这项工作得到了热带 AgriSciences 学院内部赠款机构的支持 (项目编号: 20175013) 和 CIGA 20182023 两项赠款, 来自捷克生命科学大学。

1. 肉类制剂
<ol><li>从当地的屠杀中获得一小块牛肉 (从股二头肌中的新鲜牛肉), 转移到实验室。 注: 建议在常温下 (20-25 °c) 运输牛肉的腰部, 在密封袋内不超过20分钟。</li>
	<li>为了对牛肉肌肉的外层进行消毒, 在层流安全柜中, 用500毫升的挤压瓶喷洒 70% (v/v) 乙醇, 以洗涤肌肉。应用0.025 克乙醇每1厘米的肌肉表面2 。</li>
	<li>用小刀除去肉灌装的外表面, 以避免在肌肉内部残留乙醇。去掉大约3毫米的肌肉内部, 以保持肌肉的表面均匀。</li>
	<li>将肌肉包装在塑料密封袋中, 并将其转移到冰箱。</li>
	<li>储存的肌肉在-18 °c 1 天。然后, 解冻4摄氏度的冰冻肌肉6小时。 注: 对于解冻, 建议将肌肉从冰箱移动到冰箱。</li>
	<li>在层流安全柜中, 用切肉机将每块肌肉切成0.5 厘米厚的切片。然后, 用小刀把它切成小 5 x 2.5 厘米2矩形样品。</li>
	<li>将长方形的肉类样品包装在塑料袋中, 并将其存放在-18 摄氏度的冷藏库中供以后使用。</li>
</ol>2. 在层流安全柜中编制标准化的接种和接种程序
<ol><li>准备标准化的接种 (1.5 x 108 CFU/毫升) 的大肠杆菌ATCC 25922, 以接种肉类样品。<ol>
<li>为准备库存接种, 首先将冻干细菌培养 (由供应商交付) 转化为15毫升灭菌管, 预填充10毫升消毒的米勒韩丁汤 (BMHB)。在37摄氏度培养24小时的悬浮液。<ol>
<li>准备细菌库存解决方案如下: 采取大约 0.1-0.2 毫升的细菌悬浮和稀释成一个20毫升瓶封闭与橡胶塞子与铝帽预填充与15毫升的灭菌 BMHB。在37摄氏度培养24小时的悬浮液。</li>
			<li>存放在冰箱内的4°c 为标准化接种的准备。</li>
		</ol>
</li>
		<li>从库存解决方案 (见步骤 2.1.1) 的大肠杆菌采取大约 0.1-0.2 毫升的细菌悬浮和稀释在15毫升塑料灭菌管预填充10毫升消毒的韩丁汤 (BMHB)。在37摄氏度的24小时内孵化试管。</li>
		<li>为编制标准化的接种 (1.5 x 108 CFU/毫升), 添加少量此悬浮在15毫升灭菌管预填充与10毫升的灭菌 BMHB。</li>
		<li>彻底涡旋混合物, 并测量光学密度 (OD) 在600毫微米由密度计32。</li>
		<li>重复步骤 2.1.3 2.1.4, 直到表示为麦克法兰值的 OD 比清洁 BMHB 的值增加0.5。</li>
	</ol>
</li>
	<li>为接种做法, 安置长方形肉样品在二个不同的铝箔 (20 cm x 30 cm), 一个为控制样品和第二为被接种的肉样品。<ol>
<li>在第二个铝箔, 接种原始的长方形肉样品与800µL 细菌悬浮的选定的菌株 (这对应于 1.2 x 108 CFU 每肉样品) 通过均匀地分布接种在表面。<ol>
<li>吸管400µL 在样品的一边和轻轻地传播使用无菌的细胞摊铺机在表面上。让他们干10分钟. 在样品的另一侧对其余的悬浮液重复相同的过程。</li>
		</ol>
</li>
	</ol>
</li>
</ol>3. 干燥和特奥应用
<ol><li>将含有矩形肉类样品的铝箔从层流安全柜转移到烘干机: 用铝箔盖住, 然后将样品放在烘干机内。</li>
	<li>在标准的实验室烘干机中进行干燥。 注: 首先, 预热烤箱到摄氏55摄氏度。此过程可以持续20分钟。<ol>
<li>干燥控制样品6小时在55°c, 与干燥空气相对湿度值从 30-45% 不等。 注: 干燥空气相对湿度值根据液体从肉类中蒸发的速率而变化。</li>
	</ol>
</li>
	<li>计算所应用的特奥体积, 并将必要的油浓度表示为每烘干机容积 (mL/升空气) 的特奥量。例如, 1.5 毫升的特奥在53升 (干燥机容积) 的剂量导致浓度为0.028 毫升/升空气。要确定特奥的 MIC 为大肠杆菌, 使用剂量1.5 毫升 (0.028 毫升/升空气), 1 毫升 (0.019 毫升/升空气) 和0.75 毫升 (0.014 毫升/升空气)。</li>
