,1, 姚妙爱1, 闾怀中1, 胡佩红2, 董静1Antibacterial Mechanism of Cold Plasma Against Listeria monocytogenes
DOU Yong,1, YAO MiaoAi1, Lü HuaiZhong1, HU PeiHong2, DONG Jing1通讯作者:
责任编辑: 赵伶俐
收稿日期:2020-04-17接受日期:2020-06-10网络出版日期:2020-12-16
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Received:2020-04-17Accepted:2020-06-10Online:2020-12-16
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窦勇, 姚妙爱, 闾怀中, 胡佩红, 董静. 冷等离子体对单核增生李斯特菌的杀菌机理[J]. 中国农业科学, 2020, 53(24): 5104-5114 doi:10.3864/j.issn.0578-1752.2020.24.013
DOU Yong, YAO MiaoAi, Lü HuaiZhong, HU PeiHong, DONG Jing.
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0 引言
【研究意义】单核增生李斯特菌（Listeria monocytogenes,LM）是一种常见的人畜共患病革兰氏阳性致病菌,因耐低温（4℃）、抗碱盐以及抗高渗透压特性使其普遍存在于食品表面和环境中,对食品安全和人类健康造成了巨大的威胁[1]。传统的热杀菌技术包括巴氏杀菌、高温瞬时杀菌和微波杀菌法等是食品行业中常用的控制LM污染的方法,然而,这些热杀菌技术由于温度的升高,对食品的营养、质构和风味等造成了一定的影响[2]。因此,寻找一种有效的冷杀菌技术,研究其潜在的抗菌机制,对食品行业中LM的控制有重要的意义。【前人研究进展】目前,已经有研究人员应用不同的冷杀菌技术控制食品中微生物的繁殖,包括脉冲强光[3]、超高压[4]、超声波[5]和冷等离子体技术等,其中,冷等离子体杀菌技术因操作装置简单,不会引起处理样品升温,已在食品领域取得了广泛的应用[6]。冷等离子体对LM的杀菌效果已经得到了很多研究的验证,如KIM等[7]利用介质阻挡放电冷等离子体技术处理猪排,发现处理后猪排中的李斯特菌数量明显降低;李兆杰等[8]发现,在功率为120 W、电压10 kV和频率30—40 kHz条件下,4 min的冷等离子处理可以彻底杀灭LM。此外,冷等离子体对微生物作用机制也得到了初步研究,如LIN等[9]发现冷等离子体的活性成分能够抑制鼠伤寒沙门氏菌的新陈代谢、胞内蛋白、多糖和DNA的含量,进而导致细菌的死亡。细胞膜是隔离细菌与外部环境的重要屏障,对维持细菌的生理功能和外观形态起着重要的作用。遭遇外部压力和抗菌处理时,细菌细胞膜作为主要的作用位点,完整性、流动性和通透性会发生明显的改变,从而影响胞内物质如DNA、ATP、蛋白质和酶以及物质运输与信息交流[10]。同时,细胞膜是胞内活性氧产生的主要位点,一些环境因素如辐照、紫外线处理和脉冲照射等都会刺激胞内活性氧的产生,从而激活氧化压力造成细胞膜的氧化损伤[11]。为了感知和应对胞内活性氧造成的氧化损伤,LM已经进化出一些复杂的氧化还原调节基因。【本研究切入点】尽管冷等离子体对LM的杀菌效果已经得到了广泛的验证,但是冷等离子体对LM的杀菌机制却鲜有研究,尤其是冷等离子体基于细胞膜层面的破坏机制还未见报道。【拟解决的关键问题】本研究以LM为研究对象,优化冷氮源等离子体对LM的最佳抑制条件,从细胞膜层面揭示冷氮源等离子体的杀菌机制,研究其对细胞膜完整性、流动性和通透性的影响（图1）。此外,通过研究冷等离子体对细胞膜引起的氧化损伤以及对氧化还原相关基因表达的影响,为拓展冷等离子体的抗菌应用提供参考。图1
新窗口打开|下载原图ZIP|生成PPT图1冷等离子体对细胞膜的作用机制示意图
Fig. 1Graphical abstract of mechanism of cold plasma on cytomembrane
1 材料与方法
1.1 材料与试剂
CMCC（B）54002单核增生李斯特菌由丰晖生物科技有限公司提供,斜面保藏的细菌接种至液体蛋白胨酵母浸膏葡萄糖培养基中（PYG）,37℃培养48 h到达对数期;蛋白检测试剂盒、细菌基因组DNA提取试剂盒,美国Sigma公司;8-苯胺-1-萘磺酸（ANS）、碘化丙啶（PI）、2',7'-二氯荧光黄双乙酸盐（DCFH- DA）、5,5-二甲基-1-吡咯啉-N-氧化物（DMPO）、邻-硝基苯-β-d-半乳糖苷（ONPG）,上海阿拉丁生物科技有限公司;磷酸二氢钾、磷酸氢二钠、氯化钠,国药集团化学试剂有限公司。1.2 仪器与设备
介质阻挡放电（DBD）冷等离子体装置,苏州ATV电子技术有限公司;透射电子显微镜,德国蔡司公司;蛋白电泳仪、核酸电泳仪,北京六一生物科技有限公司;紫外可见分光光度计,上海精密仪器仪表有限公司;倒置荧光显微镜,日本奥林巴斯;电子自旋共振仪,德国布鲁克公司;气质联用仪,美国安捷伦公司。1.3 方法
1.3.1 冷等离子体最佳抑制条件的优化 将对数期的LM菌悬液稀释后,加入到无菌PYG液体培养基中（总体积5 mL）,菌体初始浓度为5.6 CFU/mL。随后立即置于冷等离子体设备中进行处理。使用氮气为激发气体,功率（180、200和220 W）、处理时间（50、60和70 s）和氮气流速（40、50和60 cm3?min-1）为因素,残存菌数为因变量,应用Design Expert软件进行冷等离子体最佳抑菌条件（处理前后菌落数量变化最小）的优化,优化后的条件用于后续试验。1.3.2 细胞膜的完整性
1.3.2.1 透射电子显微镜观察 冷等离子体处理后,对数期的LM菌悬液（试验组,~108—109 CFU/mL）滴于铜网上面,与3%（v﹕v）的磷钨酸溶液混合染色5 min,晾干,使用透射电子显微镜观察冷等离子体对LM细胞膜完整性的影响[12]。未经冷等离子体处理的LM菌悬液作为对照组。
1.3.2.2 胞内蛋白质的泄漏 细胞膜的完整性也可以用胞内物质如蛋白质和DNA的泄漏来反映。蛋白质的泄漏使用聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质浓度的变化来检测。冷等离子体处理后,对数期的LM菌悬液（~108—109 CFU/mL）离心、洗涤,收集菌体沉淀后重悬在1 mL的无菌PBS中。重悬液使用超声波细胞破碎仪进行破胞,离心,取上清液与4×上样缓冲液沸水浴中煮沸10 min。冷却后,加样到蛋白电泳仪中,80 V条件下泳动20 min后,调整电压至120 V进行SDS-PAGE试验[13]。至于蛋白质浓度的变化,将经过冷等离子体处理后的LM菌悬液离心、洗涤和重悬后,其蛋白质含量使用蛋白质检测试剂盒进行测定。未经冷等离子体处理的LM菌悬液作为对照组。
1.3.2.3 胞内DNA的泄漏 冷等离子体处理（试验组）和未处理（对照组）的LM菌悬液（对数期,~108—109 CFU/mL）经离心、洗涤和重悬后,用基因组DNA提取试剂盒提取其中的DNA。使用紫外可见分光光度计在260 nm波长处测定不同样品中DNA含量的变化[14]。
1.3.3 细胞膜的流动性
