,1, 徐幸莲1, 彭斌3Lethal Effect of Meat-Borne Pseudomonas fluorescens to Acidic Electrolyzed Water
CAI LinLin1, HU HaiJing1, YI XiaoKun2, WANG HuHu,1, XU XingLian1, PENG Bin3通讯作者:
收稿日期:2018-09-4接受日期:2019-01-31网络出版日期:2019-05-01
| 基金资助: |
Received:2018-09-4Accepted:2019-01-31Online:2019-05-01
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蔡林林, 胡海静, 衣晓坤, 王虎虎, 徐幸莲, 彭斌. 酸性电解水对肉源性荧光假单胞菌的致死效应[J]. 中国农业科学, 2019, 52(9): 1614-1623 doi:10.3864/j.issn.0578-1752.2019.09.012
CAI LinLin, HU HaiJing, YI XiaoKun, WANG HuHu, XU XingLian, PENG Bin.
0 引言
【研究意义】随着经济的发展和人民生活水平的日益提高,食品安全问题也越来越受重视,而微生物引起的腐败变质是影响食品安全的最主要因素。荧光假单胞菌（Pseudomonas fluorescens）是属于假单胞菌属（Pseudomonas）的嗜冷微生物,它具有极强的腐败潜能[1,2],可在低温环境下生长繁殖。冷藏的高蛋白类和脂类丰富的食品一旦污染荧光假单胞菌,荧光假单胞菌便会大量繁殖,逐渐演变为优势菌群,导致食品腐败[3,4]。同时,随着制冷设备的普及,冷藏食品在人类的饮食构成中占据了极大的比例,嗜冷的荧光假单胞菌对于人类而言便是一种潜在的威胁。目前已在猪肉[5]、牛肉[6]和鸡肉[7]等冷藏肉制品中检测到了大量的假单胞菌污染,严重影响产品货架期。因此做好肉源性荧光假单胞菌的防范与控制对保障食品卫生与安全具有重要意义。【前人研究进展】目前对加工车间及肉制品胴体表面等采用的消毒防控手段主要通过次氯酸钠等化学消毒剂来进行,尤其是在中国,多数采用100—200 mg?L-1高浓度次氯酸钠清洗5—10 min[8]。有研究报道高浓度次氯酸钠可有效降低多种肉源性致病菌和包括假单胞菌在内的腐败菌的活菌数[9,10,11,12],但是在经次氯酸钠消毒过后,其洁净区和胴体等仍然有大量假单胞菌检出[13,14]。此外,最近研究发现次氯酸钠存在毒副作用,其与食品中的有机质反应可产生有害的含氯消毒副产物[15],已不能满足环保和高效杀菌的需要。酸性电解水是指含电解质的水溶液（生产实践中常为氯化钠）在电场作用下,消耗微量电能电解而成的具有杀菌功效的功能水,具有储藏稳定性好、杀菌范围广、制取方便、价格低廉等应用优势,室温暴露时的不稳定、短寿和低浓度的活性氯会在高效的杀菌处理后很快还原为普通水,因而绿色安全,近年来已广泛应用于食品消毒、医疗卫生和农业保鲜等领域 [16]。目前酸性电解水杀菌效果的研究主要还是集中于对食品和加工接触表面的食源性致病菌大肠杆菌[17,18]、单增李斯特菌、沙门氏菌[19,20]、金黄色葡萄球菌[21,22]、副溶血性弧菌[23]以及菌落总数的作用效果[24,25]。【本研究切入点】虽有较多酸性电解水杀菌的研究报道,但是酸性电解水对肉源性腐败菌假单胞菌的杀菌效果及假单胞菌的耐受响应尚不清楚。【拟解决的关键问题】本试验以典型腐败菌肉源性荧光假单胞菌为作用对象,从细胞水平上研究酸性电解水对肉源性荧光假单胞菌的抑制作用,为酸性电解水在食品中的应用及新型杀菌剂的研发提供一定的理论支持,为腐败菌的控制措施提供新的思路。1 材料与方法
试验于2018年3—6月在南京农业大学国家肉品质量安全控制工程技术研究中心和生命科学实验中心进行。1.1 材料与试剂
荧光假单胞菌由笔者实验室分离自腐败鸡肉,且在前期研究中发现该菌在不锈钢表面的粘附性较高,具有较强的交叉污染能力[26];胰蛋白胨大豆琼脂（TSA）、胰蛋白胨大豆肉汤（TSB）购于青岛海博责任有限公司;LIVE/DEAD? BacLightTM细菌活性检测试剂盒、Baclight Bacterial Membrane Potential Kit细菌膜电位检测试剂盒、Pierce BCA Protein Assay Kit 蛋白检测试剂盒和Intracellular pH Calibration Buffer Kit 胞内pH校准缓冲液试剂盒购于赛默飞世尔科技公司;BacTiter-GloTM Microbial Cell Viability Assay ATP检测试剂盒购于普洛麦格生物技术（北京）有限公司;BCECF-AM荧光探针购于东仁化学科技（上海）有限公司;4-羟乙基哌嗪乙磺酸（HEPES）购于北京Coolaber试剂公司。1.2 仪器与设备
酸性氧化电位水生成器（新宇光,山西）;Scan 1200自动影像分析菌落计数仪（Interscience,法国）;生物安全柜（Baker,美国）;高压灭菌锅（Hirayama,日本）;生化培养箱（博讯,上海）;BS223S电子天平（赛多利斯,北京）;高速冷冻离心机（Beckman,美国）;S-3000N扫描电子显微镜（Hitachi,日本）;M2e多功能酶标仪（IKA,德国）;流式细胞仪（BD,美国）;pH计（梅特勒-托利多,上海）;便携式有效氯检测仪（Hach,美国）等。1.3 方法
1.3.1 菌悬液制备 将冻存于-80℃的肉源性荧光假单胞菌采用划线法在TSA平板上活化,随后挑取单菌落接种于6 mL TSB中28℃培养24 h。活化好的肉源性荧光假单胞菌以0.9%的生理盐水稀释至104 CFU/mL,取1.5 mL接种于150 mL TSB培养基中,置于20℃下培养5 d。培养后的菌液经离心（6 000×g、15 min、4℃）去除上清液,清洗两次后重新悬浮制成约108 CFU/mL的菌悬液。1.3.2 酸性电解水制备 在酸性氧化电位水生成器中加入不同浓度的NaCl溶液,结合电流调节,制成不同浓度的酸性电解水,随后立即采用便携式有效氯检测仪测定有效氯浓度（ACC）,氧化还原电位值（ORP）采用pH计在转换电极后利用电位测定功能进行测定。所制备电解水的理化特性见表1。
Table 1
表1
表1酸性电解水的理化特性
Table 1
| NaCl浓度 NaCl concentration (%) | 电流Electricity (A) | 有效氯浓度 ACC (mg?L-1) | 氧化还原电位 ORP (mV) | pH |
|---|---|---|---|---|
| 2.0 | 16 | 20.33±0.47c | 1116.00±2.45c | 2.91±0.01a |
| 3.5 | 18 | 40.33±0.47b | 1152.67±1.70b | 2.78±0.00b |
| 4.6 | 19 | 61.33±0.47a | 1167.67±0.94a | 2.69±0.00c |
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1.3.3 细菌存活数量 将0.5 mL上述制备的菌悬液加入4.5 mL酸性电解水中,充分混匀,分别处理5、10、15、20、25和30 min后,迅速吸取0.5 mL加入到4.5 mL中和剂（含0.8%硫代硫酸钠的PBS缓冲液）中作用1 min。混合均匀后采用灭菌生理盐水进行梯度稀释。本试验吸取0.5 mL菌液加入4.5 mL 0.9%生理盐水代替酸性电解水作为对照。取100 μL的稀释液涂布于TSA平板上,28℃条件下培养24 h后计数。每组试验重复5次。
1.3.4 菌体微观结构 取1.3.3处理后的菌液,离心（3 000×g、15 min、4℃）去除上清液,使用2.5%的戊二醛固定液悬浮细菌并4℃静置12 h,随后30%、50%、70%、80%和90%梯度酒精脱水。最后使用100%酒精悬浮菌体。对处理好的样品进行喷金,用扫描电子显微镜进行观察、拍照。
1.3.5 细胞膜完整性 取上述菌液1 mL,调整菌浓度为107 CFU/mL,将3 μL SYTO9和PI的混合染料加入到菌液中,充分混匀。室温避光孵育15 min,使用流式细胞仪检测荧光强度,其中绿色荧光染料SYTO9的激发波长为480 nm,发射波长为500 nm。红色荧光染料PI的激发波长为490 nm,发射波长为635 nm。每组试验重复3次。
1.3.6 细胞膜电位 取上述菌液1 mL,向菌液中加入10 μL 30 mmol?L-1的荧光染料DiOC2 (3),充分混匀,室温避光孵育15 min后,立即用流式细胞仪测定荧光强度。每组试验重复3次。