	<li>在烘干之前, 为应用特奥蒸气 (以麝香作为主要化合物 79%), 浸泡一张滤纸 (12 cm x 20 cm) 与1.5 毫升药量的特奥并且安置入烘干机在风扇前面。</li>
	<li>使用与对照样品相同的程序 (步骤3.1 和 3.2) 将特奥特处理的肉类样品烘干。 注: 干燥过程结束后, 样品被除去, 在烤箱上打开3小时, 在80摄氏度, 并设置空气阀指示100% 排气口, 以清洁的基本油残渣从烤箱。</li>
</ol>4. 微生物分析
<ol><li>在肉接种与细菌之前, 检查肉样品为任何掺假。粘液的出现和发现任何强烈和刺鼻的气味都表明了肉类的腐烂。如果纹理感觉黏糊糊的, 细菌可能已经开始在肉的表面繁殖。</li>
	<li>为了评估接种效率, 对原接种的样品进行试验, 检测是否存在大肠杆菌ATCC 25922, 并将其与未接种的对照样品进行比较。为此目的:<ol>
<li>清洗每个肉类样本 (2 个控制样本和2种接种样本)。用缓冲的蛋白胨水 (8.5 克氯化钠) 将每个肉类样品挂在消毒瓶中. 1 克的蛋白胨, 5 片磷酸盐缓冲盐水, 1 克的吐温80在1升水) 的比率为 1:10 (瓦特/v), pH 值范围从 7-7.3。在室温下用 140 rpm 的摇床摇动10分钟。 注: 在接种程序后立即冲洗。</li>
		<li>用经调整的 6 x 6 滴板程序, 对钢板计数琼脂 (PCA) 和麦康凯琼脂 (MCA) 中的陈、Nace 和欧文33进行总结, 评估细菌数量。 注: 6 x 6 滴法采用发酵液微稀释法, 用多通道吸管制备研究样品的10倍系列稀释, 比常规方法33更节省劳动密集型和更经济性34。</li>
		<li>用 6 x 6 稀释法培养10倍的系列样品, 用于评价大肠杆菌。 注: 特别为 6 x 6 下落方法, 为耕种使用六5µL 下落, 从六选择的稀释被调查的样品与多通道吸管。在适当干燥的培养皿上, 滴入琼脂迅速吸收, 这种方法的种植非常方便和易于管理的34。</li>
		<li>孵育培养皿在37°c 为24小时。在培养期后, 根据第5节所述, 评估培养皿 (CFU g-1 ) 中的大肠杆菌的菌落数量。</li>
	</ol>
</li>
	<li>干燥后, 取两个接种后的干样品, 并将其与两个干燥的非接种对照样品分别进行比较, 以进行可行的大肠杆菌。要确定此四samples 的存在或不存在大肠杆菌, 请按照以下步骤对每个肉类样品进行预浓缩过程:<ol>
<li>将每个肉类样品挂在带有缓冲的蛋白胨水的消毒瓶中 (请参阅步骤 4.2.1), 并在室温下使用 140 rpm 10 分钟的振动筛进行摇动. 然后在6小时内孵化每瓶37摄氏度以进行预富集。</li>
		<li>对于细菌的评价和培养, 按照步骤 4.2.2 4.2.4 中所述的相同步骤进行。</li>
	</ol>
</li>
</ol>5. 审查结果
<ol><li>孵化完成后, 从孵化器中取出培养皿, 并按如下方式检查结果:<ol>
<li>为了评估菌落总数, 在 MCA 上检查温有氧细菌在 PCA (白点) 和典型的大肠杆菌菌落 (红到深粉色) 上存在的板块。如果病原体缺失, 两 agars 均无生长。</li>
		<li>计算菌落数并确定大肠杆菌(CFU/g-1干肉) 的数量。 注意: 在连续的两个稀释 (图 1) 中, 计算30个或更少的菌落数 (N)。CFU/g-1的干肉的编号N确定如下35 <img alt="Equation" src="/files/ftp_upload/57054/57054eq1.jpg"> 在其中, C是计数的所有水滴的菌落总数, v是每滴使用的样本稀释量 (这里, 0.05 毫升), n1 是第一稀释时使用的水滴数, n2 是滴数用于第二次稀释, d表示从其中捕获第一个计数的稀释。</li>
	</ol>
</li>
	<li>要分析微生物数据, 请将菌落数转换为日志 CFU g-1 , 并将其与治疗的主要效果36的方差分析 (方差计算) 进行比较。<ol>
<li>对多个平均比较36执行 Tukey 诚实显著差异测试 (Tukey HSD), 并确定治疗之间的显著差异。</li>
	</ol>
</li>
</ol>