1.3.3.1 细胞膜脂肪酸的变化 试验组和对照组的LM（对数期,~108—109 CFU/mL）经离心洗涤后收集菌体沉淀,菌体沉淀置于冷冻干燥机干燥24 h。称取1 g干燥后LM加入35 mL的甲醇/氯仿（v﹕v,1﹕2）溶液,超声30 min,0.45 μm有机滤膜过滤,滤液旋转蒸发浓缩后进行氮吹,所得提取物即为磷脂。提取得到的磷脂加入2 mL的甲基化试剂（2 mol?L-1 NaOH-CH3OH）,75℃加热20 min,将磷脂中的脂肪酸甲酯化[15]。脂肪酸甲酯用正己烷萃取,加样到气质联用仪（GC-MS）中,分析脂肪酸组分和含量的变化。GC-MS条件为：注射温度250℃,检测温度260℃,程序升温60℃保持3 min,5℃?min-1加热到160℃,2℃?min-1加热至220℃,然后10℃?min-1加热到230℃并保持2 min。
1.3.3.2 1-苯胺-8-萘磺酸 （ANS）荧光 4 mL冷等离子体处理的LM菌悬液（试验组,~106 CFU/mL）与20 μL的ANS溶液（8 mmol?L-1）混合,置于暗处反应30 min。反应结束后,置于荧光分光光度计中,385 nm的激发波长和473 nm的发射波长处检测荧光强度[16]。未经等离子体处理的LM作为对照组。
1.3.4 细胞膜的通透性
1.3.4.1 碘化丙啶（PI）荧光 20 μL的PI溶液（20 mmol?L-1）与4 mL冷等离子体处理的LM菌悬液（~106 CFU/mL）混合,置于暗处反应30 min。使用倒置荧光显微镜获取不同样品的PI荧光照片,激发波长为400 nm,发射波长为615 nm[17]。未经等离子体处理的LM作为对照组。
1.3.4.2 电导率 不同处理的LM菌悬液（对数期,~108—109 CFU/mL）离心后收集上清液。不同样品上清液的电导率由电导仪进行检测。
1.3.4.3 β-半乳糖苷酶活性 使用乳糖类似物邻-硝基苯-β-d-半乳糖苷（ONPG）检测细胞膜上β-半乳糖苷酶活性的变化。将不同的LM菌悬液（对数期,~108—109 CFU/mL）离心、洗涤和重悬后,加入ONPG溶液（30 mmol?L-1,体积比1﹕20）,置于暗处反应30 min。在420 nm波长处检测不同样品的紫外分光光度值[18]。
1.3.5 细胞膜的氧化损伤
1.3.5.1 胞内活性氧（ROS）荧光照片和荧光强度 使用2',7'-二氯荧光黄双乙酸盐（DCFH-DA）检测胞内活性氧的产生。5 mL的LM菌悬液（对数期,~108—109 CFU/mL）经离心洗涤后收集菌体沉淀。菌体沉淀重悬于2 mL的DCFH-DA（10 μmol?L-1）溶液中,置于暗处反应30 min,反应结束后立即置于冷等离子体装置中处理（试验组）。处理结束后,离心洗涤并重悬于等体积的PBS溶液中。使用倒置荧光显微镜在488 nm的激发波长和525 nm的发射波长处获取不同样品的荧光照片。不同样品的ROS荧光强度则用荧光分光光度计在488 nm的激发波长和525 nm的发射波长处检测。
1.3.5.2 电子自旋共振（ESR） 使用电子自旋共振仪检测ROS的种类。冷等离子体处理后,100 μL的LM菌悬液（对数期,~108—109 CFU/mL）与20 μL 5,5-二甲基-1-吡咯啉-N-氧化物（DMPO）混合,置于暗处反应30 min。反应结束后移入毛细管中,在ESR仪器中检测。检测条件为：中心场强3 510 G,扫描宽度100 G,频率9.85 GHz[19]。
1.3.5.3 实时定量聚合酶链反应（qRT-PCR）使用qRT-PCR检测氧化还原相关基因perR、recA和sigB表达量的变化。不同LM样品的总RNA用RNA提取试剂盒提取。提取的RNA用逆转率试剂盒来合成cDNA。合成的cDNA作为qRT-PCR反应的模板来检测相关基因表达量的变化[20]。qRT-PCR反应条件为：95℃预变性15 min,95℃变性10 s,60℃退火20 s,72℃延伸30 s,40个循环。16S内参基因和相关基因的引物序列如表1所示。
Table 1
表1
表1不同基因的引物序列
Table 1
| 基因名称 Gene name | 前引物（5'-3'） Forward primer (5'-3') | 后引物（5'-3'） Reverse primer (5'-3') |
|---|---|---|
| 16S RNA | ACCGTCAAGGGACAAGCA | GGGAGGCAGCAGTAGGGA |
| perR | CAACTGTATATAATAATTTACGCGT | TGCTGCGAAATGTTCTACTT |
| recA | CAAGCACAAGGCGGAACAG | CAACGGAGTCAATTACTAGCATAT |
| sigB | TATTTGGATTGCCGCTTACC | TTTCGGACCTAACTCTTTGATT |
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1.4 数据分析
所有的数据在3次测试后以平均值±标准差的形式表示。使用SPSS软件中的单因素方差分析进行显著性分析,P<0.05为差异显著。使用Design Expert软件进行响应面结果的处理。2 结果
2.1 冷等离子体最佳抑制条件的优化和细胞膜完整性变化
由响应面分析法得出的最佳抑制条件是：功率182 W,处理时间67 s,氮气流速52 cm3?min-1（图2）,用此条件作为后续的全部试验。图2
新窗口打开|下载原图ZIP|生成PPT图2功率、处理时间和气体流速影响等离子体抑制效果的响应面设计
Fig. 2Response surface of the effects of power, treatment time and flow rate on the inhibitory effect of cold plasma
未经冷等离子体处理的LM表面光滑,饱满圆润,有完整的细胞膜包裹在细菌的外面（图3-A）。但经过冷等离子体处理后,LM细胞膜表面观察到破损变形的结构和泄漏的胞内物质（图3-B）,说明冷等离子体能够导致LM细胞膜完整性的破坏和变形。
图3
新窗口打开|下载原图ZIP|生成PPT图3冷等离子体处理对细胞膜完整性的影响
A：对照组的TEM图;B：试验组的TEM图;C：不同样品的SDS-PAGE结果;D：不同样品的蛋白质含量变化;E：不同样品DNA含量变化。*表示试验组和对照组差异显著（P<0.05）。下同
Fig. 3Effect of cold plasma treatment on cell membrane integrity
A: TEM results of control; B: TEM results of CP treatment; C: SDS-PAGE results; D: Protein concentration; E: DNA concentration. * means significant difference (P<0.05). The same as below
胞内物质的泄漏也可以反映细胞膜完整性的破坏。由图3-C可知,经冷等离子体处理后（试验组）,蛋白质条带的亮度低于对照组,说明冷等离子体导致了胞内蛋白质的泄漏。经等离子体处理后,LM内部的蛋白质含量从157 mg?mL-1（对照组）下降到89 mg?mL-1（试验组）,胞内蛋白质出现了明显的泄漏（图3-D）。同样,经过冷等离子体处理后,胞内DNA的含量也发生了明显的变化。DNA含量从35 μg?mL-1（对照组）下降到21 μg?mL-1（试验组）。以上LM形态观察和胞内物质的检测说明冷等离子体破坏了细胞膜的完整性。