1.3.7 胞内ATP 取上述菌液100 μL加入到黑色酶标板中,随后加入100 μL检测工作液,充分混匀,25℃避光孵育5 min,使用多功能酶标仪检测化学发光强度。不含有菌悬液的生理盐水为背景空白组,各试验组的化学发光强度均减去背景空白组化学发光强度。每组试验重复3次。
1.3.8 胞内pH 取上述菌液使用HEPES缓冲溶液洗涤菌体两次,菌悬液中加入1 mmol?L-1 BCECF-AM荧光染料使其终浓度为3 mmol?L-1,37℃避光孵育30 min,最后用HEPES缓冲溶液清洗并悬浮菌体。试验设置标准曲线组：分别使用pH为4.0、5.0、6.0、7.0和8.0的标定液,将8 μL尼日利亚菌素和缬胺霉素混合液添加到8 mL的pH标定液中,混匀。菌液离心去上清,以标定液悬浮菌体,37℃避光孵育10 min。将样品及标准曲线组添加于黑色酶标板中,使用多功能酶标仪检测样品的荧光强度,设定激发波长为490 nm,发射波长526 nm,绘制标准曲线并计算样品的胞内pH。每组试验重复3次。
1.3.9 胞外聚合物 取1.3.3处理5 min菌液,生理盐水清洗3次（5 000×g、10 min、4℃）,第一次上清液作为松散胞外聚合物（L-EPS）,收集沉淀。生理盐水重悬沉淀至浓度为5 mg?mL-1（菌体湿重/生理盐水体积）,加入等体积2% EDTA-Na2溶液,混匀后4℃静置3 h,离心取上清（14 000×g、20 min、4℃）,0.22 μm滤膜过滤,分子截流量透析袋透析（3 500 Da、4℃、24 h）所得即为紧密胞外聚合物（B-EPS）。L-EPS和B-EPS中蛋白含量测定采用BCA试剂盒,总糖含量测定采用苯酚-硫酸法。以蛋白或总糖浓度（μg?mL-1）/菌体湿重（mg?mL-1）得到最终浓度（μg?mg-1）。每组试验重复3次。
1.3.10 胞外聚合物对酸性电解水理化特性影响 胞外聚合物提取同1.3.7,取B-EPS 0.5 mL加入到4.5 mL电解水中,处理5 min后分别测定酸性电解水有效氯浓度（ACC）、氧化还原电位（ORP）和pH。每组试验重复3次。
1.4 数据分析
结果以平均值±标准差的形式表示,采用Excel软件进行作图分析,SPSS 13.0软件对数据进行统计分析,对酸性电解水致死效果、膜完整性、膜电位、胞内ATP和胞内pH数据采用Duncan’s ANOVA进行多重比较分析,对胞外聚合物含量变化和胞外聚合物对酸性电解水理化特性影响数据进行t检验比较分析。2 结果
2.1 酸性电解水对肉源性荧光假单胞菌的致死效果
酸性电解水对肉源性荧光假单胞菌具有显著的杀菌效果（P<0.05）,20和40 mg?L-1的酸性电解水在5 min达到相同的致死效果,曲线重叠,而在60 mg?L-1的酸性电解水处理5 min后,肉源性荧光假单胞菌活菌数已在检出限之下（图1）。图1
新窗口打开|下载原图ZIP|生成PPT图1酸性电解水对肉源性荧光假单胞菌的致死效果
黑色虚线：检出限,1.32 log（CFU/mL）。不同小写字母表示差异显著（P<0.05）。下同
Fig. 1Bactericidal effect of acidic electrolyzed water on meat-borne P. fluorescens
Dash line represents detection limit of 1.32 log (CFU/mL). Different lowercase letters indicate significant differences (P<0.05). The same as below
2.2 菌落形态分析
由图2-A可以看出,正常的肉源性荧光假单胞菌保持原有形态,呈杆状,细胞完整,菌体表面饱满光滑、无褶皱。而经酸性电解水处理后（图2-B、C、D）,菌体结构遭到破坏,大量细胞表面粗糙,凹凸不平,÷出现褶皱甚至变形,细菌细胞的受损程度随着处理浓度的增加而增大。图2
新窗口打开|下载原图ZIP|生成PPT图2肉源性荧光假单胞菌SEM图
A：对照;B：20 mg?L-1酸性电解水处理;C：40 mg?L-1酸性电解水处理;D：60 mg?L-1酸性电解水处理
A: Control; B: Treatment with 20 mg?L-1 acidic electrolyzed water; C: Treatment with 40 mg?L-1 acidic electrolyzed water; D: Treatment with 60 mg?L-1 acidic electrolyzed water
Fig. 2SEM observation of meat-borne P. fluorescens
2.3 细胞膜完整性的变化
由图3可见,经酸性电解水处理后,肉源性荧光假单胞菌的膜完整性显著降低（P<0.05）,经20 mg?L-1的酸性电解水处理的肉源性荧光假单胞菌细胞膜完整的比例仅有2.26%,经40和60 mg?L-1的酸性电解水处理的肉源性荧光假单胞菌细胞膜完整的比例则显著降低至1.87%和1.20%。图3
新窗口打开|下载原图ZIP|生成PPT图3酸性电解水对肉源性荧光假单胞菌膜完整性的影响
Fig. 3Effect of acidic electrolyzed water on the membrane integrity of meat-borne P. fluorescens
2.4 细胞膜电位的变化
由图4可见,经酸性电解水处理后,红色荧光/绿色荧光强度呈现下降趋势,说明酸性电解水对细胞膜产生了损害,膜电位消散。相比于对照,20 mg?L-1酸性电解水处理下红/绿荧光比值降低了40%,而随着处理浓度的增加,红/绿荧光比值显著降低,40 mg?L-1和60 mg?L-1酸性电解水红/绿荧光比值均降低到对照的50%以内,酸性电解水浓度越增加,膜电位降低越显著。图4
新窗口打开|下载原图ZIP|生成PPT图4酸性电解水对肉源性荧光假单胞菌膜电位的影响
Fig. 4Effect of acidic electrolyzed water on the membrane potential of meat-borne P. fluorescens
2.5 胞内ATP的变化
由图5可见,酸性电解水可显著降低肉源性荧光假单胞菌胞内ATP含量（P<0.05）。5 min内化学发光值急剧下降,40和60 mg?L-1电解水处理对荧光假单胞菌胞内pH的降低无显著影响,无明显改变,验证了酸性电解水对肉源性荧光假单胞菌细胞膜的破坏作用。图5
新窗口打开|下载原图ZIP|生成PPT图5酸性电解水对肉源性荧光假单胞菌胞内ATP的影响
Fig. 5Effect of acidic electrolyzed water on the intracellular ATP of meat-borne P. fluorescens
2.6 胞内pH的变化
由图6可见,酸性电解水可显著降低肉源性荧光假单胞菌胞内pH,高浓度酸性电解水的处理导致了胞内pH更显著的降低（P<0.05）。肉源性荧光假单胞菌初始胞内pH为7.5,经20、40和60 mg?L-1酸性电解水处理后,胞内pH分别降低了0.97、1.60和1.67。40和60 mg?L-1电解水处理对肉源性荧光假单胞菌胞内pH的降低无显著影响。图6
新窗口打开|下载原图ZIP|生成PPT图6酸性电解水对肉源性荧光假单胞菌胞内pH的影响
Fig. 6Effect of acidic electrolyzed water on the intracellular pH of meat-borne P. fluorescens
2.7 胞外聚合物含量的变化
酸性电解水处理下EPS主要成分变化如表2所示。由表中可以看出,酸性电解水处理显著降低了L-EPS和B-EPS中蛋白和总糖含量（P<0.05）。相比对照,L-EPS中蛋白、总糖含量分别降低了71.08%和62.29%,而B-EPS中蛋白、总糖含量则分别降低了99.32%和40.62%。同时,L-EPS和B-EPS中蛋白总糖的比值分别降低了23.46%和99.00%。Table 2
表2
表2酸性电解水对肉源性荧光假单胞菌胞外聚合物中蛋白和总糖含量影响
Table 2
| 类型 Type | 处理 Treatment | 蛋白 Protein (μg?mg -1) | 总糖 Carbohydrate (μg?mg -1) | 蛋白/总糖 Protein/Carbohydrate ratio |
|---|---|---|---|---|
| 松散胞外聚合物L-EPS | 0 | 583.68±72.05a | 112.33±0.73a | 5.20±0.18a |
| 40 mg?L-1 AEW | 168.78±2.96b | 42.36±0.14b | 3.98±0.08b | |
| 紧密胞外聚合物B-EPS | 0 | 59.23±1.28a | 19.72±0.66a | 3.01±0.15a |