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Food Agric. 97 (1), 82-87 (2017).</li><li>Garc&#237;a-D&#237;ez, J., Alheiro, J., Falco, V., Fraqueza, M. J., Patarata, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+characterization+and+antimicrobial+properties+of+herbs+and+spices+essential+oils+against+pathogens+and+spoilage+bacteria+associated+to+dry-cured+meat+products.">Chemical characterization and antimicrobial properties of herbs and spices essential oils against pathogens and spoilage bacteria associated to dry-cured meat products.</a> J. Essent. Oil Res. 29 (2), 117-125 (2017).</li><li>Cavanagh, H. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+Activity+of+the+Volatile+Phase+of+Essential+Oils:+A+Brief+Review.">Antifungal Activity of the Volatile Phase of Essential Oils: A Brief Review.</a> Nat. Prod. Commun. 2 (12), 1297-1302 (2007).</li><li>Tajkarimi, M. M., Ibrahim, S. A., Cliver, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+herb+and+spice+compounds+in+food.">Antimicrobial herb and spice compounds in food.</a> Food Control. 21 (9), 1199-1218 (2010).</li><li>Nedorostova, L., Kloucek, P., Kokoska, L., Stolcova, M., Pulkrabek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+properties+of+selected+essential+oils+in+vapour+phase+against+foodborne+bacteria.">Antimicrobial properties of selected essential oils in vapour phase against foodborne bacteria.</a> Food Control. 20 (2), 157-160 (2009).</li><li>Burt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+oils:+Their+antibacterial+properties+and+potential+applications+in+foods+-+A+review.">Essential oils: Their antibacterial properties and potential applications in foods - A review.</a> Int. J. Food Microbiol. 94 (3), 223-253 (2004).</li><li>Ramanathan, L., Das, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+control+of+lipid+oxidation+in+ground+fish+by+some+polyphenolic+natural+products.">Studies on the control of lipid oxidation in ground fish by some polyphenolic natural products.</a> J. Agric. Food Chem. 40 (1), 17-21 (1992).</li><li>Yamazaki, K., Yamamoto, T., Kawai, Y., Inoue, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+antilisterial+activity+of+essential+oil+constituents+by+nisin+and+diglycerol+fatty+acid+ester.">Enhancement of antilisterial activity of essential oil constituents by nisin and diglycerol fatty acid ester.</a> Food Microbiol. 21 (3), 283-289 (2004).</li><li>Garc&#237;a-D&#237;ez, J., Alheiro, J., Falco, V., Fraqueza, M. J., Patarata, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+activity+of+essential+oils+from+herbs+and+spices+used+on+meat+products+against+food+borne+pathogens.">Synergistic activity of essential oils from herbs and spices used on meat products against food borne pathogens.</a> Nat. Prod. Commun. 12 (2), 281-286 (2017).</li><li>Hussein Hamdy Roby, M., Atef Sarhan, M., Abdel-Hamed Selim, K., Ibrahim Khalel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+antioxidant+activity,+total+phenols+and+phenolic+compounds+in+thyme+(Thymus+vulgaris+L.),+sage+(Salvia+officinalis+L.),+and+marjoram+(Origanum+majorana+L.)+extracts.">Evaluation of antioxidant activity, total phenols and phenolic compounds in thyme (Thymus vulgaris L.), sage (Salvia officinalis L.), and marjoram (Origanum majorana L.) extracts.</a> Ind. Crops Prod. 43, 827-831 (2013).</li><li>Gouveia, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Antimicrobial+Effect+of+Essential+Oils+Against+Listeria+monocytogenes+in+Sous+vide+Cook-Chill+Beef+during+Storage.">The Antimicrobial Effect of Essential Oils Against Listeria monocytogenes in Sous vide Cook-Chill Beef during Storage.</a> J. Food Process. Preserv. 41 (4), (2017).</li><li>Chen, C., Nace, G., Irwin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+6+x+6+drop+plate+method+for+simultaneous+colony+counting+and+MPN+enumeration+of+Campylobacter+jejuni,+Listeria+monocytogenes,+and+Escherichia+coli.">A 6 x 6 drop plate method for simultaneous colony counting and MPN enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli.</a> J. Microbiol. Methods. 55 (2), 475-479 (2003).</li><li>Herigstad, B., Hamilton, M., Heersink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+optimize+the+drop+plate+method+for+enumerating+bacteria.">How to optimize the drop plate method for enumerating bacteria.</a> J. Microbiol. Methods. 44 (2), 121-129 (2001).</li><li>. Practical food microbiology Available from: <a target="_blank" href="https://drive.google.com/file/d/0BzyVOLllJ0B1YmlEemZ5M1RZekU/view?ts=590d8019">https://drive.google.com/file/d/0BzyVOLllJ0B1YmlEemZ5M1RZekU/view?ts=590d8019</a> (2003)</li><li>Smith-Palmer, A., Stewart, J., Fyfe, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+properties+of+plant+essential+oils+and+essences+against+five+important+food-borne+pathogens.">Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens.</a> Lett. Appl. Microbiol. 26 (2), 118-122 (1998).</li><li>Burt, S. a., Reinders, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+activity+of+selected+plant+essential+oils+against+Escherichia+coli+O157:H7.">Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7.</a> Lett. Appl. Microbiol. 36 (3), 162-167 (2003).</li></ol>