2.2 细胞膜的流动性
2.2.1 细胞膜脂肪酸分析 细胞膜的流动性对细菌的活性和生命力起到了至关重要的作用。细胞膜磷脂中不饱和脂肪酸含量的变化是预测细胞膜流动性变化的重要指标,细胞膜中不饱和脂肪酸含量越高,细胞膜的流动性也就越大[21]。由表2可知,经过冷等离子体处理后（试验组）,细胞膜中不饱和脂肪酸的含量达到53.91%,比对照组高40.17%。同时,经过冷等离子体处理后,饱和脂肪酸的含量从53.68%下降到41.57%。说明经冷等离子体处理后,LM细胞膜的流动性明显增加。此外,经冷等离子体处理后,短链脂肪酸（C12和C14）的含量增加到8.13%。有研究表明短链脂肪酸含量的增加能够提高细胞膜的流动性[22],本研究结果说明冷等离子体处理提高了LM的膜流动性。Table 2
表2
表2不同样品的脂肪酸成分和含量
Table 2
| 脂肪酸成分 Fatty acid composition | 样品Sample | |
|---|---|---|
| 试验组 Test group | 对照组 Control group | |
| C12:0 (%) | 2.12±0.03a | 1.43±0.02b |
| C14:0 (%) | 6.01±0.13a | 5.45±0.07b |
| C16:0 (%) | 16.45±0.75a | 19.48±1.03b |
| C16:1 (%) | 27.27±1.25a | 21.26±0.77b |
| C17:0 (%) | 2.73±0.05a | 2.84±0.02a |
| C18:0 (%) | 14.26±0.93a | 24.48±1.02b |
| C18:1 (%) | 26.64±0.16a | 18.91±0.84b |
| 不饱和脂肪酸 Unsaturated fatty acid (%) | 53.91±1.76a | 40.17±0.78b |
| 饱和脂肪酸 Saturated fatty acid (%) | 41.57±0.82a | 53.68±1.64b |
| 短链脂肪酸 Short chain fatty acid (%) | 8.13±0.09a | 6.88±0.15b |
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2.2.2 ANS荧光 ANS荧光探针可以用来测定细胞膜的流动性。ANS能够和细胞膜的疏水区域结合,细胞膜的流动性越小,ANS就越容易与细胞膜结合,进而荧光强度也越高[23]。本研究利用ANS荧光强度的变化来反映细胞膜流动性的改变,由图4可知,试验组的荧光强度明显低于对照组,从8.99下降到3.73,说明冷等离子体处理后细胞膜的流动性增加。
图4
新窗口打开|下载原图ZIP|生成PPT图4不同样品的ANS荧光
Fig. 4ANS fluorescence intensity of different samples
2.3 细胞膜的通透性
2.3.1 PI荧光 碘化丙啶（PI）是一种膜不透性的荧光探针,能够与细胞内的遗传物质结合,发射出红色荧光。PI不能穿透完整的细胞膜,只有细胞膜的通透性提高后,PI才能进入细胞并与遗传物质结合[24]。因此,通过检测PI荧光反映细胞膜通透性的变化。图5-A中没有检测到红色的荧光,说明PI不能进入完整的细胞膜,进而不能和遗传物质结合。图5-B中观察到大量的红色荧光,说明冷等离子体处理后,细胞膜的通透性提高,大量的PI进入细胞内与遗传物质结合发射出红色的荧光。图5
新窗口打开|下载原图ZIP|生成PPT图5冷等离子体处理对LM细胞膜透性的影响
A：对照组的PI荧光图;B：试验组的PI荧光图;C：电导率和β-半乳糖苷酶活性的变化
Fig. 5Effect of cold plasma treatment on cell membrane permeability
A: PI fluorescent images of control; B: PI fluorescent images of CP treatment; C: Changes of conductivity and β-galactosidase
2.3.2 电导率和β-半乳糖苷酶活性 细胞膜通透性的提高能够促进电解质和胞内离子的泄漏,进而提高菌悬液的电导率。由图5-C可以看出,对照组的电导率为0.15 mS?cm,低于试验组的0.33 mS?cm,说明冷等离子体处理后细胞膜通透性的提高。β-半乳糖苷酶是一种位于细胞膜上的水解酶,能够水解乳糖生成半乳糖和葡萄糖。ONPG是一种乳糖类似物,能够被β-半乳糖苷酶水解成半乳糖和O-硝基酚（ONPE）,ONPE在420 nm波长处有特定的紫外吸收。通常情况下,ONPG透过细胞膜的速率很慢,但当细胞膜的通透性改变时,大量的ONPG迅速透过细胞膜,导致420 nm处紫外强度的增加[25]。对照组的OD420nm为0.274,而试验组的OD420nm为0.683,增加的紫外强度说明了细胞膜通透性的提高。
2.4 细胞膜的氧化损伤
2.4.1 ROS荧光照片和荧光强度 本试验中,非荧光探针DCFH-DA用来检测细胞内的ROS水平。DCFH-DA可以被ROS氧化成绿色荧光物质 2',7'-二氯荧光素（DCF）。由图6可以看出,经过冷等离子体处理后,LM细胞内产生大量绿色荧光,这些绿色荧光是DCFH-DA被ROS氧化后产生的,说明冷等离子体刺激LM产生了大量的ROS,这些ROS会对细胞膜造成巨大的氧化损伤。由图6-D可以看出,对照组中的荧光强度在40 min内几乎没有变化,而试验组中的荧光强度从0.45增长到2.48（处理后20 min）和2.73（处理后40 min）。图6
新窗口打开|下载原图ZIP|生成PPT图6冷等离子体处理对细胞荧光强度的影响
A：对照组ROS荧光图片;B：CP处理20 min的荧光图片;C：CP处理40 min的荧光图片;D：ROS荧光强度
Fig. 6Effect of cold plasma treatment on cell fluorescence intensity
A: ROS fluorescent images of control; B: Fluorescent images of CP treatment for 20 min; C: Fluorescent images of CP treatment for 40 min; D: ROS fluorescence intensity
2.4.2 ESR和qRT-PCR ESR仪可以检测未成对的电子,因此,可以利用ESR技术鉴定LM细胞内产生的ROS种类。由图7-A可以看出,对照组的ESR谱图里没有任何明显峰,说明对照组中没有检测到ROS的存在。试验组的ESR谱图中检测到6个明显峰（用*标记）,这6重峰由烷基过氧化自由基、烷氧基自由基、烃基自由基和超氧自由基叠加造成[26]。
图7
新窗口打开|下载原图ZIP|生成PPT图7ESR谱图（A）及perR、recA和sigB表达量（B）的变化
