Strategies to understand and reduce the spoilage potential of low and non-alcoholic beverages

发酵饮料被认为是“人类食用安全”的产品，主要原因是病原体与最终产品无关。然而，近年来，由于消费者的喜好，“不含酒精或低度酒精饮料”(NABLAB‘s)越来越受欢迎。这一类别反映了酿造行业内快速增长的市场，从2015年占全球所有产品的0.1%扩大到目前的5%，到2025年进一步增长预计将达到约33%。虽然NABLAB的设计通常是为了模仿酒精饮料的风味特征，但它们在组成方面有根本的不同，并且由于营养物质浓度相对较高、pH值较高、碳化程度较温和以及乙醇含量较低而面临更高的微生物污染风险。因此，NABLAB产品变质的报告大幅增加，帝亚吉欧最近的一次重大产品召回突显了这一问题。在这个项目中，我们的目标是全面了解在整个酿造过程中与生产NABLAB相关的微生物风险，包括被以前被认为是低威胁的微生物污染的可能性。我们的目标是选择能够在NABLAB环境中茁壮成长的生物，并分析它们的生理和生长偏好，以期通过有针对性的方法防止污染。这一主题将通过以下4个工作包来解决：WP1--关键污染物的识别(0-12个月)这将通过一系列现场调查进行，重点是原材料、制造过程、产品的储存和运输以及销售点的货物。为了实现这一目标，我们将利用UON酿造小组目前的专业知识，开发一种基于元基因组的策略(MINION)，以在每个过程阶段完全捕获微生物组。还将使用WP2的标准微生物技术来分离微生物。WP2-微生物特征(9-18个月)为了了解腐败潜力，将使用Biolog表型微阵列分析来评估NABLAB在一系列条件下的微生物生长。其目的是了解微生物的生物学，包括它们对抗菌剂(啤酒花化合物、亚硫酸盐和苯甲酸盐)的耐受性，它们对啤酒特性(酒精、pH值)的耐受性，以及它们的营养需求(糖和必需离子)。我们已经在应用Biolog PM技术分析生长抑制剂的效果和模拟微生物种群动态方面拥有广泛的专业知识2-3。WP3-产品和腐败潜力之间的关系(18-27个月)这里我们的目标是确定是否所有NABLAB产品都具有相同的腐败潜力，并确定增强或防止污染的关键属性(营养或成分)。我们将使用在WP1中开发的元基因组方法来跟踪随时间的腐败，并通过种群监测来确定随着时间的推移多种生物对腐败的影响。WP4-最大限度地发挥防腐战略的影响(24-36个月)WP3和WP4的收益将应用于关键产品，以便使用有针对性的方法加强当前的保鲜战略。其目标是减少对人工防腐剂的依赖，并增强自然特性，以防止微生物生长和腐败。

Fermented beverages are considered to be products which are 'safe for human consumption', for the primary reason that pathogenic organisms are not associated with the final product. However, in recent years, consumer preferences have led to the growth in popularity of 'no or low alcohol beverages' (NABLAB's). This category reflects a rapidly growing market within the brewing sector, expanding from <0.1% of all products in 2015 to 5% global consumption at the current time and further growth expected to reach ~33% by 2025. Although often designed to mimic alcoholic beverage flavour profiles, NABLAB's are fundamentally different in terms of their composition and are at higher risk of microbial contamination due to the relatively high concentration of nutrients, elevated pH, milder carbonation levels and reduction in ethanol1. Consequently, reports of spoilage in NABLAB products have dramatically increased, an issue highlighted by a recent major product recall by Diageo. In this project we aim to fully understand the microbial risks associated with production of NABLAB's across the brewing process, including the potential for contamination by organisms previously considered to be of low threat. We aim to select for organisms capable of thriving in the NABLAB environment and analyse their physiology and growth preferences with the ambition of preventing contamination via a targeted approach. This topic will be addressed through 4 work packages as described below: WP1 - Identification of key contaminants (0-12 months) This will be conducted via a series of site surveys focused on raw materials, the manufacturing process, storage and transport of products, and goods at point of sale. To achieve this, we will develop a metagenomic based strategy (MinION) using current expertise within the brewing group at UoN, to fully capture the microbiome at each process stage. Microbes will also be isolated using standard microbiological techniques for WP2. WP2 - Characterisation of microbes (9-18 months) To understand spoilage potential, BioLog Phenotype Microarray analysis will be used to assess microbial growth in NABLAB's under a range of conditions. The aim is to understand the biology of microorganisms, including their resilience to antimicrobials (hop compounds, sulphites and benzoates), their tolerances to beer properties (alcohol, pH) and their nutritional requirements (sugars and essential ions). We already have extensive expertise in the application of Biolog PM technology to analyse for the effect of growth inhibitors and in modelling microbial population dynamics2-3. WP3 - Relationship between product and spoilage potential (18-27 months) Here we aim to determine if all NABLAB products have the same spoilage potential and to identify key attributes (nutritional or compositional) which enhance or prevent contamination. We will use the metagenomic approach developed in WP1 to track spoilage over time and to determine the impact of multiple organisms on spoilage over time through population monitoring. WP4 - Maximising the impact of preservative strategies (24-36 months)Learnings from WP3 and WP4 will be applied to key products in order to enhance current preservation strategies using a targeted approach. The goal is to reduce dependency on artificial preservatives and to enhance natural characteristics to prevent microbial growth and spoilage.