作者没有什么可透露的。

先前的研究表明, 导致食源性疾病的微生物在干燥过程中生存了10。因此, 必须在干燥前应用防腐剂, 以保证食品安全。在这项研究中, 我们专注于使用特奥。其原因有两个方面: 第一, 消费者对使用天然产品作为替代添加剂以提高食品质量的需求很高16;其次, 前一项研究表明, 在肉类干燥过程中使用 OEO 的结果为22。因此, 在肉类干燥过程中应用 OEO...

干燥作为传统的保存食物的方法已经被使用了从远古时代。如今, 对干燥的兴趣越来越浓厚, 作为一种有效的食品保鲜方法1,2,3。它是用来制造各种特殊加工的肉类。其中最知名的是干。
干法是肉类保鲜的最古老的方法之一, 其基础是固化和干燥, 以降低水分活动, 从而延长其保质期4。如今, 干腌肉仍然很受欢迎, 食品的安全性、风味和质地是必不可少的。干制剂可用于几乎任何类型的肉类, 包括牛肉, 猪肉, 家禽, 或游戏5, 它需要切肉在瘦肉条和干燥。通常, 浸泡在固化溶液中的肉或吸烟与干燥一起使用, 以使干它的特色风味6。
尽管干燥的巨大兴趣, 以真正保存食物, 食源性暴发的风险由大肠杆菌从干燥肉是关键的, 需要控制。有一些研究报告食源性胃肠炎暴发, 特别是与大肠杆菌O157:H7, 归因于不充分的热处理在家庭干燥。即使在商业准备的生涩的7、8、9中也发生类似情况。莱文et al。10建议, 食源性微生物可以在干燥条件 (约60°c) 的情况下生存。大肠杆菌O157:H7 二十世纪九十年代中旬的食源性疾病暴发归因于地面干肉制品6,11。有趣的是, 在以前的所有病例中, 主要的风险是由被确认为可行但非可培养 (VBNC) 的细菌病原体引起的。在诸如温度变化或饥饿等各种压力下,大肠杆菌单元可以进入一个称为 VBNC 状态1213的特定状态。VBNC 细胞可以通过暴露在适当的条件下恢复到可培养细胞, 然后由于食源性污染而对人类健康构成威胁 14, 15.这意味着, 如果肉类是在干燥后立即消耗的产品, 它是安全的。但是, 如果储存不足, 如湿度增加, 病原体和微生物生长的复发风险很高。
除了干燥和腌料的方法, 消费者有很高的需求, 使用天然产品作为替代添加剂, 以改善食品质量16,17。对肉类天然食品添加剂的应用特别感兴趣, 而不是传统的合成防腐剂18,19,20,21。尽管在干燥肉类时使用精油缺乏足够的实验证据, 但该领域早期的研究已经显示出积极的结果22,23。
自中世纪以来, 人们已经认识到必需的油脂化合物 (EOCs) 的抗菌剂, 杀虫, 和寄生虫特征24,25,26。今天, EOCs 是最重要的一组生物活性天然化合物的一部分。在不同的 EOCs 中, 麝香是其中一个最知名的。它由85% 以上的特奥23组成。这种苯酚能防止微生物和化学物质在食物中变质。此外, 它的抗菌性能可能会改善与其他天然防腐剂2,27,28,29,30。现在, 百里香 (胸腺), 一种属于唇形家族的药草, 已被公认为调味剂, 也是一种非常有效的肉类防腐剂 31.由加西亚-Díez et进行的一项研究。30在肉类产品上发现, 与其他精油相比, 特奥对食源性致病菌的抑制作用更大。因此, 在干燥过程中施用精油, 有机会增加干肉的价值, 减少食源性疾病的风险。
在本协议中, 我们提出了一种新的方法来应用特奥在肉类干燥, 特别是使用它在蒸气形式直接在干燥室。为了评估, 我们使用麦克风来确定治疗样本中的致病菌与原生细菌的缺失。初步结果表明, 该方法是一种高效的合成防腐剂替代品, 大大减少了干肉中的微生物负荷。