Fig. 7ESR spectrogram of different samples (A), Changes of expression of perR, recA and sigB genes (B)
利用qRT-PCR技术检测perR、recA和sigB表达量的变化,从基因层面揭示细胞内ROS的产生机制。perR、recA和sigB对细胞内的氧化还原平衡具有重要影响。经过冷等离子体处理后,perR和recA相对表达量下降明显,分别比对照下降了43.29%和52.71%;而sigB的相对表达量上调了89.42%（图7-B）。说明冷等离子体通过调节相关基因的表达破坏了LM细菌内的氧化还原平衡,进而刺激了ROS的大量产生。
3 讨论
细胞膜是保证细菌生存的重要屏障,对细菌的物质交流和信息交换起着重要的作用。很多抗菌剂和杀菌装置的设计都以细胞膜作为主要的作用位点。细胞膜的完整性对保护细菌的胞内物质如蛋白质和DNA等有重要意义。一旦细胞膜的完整性遭到破坏,细胞的形态会发生变化,胞内物质发生泄漏。OLATUNDE等[27]发现冷等离子体处理破坏了一些革兰氏阴性菌的细胞膜,进而加速了胞内物质如脂肪、蛋白质和DNA的泄漏。本研究透射电镜图片显示,经冷等离子体处理后,LM细胞膜表面可观察到明显的破损结构,细胞膜的破损导致了蛋白质和DNA的泄漏,使胞内蛋白含量和DNA含量下降。当遭遇外部极端环境和抗菌处理时,细菌细胞通常会主动改变细胞膜中脂肪酸含量和类型,进而改变细胞膜的流动性。不饱和脂肪酸有较低的熔融温度,因此,细胞膜中不饱和脂肪酸含量越高,细胞膜的流动性就越大。脂肪酸的成分和含量分析结果证明,冷等离子体处理通过刺激不饱和脂肪酸和短链脂肪酸含量的增加以及饱和脂肪酸含量的下降,间接增大了LM细胞膜的流动性。然而ZHAO等[21]报道了不一样的结果,他们发现酿酒酵母细胞膜经脉冲电场处理后,不饱和脂肪酸含量从71.14%下降到60.56%,饱和脂肪酸含量从23.6%增长到30.27%,说明脉冲电场降低了细胞膜的流动性。这种差异可能由菌种不同和处理方式不同引起,ANS荧光强度测定也证明了这个结果。冷等离子体处理后,荧光强度从8.99下降到3.73。细胞膜的流动性越大,ANS就越不容易与细胞膜的疏水区域结合,进而荧光强度也就越低。细胞膜的流动性对维持细胞的物质运输和信息交流起到了至关重要的作用,流动性的增大会导致细胞内外渗透压的失衡,加速胞内重要物质的泄漏,影响细菌细胞的生理活性。细胞膜流动性的增大提升了细胞膜的通透性,导致膜不透性的荧光探针碘化丙啶可以自由地穿过细胞膜,与细胞内的遗传物质结合并产生红色荧光。此外,通透性的增加导致了电解质和胞内离子的泄漏,菌悬液的电导率也随之提高。
细胞膜是胞内产生活性氧的主要位点,主要通过氧化呼吸链在细胞膜上进行积累。很多处理条件如紫外线处理、辐射等都会促进细菌细胞产生超过自身承受极限的ROS,从而破坏氧化还原平衡,造成细胞膜的氧化损伤[28]。REN等[29]发现抗菌剂蝶芪处理会刺激大肠杆菌和金黄色葡萄球菌细胞产生大量的ROS,这些ROS的产生与氧化应激基因DinF的上调有关。荧光图片显示,经冷等离子体处理后,出现了大量的绿色荧光,这些绿色荧光由DCFH-DA经ROS氧化后产生的2',7'-二氯荧光素（DCF）导致,荧光强度增长,说明冷等离子体处理刺激了细胞膜上ROS 的产生和积累,改变了细胞膜完整性、流动性和通透性。ESR技术显示这些自由基可能是烷基过氧化自由基、烷氧基自由基、烃基自由基和超氧自由基,这些自由基的共同作用给细胞膜造成巨大的氧化损伤。
perR、recA和sigB是LM细胞内重要的氧化还原相关的基因,对维持LM的氧化还原平衡起着重要的作用。perR通过调节ROS敏感的转录抑制子的产生,从而解毒细胞内产生的内源性过氧化物和其他ROS[30]。而recA编码的活化剂能够催化细胞内的SOS反应,进而可以清除ROS和修复ROS造成的损伤[31]。同时,sigB也对ROS造成的氧化损伤起到一定的调节作用[32]。qRT-PCR结果显示冷等离子体处理下调了perR和recA,上调了sigB,说明ROS的产生与冷等离子体对这些氧化还原相关基因的调控有关,从基因层面揭示了ROS的产生机理。
4 结论
通过响应面试验设计,采用功率182 W,处理时间67 s,氮气流速52 cm3?min-1的冷等离子体最佳抑制条件研究其对LM细胞膜完整性、流动性、通透性和氧化损伤的影响。经冷等离子体处理后,细胞膜表面可以观察到破损变形的结构,胞内蛋白质含量和DNA含量下降,细胞膜完整性被破坏。同时,经冷等离子体处理后,细胞膜磷脂不饱和脂肪酸含量增加,饱和脂肪酸含量降低,ANS荧光强度降低,细胞膜流动性增加。此外,冷等离子体增加了细胞膜的通透性,冷等离子体通过调节perR、recA和sigB的相对表达量打破LM的氧化还原平衡,刺激了细胞膜上ROS的产生。参考文献 原文顺序
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URL [本文引用: 1]
【Objective】 A multiple polymerase chain reaction (PCR) system was developed to detect Listeria monocytogenes (LM),the more important food born pathogens, and applied to detect LM from food samples. 【Method】 Template DNA was obtained by thermolysis broth cultured bacteria and thermolysis colony. Primers were designed according to the 16S rRNA, inlAB, iap, hly genes. The concentration of primers and annealing temperature were examined and optimized. 【Result】 The PCR products were not detected in other Listeria spp, including L. innocua, L. seeligeri, L.welshimeri, L. ivanovii, L. grayi and in Vibrio parahemolyticus, indicating that this method was highly specific for L. monocytogenes. The detection limit of the PCR assay was 102 CFU/mL of pure cell culture. The PCR assay could detect no more than 0.4 CFU/g of L. monocytogenes in the contaminated pork. 【Conclusion】 The results show that the colony PCR method is better ,the method assay is highly sensitive, specific, rapid and accurate and can be used for rapid detection of L. monocytogenes in food.