| 40 mg?L-1 AEW | 0.40±0.28b | 11.71±1.23b | 0.03±0.02b |
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2.8 胞外聚合物对酸性电解水理化特性影响
由图7可见,在胞外聚合物的添加下,各浓度酸性电解水ACC基本不变,没有显著影响（P>0.05）。20和40 mg?L-1的酸性电解水相比于对照没有显著差异（P>0.05）,而胞外聚合物添加到60 mg?L-1酸性电解水后pH显著上升。胞外聚合物的添加显著降低各浓度电解水的ORP（P<0.05）。图7
新窗口打开|下载原图ZIP|生成PPT图7肉源性荧光假单胞菌胞外聚合物对酸性电解水有效氯浓度（A）、氧化还原电位（B）和pH（C）的影响
Fig. 7Effect of EPS of meat-borne P. fluorescens on the available chlorine concentration (A), oxidation- reduction potential (B) and pH (C) of acidic electrolyzed water
3 讨论
酸性电解水对肉源性荧光假单胞菌具有极强的杀菌效果,3种浓度在5 min内的细菌杀灭对数值均>5,而浓度的增加显著增强杀菌效果,表明酸性电解水是一种有效的杀菌剂。细胞膜的完整性是菌体正常生长代谢的一个主要因素,是细胞内外物质交换的主要屏障和介质,细胞膜不完整的细胞通常不能维持或产生稳定的膜电位,并且胞内结构也暴露于外部环境之下,因此对于外部胁迫表现更为脆弱,通常也可以判断为细胞死亡[27]。研究发现5 min处理下的细胞膜完整性急剧降低,且酸性电解水浓度越高,完整性越低。相似的结果也有报道[28,29],研究发现存在大量的胞内物质泄露的现象,例如离子、蛋白、核酸等,表明细胞膜可能是酸性电解水的作用位点之一。有效氯是酸性电解水中的有效杀菌成分,其中HClO和ClO-发挥不同的作用。HClO由于其电中性及合适的分子大小可以先行透过细胞膜进入到细胞内部进行作用,抑制糖代谢进程,而离子态的ClO-无法直接通过细胞膜,会首先作用于细胞膜上的功能蛋白等成分,进而导致细胞膜运输等功能的退化失效[16],从而影响细胞膜完整性和通透性。细胞膜完整性所对应的细胞死亡程度低于平板计数结果,极少数细胞处于存活但不可计的状态是可能原因。未经酸性电解水处理的细胞膜两侧外正、内负的状态称为膜的极化状态,此时Na+与K+泵关闭。Na+是酸性电解水的主要成分之一,经酸性电解水作用后,Na+泵打开,胞内Na+浓度升高,肉源性荧光假单胞菌的膜电位降低,即表示细胞去极化。除了离子的透膜移动,环境pH的变化也会引起膜电位的改变[30,31]。酸性电解水处理下细胞从中性环境到酸性环境的改变也会使细胞膜呈现去极化状态。膜电位对大量细胞分裂蛋白的定位具有很重要的作用,而去极化则会导致细胞壁自溶酶的释放进而引起细胞自溶[32]。同时,细胞膜完整性的下降导致了胞内ATP的释放,而Na+和H+等离子的跨膜主动运输也对胞内ATP产生了大量消耗,从而引起了胞内ATP含量的降低,相似的结果前期[31,33]等也有报道。酸性电解水有效成分的进入可能降低胞内ATP合成速度的降低或加速ATP合成酶的水解,同样也会导致胞内ATP含量的降低。胞内pH对胞内DNA转录、酶活和蛋白合成等起着很重要的控制作用[34]。由于培养条件和菌种的不同,胞内pH往往在5.6—9.0。酸化的胞内环境可以极大的提高杀菌剂的效果而导致细胞死亡[31]。研究发现胞内pH的降低与酸性电解水的浓度成正比。酸性电解水较低的pH带来的大量H+流入是胞内pH降低的可能原因,胞内pH的降低会干扰正常的胞内代谢进程,也会导致细胞对有效氯更为敏感。当面临酸性或碱性外在胁迫时细菌有多种机制来维持质子稳态,例如H+-ATPase的合成,但是当这些机制或质子泵外排能力被质子进入能力超过时,胞内pH最终将酸化到某个程度而导致胞内关键进程的瓦解,加速细胞的死亡。
EPS广泛存在于细胞表面,表面吸附性强,可以形成保护层抵御杀菌剂和有毒物质的危害,其渗透阻碍性是细胞对杀菌剂的基础抗性机制之一,因此杀菌剂能否穿透或破坏EPS从而接触到活菌菌体对其杀菌效果具有很重要的影响[35]。EPS中的成分及其含量受菌种和培养环境的影响,相比于前期的一些研究,本研究中肉源性荧光假单胞菌产生的蛋白和总糖含量较高[36,37]。研究发现酸性电解水处理下EPS产生了一定的消耗,其中蛋白含量的降低远高于总糖,表明EPS确实起到了一定的阻碍作用,相比于总糖,蛋白对酸性电解水杀菌效果的降低可能起到更大的作用。需氧微生物生活的环境中ORP通常为200—800 mV[38],酸性电解水的ORP超过1 000 mV,其可改变微生物细胞的电子流,影响物质运输、信息传递,引起细胞表面巯基混合物氧化,干扰胞内细胞代谢通路,从而影响其正常代谢和ATP合成,最终使其死亡。本研究发现EPS可以显著降低酸性电解水的ORP,从而可能对酸性电解水的杀菌效果起削弱作用。EPS未能显著降低酸性电解水的ACC和提高其pH,可能与其较低的添加量有关。
4 结论
酸性电解水可有效控制肉源性荧光假单胞菌,影响其细胞形态,降低细胞膜的完整性、胞内pH和胞内ATP的含量,引起细胞膜去极化。同时,胞外多聚物对酸性电解水具有一定的阻碍作用,削弱其致死效果。参考文献 原文顺序
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被引期刊影响因子
DOI:10.1016/j.foodres.2017.12.033URLPMID:29433294 [本文引用: 1]
Meat spoilage greatly depends on meat composition and storage conditions. Microbial and biochemical changes in minced pork (100-g portions) wrapped with a polyvinyl chloride film during a 4-day refrigerated storage were studied. As glucose is the first substrate used by spoilage bacteria and when it is depleted bacteria could generate undesirable volatiles, the effect of the addition of... [Show full abstract]
[本文引用: 1]
[本文引用: 1]
DOI:10.13386/j.issn1002-0306.2015.16.068URL [本文引用: 1]
荧光假单胞菌(Pseudomonas Fluorescens)隶属于假单胞菌属,是食品中常见的一种嗜冷致病微生物。它能够产生极其耐热的蛋白酶和脂肪酶,这些酶在高温处理后仍有残留,并在食品储藏过程中继续分解其中的脂肪和蛋白质,导致产品的风味和质地发生变化。因此,研发出一种既快速又实用的荧光假单胞菌检测方法成为食品行业关注的焦点。本文对食品中荧光假单胞菌的危害特点及国内外的一些快速检测方法,如聚合酶链式反应(PCR)、荧光定量PCR、荧光原位杂交、流式细胞术等方法进行了综述,比较各方法的优缺点并展望了荧光假单胞菌快速检测方法的发展趋势。
DOI:10.13386/j.issn1002-0306.2015.16.068URL [本文引用: 1]
荧光假单胞菌(Pseudomonas Fluorescens)隶属于假单胞菌属,是食品中常见的一种嗜冷致病微生物。它能够产生极其耐热的蛋白酶和脂肪酶,这些酶在高温处理后仍有残留,并在食品储藏过程中继续分解其中的脂肪和蛋白质,导致产品的风味和质地发生变化。因此,研发出一种既快速又实用的荧光假单胞菌检测方法成为食品行业关注的焦点。本文对食品中荧光假单胞菌的危害特点及国内外的一些快速检测方法,如聚合酶链式反应(PCR)、荧光定量PCR、荧光原位杂交、流式细胞术等方法进行了综述,比较各方法的优缺点并展望了荧光假单胞菌快速检测方法的发展趋势。
DOI:10.1016/j.meatsci.2016.02.034URLPMID:26943946 [本文引用: 1]