<table><tbody><tr><td>Meat cutter</td><td>Kalorik</td><td>KP 3530</td><td>from Miami Gardens, FL, USA</td></tr><tr><td>Laminar safety cabinet</td><td>Faster s.r.l</td><td></td><td>from Italy</td></tr><tr><td>Squeeze bottle of 500 mL</td><td>Merci</td><td>632 524 325 025</td><td>from CZ</td></tr><tr><td>Standard laboratory drier UFE 400</td><td>Memmert</td><td>DE 66812464</td><td>from Germany</td></tr><tr><td>Incubator</td><td>BT 120</td><td>N/A</td><td>from CZ</td></tr><tr><td>Refrigerator and Freezer</td><td>Bosch</td><td>KGN34VW20G</td><td>from DE</td></tr><tr><td>Densitometer</td><td>Biosan</td><td>220 000 050 122</td><td>Latvia; supplier Merci, CZ</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; ATCC 25922</td><td>Oxoid</td><td>CL7050</td><td>from CZ</td></tr><tr><td>Vortex</td><td>Chromservis</td><td>22008013</td><td>from CZ</td></tr><tr><td>Sterilized plastic tubes 15 mL</td><td>Gama</td><td>331 000 020 115</td><td>from CZ, supplier Merci</td></tr><tr><td>20 mL injection vial</td><td>Healthy vial</td><td>hvft169</td><td>from China</td></tr><tr><td>20 mm sterile butyl rubber stopper</td><td>Merci</td><td>22008013</td><td>from CZ</td></tr><tr><td>20 mm aluminum cap</td><td>Healthy vial</td><td>N/A</td><td>from China</td></tr><tr><td>Thyme essential oil</td><td>Sigma Aldrich</td><td>W306509</td><td>from St Louis, MO, USA</td></tr><tr><td>Mueller Hinton Broth</td><td>Oxoid</td><td>CM0337</td><td>from CZ</td></tr><tr><td>NaCl</td><td>Penta</td><td>16610-31000</td><td>from CZ</td></tr><tr><td>Peptone</td><td>Oxoid</td><td>LP0034</td><td>from CZ</td></tr><tr><td>Phosphate-buffered saline</td><td>Sigma Aldrich</td><td>P4417</td><td>from CZ</td></tr><tr><td>Polysorbate 80 (Tween 80)</td><td>Roth</td><td>T 13502</td><td>from DE, supplier P-lab</td></tr><tr><td>Shaker SHO-1D</td><td>Verkon</td><td>DH.WSR04020</td><td>from