Efficacy of natural antimicrobial systems used in foods; bacterial stress response and mode of action

尽管加拿大被认为拥有相当安全的食品供应，但加拿大公共卫生局估计，每年有400万人(每八个加拿大人中就有一个)因国内获得的食源性疾病而患病，而肉类仍然是最常见的被牵连的食物。2008年与受污染的加工肉类有关的李斯特菌病危机提醒我们，微生物是如何具有破坏性的。去年，在大肠杆菌O157：H7污染肉类事件发生后，加拿大自己的公司XL Foods被出售给一个巴西财团的美国分公司。这些事件是加拿大人面临的卫生问题的主要例子，我们的实验室正在这项提案中解决这一问题。此外，消费者对添加剂较少但仍易于制备的最低加工食品的需求，向食品科学家提出了挑战，要求他们找到创新的方法来保持我们食品供应的安全性和微生物质量。在挑战研究中，传统上通过固体生长介质上的细胞计数来评估抗菌系统的有效性。然而，过于紧张或受到伤害的活生物体不会在固体介质上直接生长以形成不同的菌落。这些可存活但不可培养的生物体在食品中的储存期可能会恢复并恢复生长。因此，如果仅仅基于细胞计数来确定抗菌治疗的疗效，可能被高估了。因此，为了深化对食品供应中微生物的有效控制，我们的干预措施需要建立在可靠的科学数据基础上。我们必须在分子水平上了解抗菌系统的有效性和微生物对它们的生理反应，以便开发有效和实用的微生物控制手段。蛋白质组学、转录组学、RT-PCR等强大的分子工具已经被用来更好地了解大肠杆菌对烹饪温度的生理反应。利用微阵列分析已经确定了几个潜在的热处理疗效的生物标志物。当细胞暴露在剧烈的热处理中时，必须确定从这些感兴趣的基因转录的mRNA的稳定性和功能性。此外，实时定量聚合酶链式反应(Real-time PCR)对热休克基因表达的定量检测显示，在60℃以上，dnaK和GroEL基因的表达水平显著下降，达到与37℃下对照细胞相似的水平，这表明在60℃以上，细胞不再能够适应，治疗开始有效。我们寻求新的替代品来控制微生物腐败和病原体，这促使我们研究了来自各种来源的天然抗菌剂(如植物精油、乳链菌素、蟹水解物中的多肽)在肉类产品中的有效性。仅在魁北克省，每年就有15000吨洋葱因各种原因被拒绝并不适合人类食用。在食品或饲料中有效利用从植物副产品中提取的多酚符合可持续农业和经济效率的全球愿景。但是，为了在肉类等复杂的食品基质中充分利用这些新型抗菌剂的潜力，必须在分子水平上深入了解细菌的应激反应和作用模式，并将在本提案中进行研究。

Although Canada is recognized to have a rather safe food supply, the Public Health Agency of Canada estimates that four million people (one in eight Canadians) get sick every year due to domestically acquired foodborne illnesses and meat remains the most often incriminated food. The 2008 listeriosis crisis related to tainted processed meat reminds us how microorganisms can be devastating and last year, XL Foods, a Canadian own company, was sold to the US branch of a Brazilian consortium following the Escherichia coli O157:H7 contaminated meat incident. Those events are prime examples of sanitary problems Canadians are facing, and that our laboratory is addressing in this proposal. Furthermore, consumer's demand for minimally processed foods with fewer additives, but that remain easy to prepare, is challenging food scientists to find innovative ways to maintain the safety and microbial quality of our food supply. The efficacy of antimicrobial systems is traditionally evaluated by cell enumeration on solid growth medium during challenge studies. However, viable organisms too stressed or injured do not grow directly to form distinct colonies on solid media. These viable but not culturable organisms may recover during the storage period in foods and resume growth. Hence, determination of the efficacy of an antimicrobial treatment can be overestimated if solely based on cell counts. Therefore, to deepen the control of microorganisms in our food supply effectively, our interventions need to be based on sound scientific data. We must understand the efficacy of antimicrobial systems and the microbial physiological response to them at the molecular level in order to develop effective and practical means of microbial control. Powerful molecular tools for proteomics, transcriptomics, RT-PCR, etc. have been used to better understand the physiological response of Escherichia coli to cooking temperatures. Several potential biomarkers of heat treatment efficacy have been identified using microarray analysis. The stability and functionality of the mRNA transcribed from those genes of interest when cells are exposed to severe heat treatment must be established. Also, quantification of heat shock gene expression by real-time PCR revealed that dnaK and groEL mRNA levels decreased significantly above 60°C to reach levels similar to those of control cells at 37°C suggesting that above 60°C, the cell is no longer able to adapt and that the treatment starts to be effective. Our search for new alternatives to control microbial spoilage and pathogens has leaded us to study the efficacy of natural antimicrobials from various sources (e.g., essential oils of plant origins, nisin, peptides from crab hydrolysate) in meat products. In Quebec alone, 15 000 tonnes of onion per year are rejected and found unfit for human consumption for various reasons. Effective use of polyphenols extracted from by-products of plant productions in food or feed is in line with a global vision for sustainable agriculture and economic efficiency. But in order to use those novel antimicrobials to their full potential in a complex food matrix such as meat, a deep understanding of the bacterial stress response and mode of action at the molecular level is mandatory and will be investigated in this proposal.