The aim of this study was to disentangle the microbial diversity on porcine musculature. The hypervariable V1–V2 region of the 16S rRNA gene was amplified from DNA samples of clinically healthy slaughter pigs (n=8). Pyrosequencing yielded 37,000 quality-controlled reads and a diverse microbiome with 54–159 OTUs per sample was detected. Interestingly, 6 out of 8 samples were strongly dominated by 1–2 highly abundant OTUs (best hits of highly abundant OTUs:Serratia proteamaculans,Pseudomonas syringae,Aeromonas allosaccharophila,Brochothrix thermosphacta,Acidiphilium cryptumandEscherichia coli). In 1g musculature scraping, 3.20E+06 16S rRNA gene copies and 4.45E+01EnterobacteriaceaerRNA gene copies were detected with qPCR. We conclude that i.) next-generation sequencing technologies help encompass the full content of complex, bacterial contamination, ii.) psychrophile spoilers dominated the microbiota and iii.)E. coliis an effective marker species for pork contamination, as it was one of very few abundant species being present in all samples.
DOI:10.1016/j.fm.2016.08.003URL [本文引用: 1]
DOI:10.3389/fmicb.2017.02588URLPMID:5743932 [本文引用: 1]
The consumption of yellow-feathered broiler has been advocated for purchasing with chilled meat rather than live broilers in Asia due to the outbreaks of animal influenza. Here, the microbial community of chilled yellow-feathered broiler response to modified-air packaging (MAP, 80% CO2/20% N2) and penetrated-air packaging (PAP, air-filling) during storage was revealed by a combination of whole-metagenome shotgun sequencing and traditional isolation methods, and the volatile organic compounds and proteolytic activity of representative dominant isolates were also accessed. The results revealed that MAP prolonged shelf life from 4 to 8 days compared to PAP, when the numbers of total viable counts and lactic acid bacteria reached more than 7 log CFU/g.Aeromonas,Acinetobacter,Escherichia, andStreptococcusoccupied the bacteria communities in initial broiler carcasses. MAP dramatically increased the bacteria diversity during storage compared to PAP. Clear shifts of the dominant bacteria species were obviously observed, with the top genera ofAeromonas,Lactococcus,Serratia, andShewanellain MAP, whereas the microbial communities in PAP were largely dominated byPseudomonas. The isolates ofPseudomonasfrom PAP carcasses andAeromonasfrom MAP carcasses displayed strong proteolytic activities. Meanwhile, the principal component analysis based on the volatile organic compounds indicated that the metabolic profiles greatly varied between each treatment, and no link between the natural odor of spoilage meatin situand the volatile odor of the dominant isolates incubated in standard culture was found. These data could lead to new insights into the bacteria communities of yellow-feathered broiler meat during storage and would benefit the development of novel preservative approaches.
DOI:10.1292/jvms.17-0089URLPMID:5487776 [本文引用: 1]
An alkaline agent, namely food additive grade calcium hydroxide (FdCa(OH)2) in solution at 0.17%, was evaluated for its bactericidal efficacies in chiller water with sodium hypochlorite (NaOCl) at a concentration of 200 ppm total residual chlorine. Without organic material presence, NaOCl could inactivateSalmonellaInfantis andEscherichia coliwithin 5 sec, but in the presence of fetal bovine serum (FBS) at 0.5%, the bactericidal effects of NaOCl were diminished completely. FdCa(OH)2solution required 3 min to inactivate bacteria with or without 5% FBS. When NaOCl and FdCa(OH)2were mixed at the final concentration of 200 ppm and 0.17%, respectively, the mixed solution could inactivate bacteria at acceptable level (103reduction of bacterial titer) within 30 sec in the presence of 0.5% FBS. The mixed solution also inhibited cross-contamination withS.Infantis orE. colion chicken meats. It was confirmed and elucidated that FdCa(OH)2has a synergistic effect together with NaOCl for inactivating microorganisms.