CZ,&amp;nbsp; 10 - 300 rpm. 350 x 350 mm with a platform for flasks</td></tr><tr><td>Ethanol 70%</td><td>Bioferm</td><td>N/A</td><td>from CZ</td></tr><tr><td>MacConkey Agar</td><td>Oxoid</td><td>CM007</td><td>from CZ</td></tr><tr><td>Plate Count Agar</td><td>Oxoid</td><td>CM0325</td><td>from CZ</td></tr><tr><td>Filter paper</td><td>Merci</td><td>480 622 080 040</td><td>from CZ</td></tr><tr><td>Erlenmeyer flasks 250 mL</td><td>Simax</td><td>610 002 122 636</td><td>from CZ; supplier Merci CZ</td></tr><tr><td>Multichannel pipette</td><td>Socorex</td><td>S852820</td><td>from Switzerland; supplier P lab, CZ</td></tr><tr><td>Microtiter plate</td><td>Gamma</td><td>V400916</td><td>CZ</td></tr><tr><td>Microlitre pipette 100-1000 &amp;mu;L</td><td>Eppendorf</td><td>333 120 000 062</td><td>from Germany; supplier Merci, CZ</td></tr></tbody></table>

the effect of the application of thyme essential oil on microbial load during meat drying

肉类是一种高蛋白的膳食, 用于配制干, 一种流行的食品零食, 在那里保存和安全是重要的。为了保证食品安全和延长肉类和肉类产品的保质期, 使用合成或天然防腐剂来控制和消除食源性细菌。越来越多的人对天然食品添加剂在肉类中的应用增加了兴趣。微生物, 如大肠杆菌, 污染肉类和肉类产品, 造成食源性疾病. 因此, 有必要改进肉类的保护过程。然而, 在肉类干燥时使用精油还没有深入研究。在这方面, 有机会增加干肉的价值, 并通过在干燥过程中应用精油降低食源性疾病的风险。在本议定书中, 我们提出了一种新的方法来应用百里香精油 (特奥) 在肉类干燥, 特别是在蒸气形式直接在干燥室。为了评估, 我们使用最小的抑制浓度 (MIC) 来检测样本中有害细菌的数量与原样品相比。初步结果表明, 该方法是一种可行的、可替代的合成防腐剂, 大大减少了干肉中的微生物负荷。

我们首先开发了这种方法, 通过使用牛至精油 (OEO), 以提高食品安全和提高干肉的价值。一般而言, 上述实验表明, 在干燥过程中,大肠杆菌进入 VBNC 状态作为一种生存策略。这是由事实证明, 在干燥完成后没有可培养细菌 22.因此, 6 小时的预浓缩过程是必要的, 以允许计数的应变。在较短的时期内, 生长细胞的数量仍然非常低。结果是在预富集过程后, ...

Watch this Scientific Journal Video about The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying at JoVE.com

The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

百里香精油应用于肉类干燥过程中微生物负荷的影响

微生物如大肠杆菌污染肉类产品会引起食源性疾病. 精油在肉类干燥过程中的应用尚未深入研究。在此, 我们提出了一种新的方法, 将百里香精油应用于肉类干燥过程中, 以减少干肉中的微生物负荷。

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food ...