URL [本文引用: 1]
【Objective】 A multiple polymerase chain reaction (PCR) system was developed to detect Listeria monocytogenes (LM),the more important food born pathogens, and applied to detect LM from food samples. 【Method】 Template DNA was obtained by thermolysis broth cultured bacteria and thermolysis colony. Primers were designed according to the 16S rRNA, inlAB, iap, hly genes. The concentration of primers and annealing temperature were examined and optimized. 【Result】 The PCR products were not detected in other Listeria spp, including L. innocua, L. seeligeri, L.welshimeri, L. ivanovii, L. grayi and in Vibrio parahemolyticus, indicating that this method was highly specific for L. monocytogenes. The detection limit of the PCR assay was 102 CFU/mL of pure cell culture. The PCR assay could detect no more than 0.4 CFU/g of L. monocytogenes in the contaminated pork. 【Conclusion】 The results show that the colony PCR method is better ,the method assay is highly sensitive, specific, rapid and accurate and can be used for rapid detection of L. monocytogenes in food.
DOI:10.1016/j.lwt.2013.12.036URL [本文引用: 1]
DOI:10.1016/j.foodcont.2019.106751URL [本文引用: 1]
DOI:10.3864/j.issn.0578-1752.2018.07.014URL [本文引用: 1]
【Objective】 The optimum conditions of ultra-high pressure (UHP) sterilization for kiwi fruit pulp and the effects of sterilization during storage period after ultrahigh pressure treatment were investigated by using Hayward Kiwifruit as raw materials. This study will provide an experimental reference for the non-thermal processing of kiwi fruit and its products development. 【Method】 The kiwi fruit pulp was obtained by cold crushing technology, the response surface method (RSM) was used to establish the mathematical model of ultra-high pressure sterilization conditions for kiwi fruit pulp to obtain the optimum conditions, and the total number of colonies, Vitamin C and browning degree were used as the evaluation indexes. The changes of the total number of colonies, mold and yeast and Escherichia coli group of kiwi fruit pulp were studied with microbiological analysis methods during the storage period at temperatures of 4℃ and -20℃ after ultrahigh pressure treatment. 【Result】 The optimum sterilization conditions of UHP treatment on kiwi fruit pulp were pressure 497 MPa, temperature 27℃, holding time 24 min through the single factor test and the response surface analysis of Box-Behnken model. Under this optimal sterilization condition, the total lethality rates of total number of colonies, Escherichia coli group and mold and yeast were 73.18%, 97.46% and 100.00%, respectively, in kiwi fruit pulp after UHP treatment. Under the standard range, the increments of total number of colonies in kiwi fruit pulp was bigger, which increased significantly of 97.19% and 85.98% respectively after storage for 6 and 14 weeks at 4℃, -20℃, compared with the first day of storage, but the growth rate of total number of colonies was small. While the increments rates of Escherichia coli group, mold and yeast in kiwi fruit pulp after UHP treatment were relatively small, the content of Escherichia coli group, mold and yeast only was 1.36, 0.67 and 0.32, 0.35 lg (CFU/mL) after stored 6 and 14 weeks at 4℃, -20℃, respectively. 【Conclusion】 As a new type of non-thermal sterilization method, ultra-high pressure technology had good sterilization effects for the heat sensitivity materials of kiwi fruit pulp. The kiwi fruit pulp which was used as processing materials and sterilized by the ultra-high pressure technology could be stored 14 weeks at -20℃ still to meet the commercial aseptic requirements, so the ultra-high pressure sterilization combined with the low temperature storage will benefit the further storage, process and utilization of kiwi fruit pulp produced by cold crushing.
DOI:10.3864/j.issn.0578-1752.2018.07.014URL [本文引用: 1]
【Objective】 The optimum conditions of ultra-high pressure (UHP) sterilization for kiwi fruit pulp and the effects of sterilization during storage period after ultrahigh pressure treatment were investigated by using Hayward Kiwifruit as raw materials. This study will provide an experimental reference for the non-thermal processing of kiwi fruit and its products development. 【Method】 The kiwi fruit pulp was obtained by cold crushing technology, the response surface method (RSM) was used to establish the mathematical model of ultra-high pressure sterilization conditions for kiwi fruit pulp to obtain the optimum conditions, and the total number of colonies, Vitamin C and browning degree were used as the evaluation indexes. The changes of the total number of colonies, mold and yeast and Escherichia coli group of kiwi fruit pulp were studied with microbiological analysis methods during the storage period at temperatures of 4℃ and -20℃ after ultrahigh pressure treatment. 【Result】 The optimum sterilization conditions of UHP treatment on kiwi fruit pulp were pressure 497 MPa, temperature 27℃, holding time 24 min through the single factor test and the response surface analysis of Box-Behnken model. Under this optimal sterilization condition, the total lethality rates of total number of colonies, Escherichia coli group and mold and yeast were 73.18%, 97.46% and 100.00%, respectively, in kiwi fruit pulp after UHP treatment. Under the standard range, the increments of total number of colonies in kiwi fruit pulp was bigger, which increased significantly of 97.19% and 85.98% respectively after storage for 6 and 14 weeks at 4℃, -20℃, compared with the first day of storage, but the growth rate of total number of colonies was small. While the increments rates of Escherichia coli group, mold and yeast in kiwi fruit pulp after UHP treatment were relatively small, the content of Escherichia coli group, mold and yeast only was 1.36, 0.67 and 0.32, 0.35 lg (CFU/mL) after stored 6 and 14 weeks at 4℃, -20℃, respectively. 【Conclusion】 As a new type of non-thermal sterilization method, ultra-high pressure technology had good sterilization effects for the heat sensitivity materials of kiwi fruit pulp. The kiwi fruit pulp which was used as processing materials and sterilized by the ultra-high pressure technology could be stored 14 weeks at -20℃ still to meet the commercial aseptic requirements, so the ultra-high pressure sterilization combined with the low temperature storage will benefit the further storage, process and utilization of kiwi fruit pulp produced by cold crushing.