DOI:10.11726/j.issn.1001-7658.2017.06.005URL [本文引用: 1]
目的观察次氯酸钠对猪源性病原菌的杀灭效果及影响因素。方法采用悬液定量杀菌试验方法,对次氯酸钠杀灭分离自猪体的大肠埃希菌和金黄色葡萄球菌的效果进行观察。结果常温条件下用有效氯138 mg/L的次氯酸钠消毒液对悬液内猪源大肠埃希菌和金黄色葡萄球菌作用5 min,杀灭率均达100%。随作用温度升高,次氯酸钠对猪源性致病菌的杀灭效果明显增强（P〈0.01）;随有机物浓度增加,杀灭效果明显降低（P〈0.01）。结论次氯酸钠在常温条件下对猪源大肠埃希菌和金黄色葡萄球菌杀灭效果较好,试验温度和有机干扰物对杀菌效果存在明显影响。
DOI:10.11726/j.issn.1001-7658.2017.06.005URL [本文引用: 1]
目的观察次氯酸钠对猪源性病原菌的杀灭效果及影响因素。方法采用悬液定量杀菌试验方法,对次氯酸钠杀灭分离自猪体的大肠埃希菌和金黄色葡萄球菌的效果进行观察。结果常温条件下用有效氯138 mg/L的次氯酸钠消毒液对悬液内猪源大肠埃希菌和金黄色葡萄球菌作用5 min,杀灭率均达100%。随作用温度升高,次氯酸钠对猪源性致病菌的杀灭效果明显增强（P〈0.01）;随有机物浓度增加,杀灭效果明显降低（P〈0.01）。结论次氯酸钠在常温条件下对猪源大肠埃希菌和金黄色葡萄球菌杀灭效果较好,试验温度和有机干扰物对杀菌效果存在明显影响。
DOI:10.1093/ofid/ofx163.1817URLPMID:29330015 [本文引用: 1]
Preterm infants are at high risk for extended spectrum β-lactamase producingEnterobacteriaceae(ESBL-E) sepsis and neonatal intensive care unit (NICU) outbreaks. There is no consensus regarding surveillance of pregnant women for ESBL-E colonization. However, neonatal screening is accepted in order to prevent NICU’s transmissions by contact isolation. We hypothesized that a significant proportion of ESBL-E colonized infants in our NICU were due to mother-to-child transmission. The aim of this study was to molecularly identify pairs of mothers and offspring ESBL-E colonization. The Soroka University Medical Center serves the entire population of southern Israel with an estimated 17,000 deliveries per year. This was a one year analysis from an ongoing, prospective, active rectal ESBL-E colonization surveillance of mothers of premature infants and their offspring. Pairs of mothers and infants colonized with the same bacteria underwent molecular identification by pulse-field gel electrophoresis (PFGE). Between January 2015 and February 2016, 311(76%) out of 407 mothers and all 477(100%) infants were screened for ESBL-E colonization; carriage rates were 21.5% and 14.9%, respectively. Four (5.6%) carrier infants developed late onset sepsis and 2 (2.8%) died. Mothers of 47% (28) ESBL-E carrier infants were colonized at delivery; comparison of the characteristics between colonized infants of positively and negatively screened mothers is presented in Table021. Twenty-three (34.3%) colonized mothers delivered 25 infants who were found to be colonized with the same bacterial strain according to species’ identification and antibiogram. A02subgroup of 10 mother-infant dyads underwent PFGE and 70% shared identical molecular fingerprint patterns. No similarities were found between isolates recovered from unrelated neonates and mothers. High ESBL-E carriage rates in mothers and NICU’S infants, in our region, with a non-negligible maternal-neonatal ESBL-E colonization transmission illuminates the importance of maternal ESBL colonization surveillance and further consideration regarding infectious control preventive measurements. All authors:No reported disclosures.
DOI:10.1016/j.prosdent.2017.09.003URL [本文引用: 1]
AbstractStatement of problemImpression materials must be disinfected to avoid cross-contamination before they are sent to the dental laboratory. However, whether aqueous state disinfectants affect material wettability is unclear.PurposeThe purpose of this in vitro study was to compare the efficacy of gaseous ozone and sodium hypochlorite (NaOCl) in disinfecting light-body consistency hydrophilized polyvinyl siloxane (PVS) impression specimens inoculated with a cocktail of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis. The effect of both disinfectants on the wettability of the material was also evaluated on uninoculated specimens.Material and methodsDisk-shaped specimens (N=140) were subjected to the either gaseous ozone or NaOCl for up to 30 minutes at room temperature. In the ozone group, the specimens were ozonated under a continuous stream of gaseous ozone at a concentration of 12.8 mg/L, while the specimens in the NaOCl group were immersed into 0.5% NaOCl solution. Reductions in the size of the bacterial population at the end of the exposure times were determined by the plate count technique. The contact angle measurements on the impression surface were used to determine the wettability of the specimens. Results were analyzed with 1-way ANOVA followed by the Tukey post hoc test ( =.05).ResultsThe material exposed to both disinfectants for 30 minutes demonstrated a reduction in the number of bacteria of up to more than 3 log. The contact angle of water on the material increased significantly (P<.001) after only 5 minutes of contact with the NaOCl solution. However, the treatment with gaseous ozone for 30 minutes resulted in a reduced contact angle (P<.001).ConclusionsGaseous ozone treatment was identified as a promising method of disinfecting polymerized PVS impression materials because of its positive effect on the wettability of the material.
DOI:10.5005/jp-journals-10024URL [本文引用: 1]
DOI:10.1016/j.lwt.2017.01.038URL [本文引用: 1]
The knowledge on the microorganisms present in an industrial process is crucial to delineate the best strategy for their effective control. The aims of the present work were to isolate, identify and characterize (in terms of production of proteases, gelatinases and siderophores, quorum-sensing inhibition and biofilm formation) the resident heterotrophic bacteria present in a minimally processed vegetables (MPV) plant where sodium hypochlorite was used for decontamination. A total of 47 isolates were obtained with 49% belonging to thegenera. Twenty different bacterial species were identified and the conveyor belt in the high care area was found to be a significant source of contamination. Most of the isolates were capable of producing virulence related molecules and all isolates were able to form biofilm.was the genera with the highest biofilm formation ability, being the predominant microflora along the process chain. Even if no relevant foodborne pathogen was isolated, the results clearly propose that improvements in decontamination during processing are required to effectively control microbial presence in the final product.
URL [本文引用: 1]
采用乳酸、乙酸、柠檬酸、次氯酸钠、酸化亚氯酸钠、二氧化氯六种减菌剂,在不同的浓度的情况下,运用简易的手持式喷淋设备对人工接种的优势腐败菌液(假单胞菌属、大肠菌群、葡萄球菌、乳酸菌)的牛肉表面进行喷淋,通过喷淋前后牛肉表面细菌的计数,来检测减菌剂的减菌效果。结果表明,有机酸的减菌效果大于含氯化合物;2%的三种有机酸中,乳酸的减菌效果最好,三种含氯化合物中,酸化的亚氯酸钠溶液减菌效果最好;2%的乳酸对人工接种的牛肉表面喷淋减少菌落总数和腐败菌分别为5.19 lg CFU/cm2和3.37~4.71 lg CFU/cm2;由于2%乳酸较好的减菌效果,性价比高,使用方便,所以最适合在小型屠宰厂对牛胴体喷淋。
.
URL [本文引用: 1]
采用乳酸、乙酸、柠檬酸、次氯酸钠、酸化亚氯酸钠、二氧化氯六种减菌剂,在不同的浓度的情况下,运用简易的手持式喷淋设备对人工接种的优势腐败菌液(假单胞菌属、大肠菌群、葡萄球菌、乳酸菌)的牛肉表面进行喷淋,通过喷淋前后牛肉表面细菌的计数,来检测减菌剂的减菌效果。结果表明,有机酸的减菌效果大于含氯化合物;2%的三种有机酸中,乳酸的减菌效果最好,三种含氯化合物中,酸化的亚氯酸钠溶液减菌效果最好;2%的乳酸对人工接种的牛肉表面喷淋减少菌落总数和腐败菌分别为5.19 lg CFU/cm2和3.37~4.71 lg CFU/cm2;由于2%乳酸较好的减菌效果,性价比高,使用方便,所以最适合在小型屠宰厂对牛胴体喷淋。
DOI:10.1007/s11270-014-1921-1URL [本文引用: 1]
Continuous seawater disinfection by chlorine dioxide (ClO 2 ) was studied at residual concentrations of 0.2 and 0.4 mg ClO 2 L 611 and compared with sodium hypochlorite (NaClO) disinfection at 1 mg L 611 of free chlorine. The results revealed that both disinfectants decrease the biological activity and cell counts in seawater. When NaClO was used, both the cell counts and the adenosine triphosphate (ATP) level were diminished (1.8 log and 76 %, respectively); however, when ClO 2 was used, the ATP level decreased to the same level as with NaClO (78–84 %), but the cell counts were reduced only weakly (~0.1 log). The biofilm concentration in seawater without disinfectants reached 700 pg ATP cm 612 after 40 days, whereas in the treated lines, the biofilms remained below 1 pg ATP cm 612 irrespective of the disinfectant and dose used. ClO 2 generated much less trihalomethanes than NaClO (<1 vs. 154 μg L 611 ). Bromoform (77–96 %) was the predominant chemical species found in disinfected seawater.