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Research

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Environment

9.2K 次观看.  Czech University of Life Sciences Prague. 微生物如大肠杆菌污染肉类产品会引起食源性疾病. 精油在肉类干燥过程中的应用尚未深入研究。在此, 我们提出了一种新的方法, 将百里香精油应用于肉类干燥过程中, 以减少干肉中的微生物负荷。

视频: The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

Preparation of Homogeneous MALDI Samples for Quantitative Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

定量应用同质MALDI样品的制备

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

科研

生物化学

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

样品制备<em&gt;结核分枝杆菌</em提取物对核磁共振的代谢组学研究

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

免疫与感染

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental stages. A specified number of spores are spread on an agar plate. This agar plate is incubated for a defined period to allow the fungi to reach the expected developmental stage, except for spores where incubation is not required. Agar plugs covered by spores, hyphae, or mycelium are next withdrawn and transferred onto agar media containing the antifungal compound to be tested either placed at a distance from the fungi or in contact. This method is applicable to test both liquid extracts and solid samples (powders). It is particularly well suited for quantifying the relative contributions of volatile and non-volatile agents in bioactive mixtures and for determining their effects, specifically on spores, early hyphae, and mycelium.
The method is highly relevant for the characterization of the antifungal activity of biocontrol products, notably plant-derived products. Indeed, for plant treatment, the results can be used to guide the choice of mode of application and to establish the trigger thresholds.

We describe a modified agar-based method designed to quantify the antifungal effects of plant-derived products. Both volatile and non-volatile contributions to the antifungal activity can be assessed through this protocol. In addition, efficacy against fungi can be measured at key developmental stages in a single experimental setup.

测量生物控制产品的挥发性和非挥发性抗真菌活性

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental ...

环境科学

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

通过成像质谱分析 研究 感染期间琼脂和组织中生长的细菌菌落的微生物合作

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical ...

Therapeutic Effectiveness of a Dietary Supplement for Management of Halitosis in Dogs

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized placebo-controlled crossover clinical evaluation, we assessed&#160;the effectiveness of a dedicated dietary supplement to improve chronic halitosis in 32 dogs of different breeds and ages. This protocol describes how to evalute the presence of oral volatile suphur compunds, e.g. methyl mercaptan, hydrogen sulfide and dimethyl sulfide, by means of a portable gas chromatograph device coupled with a syringe, which was used to collect the breath, and a dedicated software, which allows the operator to monitor each compound concentration during each measurement, in a relatively short time (8 min).
A significant modification of halitosis parameters was observed after 30 days since the beginning of treatment (p &#60;0.05), while a long-lasting effect was still observed even 20 days after the suspension of the treatment. Portable gas chromatograph, which is also widely used in clinical practice, can be therefore used to confirm&#160;and control halitosis in humans and animals. Even though human and animal species present some differences,&#160;this innovative and alternative therapy for halitosis management might be extended to human clinical practice as an adjuvant dietary approach.

We describe a simple approach for diagnosis of halitosis in dogs as well as a dietary approach for its management. This protocol may be extended to the management of halitosis in humans in the near future.

膳食补充剂的口臭犬管理疗效

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized ...

医学

Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as an&#160;innovative source of such new compounds. One bottleneck in the process is typically the elucidation of their mode-of-action. We recently established isothermal microcalorimetry as part of a routine profiling pipeline. This technology allows for investigating the effect of antibiotic exposure on the total bacterial metabolic response, including processes that are decoupled from biomass formation. Importantly, bacteriostatic and bactericidal effects are easily distinguishable without any user intervention during the measurements. However, isothermal microcalorimetry is a rather new approach and applying this method to different bacterial species usually requires pre-evaluation of suitable measurement conditions. There are some reference thermograms available of certain bacteria, greatly facilitating interpretation of results. As the pool of reference data is steadily growing, we expect the methodology to have increasing impact in the future and expect it to allow for in-depth fingerprint analyses enabling the differentiation of antibiotic classes.

The elucidation of the mode-of-action of a novel antibiotic is a challenging task in the drug discovery process. The goal of the method described here is the application of isothermal microcalorimetry using calScreener in antibacterial profiling to provide additional insight into drug-microbe interactions.

