenzyme production by twofold and increased attachment and biofilm formation. Further, studies on the effect of a synthetic quorum sensing inhibitor, furanone C-30 on protease and lipase production and biofilm formation, revealed that it inhibited exoenzyme production and bacterial attachment. This study adds to our understanding that quorum sensing plays a role in the low temperature spoilage of foods by psychrotrophic Pseudomonads and quorum sensing inhibitors such as furanones can be developed as new intervention techniques and food preservatives.     

Combined Effect of Dimethyl Dicarbonate (DMDC) and Nisin on Indigenous Microorganisms of Litchi Juice and its Microbial shelf life

The individual and combined influences of dimethyl dicarbonate (DMDC) and nisin (200 IU/mL) at mild heat on the inactivation of indigenous microorganisms in litchi juice, including bacteria, molds and yeasts (M&Y), were investigated. The fresh litchi juice with or without nisin were exposed to 250 mg/L DMDC at 30, 40, or 45 °C for 0.5, 1, 2, 3, 4, or 6 h. A complete inactivation of M&Y in the litchi juice with or without nisin was achieved as exposed to 250 mg/L DMDC at 30, 40, or 45 °C for 0.5 h. The bacteria, especially Bacillus sp. and Leuconstoc mesenteroides showed higher resistance than M&Y in the litchi juice. Bacillus sp. and Leuconstoc mesenteroides in the litchi juice was not completely inactivated by 250 mg/L DMDC at 30, 40, or 45 °C. However, nisin addition can enhanced the inactivation of these bacteria by DMDC, and nisin and DMDC also showed a synergistic effect on the inactivation of bacteria. M&Y and bacteria were not detected in the litchi juice added with 200 IU/mL nisin as exposed to 250 mg/L DMDC at 45 °C for 3 h. In addition, microbial shelf life of the litchi juice during storage at 4 °C also was evaluated as treated by 250 mg/L DMDC or combination with nisin at 45 °C for 3 h.     

Biofilm Formation Characteristics of Pseudomonas lundensis Isolated from Meat

Biofilms formations of spoilage and pathogenic bacteria on food or food contact surfaces have attracted increasing attention. These events may lead to a higher risk of food spoilage and foodborne disease transmission. While Pseudomonas lundensis is one of the most important bacteria that cause spoilage in chilled meat, its capability for biofilm formation has been seldom reported. Here, we investigated biofilm formation characteristics of P. lundensis mainly by using crystal violet staining, and confocal laser scanning microscopy (CLSM). The swarming and swimming motility, biofilm formation in different temperatures (30, 10, and 4 °C) and the protease activity of the target strain were also assessed. The results showed that P. lundensis showed a typical surface‐associated motility and was quite capable of forming biofilms in different temperatures (30, 10, and 4 °C). The strain began to adhere to the contact surfaces and form biofilms early in the 4 to 6 h. The biofilms began to be formed in massive amounts after 12 h at 30 °C, and the extracellular polysaccharides increased as the biofilm structure developed. Compared with at 30 °C, more biofilms were formed at 4 and 10 °C even by a low bacterial density. The protease activity in the biofilm was significantly correlated with the biofilm formation. Moreover, the protease activity in biofilm was significantly higher than that of the corresponding planktonic cultures after cultured 12 h at 30 °C.     

食源性混合菌生物被膜的形成和相互作用机制

在食品加工中,食源性病原菌生物被膜的存在会引发大量食品污染等安全问题。目前,国内外学者深入研究了单一菌生物被膜,然而对混合菌生物被膜的研究较少。本文在现有的研究基础上,归纳总结了食源性混合菌生物被膜的形成规律及混合菌生物膜中菌种间的相互作用,初步探讨了群体感应在混合菌生物膜中的作用,以期为食品领域应对和破解食源性混合菌生物被膜污染问题提供思路。     

大黄鱼源腐败菌的黏附特性与生物膜特性分析

以分离纯化自冰鲜大黄鱼的3 株希瓦氏菌（MA1-5、MA1-7、MA1-13）和3 株假单胞菌（R3-1、R3-2、R3-5）为供试菌株,研究鱼源腐败菌的表面疏水性、自凝聚能力、生物膜和腐败菌对鱼体（鱼肠、鱼鳃和体表）黏液的黏附能力,探明黏附特性与不同部位黏附能力的相关性,并进一步研究生物膜在不同因素下的形成特性。研究表明,希瓦氏菌具有更强的表面疏水性和自凝聚能力,对鱼体黏液的黏附性强,并具有更强的生物膜形成能力。主成分分析和聚类分析说明黏附能力在属间存在差异性,属内具有相似性。腐败菌对鱼肠和鱼鳃的黏附能力与自凝聚能力和生物膜形成能力具有较高的相关性,对体表黏附能力只与生物膜形成能力有关。腐败菌生物膜的形成受初始菌浓度、培养时间、温度、pH值、盐含量等多种环境因素影响。希瓦氏菌在初始菌浓度106 CFU/mL以上、37 ℃、pH 7～8、0.8% NaCl时形成的生物膜量最多;生物膜在12～24 h期间快速形成,并在36 h达到峰值后,随培养时间延长而逐渐下降。经鱼体黏液包被后,生物膜形成量受到显著影响,鱼鳃黏液促进生物膜形成,鱼肠黏液抑制生物膜形成。