DOI:10.1111/1541-4337.12200URL [本文引用: 2]
Electrolyzed water (EW) has gained immense popularity over the last few decades as a novel broad-spectrum sanitizer. EW can be produced using tap water with table salt as the singular chemical additive. The application of EW is a sustainable and green concept and has several advantages over traditional cleaning systems including cost effectiveness, ease of application, effective disinfection, on-the-spot production, and safety for human beings and the environment. These features make it an appropriate sanitizing and cleaning system for use in high-risk settings such as in hospitals and other healthcare facilities as well as in food processing environments. EW also has the potential for use in educational building, offices, and entertainment venues. However, there have been a number of issues related to the use of EW in various sectors including limited knowledge on the sanitizing mechanism. AEW, in particular, has shown limited efficacy on utensils, food products, and surfaces owing to various factors, the most important of which include the type of surface, presence of organic matter, and type of tape water used. The present review article highlights recent developments and offers new perspectives related to the use of EW in various areas, with particular focus on the food industry. Practical Application From an industrial viewpoint, this publication can be used for the comparison and improvement of electrolyzed water (EW) generators. From a scientific standpoint, this publication can help in understanding the role of various parameters and can provide insight into EW producing systems and its applications for further research and development.
DOI:10.1016/j.foodcont.2014.09.027URL [本文引用: 1]
61Bactericidal ability of combination of AlEW and AcEW was higher than the single.61Results were also demonstrated microcosmically by scanning electron microscopy.61Combination of EW has stronger disinfection ability than the single on natural flora.61Combination of AlEW and AcEW may be also a better choice in fresh-cut produce.
DOI:10.1016/j.ijfoodmicro.2011.12.031URLPMID:22342456 [本文引用: 1]
The food industry has recognized electrolyzed oxidizing water (EOW) as a promising alternative decontamination technique. However, there is not a consensus about the sanitizing mechanism of EOW. In this study, we evaluated the disinfection efficacy of different types of EOW on Escherichia coli. Based on the hypothesis of hydroxyl radicals existing in EOW, in the present study, the hydroxyl radicals existed in slightly acidic electrolyzed water (SAEW) and acidic electrolyzed water (AEW) diluted to different levels were detected quantitatively. An ultraviolet (UV) spectrophotometer was used to scan EOW with different pH values. Accounting for the results of UV scanning to EOW with different pH value and the disinfection efficacy of different types of EOW, it can be concluded that considering the lower chlorine concentration of EOW compared with traditional chlorine disinfectants, the existing form of chlorine compounds rather than the hydroxyl radicals played important role in the disinfection efficacy of EOW.
DOI:10.1016/j.postharvbio.2011.04.001URL [本文引用: 1]
http://linkinghub.elsevier.com/retrieve/pii/S0925521411000937
[本文引用: 1]
URL [本文引用: 1]
为了阐明不同性质的电解水在控制金黄色葡萄球菌生物被膜中的作用,本研究分别使用酸性、碱性和中性电解水处理生物被膜,并测定处理前后生物被膜生物量、活性、活菌数和构造的变化。结果表明,酸性电解水能够有效杀死生物被膜中的菌体,但不能有效去除生物被膜;其有效氯含量与杀菌作用呈正相关。碱性电解水能够有效去除生物被膜,其pH值直接影响去除能力。中性电解水的作用与酸性电解水相似。不锈钢表面的生物被膜用碱性、酸性和中性电解水处理10min后,活菌数从7.5lgCFU/cm2分别减少至5.3,2.3和3.2lgCFU/cm2。电解水有潜力成为生物被膜杀菌剂和去除剂。
URL [本文引用: 1]
为了阐明不同性质的电解水在控制金黄色葡萄球菌生物被膜中的作用,本研究分别使用酸性、碱性和中性电解水处理生物被膜,并测定处理前后生物被膜生物量、活性、活菌数和构造的变化。结果表明,酸性电解水能够有效杀死生物被膜中的菌体,但不能有效去除生物被膜;其有效氯含量与杀菌作用呈正相关。碱性电解水能够有效去除生物被膜,其pH值直接影响去除能力。中性电解水的作用与酸性电解水相似。不锈钢表面的生物被膜用碱性、酸性和中性电解水处理10min后,活菌数从7.5lgCFU/cm2分别减少至5.3,2.3和3.2lgCFU/cm2。电解水有潜力成为生物被膜杀菌剂和去除剂。
URL [本文引用: 1]
研究酸性氧化电解水对金黄色葡萄球菌生物被膜的清除效果。通过观察放大5000倍的金黄色葡萄球菌生物被膜的扫描电镜照片发现，经酸性氧化电解水处理后，细菌外部基质基本被破坏，细胞破裂严重，生物被膜态细菌量下降，清除效果明显。增加保存酸性氧化电解水的时间、提高保存温度以及存在有机干扰物时均会显著降低清除效果；闭口储存的电解水清除生物被膜的能力高于敞口保存。有效氯、pH值和氧化还原电位对清除效果具有协同作用：有效氯含量较低时，电解水清除能力影响明显，有效氯含量较高时，清除效果无明显影响；低pH值备件下，清除效果较好；氧化还原电位与清除效果有明显正相关关系。
URL [本文引用: 1]
研究酸性氧化电解水对金黄色葡萄球菌生物被膜的清除效果。通过观察放大5000倍的金黄色葡萄球菌生物被膜的扫描电镜照片发现，经酸性氧化电解水处理后，细菌外部基质基本被破坏，细胞破裂严重，生物被膜态细菌量下降，清除效果明显。增加保存酸性氧化电解水的时间、提高保存温度以及存在有机干扰物时均会显著降低清除效果；闭口储存的电解水清除生物被膜的能力高于敞口保存。有效氯、pH值和氧化还原电位对清除效果具有协同作用：有效氯含量较低时，电解水清除能力影响明显，有效氯含量较高时，清除效果无明显影响；低pH值备件下，清除效果较好；氧化还原电位与清除效果有明显正相关关系。
DOI:10.1016/j.foodres.2011.07.041URL [本文引用: 1]
78 AEW used at 50°C showed the best bactericidal activity. 78 The ORP and ACC of AEW used at 50°C showed a remarkably decrease. 78 AEW stored at –18 °C was better than that of stored at 25 °C.
