Elucidating the role of low molecular weight peptides (< 1 kDa) as Maillard reactant flavour precursors in selected cooked white and red meats using a flavouromics approach

肉味是通过生肉中的水和脂溶性风味前体之间的化学反应加热形成的。虽然游离氨基酸、还原糖和磷酸化糖在美拉德反应中起主要作用，但小肽在肉和相关产品中作为美拉德反应物的作用有待阐明。将研究低分子量肽作为各种肉类中关键风味前体的作用，它们的形成、鉴定和对关键挥发性有机化合物的产生的贡献，这些挥发性有机化合物对熟肉的香气和风味有重大影响。肉类提取物（鸡胸肉、鸡腿肉、牛肉和野牛肉）将使用一种开发的水提取方法制备，使用1 K Da的截止膜尺寸过滤，所得滤液将使用最近开发的高分辨率液相色谱质谱技术进行分析，以确定和量化生肉中存在的所有小尺寸化合物。模型系统包含天然浓度的已识别肽、还原糖和氨基酸（购买或合成），将用于研究加热后产生的美拉德产物。液相色谱（LC）四倍飞行时间（QTOF）质谱（MS）和气相色谱-MS/MS将用于鉴定中间化合物（非挥发性和半挥发性）和加热过程中产生的挥发性有机化合物。稳定的同位素氨基酸、多肽和糖（购买或合成）将用于阐明单个美拉德前体对关键挥发性有机化合物产生的贡献。将研究添加选定肽的模型系统和肉类样品的感官评估，以进一步阐明每种肽所起的具体作用。多元回归统计分析和偏最小二乘（PLS）统计检验将阐明肽，还原糖，氨基酸和训练后的感觉面板产生的感觉属性之间的关系。具体目标是：(1)利用高分辨率质谱技术研究肉提取物（鸡胸肉和鸡腿肉、牛肉、野牛和鱼）中低分子量（MW）肽（< 1 KDa）的形成、鉴定和定量。(2)利用稳定同位素稀释质谱技术鉴定和定量含有天然浓度选定肽和关键还原糖和磷酸化糖的模型系统中产生的挥发性有机化合物（VOCs）。(3)通过消费者可接受性测试阐明选择性小肽对选定肉类物种的熟肉香气和风味的总体可接受性的个体贡献。(4)使用训练过的感官面板确定烹饪前添加选定肽到生肉中产生的感官属性；(5)澄清不同烹饪方法对目标1至4中获得的数据的影响。新颖的建议：天然蛋白水解酶在死后肌肉中具有活性，随着时间的推移，小尺寸肽的不断产生，导致肉的嫩度显著增加，小尺寸肽作为美拉德反应物的可用性对香气和风味有潜在的重大影响。因此，这一提议在寻求提供数据来阐明低分子量肽作为美拉德反应物在肉类风味形成中的作用方面是非常新颖的。意义：许多食物中富含蛋白水解酶，产生数百种低分子量肽，在美拉德反应中充当NH2供体。正确理解多肽影响肉类和其他复杂食品风味的机制，可以开发出可接受的含有具有选择性功能特性的多肽的营养配方。

Meat flavour is formed upon heating via chemical reactions between water and fat-soluble flavour precursors present in raw meat. Although free amino acids, reducing and phosphorylated sugars play a major role via the Maillard reaction, the role of small peptides as Maillard reactants in meat and related products need to be elucidated. The role of low MW peptides as key flavour precursors in various types of meat, their formation, identification and contribution to the generation of key VOCs with a significant impact on cooked meat aroma and flavour will be investigated. Meat extracts (chicken breast, thigh, beef and bison) will be prepared using a developed water extraction method, filtered using a 1 K Da cutoff membrane size and the resulting filtrate will be analyzed using a recently developed high resolution liquid chromatography mass spectrometry technique to identify and quantify all small size compounds present in raw meat. Model systems containing natural concentrations of identified peptides, reducing sugars and amino acids (purchased or synthesized) will be used to investigate the Maillard products generated upon heating. Liquid chromatography (LC) quadruple time of flight (QTOF) Mass Spectrometry (MS) and gas chromatography-MS/MS will be used to identify the intermediary compounds (non and semi-volatiles) and the VOCs generated during heating. Stable isotope amino acids, peptides and sugars (purchased or synthesized) will be used to elucidate the contribution of individual Maillard precursors to generation of key VOCs. Sensory evaluation of model systems and meat samples with added selected peptides will be investigated to further clarify the specific role played by each peptide. Multiple regression statistical analysis and partial least squares (PLS) statistical tests will be performed to elucidate the relationship between peptides, reducing sugars, amino acids and the sensory attributes generated by trained sensory panels. Specific objectives are to: (1) Investigate the formation, identification and quantification of low molecular weight (MW) peptides (< 1 KDa) in meat extracts (chicken breast and thigh, beef, bison and fish) using high resolution mass spectrometry techniques (2) Identify and quantify the volatile organic compounds (VOCs) generated in a model system containing natural concentrations of selected peptides and key reducing and phosphorylated sugars using stable isotope dilution mass spectrometry techniques. (3) Elucidate the individual contribution of selective small peptides to the overall acceptability of cooked meat aroma and flavour in selected meat species using consumer acceptability tests. (4) Determine the sensory attributes generated with added selected peptides to raw meat prior to cooking using a trained sensory panel and (5) Clarify the effect of different cooking methods on data obtained in objectives 1 to 4. Novelty of proposal: Natural proteolytic enzymes are active in muscle post-mortem with a continuous generation of small size peptides over time resulting in a significant increase in meat tenderness and availability of small size peptides as Maillard reactants with a potential significant impact on aroma and flavour. Therefore, this proposal is highly novel in seeking to provide data to elucidate the role played by low MW peptides as Maillard reactants in flavour formation in meat. Significance: Proteolytic enzymes are abundant in many foods generating hundreds of low MW peptides that can act as NH2 donor in the Maillard reaction. A proper understanding of the mechanism by which peptides affect flavour in meat and other complex foods could enable the development of acceptable nutritional formulations containing peptides with selective functional properties.