使用等温微计算法确定新天然产物的杀菌或杀菌作用的代谢分析

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as ...

生物工程

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers&#8217; safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 &#176;C, 85 &#176;C, or 95 &#176;C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 &#181;L of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of &#60;1 log10eCFU/mL (i.e., &#60;10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

A Tuberculosis Molecular Bacterial Load Assay TB-MBLA

We describe a tuberculosis molecular bacterial load assay test performed after heat inactivation of sputum. Heat inactivation renders sputum samples noninfectious and obviates the need for containment level 3 laboratories for tuberculosis molecular tests.

结核病分子细菌负荷测定（TB-MBLA）

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

用化学干燥进行扫描电镜 (sem) 形态分析的原核生物和真核生物的制备

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

生物学

纳米草本涂层织物的抗菌功效

An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and burns. These cotton-based protective coats provide ideal conditions for microbial growth and attachment sites due to their porous nature, moisture-holding capacity, and retention of warmth from the user&#39;s body. Several studies have demonstrated the survival of pathogenic bacteria on hospital garments and lab coats, acting as vectors of microbial transmission.
A common approach to fix these problems is the application of antimicrobial agents in textile finishing, but concerns have been raised due to the toxicity and environmental effects of many synthetic chemicals. The ongoing pandemic has also opened a window for the investigation of effective antimicrobials and eco-friendly and toxic-free formulations. This study uses two natural bioactive compounds, carvacrol and thymol, encapsulated in chitosan nanoparticles, which guarantee effective protection against four human pathogens with up to a 4-log reduction (99.99%). These pathogens are frequently detected in lab coats used in biohazard laboratories.
The treated fabrics also resisted up to 10 wash cycles with 90% microbial reduction, which is sufficient for the intended use. We made modifications to the existing standard fabric tests to better represent the typical scenarios of lab coat usage. These refinements allow for a more accurate evaluation of the effectiveness of antimicrobial lab coats and for the simulation of the fate of any accidental microbial spills that must be neutralized within a short time. Further studies are recommended to investigate the accumulation of pathogens over time on antimicrobial lab coats compared to regular protective coats.

作者聚焦：使用精油纳米草药封装的抗菌织物  

Antimicrobial lab coats prevent the cross-contamination of pathogen accumulation and accidental bio-spills. Here, we describe the protocol for developing a skin-friendly antimicrobial fabric using nano-herbal encapsulation and modified standard tests to precisely evaluate the efficacy and suitability for typical usage of the lab coat.

一种使用纳米草本封装精油的抗菌织物

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and ...

百里香精油应用于肉类干燥过程中微生物负荷的影响

摘要

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此视频中的章节

定量应用同质MALDI样品的制备

样品制备

测量生物控制产品的挥发性和非挥发性抗真菌活性

通过成像质谱分析研究感染期间琼脂和组织中生长的细菌菌落的微生物合作

膳食补充剂的口臭犬管理疗效

使用等温微计算法确定新天然产物的杀菌或杀菌作用的代谢分析

结核病分子细菌负荷测定（TB-MBLA）

用化学干燥进行扫描电镜 (sem) 形态分析的原核生物和真核生物的制备

一种使用纳米草本封装精油的抗菌织物

一种使用纳米草本封装精油的抗菌织物

百里香精油应用于肉类干燥过程中微生物负荷的影响

摘要

探索更多视频

此视频中的章节

定量应用同质MALDI样品的制备

样品制备

测量生物控制产品的挥发性和非挥发性抗真菌活性

通过成像质谱分析 研究 感染期间琼脂和组织中生长的细菌菌落的微生物合作

膳食补充剂的口臭犬管理疗效

使用等温微计算法确定新天然产物的杀菌或杀菌作用的代谢分析

结核病分子细菌负荷测定（TB-MBLA）

用化学干燥进行扫描电镜 (sem) 形态分析的原核生物和真核生物的制备

一种使用纳米草本封装精油的抗菌织物

一种使用纳米草本封装精油的抗菌织物

通过成像质谱分析研究感染期间琼脂和组织中生长的细菌菌落的微生物合作