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[本文引用: 1]
[本文引用: 1]
[本文引用: 1]
DOI:10.1016/j.cap.2013.04.021URL [本文引用: 1]
This study aimed to evaluate the use of a dielectric barrier discharge (DBD) plasma system to improve the safety of pork loins. When pork loin was exposed to DBD plasma with the input gases He and He + O-2, the population of Escherichia coli was reduced by 0.26 and 0.50 log cycles following a 5-min treatment and by 0.34 and 0.55 log units following a 10-min treatment, respectively. That of Listeria monocytogenes was also reduced from 0.17 to 0.35 and 0.43 to 0.59 log cycles when the samples were exposed to DBD for 5 and 10 min using He and He + O-2, respectively. The pH and L*-values (lightness) of the samples decreased significantly with DBD plasma treatment, but a*- (redness) and b*-values (yellowness) exhibited no obvious changes. Lipid oxidation, measured by TBARS values, was greater in samples with He + O-2 than in other samples. Significant reductions in sensory quality parameters (appearance, color, odor, acceptability, etc.) were observed in DBD-treated samples. These results indicate that the DBD plasma system has potential for use in sanitizing pork loins by inactivation of foodborne pathogens, although the effect was limited. In order to meet market requirements, however, a method to overcome sensory deterioration of pork loins should be developed and applied. (C) 2013 Elsevier B.V.
DOI:10.7506/spkx1002-6630-201511032URL [本文引用: 1]
Low temperature plasma glow discharge sterilizer was developed and used to study the bactericidal kinetics of sixmicroorganisms including Escherichia coli, Shigella sonnei, Staphylococcus aureus, Listeria monocytogenes, Aspergillusflavus and Candida albicans. Moreover, the bactericidal mechanisms were studied by scanning electron microscopy,transmission electron microscopy, DNA electrophoresis and spectroscopy analysis. It was shown that low temperature glowdischarge plasma could kill E. coli, S. sonnei, S. aureus, L. monocytogenes, A. flavus and C. albicansi effectively in 2, 1.5, 3,4, 10 and 10 minutes, respectively. According to the bactericidal kinetic curves, the six microorganisms were classified intothree categories, namely gram-negative bacteria, gram-positive bacteria and fungi, which may be resulted from the differentstructures of cell walls of microorganisms. Furthermore, the charged particles with high energy produced by low temperatureglow discharge plasma played a dominant role in plasma-induced bacterial spore inactivation by destroying bacterial cellwalls. This study may provide an important method basis for the application of low temperature glow discharge plasma insterilization of microorganisms.
DOI:10.7506/spkx1002-6630-201511032URL [本文引用: 1]
Low temperature plasma glow discharge sterilizer was developed and used to study the bactericidal kinetics of sixmicroorganisms including Escherichia coli, Shigella sonnei, Staphylococcus aureus, Listeria monocytogenes, Aspergillusflavus and Candida albicans. Moreover, the bactericidal mechanisms were studied by scanning electron microscopy,transmission electron microscopy, DNA electrophoresis and spectroscopy analysis. It was shown that low temperature glowdischarge plasma could kill E. coli, S. sonnei, S. aureus, L. monocytogenes, A. flavus and C. albicansi effectively in 2, 1.5, 3,4, 10 and 10 minutes, respectively. According to the bactericidal kinetic curves, the six microorganisms were classified intothree categories, namely gram-negative bacteria, gram-positive bacteria and fungi, which may be resulted from the differentstructures of cell walls of microorganisms. Furthermore, the charged particles with high energy produced by low temperatureglow discharge plasma played a dominant role in plasma-induced bacterial spore inactivation by destroying bacterial cellwalls. This study may provide an important method basis for the application of low temperature glow discharge plasma insterilization of microorganisms.
DOI:10.1016/j.lwt.2020.109340URL [本文引用: 1]
DOI:10.3864/j.issn.0578-1752.2020.05.015URL [本文引用: 1]
【Objective】Effects of heat stress on cell membrane and membrane protein of Escherichia coli were investigated with ATCC43889 as the test microorganism in this work, which would provide a theoretical basis for the application of high temperature sterilization technology in the food industry. 【Method】 The changes of the cell membrane and membrane protein for three heat-resistant ATCC43889 strains which were treated with 10 times of heat stress treatments at 50℃, 60℃, and 70℃ for 15 min, transferred into TSB and incubated again were studied, respectively. The morphologic changes of original control strain and the three heat-resistant strains of ATCC43889 were observed using scanning electron microscopy. The biofilm-forming ability for each strain was determined using a 96-well microplate method. The changes and differences of cell membrane fatty acid composition for each strain were monitored using gas chromatography. The changes of phase transition temperature of cell membrane phospholipid for each strain were measured using differential scanning calorimetry (DSC). The changes of outer membrane protein expression for each strain were examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). 【Result】 The experimental results showed that the individual morphology of ATCC43889 changed evidently after 10 times of heat stress treatments at 50℃, 60℃, and 70℃, transfers and incubation again, respectively. The individual morphology for a part of the cells changed from spheroids to long rods after heat stress treatment at 50℃. The individual morphology of the cells upon heat stress treatment at 60℃ was thinner and longer than that of the cells upon heat stress treatment at 50℃. Upon heat stress treatment at 70℃, most of the cells became longer rods, a large number of cells gathered together, and the surface of the cells was irregular and uneven. The vitality and ability of biofilm-forming for the three heat-resistant ATCC43889 strains enhanced with increasing heat stress temperature. The ability of biofilm-forming for the three ATCC43889 heat-resistant strains significantly (P<0.05) differ from that of the control group, while the ability of biofilm-forming for the heat-resistant strain at 60℃ was not significantly (P>0.05) different from that of the heat-resistant strain at 70℃. Among the fatty acids identified, three fatty acids were absent after 10 times of heat stress treatments at 50℃, 60℃ and 70℃ compared to the original control strain, which were C18:1n9c, C18:3n3, and C21:0, respectively. The amount of saturated fatty acid such as C13:0, C16:0, C17:0 and their total amounts increased, while the amount of unsaturated fatty acid such as C14:1, C16:1, C17:1, C18:1n9t, C18:2n6t and their total amounts in the cell membrane for the three heat-resistant strains decreased with the increase of heat stress temperature. The ratio of saturated to unsaturated fatty acids (SFA/USFA), the melting point (Tm) and the phase transition temperature of cell membrane phospholipids increased as the heat stress temperature increased. These changes in membrane fatty acid composition and the phase transition temperature of cell membrane phospholipids resulted in decreased membrane fluidity of the three heat-resistant strains. The protein band color of molecular weight about 63 and 75 kD deepened with the increase of heat stress temperature. The increase of heat-resistance for the two heat-resistant strains after 10 times of heat stress treatments at 60℃ and 70℃ was accompanied by synthesis of specific outer membrane proteins of molecular weight about 48 to 75 kD. These results indicated that synthesis and increased amounts of some specific outer membrane proteins could induce heat-resistance for E. coli ATCC43889. 【Conclusion】The longer individual morphology, the enhanced ability of biofilm-forming, the increased ratio of saturated to unsaturated fatty acids, the increased phase transition temperature of cell membrane phospholipid and the increased expression of some outer membrane proteins correlated with the greater heat-resistance of E. coli ATCC43889. These changes contributed to the adaptation of ATCC43889 to heat stress environment and improvement of cellular survival viability.