URL [本文引用: 1]
强酸性电解水用于冷鲜鸡的减菌化处理,在单因子试验的基础上采用二次通用旋转实验设计,对减菌化条件进行优化。结果表明:所得回归方程与实际情况拟合良好,影响冷鲜鸡减菌率的主次顺序为有效氯浓度用液量浸泡时间,减菌率的理论极值为97.84%,最佳工艺条件为强酸性电解水中的有效氯浓度为130mg/L、浸泡时间为11.64min、用液量为4.43倍。
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URL [本文引用: 1]
强酸性电解水用于冷鲜鸡的减菌化处理,在单因子试验的基础上采用二次通用旋转实验设计,对减菌化条件进行优化。结果表明:所得回归方程与实际情况拟合良好,影响冷鲜鸡减菌率的主次顺序为有效氯浓度用液量浸泡时间,减菌率的理论极值为97.84%,最佳工艺条件为强酸性电解水中的有效氯浓度为130mg/L、浸泡时间为11.64min、用液量为4.43倍。
URL [本文引用: 1]
采用酸性电解水对熟制虾仁进行 抑菌处理,考察了酸性电解水的pH、料液比、浸泡时间对虾仁抑菌效果的影响。通过单因素和正交试验确定了最佳的抑菌工艺参数为:pH 3.0,料液比1︰2(g/mL),浸泡时间25 min。在此条件下,经过抑菌处理的虾仁细菌总数可降至1.94 lg CFU/g。
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URL [本文引用: 1]
采用酸性电解水对熟制虾仁进行 抑菌处理,考察了酸性电解水的pH、料液比、浸泡时间对虾仁抑菌效果的影响。通过单因素和正交试验确定了最佳的抑菌工艺参数为:pH 3.0,料液比1︰2(g/mL),浸泡时间25 min。在此条件下,经过抑菌处理的虾仁细菌总数可降至1.94 lg CFU/g。
[本文引用: 1]
DOI:10.1080/09670262.2012.687144URL [本文引用: 1]
The aim of our study was to investigate the intracellular toxicity mechanisms of the photoactive, potentially anti-cyanobacterial agent hydrogen peroxide (H2O2) in Microcystis aeruginosa, which represents one of the most significant toxin-producing cyanobacterial species in European water bodies. Metabolic activity and cell membrane integrity were evaluated by flow cytometry in cyanobacteria exposed to H2O2 in the dark or light; the relationships between exposure effects and the kinetics of hydrogen peroxide decomposition were studied. In the light (irradiance 1406508mol m61265s611), cyanobacteria were exposed to initial H2O2 concentrations of 0.00 (control), 0.75, 2.00, and 4.0065mg65l611 respectively, while in the dark concentrations were ten times higher. Flow cytometry and chlorophyll a fluorescence measurements suggested that hydrogen peroxide exposure elicits an immediate decline of metabolic (esterase) activity, measured as a decrease in fluorescein fluorescence after hydrolysis of fluorescein diacetate (FDA), and immediate changes of chlorophyll a fluorescence parameters, followed later by an increase in the percentage of membrane-compromised (SYTOX Green positive) cells. When the concentration of H2O2 used was lethal (in the two highly exposed treatments in the light), a significant drop in total cell counts was detected, whereas in other treatments no drop was observed during the entire experimental period (7265h). Our study also confirmed that light is one of the critical factors affecting H2O2 decomposition and thus greatly influences its toxicity. Whereas in the light, M. aeruginosa exposed to 0.7565mg65l611 H2O2 recovered after all the H2O2 had decomposed, in the dark H2O2 decomposed relatively slowly and its toxic effects on the cyanobacteria were observed over the whole 72-h period, though without cell lysis in any experimental concentration.
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[本文引用: 1]
URL [本文引用: 1]
为了解细胞膜氢离子泵的电生理特性，本实验用膜片箝法研究了大麦根木质部薄壁细胞质膜上的生电泵在无一价阳离子条件下的生电活性与阴离子通道电导性的关系．结果显示：在大约40％的所测定的原生质体中观察到了由离子泵活动导致的膜电位超极化；添加H＋泵激活剂壳梭孢菌素导致在零电位的越膜电流密度的增加和膜电位大约-50V的超极化；相比之下，添加H＋泵抑制剂DCCD（Dicylohexylcarbondiimid）则导致膜电位的去极化作用和零电位越膜电流密度的减少；没有观察到钙离子通道抑制剂赤藓红B对生电泵活性的作用；细胞外溶液的pH值从5．8升高到8．8时，由于氢离子浓度梯度的加大导致泵的激活．这些特征都表明所测到的生电泵活性是H＋泵的作用而不是Ca2＋泵．负离子通道抑制剂DIDS（4，4’-Diisothiocyano-2，2’-stilbenedisulfonic acid）能导致膜电位的强烈超极化，暗示负离子通道的活性导致的短路效应强烈干扰和影响了H’泵的电活性的测定．这可能是植物根木质部细胞质膜上的H＋泵的电活性用膜片箝法难以检测的原因．尽管非特异性Ca2＋通道抑制剂La3＋也对泵的活性有明显影响，但在添加DIDS后添加的钙离子通道抑制剂nifidepin没有显示出对泵活性的影响，表明钙离子泵的活性在钳制的原生质体膜的膜片上几乎看不到．
URL [本文引用: 1]
为了解细胞膜氢离子泵的电生理特性，本实验用膜片箝法研究了大麦根木质部薄壁细胞质膜上的生电泵在无一价阳离子条件下的生电活性与阴离子通道电导性的关系．结果显示：在大约40％的所测定的原生质体中观察到了由离子泵活动导致的膜电位超极化；添加H＋泵激活剂壳梭孢菌素导致在零电位的越膜电流密度的增加和膜电位大约-50V的超极化；相比之下，添加H＋泵抑制剂DCCD（Dicylohexylcarbondiimid）则导致膜电位的去极化作用和零电位越膜电流密度的减少；没有观察到钙离子通道抑制剂赤藓红B对生电泵活性的作用；细胞外溶液的pH值从5．8升高到8．8时，由于氢离子浓度梯度的加大导致泵的激活．这些特征都表明所测到的生电泵活性是H＋泵的作用而不是Ca2＋泵．负离子通道抑制剂DIDS（4，4’-Diisothiocyano-2，2’-stilbenedisulfonic acid）能导致膜电位的强烈超极化，暗示负离子通道的活性导致的短路效应强烈干扰和影响了H’泵的电活性的测定．这可能是植物根木质部细胞质膜上的H＋泵的电活性用膜片箝法难以检测的原因．尽管非特异性Ca2＋通道抑制剂La3＋也对泵的活性有明显影响，但在添加DIDS后添加的钙离子通道抑制剂nifidepin没有显示出对泵活性的影响，表明钙离子泵的活性在钳制的原生质体膜的膜片上几乎看不到．
[本文引用: 3]
DOI:10.1111/j.1365-2958.2012.08038.xURLPMID:3839633 [本文引用: 1]
SummaryBacillus subtilis SDP is a peptide toxin that kills cells outside the biofilm to support continued growth. We show that purified SDP acts like endogenously produced SDP; it delays sporulation, and the SdpI immunity protein confers SDP resistance. SDP kills a variety of Gram-positive bacteria in the phylum Firmicutes, as well as Escherichia coli with a compromised outer membrane, suggesting it participates in defence of the B. subtilis biofilm against Gram-positive bacteria as well as cannibalism. Fluorescence microscopy reveals that the effect of SDP on cells differs from that of nisin, nigericin, valinomycin and vancomycin-KCl, but resembles that of CCCP, DNP and azide. Indeed, SDP rapidly collapses the PMF as measured by fluorometry and flow cytometry, which triggers the slower process of autolysis. This secondary consequence of SDP treatment is not required for cell death since the autolysin-defective lytC, lytD, lytE, lytF strain fails to be lysed but is nevertheless killed by SDP. Collapsing the PMF is an ideal mechanism for a toxin involved in cannibalism and biofilm defence, since this would incapacitate neighbouring cells by inhibiting motility and secretion of proteins and toxins. It would also induce autolysis in many Gram-positive species, thereby releasing nutrients that promote biofilm growth.