DOI:10.3864/j.issn.0578-1752.2020.05.015URL [本文引用: 1]
【Objective】Effects of heat stress on cell membrane and membrane protein of Escherichia coli were investigated with ATCC43889 as the test microorganism in this work, which would provide a theoretical basis for the application of high temperature sterilization technology in the food industry. 【Method】 The changes of the cell membrane and membrane protein for three heat-resistant ATCC43889 strains which were treated with 10 times of heat stress treatments at 50℃, 60℃, and 70℃ for 15 min, transferred into TSB and incubated again were studied, respectively. The morphologic changes of original control strain and the three heat-resistant strains of ATCC43889 were observed using scanning electron microscopy. The biofilm-forming ability for each strain was determined using a 96-well microplate method. The changes and differences of cell membrane fatty acid composition for each strain were monitored using gas chromatography. The changes of phase transition temperature of cell membrane phospholipid for each strain were measured using differential scanning calorimetry (DSC). The changes of outer membrane protein expression for each strain were examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). 【Result】 The experimental results showed that the individual morphology of ATCC43889 changed evidently after 10 times of heat stress treatments at 50℃, 60℃, and 70℃, transfers and incubation again, respectively. The individual morphology for a part of the cells changed from spheroids to long rods after heat stress treatment at 50℃. The individual morphology of the cells upon heat stress treatment at 60℃ was thinner and longer than that of the cells upon heat stress treatment at 50℃. Upon heat stress treatment at 70℃, most of the cells became longer rods, a large number of cells gathered together, and the surface of the cells was irregular and uneven. The vitality and ability of biofilm-forming for the three heat-resistant ATCC43889 strains enhanced with increasing heat stress temperature. The ability of biofilm-forming for the three ATCC43889 heat-resistant strains significantly (P<0.05) differ from that of the control group, while the ability of biofilm-forming for the heat-resistant strain at 60℃ was not significantly (P>0.05) different from that of the heat-resistant strain at 70℃. Among the fatty acids identified, three fatty acids were absent after 10 times of heat stress treatments at 50℃, 60℃ and 70℃ compared to the original control strain, which were C18:1n9c, C18:3n3, and C21:0, respectively. The amount of saturated fatty acid such as C13:0, C16:0, C17:0 and their total amounts increased, while the amount of unsaturated fatty acid such as C14:1, C16:1, C17:1, C18:1n9t, C18:2n6t and their total amounts in the cell membrane for the three heat-resistant strains decreased with the increase of heat stress temperature. The ratio of saturated to unsaturated fatty acids (SFA/USFA), the melting point (Tm) and the phase transition temperature of cell membrane phospholipids increased as the heat stress temperature increased. These changes in membrane fatty acid composition and the phase transition temperature of cell membrane phospholipids resulted in decreased membrane fluidity of the three heat-resistant strains. The protein band color of molecular weight about 63 and 75 kD deepened with the increase of heat stress temperature. The increase of heat-resistance for the two heat-resistant strains after 10 times of heat stress treatments at 60℃ and 70℃ was accompanied by synthesis of specific outer membrane proteins of molecular weight about 48 to 75 kD. These results indicated that synthesis and increased amounts of some specific outer membrane proteins could induce heat-resistance for E. coli ATCC43889. 【Conclusion】The longer individual morphology, the enhanced ability of biofilm-forming, the increased ratio of saturated to unsaturated fatty acids, the increased phase transition temperature of cell membrane phospholipid and the increased expression of some outer membrane proteins correlated with the greater heat-resistance of E. coli ATCC43889. These changes contributed to the adaptation of ATCC43889 to heat stress environment and improvement of cellular survival viability.
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DOI:10.1016/j.foodchem.2015.04.108URL [本文引用: 1]
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DOI:10.3382/ps/pey370URLPMID:30101285 [本文引用: 1]
As the most abundant protein in chicken eggs, ovalbumin plays an important role in the processing of high value-added poultry products. The present study investigated the effects of hydroxyl radical-induced early stage oxidation on the physicochemical and interfacial properties of chicken egg white ovalbumin. Protein carbonyl content of ovalbumin increased (from 0.78 to 1.13 nmol/mg) with the oxidation time (0-5 h), while free sulfhydryl content (from 0.43 to 0.09 nmol/mg) and free amino group content (from 0.49 to 0.43 nmol/mg) decreased. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed that the exposure of ovalbumin to hydroxyl radicals caused self-cross-linking and resulted in the formation of dimers and trimers. Accompanied by these changes, the surface hydrophobicity of ovalbumin was enhanced about 1.5-fold with the deepening of oxidation, and the value of zeta potential became more negative (from -7.15 to -20.51 mv). About 2 h of moderate oxidation improved the foaming and emulsifying properties of ovalbumin (1.2-fold to 1.8-fold), while excessive oxidation (3 h) decreased these interface properties. Hydroxyl radical-induced oxidation changed the surface chemical groups and structures of ovalbumin, thereby affecting the surface properties. The foaming and emulsifying properties of ovalbumin could be improved by oxidation, increasing the application possibilities of ovalbumin in the food interface system.
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DOI:10.1016/j.mimet.2016.06.030URL [本文引用: 1]
DOI:10.1016/j.foodchem.2018.09.101URLPMID:30409600 [本文引用: 1]
The generation, accumulation and decay of free radicals in six varieties of cheese, irradiated (0-4kGy) in an electron accelerator, have been studied by electron spin resonance (ESR) spectroscopy. Remarkably, the ESR spectra of all untreated cheeses showed only one singlet signal with a g-factor of 2.0064+/-0.0005. Surprisingly, the ESR spectra of irradiated samples presented a new signal with g-factor of 2.0037+/-0.0003 which was independent of the type of cheese, and which might be due to free radicals from the radiolysis of proteins. Surface regression models (P<0.0001) established the relationship among signal intensity, absorbed dose (0, 1, 2 and 4kGy) and storage time (0-180days) for the different types of cheese. Results suggested that the analysis by ESR (or electron paramagnetic resonance, EPR) is suitable to evaluate, either qualitatively or quantitatively, the irradiation treatment of different types of cheese.