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DOI:10.1002/jcb.24122URLPMID:22396185 [本文引用: 1]
To investigate reversal effects of pantoprazole (PPZ) on multidrug resistance (MDR) in human gastric adenocarcinoma cells in vivo and in vitro. Human gastric adenocarcinoma cell SGC7901 was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and antibiotics in a humidified 5% CO2 atmosphere at 3700°C. Adriamycin (ADR)-resistant cells were cultured with addition of 0.80900090008g/ml of ADR maintaining MDR phenotype. ADR was used to calculate ADR releasing index; CCK-8 Assay was performed to evaluate the cytotoxicity of anti-tumor drugs; BCECF-AM pH-sensitive fluorescent probe was used to measure intracellular pH (pHi) value of cells, whereas pH value of medium was considered as extracellular pH (pHe) value; Western blotting and immunofluorescent staining analyses were employed to determine protein expressions and intracellular distributions of vacuolar H+-ATPases (V-ATPases), mTOR, HIF-1, P-glycoprotein (P-gp), and multidrug resistant protein 1 (MRP1); SGC7901 and SGC7901/ADR cells were inoculated in athymic nude mice. Thereafter, effects of ADR with or without PPZ pretreatment were compared by determining the tumor size and weight, apoptotic cells in tumor tissues were detected by TUNEL assay. At concentrations greater than 200900090008g/ml, PPZ pretreatment reduced ADR releasing index and significantly enhanced intracellular ADR concentration of SGC7901 (P090009<0900090.01). Similarly, PPZ pretreatment significantly decreased ADR releasing index of SGC7901/ADR dose-dependently (P090009<0900090.01). PPZ pretreatment also decreased cell viabilities of SGG7901 and SGC7901/ADR dose-dependently. After 24-h PPZ pretreatment, administration of chemotherapeutic agents demonstrated maximal cytotoxic effects on SGC7901 and SGC7901/ADR cells (P090009<0900090.05). The resistance index in PPZ pretreatment group was significantly lower than that in non-PPZ pretreatment group (3.71 vs. 14.80). PPZ at concentration >100900090008g/ml significantly decreased pHi in SGC7901 and SGC7901/ADR cells and diminished or reversed transmembrane pH gradient (P090009<0900090.05). PPZ pretreatment also significantly inhibited protein expressions of V-ATPases, mTOR, HIF-1, P-gp, and MRP1, and alter intracellular expressions in parent and ADR-resistant cells (P090009<0900090.05). In vivo experiments further confirmed that PPZ pretreatment could enhance anti-tumor effects of ADR on xenografted tumor of nude mice and also improve the apoptotic index in xenografted tumor tissues. PPZ pretreatment enhances the cytotoxic effects of anti-tumor drugs on SGC7901 and reverse MDR of SGC7901/ADR by downregulating the V-ATPases/mTOR/HIF-1/P-gp and MRP1 signaling pathway. J. Cell. Biochem. 113: 24740900092487, 2012. 0008 2012 Wiley Periodicals, Inc.
DOI:10.1021/acs.est.5b04653URLPMID:5135099 [本文引用: 1]
Mechanical and structural properties of biofilms influence the accumulation and release of pathogens in drinking water distribution systems (DWDS). Thus, understanding how long-term residual disinfectants exposure affects biofilm mechanical and structural properties is a necessary aspect for pathogen risk assessment and control. In this study, elastic modulus and structure of groundwater biofilms was monitored by atomic force microscopy (AFM) and optical coherence tomography (OCT) during three months of exposure to monochloramine or free chlorine. After the first month of disinfectant exposure, the mean stiffness of monochloramine- or free-chlorine-treated biofilms was 4 to 9 times higher than those before treatment. Meanwhile, the biofilm thickness decreased from 120 ± 8 μm to 93 ± 6-107 ± 11 μm. The increased surface stiffness and decreased biofilm thickness within the first month of disinfectant exposure was presumably due to the consumption of biomass. However, by the second to third month during disinfectant exposure, the biofilm mean stiffness showed a 2- to 4-fold decrease, and the biofilm thickness increased to 110 ± 7-129 ± 8 μm, suggesting that the biofilms adapted to disinfectant exposure. After three months of the disinfectant exposure process, the disinfected biofilms showed 2-5 times higher mean stiffness (as determined by AFM) and 6-13-fold higher ratios of protein over polysaccharide, as determined by differential staining and confocal laser scanning microscopy (CLSM), than the nondisinfected groundwater biofilms. However, the disinfected biofilms and nondisinfected biofilms showed statistically similar thicknesses (t test, p > 0.05), suggesting that long-term disinfection may not significantly remove net biomass. This study showed how biofilm mechanical and structural properties vary in response to a complex DWDS environment, which will contribute to further research on the risk assessment and control of biofilm-associated-pathogens in DWDS.
DOI:10.1016/j.jelechem.2014.09.024URL [本文引用: 1]
Copper alloys, often used in cooling circuits of industrial plants using seawater as coolant, can be affected by biocorrosion induced by biofilm formation. Extracellular polymeric substances (EPS) produced by bacteria play a fundamental role in the different stages of biofilm formation, maturation and maintenance. The influence of loosely bound (LB) and tightly bound (TB) EPS, extracted from marine Pseudomonas NCIMB 2021, on the electrochemical behaviour of 70Cu 30Ni (wt.%) alloy in static artificial seawater (ASW) and on the chemical composition of oxide layers was studied by combined electrochemical measurements (polarization curves, EIS) and surface analysis (XPS, ToF-SIMS). Results were compared with those obtained in the presence of bovine serum albumin (BSA), a model protein. Compared to 70Cu 30Ni alloy in static ASW without biomolecules, for which a thick duplex oxide layer (outer redeposited Cu2O layer and inner oxidized nickel layer) is shown, the presence of BSA, TB EPS and LB EPS leads to a mixed oxide layer (oxidized copper and nickel) with a lower thickness. In the biomolecules-containing solutions, this oxide layer is covered by an adsorbed organic layer, mainly composed of proteins. A model is proposed to analyse impedance data obtained at the corrosion potential. The fitting procedure of impedance diagrams allows extracting the anodic charge transfer resistance, from which the corrosion current density can be calculated. The results show a slow-down of the anodic reaction in the presence of biomolecules (BSA, TB EPS and LB EPS), and a corrosion inhibition effect by LB EPS and to a lesser extent by BSA. No detrimental effect is evidenced with TB EPS.
DOI:10.1080/08927014.2015.1114609URLPMID:26769222 [本文引用: 1]
The chemical compositions of the surface conditioning layers formed by different types of solutions (from isolated EPS to whole culture media), involving different bacterial strains relevant for biocorrosion were compared, as they may influence the initial step in biofilm formation. Different substrata (polystyrene, glass, steel) were conditioned and analyzed by X-ray photoelectron spectroscopy. Peak decomposition and assignment were validated by correlations between independent spectral data and the ubiquitous presence of organic contaminants on inorganic substrata was taken into account. Proteins or peptides were found to be a major constituent of all conditioning layers and polysaccharides were not present in appreciable concentrations; the proportion of nitrogen which may be due to DNA was lower than 15%. There was no significant difference between the compositions of the adlayers formed from different conditioning solutions, except for the adlayers produced with tightly bound EPS extracted from D. alaskensis.
DOI:10.1016/j.foodcont.2007.08.012URL [本文引用: 1]
Electrolyzed oxidizing (EO) water has been regarded as a new sanitizer in recent years. Production of EO water needs only water and salt (sodium chloride). EO water have the following advantages over other traditional cleaning agents: effective disinfection, easy operation, relatively inexpensive, and environmentally friendly. The main advantage of EO water is its safety. EO water which is also a strong acid, is different to hydrochloric acid or sulfuric acid in that it is not corrosive to skin, mucous membrane, or organic material. Electrolyzed water has been tested and used as a disinfectant in the food industry and other applications. Combination of EO water and other measures are also possible. This review includes a brief overview of issues related to the electrolyzed water and its effective cleaning of food surfaces in food processing plants and the cleaning of animal products and fresh produce.