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DOI:10.1016/j.foodcont.2013.11.015URL [本文引用: 2]
In this study, pulsed electric fields (PEF) treatments at 20 kV/cm for 0-500 mu s were applied to Saccharomyces cerevisiae to investigate the effects of PEF on the cytomembrane and intracellular nucleic acids. The change in composition of membrane lipids characterized by decrease in the ratio of contents of unsaturated fatty acids versus saturated fatty acids induced by PEF was observed, which was related with the increase in rigidness of cytomembrane (lower cytomembrane fluidity and higher cytomembrane viscosity) after PEE PEF treatment caused no significant change in DNA but disruption of RNA of S. cerevisiae, which was also confirmed by the increase of PEF resistance and no occurrence of injured microbial cells by supplementation of RNA stabilizer, magnesium. The results showed the damage of cytomembrane and RNA were associated with the sublethally injured S. cerevisiae, suggesting cytomembrane and RNA were primary targets for PEF induced microbial damage. (C) 2013 Elsevier Ltd.
DOI:10.1016/j.lwt.2016.10.019URL [本文引用: 1]
DOI:10.1016/s0005-2736(98)00020-0URLPMID:9630653 [本文引用: 1]
The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase family of signalling cell surface molecules. Signalling by this protein is mediated through binding of epidermal growth factor to its extracellular region ultimately leading to phosphorylation of several residues on the intracellular portion of the receptor. The only means of communication between the intracellular and extracellular domains is via the transmembrane region of the protein. In this work we describe the first structural studies of a 34-residue synthetic peptide (hEGFRp), representative of the human EGFR transmembrane region, using two-dimensional and 2H wideline NMR and CD spectroscopies. In water the peptide demonstrated a lack of regular secondary structure and existed as oligomers. Addition of the lipomimetic solvent, trifluoroethanol (TFE), led to the production of monomeric structured species. Analysis of NMR spectra of the hEGFRp indicated that an alpha-helix was present between residues M626 and R647. This observation was reinforced by solid state 2H NMR studies in lipid bilayers which showed typical 'Pake' spectra indicating axially symmetric motion. The helical region in hEGFRp commences four residues later than predicted via hydrophobicity profiles, and extends to include several charged arginine residues which would lie on the cytosolic side of the membrane. These observations provide the first evidence that the transmembrane alpha-helical region in EGFR may not only traverse the membrane but may continue to the cytosolic region near T654, an important phosphorylation site.
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DOI:10.3168/jds.2019-16654URLPMID:31477300 [本文引用: 1]
beta-Galactosidase is one of the most important enzymes used in dairy industry. Here, a novel thermostable beta-galactosidase was cloned and overexpressed from Bacillus coagulans NL01 in Escherichia coli. The phylogenetic trees were constructed using neighbor-joining methods. Phylogeny and amino acid analysis indicated that this enzyme belonged to family 42 of glycoside hydrolases. The optimal pH and temperature were, respectively, 6.0 and 55 to 60 degrees C. The purified enzyme had a 3.5-h half-life at 60 degrees C. Enzyme activity was enhanced by Mn(2+). Compared with other beta-galactosidases from glycoside hydrolase family 42, B. coagulans beta-galactosidase exhibited excellent hydrolysis activity. The Michaelis constant (Km) and maximum rate of enzymatic reaction (Vmax) values for p-nitrophenyl-beta-d-galactopyranoside and o-nitrophenyl-beta-d-galactopyranoside were 1.06 mM, 19,383.60 U/mg, and 2.73 mM, 5,978.00 U/mg, respectively. More importantly, the enzyme showed lactose hydrolysis ability superior to that of the commercial enzyme. The specific enzyme activity for lactose was 27.18 U/mg. A total of 104.02 g/L lactose in whey was completely hydrolyzed in 3 h with addition of 2.38 mg of pure enzyme per gram of lactose. In view of the high price of commercial beta-galactosidase, B. coagulans beta-galactosidase could be a promising prototype for development of commercial enzymes aimed at lactose treatment in the dairy industry.
DOI:10.1016/j.lwt.2019.01.016URL [本文引用: 1]
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DOI:10.1016/j.molmed.2019.04.005URLPMID:31080142 [本文引用: 1]
Recent advances in developmental biology and biomedical engineering have significantly improved the efficiency and purity of cardiomyocytes (CMs) generated from pluripotent stem cells (PSCs). Regardless of the protocol used to derive CMs, these cells exhibit hallmarks of functional immaturity. In this Opinion, we focus on reactive oxygen species (ROS), signaling molecules that can potentially modulate cardiac maturation. We outline how ROS impacts nearly every aspect associated with cardiac maturation, including contractility, calcium handling, metabolism, and hypertrophy. Though the precise role of ROS in cardiac maturation has yet to be elucidated, ROS may provide a valuable perspective for understanding the molecular mechanisms for cardiac maturation under various conditions.
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DOI:10.1016/j.mib.2018.10.006URLPMID:30412828 [本文引用: 1]
Listeria monocytogenes (Lm) is a Gram-positive bacterium that thrives in nature as a saprophyte and in the mammalian host as an intracellular pathogen. Both environments pose potential danger in the form of redox stress. In addition, endogenous reactive oxygen species (ROS) are continuously generated as by-products of aerobic metabolism. Redox stress from ROS can damage proteins, lipids, and DNA, making it highly advantageous for bacteria to evolve mechanisms to sense and detoxify ROS. This review focuses on the five redox-responsive regulators in Lm: OhrR (to sense organic hydroperoxides), PerR (peroxides), Rex (NAD(+)/NADH homeostasis), SpxA1/2 (disulfide stress), and PrfA (redox stress during infection).
DOI:10.1099/mic.0.035196-0URL [本文引用: 1]
DOI:10.1128/aem.67.10.4454-4457.2001URLPMID:11571142 [本文引用: 1]
To determine the contribution of sigma B (sigma(B)) to survival of stationary-phase Listeria monocytogenes cells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolar sigB null mutant strain after exposure to heat (50 degrees C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45 degrees C, 5% ethanol, or pH 4.5). The DeltasigB and wild-type strains had similar viabilities following exposure to ethanol and heat, but the DeltasigB strain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the DeltasigB strain. These results suggest the existence in L. monocytogenes of both a sigma(B)-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase. sigma(B) contributed to resistance to both oxidative stress and carbon starvation in L. monocytogenes. The DeltasigB strain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the DeltasigB strain lost viability more rapidly than the parent strain. sigma(B) contributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that sigma(B) plays a role in protecting L. monocytogenes against environmental adversities.
